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Molecular Cloning and Developmeatal Expression of the

75 kDa Protein of the Rat Fibrous Sheath

Demetra Moshonas

Department of Anatomy and Cell Biology McGill University

Montreal, Quebec, Canada January, 1998

A Thesis Submitted to the Faculty ofGraduate Studies and Research in Partial F u b e n t of the Requirements for the Degree of Master of Science

0 Demetra Moshonas 1998

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Short Title: Analysis and expression of the FS 75 protein.

Abstract The fibrous sheath (FS) is a cytoskeletal stnrcturt that encases the axoneme in the

p ~ c i p a l piece of the sperm;rtozoon tail. In the rat, it is composed of several proteins of

which a 75 kDa polypeptide (FS 75) is the most prominent. The objectives of this study

were to clone and sequence this protein and to characterize its transcriptional and

translational origins during spermatogenesis. We succeeded in isolating two overlapping

cDNAs encoding half of the downstream segment of the FS 75 protein. Both clones were

obtained by screening a rat testicular phagemid cDNA library with an anti-FS 75

polyclonal antibody. The upstream portioa of the FS 75 mRNA containing the initiation

codon for translation was obtained by using the Polymerase Chain Reaction (PCR)

technique, a pair of specific primers and a Icgtl 1 cDNA rat library. The amino acid

sequence of the longest possible open reading b e of the rat FS 75 was found to be

almost identical to two previously cloned major FS polypeptides of mouse spermatozoa.

Sequence analysis of the rat FS 75 cDNA revealed two &-'inase &choring Dotein

(AKAP) domains and several kinase phosphorylation sites supporting the idea that this

protein plays a crucial role in the motility of spermatozoa The presence of a potential N-

myristoylation site suggests that this protein may bind covalently to the inner leaflet of

the plasma membrane (PM) which may explain the close relationship between the FS and

PM fiom early development in round spermatids (step 2 of spenniogmesis) to maturation

in spermatozoa Developmental Northern Blot analysis and in situ hybridization nvealed

that the FS 75 mRNA is mainly haploid expressed with an abundant level of mRNA in

round spennatids. Maximum levels of the FS 75 polypeptide, determined by

irnmunocytochemistry, comlated with a rapid decline in corresponding mRNA levels in

step 14 - 16 spematids. Since tmscriptionai termination occurs several steps earlier, the

bulk of the FS 75 mRNA appears to be translationally regulated.

La g a b fibreuse (Fibrous Sheath ou FS) cst me structure cytoskeienique qui

encercle l'axonhme de la p i b principale des queues des spermatozo'ides. Chez le rat, la

FS est composCe de plusieurs protkines parmi lesquclles la protdine de 75 kDa (FS 75) est

qmtitativement la plus importante. L'objectif du pdsent travail est de cloner et d'dtablir

la sequence en acides amin6s de cette potdine et de caract&iser sa gedse

transcriptionelle et traductionelle au cours de la spermatogenese. Nous avow kussi it

isoler dew clones de cDNA dont la composition se superpose partiellement et qui, pris

ensemble, d e n t , a l'exception d'une courte sequence en aval, la presque totalite de la

sequence en acides amink de la protbbe FS 75. Ces dew clones ont W obtenus par

triage d'une iibrairie de cDNA, il I'aide d'un anticorps polyclonal anti-FS 75. Le segment

en aval du mRNA FS 75, qui contient le codon d'initiation de la traductim, a W obtenu

par la mdthode PCR ti I'aide d'arnorces spkifiques. La sequence d'acides amines, de

lecture ouverte, la plus longue de la FS 75 est presqu' identique aux deux protkines FS

des spermatozoides de souris dejh clones. L'aualyse dquentielle de cDNA FS 75 de rat

montre 1' existence de deux sdquences de protd ine-kinases d' ancrage et plusieurs sites de

kinases de la phosphorylation ce qui supporte I'hypothere que cette potdine joue un r61e

important dam la stimulation du mouvement des spermatozoldes. De plus la pdsence

d'un site potentiel de N-rnyristoylation, indique que cene prothe FS 75 peut Otre Me au

feuillet interne de la membrane cytoplasmique. Cette observation concorde bien avec le

fait que la gaine fibreuse est dtroitement associCe B la membrane cytoplasmique des

spermatides au cours de la spermiogen&se. L'analyse par la mdthode Northern Blot et par

hybridation in situ montre que le mRNA respo~sable de la FS 75 est exprimd par les

jeunes spermatides rondes. La quantitd maximale de FS 75, ddtefminde par

immwmqtochimie se trouve dans les spermatides allongees aux &apes 14 ii 16 de la

spermiogen&e, te. au moment oh la quantitt du mRNA commence B ddcliner. Puisque la

transcription du DNA se termhe beaucoup plus tbt au cows de la spermiogedse, i.e. aux

&apes 8 et 9, il semble donc que la plus grande fhction du mRNA FS 75 est r6gularisCe

beaucoup plus tad au c o r n de la spamiogenhe.

To Costa, Mom, Dad, Anita, Paulina

and

in loving memory of Uncle George

Acknowledgements

FCAR: To all the members on this committee, you awarded me with a scholarship that allowed me to continue my education, thank you very much for giving me this opportunity.

Dr. Clermont: From our very first encounter I knew you would be the nucleus of my enthusiasm for science and I have never been disappointed. You showed me how to strive for academic excellence but more importantly you helped direct my energy and guide me in my pursuits. I will always remember you fondly snd I will always be thankful for your kindness, honesty and hospitality.

Quig Zhao: You always made yourself available to answer my questions and give me excellent advice. You exposed me to the world of molecular biology and you were the first to unravel the secrets of how to succeed in performing molecular techniques. You were my peer and confidanf I will always remember and appreciate your help in my endeavors.

To my supervisors: Dr. Carlos Morales and Dr. Richard Oko: Thank you for all your support and guidance. 'Ihsnk you for always being available to answer my questions - time is precious and every minute you allotted me wss appreciated and acknowledged More importantly, t h d you for making my graduate experience w0ndedb.l.

Dr. Mohamed El-A@: Thank you for all your help in the laboratory, especially for teaching me how to use most of the laboratory equipment.

To Mr. Nice at the Sheldon Center: Thank you for sequencing my final cDNA product.

To my husband, Constantinos, thank you for guiding me into the world of academia. You were the backbone of this endeavor and encouraged me simply by being yourself. Thanks especially for sharing with me that twisted thing you call "a sense of humor". I love you very much.

To my Mom and Dad, Anita and Paulina, you have seen and experienced (not to mention photographed) my struggle to achieve excellence. There were times when only your unconditional love, support and belief in me got me through it all. To Anita and Paulina, thank you for your prayers, secret notes on the door, silly jokes, encouragement and belief and the constant supply of cookies. Dad, thank you for always believing in me, for keeping me well nourished, for our coffee breaks and, of course, for the many card and tavli games we played. Mom, thank you for all the time you spent (studying) with me, for the greek coffee and most of all for the encouragement during those pivotal times when I just couldn't go on. You all helped me laugh thmugh it all, I love you dl very much.

This research was supported by an M.RC. grant to Dr. Carlos R Morales, Dr. Richard Oko and Dr. Yves Ckmnont.

To alI the graduate students and staff members whom I became close with, Andrea, Jam, Pauline, Stephanie, thank you for makiag this year memorable.

To Chi Chi and Karen: who were always available when I needed a lift and who flatted me to no end by laughing at my jokes (on occasion). Remember: use this.. ... not this.. ...this. Look here. .... not here .... here.

Abbreviations

AKAP: A-Kinase Anchoring Booteein ftME: beta mercaptoethanol bp: base pair(s) BSA: bovine serum albumin CAMP: cyclic adenosine monophosphate cDNA: complementary deo~bonucleic acids cGMP: cyclic guanosinemonophosphate DAB: diaminobenzidine tetrachloride d&0: distilled water dd H20: double distilled water DEPC: diethylpyrocarbonate DTT: dithiotbreitol dNTP: equimolar of dATP, dCTP, dGTP, and dTTP E, Coii: Escherichia coli EDTA: ethy lenediaminetetraacetic acid EM: electron microscope FS: fibrous sheath HCI: hydrochloric acid Hz&: hydrogen peroxide [Q -WP: radiolabeled uracil nucleotide 1%;: immunoglobulin G kb: kilo base pair(s) kDa: kilodalton(s) LiCI: lithium chloride LM: light microscope pl: micro litre pM: micromolar ml: millilitre(s) M: molar mM: mdimolar mRNA: messenger ribonuc leic acid mV. millivolts NaCl: sodium chloride NaOH: sodium hydroxide NCBI: National Center for Biotechnology Information NGS: normal goat serum nt: nucleotide PBS: phosphate buffered saline PCR: polymerase chain d o n PKA: CAMP-dependent protein kinase RL: tegulatory subunit type I RIk qplatory subunit type I[ RNA: ribnucleic acids

SDS- PAGE: sodium dodecyl sulfate- polyacrylamide gel electrophoresis TAE: Tris base, acetic acid and EDTA TBS: Tris-Base Buffered Saline TCA: trichloracetic acid Tris: hydroxymethylaminomethane TWBS: TBS containing 0.1% Tween-20 UV: ultraviolet X-Gal: 5-bromo4chlom-3 -indo ly 1-beta D-galactoside

... ............................................................................................ Abstract 111

........................................................................................... RhmC vi ......................................................................................... Dedication v

............................................................................ Acknowledgements vi .. .................................................................................... Abbreviations vu

...................................................................... INTRODUCTION 1

II . REVIEW OF THE LITERATURE ............................................. 3

1 . Organization of the Mammalian Spermatozoon .................. 3

2 . Flagellar Components ........................................................ 4 ......................................... A) General Structure of the Tail 4

...................................................... B) Connecting Piece 4 C) Axoneme ................................................................ 5

................................................... D) Outer Dense Fibers 6 E) Mitochondria1 Sheath .................................................. 7 F) Fibrous Sheath .......................................................... 8

................................................... G ) Plasma Membrane 10

......................................................................... 3 . Cell Cycle 10 ....................................................... A) Spermatogenesis 10

........................................................ B) Spenniogenesis 11

............................. 4 . Tail Formation during Spermiogenesis 11 ....................... A) Connecting Piece and Axoneme Formation 11

................................... B) Mitochondria1 Sheath Formation 12 ....................................... C) Outer Dense Fiber Formation 13

......................................... D) Fibrous Sheath Formation -13

. 5 Kinrses ............................................................................ 14 ............................. A) General Phosphorylation Mechanism 14

.......................................................... B) Cyclic AMP 14 C) Cyclic AMP Dependent Protein Kiaase: A General . . ............................................................ Descnptmn 15

........ D) Selectivity of Cyclic AMP Dependent Rotein Kinase -15 .................................... E) A-Kinase-Anchoring-Proteins -16

F) Cyclic AMP-Dependent Protein Kinase-A and their .............................................. Localization to the FS. 17 .. .............................................................. G) Mothty -17

6 . Identification of Fibrous Sheath Proteins .......................... 18 A) Historical Background ............................................... 18

...... i) Light and Electron Microscope Radioautography -18 ii) Antibody Localization ....................................... 18

B) Immunocytochemical and Irnmunofluorescent Localization of FS Proteins ......................................................... 19

C) Molecular Studies ..................................................... 19

III . FIGURES ................................................................................. 21

IV . MATERIALSANDMETHODS ................................................ 34

1 . Fibrous Sheath Extraction and Antibody Production ......... 34 A) Isolation of the Fibrous Sheath ..................................... 34

............. B) Reparation of Immune Sera against the FS 75 kDa 35 ................. C) Affinity Purification of Anti-FS 75 Antibody ..... 35

................................................... D) Peptide Sequencing 36

.......................................... 2 . Immunocytochemistry ......... .. 36 A) Tissue Preparation for Light Microscopy ......................... 36

............. ... ... B) Light Microscopy Immunocytochemistry .. .. 37 C) Tissue Preparation for Electron Microscopy ..................... -38 D) Electron Microscopy Immunocytochemistry ...... .... ....... 38

........................................................ 3 . Immunofluorescence 38

4 . Isolation of cDNA Clones .............. .. .... .. .................. 3 9

5 . Polymerase Chain Reaction Technique ............ ... ........ 39 ............................................... A) Designing Primer 39

................................................ B) PCR Technique 40 C) Analysis of PCR Products .......................................... -41

................................. i) Agarose Gel Electrophoresis 41 ................................................ ii) DNA Isolation 41

................................................ 6 . Cloning of PCR Products 42 ............................................................... A) Ligation 42

B) Transformation ....................................................... 43 C) Identification of the Positive Clones .............................. 43 D) Isolation of Recombinant Plasmid DNA ......................... 44

7 . Northern Blot .............................................................. 4 A) Total Testicular RNA Extraction ................................... 44

..................................... i) Reparation of Solutions 44 ii) Total RNA Isolation and Purification ..................... 45

..................................................... B) Analysis of RNA 46 ......................... C) Transfer of RNA tiom Gel to Membrane -46

D) Hybridization Analysis ............................................... 47

....................................................... 8 . In Situ Hybridization 47

V . RESULTS ............... .. ...... .. .................................................. 48

......................... Light Microscope Immunocytochemistry 48

Electron Microscope Immunocytocbemistry ..................... -48

Immunofluorescence .................... W

................................................. Isolation of cDNA Clones 49

...................................... PCR Cloning ................... ... -49 ....................................... A) Analysis of the PCR Products 49 ........................................ B) Ligation and Transformation 50

.............................. C) Identification of the Positive Clones -50 ..................................... D) Sequencing of the PCR Product 50

................................................... Northern Blot Analysis -51

In Situ Hybridization ....................................................... 52

V I . FIGURES ...................*........*.......................................*............ 53

.......................................................................... MI . DISCUSSION 64

............................................. .................... VIII . CONCLUSIONS .. 69

..................................... ORIGINAL C O ~ R I B ~ I O N S ...... .. 7 0

I. INTRODUCTION

With the exception of mitochondria, the flagellum (tail) of the mammalian

spermatozoa is composed of structural proteins that are synthesized and assembled during

the haploid phase of spermatogenesis (Oko and Morales, 1996). Except for certain

tubulhs (Distel et al., 1984; Hermo et al., 1991) and actins (Flaherty et al., 1983; Breed

and Leigh, 199 1, Oko et al., 1 Wl), the majority of sperm cytoskeletal proteins appear to

have no structural counterparts in somatic cells (Oko and Morales, 1996). Some

specialized cytoskeletal elements found in the spermatozoon's tail include the outer dense

fibers (ODF), the fibrous sheath (FS), the submitochondrial reticulum, and the striated

collar and capitulum of the neck piece (reviewed by Oko and Clermont, 1989).

The fibrous sheath is located in the principal piece of the tail immediately beneath

the plasma membrane and encases the outer dense fibers and axoneme. It consists of two

longitudinal columns located on opposite sides of the axoneme co~ected by ribs oriented

circumferentially. The longitudinal columns of the FS run adjacent to microtubule

doublets three and eight, replacing the two corresponding ODF. Previous investigations

indicate that the rat FS is composed of many polypeptides, of which three are major (75,

27.5 and 14.4 kDa), nine are intermediate, and the others are minor (Oko and Clermont,

1988).

Past and recent morphological and immunocytochemical studies suggest that FS

proteins are synthesized during the latter half of spermiogenesis. During this period of

synthesis, the FS proteins assemble in a distal to proximal direction along the tail (Irons

and Clermont, 19821; Oko ad Clermont, 1989; Clermont et al., 1990a). This process is

preceded by a FS anlagen that also assembles in a proximal to distal direction and is

closely associated with the plasma membrane of the spermatid tail. The anlagen appears

in the distal portion of the tail as early as step 2 of spermiogenesis (Oko and Clermont,

1988; Clermont et al., 1990a).

An mRNA encoding a major mouse FS protein has been cloned and characterized

in two different laboratories (Camra et aL, 1994; Fulcher et d., 1995). Carrera et al.

(1994) showed that the translation of the p82 cloned protein product is a precursor of

approximately 92 kDa which is processed into a 72 lrDa protein just before FS assembly.

Fulcher et al. (1995) showed that the transcript of the Fscl cloned protein appears to be

testis specific and haploid expressed. It has been suggested that the FS provides an elastic

rigidity for sperm motility. however additional bctions are being considered. Carrera et

d. (1994) found two putative A - h e Anchoring Dotein-like (AKAP) regional domains

within the major mouse FS polypeptide that normally binds a regulatory subunit (RU) of

CAMP-dependent protein kinase (PKA). This suggests that the 72 kDa polypeptide may

act as a scaffolding protein for the subcellular localization of regulatory proteins in the

tail (Carrera et al., 1994).

Determination of the molecular mass of the most abundant constituent of the FS in

rat (i.e., our 75 kDa protein) has varied between investigators, it has also been reported as

an 80 kDa or 82 kDa protein by Olson et ai. (1976) and Carrera et al. (1994) respectively.

A study conducted by Horowitz et al. (1988) showed that the RII regulatory subunit of

P K A binds an 80 kDa rat sperm flagellar protein. Funhennore, Brito et al. (1989)

showed that the major FS polypeptide in the rat is a phosphoprotein. These results and

our analysis of the rat FS 75 suggests that phosphorylation of this protein plays a crucial

role in sperm motility.

The objective of this study was to clone and deduce the amino acid sequence of a

cDNA encoding the entire rat FS 75 protein and to developmentally analyze the

transcription and translation of the FS 75 mRNA. This study includes a thorough analysis

of the cloned cDNA sequence and a complete analysis of the expression and regulation of

the rat FS 75 mRNA.

In the following section, a review of the Literature wili examine flagellar

components and their formation in spermatozoa. Particular emphasize will be placed on

the structure and location of the fibrous sheath and how this relates to its potential

function. Some focus will be place on kinases; their function, selectivity and localization

may be associated with the 75 kDa fibrous sheath protein.

11. REVIEW OF THE LITERATURE

1. Organization of the Mammalian Spermatozoon

Leeuwenhoek fint discovered the spermatozoon in 1677 and up until the 1950's.

knowledge of its structure was limited to the use of the light microscope (LM). The first

review article characterizing the major components of mammalian sperm ultrastructure

appeared in 1958, by Fawcett, soon after the advent of the transmission electron

microscope (EM). These components are illustrated in Figure I. The head of the sperm

consists of a nucleus covered by an acrosomal cap. The head is joined to the sperm tail

or figellurn by a junctional structure called the connecting piece. The flagellum is

subdivided into three segments, fkom proximal to distal these segments are known as: the

middle, principci and end piece respectively. Each segment is made up of more than one

structurai component. A centrally located structure called the axoneme extends from the

connecting piece to the tip of the tail. It is comprised of the typical arrangement of a

central pair of microtubules surrounded by a ring of nine evenly spaced doublet

microtubules (Fig. 2). This "9 + 2" arrangement of microtubules is a universal

occurrence in cilia and flagella in both the plant and animal kiagdom (Fawcett, 1975).

The middle piece extends from the connecting piece to the annulus (Fig. 1). The annulus

is a ring-like component associated with the plasma membrane. In the middle piece, the

axowme is encircled by a set of nine circumferentiaily oriented elements known as the

outer denrefibers which in turn are surrounded by another sheath made of mitochondria

called the mitochondria1 sheath (Fig. 3). Located between the annulus and the end piece

is the principal piece (Fig. 1). This piece consists of the central axoneme, a set of nine

circumferential outer dense fibers and afibrow sheath (Figs. 4 4 4b). The fibrous sheath

consists of two longitudinal columns joined by circumferentially oriented ribs (Figs. 4%

4b). The most distal segment of the flagellum is called the end piece (Fig. 1). The end

piece consists of a short terminal segment composed only of the axonema1 core. The

axonerne retains its 9 + 2 microtubule arrangement at the proximal end but becomes

dissociated at its distal end (Irons and Clennon~ 1982a). The entire elongated cell is

enclosed in a plasma membrane.

2. Flagellar Components

A) General Structure of the Tai4

A complex organization of structural components within the flagellum determines

and characterizes sperm movement. Its development is the consequence of a series of

programmed cytological events that take place in the tail during the long transformaton

fiom spermatid to spermatozoon. The length of the tail varies between species, in the rat

it is 190 pn long, in man it is considerably shorter averaging about 60 pm (Oko and

Clermont, 1990). The tail of spermatozoa cannot be equated to the cilia or flagella of

unicellular organisms. It is true that it contains a contractile axonerne composed of a

central pair of microtubules surrounded by nine peripheral doublets, but in spermatozoa,

the axoneme is also associated with many cytoskeletal components such as the outer

dense fibers and the fibrous sheath, making it unique (Oko and Clermont 1990). I will

review these and other cytoskeletal components in order to provide a better understanding

of the fibrous sheath.

B) Connecting Piece

Joining the sperm head to the tail is an articular structure known as the connecting

piece composed of the capitulum and striated collar (Fig. 5). The connecting piece (CP)

is attached to the basal plate of the head by numerous bridging filaments. The portion of

the CP that directly articulates with the head of the spermatozoon is the capitulum.

Attached to the undersurface of the capitulum are nine columns of the striated collar. The

striated collar is continuous distally with the nine outer dense fibers (ODF) of the middle

piece along which mitochondria are helically arranged (Fig. 5a) (Fawcett, 1969, 1979).

In most species, a transverse or obliquely oriented centriole known as the proximal

centriole is embedded in the substance of the CP, d i d to the capitulum. This disappears

before maturation is complete leaving an empty compartment in the CP called a vault

(Fig. 5b) (Woolley and Fawcett, 1973).

C) Axoneme

The axoneme traverses almost the entire length of the spermatozoa tail &om the

proximal tip of the middle piece to the distal tip of the end piece. The microtubules that

form the axoneme are aligned in parallel with the long axis of the flagellum (Fig. 6).

There are two centrally located microtubufes and nine peripheral doublet microtubules

(Fig. 2). In cross section, the two central microtubules appear circular and a narrow gap, '

traversed by intermittent cross-bridges, exists between them (Fig. 2). The peripheral

doublets are made up of two components; subunit A composes a complete circular

microtubule, while subunit B is C-shaped, with its ends abutted against the wall of

subunit A (Fig. 2) (Fawcett, 1965). Each microtubule is comprised of linear structures

arranged in pairs of a and P tubulin subunits that form protofilaments (Tilney et al., 1973;

Schultheiss et al., 1983). The cylindrical microtubules of the central pair and subunit A

of the doublets each contain 13 protofilaments, whereas the incomplete walls of the B

subunits are made up of l l protofilaments (Fig. 2) (Tilney et al., 1973). Subunit A

provides attachment of two a m directed toward the adjacent doublet (Fig. 2). The two

arms are comprised mainly of dynein, a Mg stimulated protein with ATPase activity

(Gibbons, 1981). These arms that extend fkom the subunit A toward subunit B are

responsible for generating ATP-dependent sliding movements between doublet

microtubules (Satir, 1979; Sale and Satir, 1977). Mutants lacking outer arms are capable

of normal motility albeit at a reduced rate (Kamiya and Okamoto, 1985; Mitchell and

Rosenbaum, 1985). Mutants lacking the inner arms are completely paralyzed (Huang et

al., 1979). This suggest that the inner arms are essential for movement of cilia and

flagella while the outer arms aid in the force and speed of axonemaf bending (Gibbons

and Gibbons, 1976). Nexin bridges provide a circde~ential connection between subunit

A of the microtubule doublet and the radial spokes, which project inward to join with a

sheath surrounding the centraf pair (Fig. 2). The shafts of the radial spokes emanate fkom

subunit A while the heads of the spokes contact the central pair. ATPase activity has

been identified at the junction between the radial spokes and the central microtubules in

human sperm (Baccetti et al., 1981; Baccetti, 1984). The primary action of the radial

spoke system, displayed by radial spdre deficient mutants, appears to convert symmetric

bending into asymmetric bending which is required for efficient swimming (Brokaw et

al., 1982). In addition to tubulin and dynein, the sperm axoneme may be composed of

telains. These proteins fom longitudinally arranged filaments possibly in or on the walls

of subunit A of the microtubule (Linck et al., 1985, 1982; Linck, 1982).

The doublets of the axoneme may be identified a c c o r ~ g to their position with

respect to the central pair, and the orientation of the arms. Thus, in the classification of

Afielius (1959), doublet number 1 lies along the plane which bisects the central pair of

microtubules (Fig. 2). The remaining doublets are numbered sequentially, in a clockwise

fashion, given that the arms on the doublets are oriented towards the neighboring doublets

in a clockwise direction (Fig. 2). This is the orientation which is seen when the flagellum

is viewed fiom its base to its tip (Phillips, 1974).

D) Outer Dense Fibers

Mammalian spermatozoa are characterized by the presence of prominent

cytoskeletal fibers called outer dense fibers (ODF) which surround the axoneme

throughout much of its length (Fawcett, 1975). In the middle piece, there is a set of nine

such fibers (Fig. 3). In the principle piece, two of these fibers (#3 and #8) are replaced by

the longitudinal columns of the fibrous sheath (Fig. 4a). Each ODF follows a longitudinal

course peripherally along side one of the axonemal doublets (Figs. 3, 4a) and is

continuous, at its proximal extremity, with the striated collar of the connecting piece (Fig.

5). At its distal end, each fiber appears to be attached to its corresponding microtubule

doublet. There is a variation in size and cross sectional shape of each fiber and each is

composed of a central medulla and an outer thin cortex (Olson and Sammons, 1980;

Fawcett, 1970). All nine fibers taper along their length in a proximal to distal direction

and those fibers designated 1, 5, and 6 are generally larger than the others (Fig. 4a)

(Fawcett, 1975). It is also observed that the fibers terminate at different levels in a

regular sequence that relates to their ioitial thiclmess. Thus fibers 3 and 8 end at the most

proximal level being replaced by the longitudinal columns of the FS, followed by 4 and 7,

then 2 and 9. Fibers 1,5, and 6 terminate most distally, extending throughout the greater

part of the principal piece ( T e h et al., 1961).

The polypeptide composition of rat ODF has been analyzed by several

investigators and found to be composed of 14 ODF polypeptides, 5 are major and 9 are

minor (Olson and Sammons, 1980; Calvin, 1979; Vera et al., 1984; Oko, 1988). Amino

acid composition analysis showed that the major rat ODF polypeptides have a high

content of lysine, arginine, cysteine, aspartate, serine, leucine and proline. Also, large

amounts of phosphate have been found bound as phosphoserine (Olson and Sammons,

1980; Vera et al., 1984). The ODF are resistant to solubilization in ionic detergents i.e.

sodium dodecyl sulfate (SDS) because of their high content of disulfide bonds (Calvin

and Bedford, 1971 ; Calvin, 1976). Zinc is a constituent of the ODF proteins and is likely

involved in regulating the extent of disulfide cross bridging (Calvin et al., 1973; CaIvin

and Bleau, 1974; Calvin et al., 1975; Calvin, 1979). Other studies have shown that the

ODF polypeptides share biochemical similarities with major cytoskeletal components of

the spermatozoa such as the connecting piece and the perinuclear theca (Oko, 1988; Oko

and Clennont, 1989). The ODF polypeptides comprise a unique family of proteins not

found in most other mammalian somatic cells (Vera et al., 1984; Longo et al., 1987; Eddy

et al., 1987).

E) Mitochondria1 Sheath

The mitochondria1 sheath (MS) demarcates the length and location of the middle

piece due to its characteristic structure (Fig. 1). The length of the middle piece varies in

different species however it remains constant within each given species. It is 80 mm long

in the rat but only 7-8 mm long in man. The MS is constructed of many condensed and

elongated mitochondria that are packed tightly around the ODF (Figs. 3, 6) (An&&

1962). The MS probably preserves a certain degree of rigidity and their helical

disposition facilitates bending of the middle piece (Fawcctt, 1970; Harris, 1976; Phillips,

1977). The shape of the mitochondria appears to remain stabile due to an SDS insoluble

shell (Calvin, 1978; Pallini et al., 1979; Calvin and Cooper, 1979; Calvin et al., 1981;

Calvin et al., 1987). This shell consists of three polypeptide chains linked by cysteine

bridges (Pallini et al., 1979). Selenium has been shown to selectively bind to one of the

polypeptides, the 20 kDa polypeptide, of the mitochondria1 capsule (Pallini et al., 1979;

Psllini and Bacci, 1979). Selenium substitutes sulphur forming selenocysteine

structures. Studies have documented that selenium deficiency in rodents cause

disoqyuhtion and configurational changes in the mitochondria which can kad to

immobility and secondary tail defects (Wu et al., 1973; Wu et a!., 1979; Wallace et al.,

1983a; Wallace et al., 1983b).

The structural and fimctional characteristics of the spermatozoon mitochondria are

unlike other mitochondria, they are adapted to meet the needs of the motile spermatozoa.

Some of these adaptations include: resistance to hypotonic conditions (Kayhani and

Storey, 1973); an inefficient uptake of ca2' (Storey and Keyhani, 1973) and an ability to

use lactate as an oxidative substrate as well as other specialized oxidative strategies

(Baccetti et al., 1975; Montamat and Blanco, 1976; Van Dop et al., 1977; Storey and

Kayne, 1978; Storey, 1980).

F) Fibrous Sheatb

Similar to the MS, the length of the FS defines the principal piece region. The

principle piece is located distal to the annulus and ends proximal to the end piece. It

contains the axoneme and the ODF and is surrounded by the FS, a cytoskeletal element

that traverses longitudinally along the flagellum (Fig. 6). In most matnmalian species, the

principle piece is the longest flagellar segment of the spermatozoa (Fig. 1). The

organization of the FS is similar in mammals and reptiles but is modikd in birds

(Baccetti and Afielius, 1976). The FS is composed of two structural elements: a pair of

longitudinal columns and several circlllllferentially oriented ribs (Figs. 4% 4b) (Fawcett,

1958, 1970; Sapsford, 1970). The two columns are positioned 180" apart and run

opposite microtubule 3 and 8, replacing both respective ODF (Figs. 4% 4b). Each column

appears closely associated with their respective outer microtubule axonemal doublet. The

ODF associated with microtubule 1, 2, 5, 6, 7 and 9 are still present. There are many

transverse ribs that bridge and connect the two longitudinal columns together (Fig. 4b).

Among mammals there are large variations in the thickness of these nis and in the

prominence of the longitudinal columns (Fawcett, 1970). In the rat, the riis have the

form of bands that decrease in width from the proximal to distal end of the sheath. It has

been suggested that the position of the longitudinal columns in the plane of the central

pair of microtubules restrict lateral bending while the periodic position and spacing of the

nis permit bending of the tail in a plane perpendicular to that of the central singlets.

Past biochemical analyses have shown that in rat spermatozoa, the FS is

composed predominantly of an 80 kDa polypeptide (Olson et al., 1976). More recent

evidence indicates that the rat FS is composed of 18 polypeptides of which 3 are major

(75, 27.5 and 14.4 ma), 9 are intermediate (67, 63, 46, 43, 38, 33, 20, 16 and 12.5 kDa)

and the others are minor polypeptides (Oko, 1988). The proteins o f the FS are stabilized

by disulfide bonds which make them resistant to solubilization in ionic detergents i.e.

SDS (Calvin and Bedford, 1971; Bedford and Calvin, 1974; Calvin, 1976). It appears

that the 75 kDa polypeptide isolated by Oko (1988) most likely corresponds to the 80 kDa

polypeptides of Olson et al. (1976).

An immunocomparative protein analysis of the ODF and the FS of the rat

spermatozoa showed that both contain a major 14.4 kDa polypeptide with common

antigenic and electrophoretic properties (Oko, 1988). Although the 14.4 kDa polypeptide

of the FS and ODF share antigenic determinants, they do not appear to be identical

because they immunolocalize differently during spenniogenesis and assemble in different

directions along the axoneme (Oko and Clermont, 1989). The FS has also been shown to

contain similar antigenic determinants, or epitopes, with a major 16 kDa polypeptide of

the perinuclear theca found in the head of the spermatozoon (Oko and Clermont, 1988).

The findings that there are common molecular weights and antigenic determinants for

some of the major cytoskeletal polypeptides of both the head and tail of spermatozoa

suggests that the cytoskeletal proteins of the germ cell may have evolved from a common

ancestral gene pool (Oko and Clermont, 1990).

Investigators have suggested that the FS contains keratin-like proteins, based on

its relative insolubility and abundant disulfide cross-linking (Bedford and Calvin, 1974;

Calvin, 1975). Immunocomparative studies show littie resemblance if any between

intermediate filaments (keratin, desmin, vimentin) of somatic cells and cytoskeletal

proteins of spermatogenic cells (Longo et al., 1987; Eddy et al., 1987; Franke et al., 1979;

van Vorstenbosch et al., 1984; Puchtler and Meloan, 1985; Kiemzenbaum et al., 1986;

Fenderson et al., 1988). These studies negate previous comparisons that have linked the

FS in the same family as keratins. This suggests that the proteins making up the FS may

be derived fiom a set of genes that are uniquely expressed during spermatogenesis.

G) Plasma Membrane

The plasma membrane (PM) of mammalian spermatozoa has been extensively

analyzed in recent years and well reviewed (Eddy, 1988). Freeze hcture replicas of the

PM overlying the tail of mature spermatozoa were used to examine its structural

characteristics. I will consider only some pertinent points in regards to these PM features.

The PM covering the principal piece of spermatozoa in guinea pig has been shown

to contain a double row of widely spaced (8-9 MI) transmembrane particles referred to as

a zipper (Fig. 7a). This zipper courses longitudinally over the ribs of the FS opposite

ODF number 1 (Fig. 7b). Identically located in the rat is a single strand of the

trammembrane particles, although it is more prominent in the guinea pig than in the rat.

In both species, the longitudinal arrays terminate before reaching the end piece.

Morphologically, this zipper has been shown to form some type of binding to the

underlying FS (Fig. 7b) (Friend and Fawcett, 1974; Friend and Rudolf, 1974; Fawcett,

1 975; Friend, 1977).

Treatment of sperm with digitonin, a detergent that disrupts sterol-rich membranes

but spares sterol-poor areas, leaves the plasma transmembrane particles of the zipper

intact while disrupting the surrounding membrane (Friend and Rudolc 1974; Elias et al.,

1 978). Subsequent treatment with Triton-X- 100 solubiiizes these digitonin resistant

proteins and permits their isolation. Polypeptides analyzed !?om such Triton-X-100

soluble bctions have revealed several polypeptides ranging from 24 to 110 kDa (Enders

et al., 1983). The kc t ion of these various iatramernbranous proteins as well as the

mechanism involved in their formation is poorly understood.

3. Cell Cycle

A) Spermatqencris

Spermatogenesis represents a complex system of cellular differentiation involving

mitotic stem cell proliferation and meiosis and has been divided into 14 stages (Fig. 8)

(Leblond and Clermont, 1 W2b; Bellve, 1979). Subsequent remodeling of haploid

spermatids produce mature spermatozoa. Remodeling includes the formation of the

amsome; condensation and shaping of the nucleus; development and dissolution of the

microtubular structures surrounding the condensing nucleus and the generation of a

motile flagellar apparatus and associated accessory structures (Phillips, 1974).

B) Spermiogenais

Spenniogenesis is the third and last phase of spermatogenesis that takes place in

the seminiferous epithelium of the testis and is responsible for developing the spurnatid

into a mature spermatozoon. Studies by Leblond and Clermont, (1952a) and Clermont

and Leblond, (1955) examined the spermatids of severai mammalian species and

subdivided spermiogenesis into phases and steps. They used, as criteria of identification,

the structural changes of the nucleus and the associated carbohydrate rich acrosomic

system viewed by use of the periodic acid-Schiff technique. The rat was used for the

final analysis and spenniogenesis was divided into four main phases each one subdivided

into a variable number of steps: the Golgi phase (step 1-3), the Cap phase (step 4-7), the

Acrosome phase (step 8-14) and the Maturation phase (step 15-19) (Fig. 9).

Spermiogenesis lasts approximately 20 days in the rat (Clennont et al., 1959; Clermont,

1972), 15 days in the mouse, hamster and monkey (Clermont and Trott, 1969) and 23

days in man (Heller and Clermont, 1964).

4. Tail Formation during Spcrmiogenesis

A) Coanecting Piece and Asoneme Formation

During the Golgi phase of spermiogenesis (step 1-3), the comecting piece (CP) is

assembled and the distal part of the axoneme actively grows in length within a thin f h

of cytoplasm. Axonemal growth of spermatids, triggered by the centrioles, occurs by the

addition of tubulins at the distal end of the microtubular singlets and doublets

(Rosenbaum et al., 1969). Due to the extreme length of the axowme, this requires the

synthetic machinery for the production of tubulins and associated proteins to be in place

and filly fimctional at the very onset of spermiogenesis. Duriag the Cap phase (step 4-3,

the centrioles become associated with the stdace of the nucleus and a thin film of

cytoplasm st i l l surrounds the axoneme (Fig. Ma). During the early steps of the

Acmsomic phase (step 8-14), centrioles are organized into structuns rrcognizable as the

capitulum and the striated colk of the CP (Figs. lob, 10c). The latter is continuous with

newly developed outer dense fibers known as the ODF anlagen (AODF), the anlagen are

classified as very fine fibers (Fig. lob). Throughout the Acrosornic phase, the developing

tail is enclosed within a thin sleeve of cytoplasm limited this time by a double plasma

membrane (Figs. lob, 10c). This is due to the fact that the centrioles that have migrated

toward the nucleus of the young spermatid are followed by the anlagen of the annulus

which is tightly attached to the plasma membrane. This long and narrow cytoplasmic

compartment surrounding the intracytoplasmic part of the tail is fire of organelles.

Cytoskeletal proteins are synthesized in the cytoplasmic lobule surrounding the proximal

segment of the tail and are transferred to their final destination along the axooeme by

passing through the narrow passage created by the annulus (Fig. 1Oc). At the onset of the

Maturation phase (step 15-1 9), the annulus migrates down the axoneme and the proximal

portion of the developing tail then becomes surrounded by the whole cytoplasm of the

spermatid (Fig. 1Od).

A more complete analysis on the formation of the axoneme fiom one of a pair of

centrioles has been described in several ultrastructural studies of rna,cnmalian

spermiogenesis (Sotelo and TrujilloCenoz, 1958; Sapsford et al., 1967; de Kretser, 1969;

Fawcett and Phillips, 1969; Yas& et al., 1972; Baccetti et al., 1978). Kirshner (1978)

and Raff (1 979) have written reviews.

B) Mitochondria1 Sheath Formation

During the early phases of spermiogenesis in the rat, the mitochondria are

generally located along the plasma membrane of the spermatid (Fig. lob). There is a

surge of mitochondrial DNA synthesis and the number of mitochondria increase however

the mitochondria are prevented from forming a sheath around the growing tail due to the

barrier of the double layer of plasma membrane. In the early stages of the Maturation

phase (step 15- 1 9) when the plasma membrane associated annulus rapidly slides down the

axoneme, the mitochondria are k e to access the developing tail and are seen migrating

toward and aligning side by side along the ODF which are now increasing in diameter

(Fig. 10d).

C) Outer Dense Fiber Formation

The formation of the outer d e w fibers (ODF) begins in step 8 spermatids

(Acrosome phase) and proceeds in a proximal to distal direction in the tail of

spermatozoa. Nine very fine fibers of the ODF, the anlagen, become associated with the

comsponding set of microtubule doublets in the most proximal portion of the axoneme

(Fig. lob) (Irons and Clermoot, 1982a; Irons and Clermont, 1982b). These fme fibers

gradually increase in thickness and length in a pmximal to distal direction throughout the

Acrosomic Phase forming the ODF (Fig. 1Oc). In steps 15-16, the ODF fibers undergo a

rapid and marked increase in diameter. This occurs following the migration of the

annulus in early step I5 from its juxtanuclear position to its f ia l position at the distal end

of the cytoplasmic lobule (Irons and Clermont, 1982a; Irons and Clermont, 1982b). By

step 18 of the Acrosomic phase, growth in size of the ODF is almost complete but

continues to increase at a reduced rate and to a smaller degree (Fig. 10d) (Irons and

CIermont, 1982b).

D) Fibrous Sheath Formation

Assembly of the FS is a complex process that proceeds in a distal to proximal

direction in the tail of spematozoa The first phase involves the formation of the anlagen

of two longitudinally oriented columns at the distal end of the flagellum. The

longitudinal column anlagen (ALC) forms in the cytoplasm between the plasma

membrane and the axoneme in step 2 of spermiogenesis (Figs. 10% 1 la, 1 lb). The ALC

are joined to the outer aspects of microtubule doublets 3 and 8, within the principal piece

(Fig. 1 1 b). These rudimentary columns immediately underlie the plasma membrane and

appear to be connected (Fig. 1 I b) (Irons and Clermont, l98Za). Throughout steps 2-1 0 of

spermiogenesis, the ALC extends over the distal portion of the principal piece in a distal

to proximal direction (Fig. I la). The anlagen continues to elongate and thicken into true

longitudinal columns (LC) by step 17 (Figs. IOb-d, 13,14a, 14b).

During steps 11-15, pain of delicate spines known as the nib dagen (RA) get

deposited chumferentially between the two longitudinal columns (Fig. 12). The RA is

also deposited in a diptal to proximal direction and appears to attach to the previously

formed longitudinal coiumns as well as to the cytoplasmic face of the plasma membrane

(Irons and Clermont, 1982a). Early in step 15 of spermiogenesis the rib anlagen is

converted into thick ribs and the longitudinal columns become increasingly thick however

the FS still has not completely formed over the entire principal piece (Fig. 13).

Completion of the FS is brought about by the gradual growth of the longitudinal columns

toward the annulus in step 17 and concumnt thickening and coalescence of the rib

anlagen to form the definitive ribs, the last of which are completed next to the annulus

late in step 17 (Figs. 14% 14b). This method of formation indicates that both components

of the FS fkmework, the columns and the ribs, form independently fiom each other

(Irons and Clemont, 1982a).

5. Kinases A) General Phospborylation Mechanism

Protein pbosphorylation is a reversible process involving two classes of signaling

enzymes: protein kiaases, which catalyze a phosphotransfer reaction, and phosphoprotein

phosphatases that catalyze dephosphorylation (Krebs, 1985; Cohen et al.. 1989). The

activities of both enzyme classes are tightly regulated and respond to fluctuations in

diffusible second messengers such as cyclic adenosine monophosphate (CAMP)

(Sutheriand. 1972; Krebs, 1985).

B) Cyclic AMP

Cyclic AMP (CAMP) has been shown to regulate microtubule dependent

processes and initiate mammalian sperm motility (Lindemann, 1978; Garbers and Kopt

1980; Brokaw, 1987; Horowitz et al., 1988; Macleod et al., 1994). Upon synthesis by

adenylate cyclase, CAMP diffuses or is transportad from the inner face or cytoplasmic

side of the plasma membrane to its site of action where it activates cAMPdependent

protein kinme (PKA). PKA is an inactive holoenyme consisting of two regulatory (R)

and two catalytic (C) subunits (Taylor, 1989; Taylor et al., 1990). Four molecules of

CAMP bind each donnant PKA holaenzyme, activating the kinase by releasing two C

subunits h m the R sub~t-cAMP complex (Hof inm et al, 1975; Corbin et al., 1975;

b b s and Beavo, 1979). Since the discovery of CAMP-regulated processes in

eukaryotes, considerable effort has been spent on charactertPn . .

g these enzymes.

C) Cyclic AMP Dependent Protein Kinase: A General Description

The CAMP-dependent protein b a s e (PKA) regulates a variety of diverse

biochemical events through the phosphorylation of target proteins (Walsh et al., 1968;

Rubin and Rosen, 1975; Krebs and Beavo, 1979; Flockhart and Corbin, 1982; Coghlan et

al., 1994). Two classes of regulatory (R) subunits exist: RI and RII, which form the

type I and type I1 PKA holoenzymes, respectively (Hohann et al., 1975; Corbin et al.,

1975). Each has a unique protein sequence, phosphorylation state, tissue distribution and

subcellular localization. The regulatory subunits of type I kinase isoforms (Ma, RIP) are

reported to be predominantly associated with the plasma membrane (Lee et al., 1983;

Horowitz et al., 1984; Clegg et al., 1988). The regulatory subunits of type I1 kinase

isoforms (RIIa, RUB) are localized predominantly in the cytoplasm and on structures

such as the: c ytoskeleton, secretory granules, Golgi apparatus and possibly the nuclei

(Hofmann et al., 1975; Corbin et al., 1975; Walter et al., 1978; Horowitz et al., 1984;

Nigg et ai., 1985% 1985b; De Camilli et ai., 1986; Leiser et al., 1986; Jahnsen et al.,

1986; Scott et al., 1987; Pariset et al., 1989; Salvatori et al., 1990; Joachim and Schwoch,

1990). The most striking difference between the R subunit isoforms are that RII can be

phosphorylated by the catalytic (C) subunit while RI cannot and this may allow for the

self-phosphorylation of some proteins (Hofmann et al., 1975; Eriichman et al., 1974;

Rosen and Erlichrnan, 1975; RangeCAldao and Rosen, 1976; Titani et al., 1984). The C

subunit isofoms of PKA, C a and CP, are ubiquitously expressed in all tissues and have

been cloned in several species. Ca and CP are 93% identical in amino-acid sequence

(Uhler et al., 1986a; Uhler et al., 1986b; Showers and Maurer, 1986; Adavani et al.,

1987). A third known C subunit isofonn (Cy) is believed to be a testis specific form

which has diverged significantly fiom C a and Cp (Beebe et al., 1990). It has been shown

that differently composed holoenzymes have different biochemical and fhctional

properties (Onen et al., 1991).

D) Selectivity of Cyclic AMP-Dependert Protein Kinase

Selectivity of PKA action has been delegated by a family of A - h e &chooring

Proteins (AKAPs) that have been identified to tether the R subunits of PKA to specific - subcellular structures (Rubin et al., 1972; Sarkar et al., 1984; Lohmann et ai., 1984;

Lieser et al., 1986; Bregman et al., 1989; Barsony and Marks, 199 1; Cam et al., 1992).

Due to its hction, each AKAP molecule must contain at least two functional domains: a

conserved region responsible for high affinity interaction with the regulatory subunit of

PKA and a unique targeting domain that tethers the entire AKAP1PK.A complex to a

subcellular structure (Fig. IS) (Coghlan et al., 1993). Although selectivity of PKA is

directed through the R subunit, signals on the C subunit may also facilitate targeting of

PKA. The amino tennhal glycine of most C subunit isoforms are myristoylated which

may promote binding to the cytoplasmic face of the plasma membrane (Clegg et al.,

1989).

A-Kinase-Anchoring-Proteins (AKAPs) represent a growing family of signaling

molecules which contain a conserved PKA binding motif that fimctions to localize the

kinase to particular subcellular sites (Lester et al., 1996). AKAPs fuaction not only in the

spatial regulation of PKA but also in the temporal regulation of kinase activity. Many

studies have examined the amino acid sequence of target domains that characterize

AKAPs. The amino acid sequence of AKAP 79 and AKAP 150 were compared, both

anchor type II PKA to post-synaptic densities, yet no striking sequence homology was

revealed between the two (Can et al., 1992). Other studies revealed that several different

AKAPs may bind to the same or overlapping site on the RII subunit of PKA (Luo et al.,

1990; Can and Scott, 1992). These results led to the examination of the RII-binding site

of each AKAP for a conserved secondary structure motif. A high probability for an

amphipathic helix was predicted for these sequence segments (Scott and McCartney,

1994). Examination of two well chcterized AKAPs, microtubule-associated protein 2

(MAP2) and AKAP 75 (a calmoduh-binding protein), revealed no significant sequence

similarity but the two did share a conformational similarity (Vallee and Bloom, 1986;

Glantz et al., 1993; Macieod et al., 1994). It seems likely, therefore, that all AKAPs must

s b some type of conserved conformational RII-binding domain. Due to the poor

conservation of primary structure between AKAPs, the principle criterion used to identifil

these proteins is their ability to bind RII (Lester et al., 1996). Two predominant

techniques used to do this are: a solid-phase RII overlay assay and screening of bacterial

expression libraries with RII as a probe to isolate the cDNA's encoding a n c h o ~ g

proteins (Carr and Scott, 1992).

It has become clear that the number of AKAPs is greater than originaliy thought.

It is estimated that individual cell types express 10- 1 5 different AKAPs (Cam and Scott,

1992). Based on these observations Scott and McCartney (1 994) have speculated that the

total number of potential anchoring sites is greater than the intracellular concentration of

type U AKAP. This suggests that all of the type LI PKA could be associated with

AKAPs. AKAPs may themselves be phosphorylated by PKA, it has been shown that

AKAPs contain Protein Kinase-A and -C phosphorylation sites for PKA (Coghlan et al.,

1993; Glantz et id., 1993). It is believed that a high level of phosphorylation is required to

maintain the integrity of the cytoskeleton (Lamb et al., 1990; Rios et al., 1992).

F) Cyclic AMP-Dependent Protein Kinase and their Localization to the FS

Two proteins from demembranated rat sperm flagella have been shown to f m l y

bind the R subunits (RUa and NIP) of type I1 cAMPdependent protein kinase (PKA).

These proteins have molecular messes of 120 and 80 kDa and were identified by an Rn

overlay/ immuno blot procedure (Horowitz et al., 1 988). Macleod et al. (1 994) showed

that these two proteins we= tightly associated with the FS of rat testis. This study also

showed that neither the 120 nor the 80 kDa FS RII-binding protein cross-reacted with

anti-serum directed against MAP2 or AKAP 75. This suggests that both the 120 and 80

kDa proteins are sperm specific and antigenically distinct fkom MAP2 and AKAP 75.

Both the 120 and 80 kDa proteins may constitute distinct members of a larger family of

AKAPs (Macieod et al., 1994). Since these MI-binding proteins are components of the

highly insoluble cytoskektal FS structure in sperm flagella, they are believed to serve as

cAMP-dependent protein b a s e anchoring sites aad may be classified as AKAPs

(Macleod et al., 1994).

G) Motility

Lindemm (1978) showed that demernbranated bovine sperm are motile in the

presence of adenosine triphosphate (ATP) and that the addition of cyclic adenosine

monophosphate (CAMP) to these motile sperm produced a marked increase in flagellar

beat. Correlation between increased levels of CAMP and increased motility in sperm has

been confirmed by Garbers and Kopf (1980). Cyclic AMP in eukaryotes stimulates

protein phosphorylation via activation of PKA. Phosphorylation of target proteins by

PKA has been shown to activate motility of spermatozoa as they are released fiom

storage in the male reproductive tract (Brokaw, 1987). The primary effect of CAMP-

dependent phosphorylation was suggested to be the activation of a regulatory mechanism

that directly controls the fnquency of flagellar oscillation in addition to the effects on

frequency caused by changes in sliding velocity (Okuno and Brokaw, 1979; Bmkaw,

1987).

6. Identification of Fibrous Sheath Proteins

A) Historical Background

i ) Light and Electron Microscope Rndioautograpby

Early light and electron radioautographic microscopic studies by Irons and

Clermont (1982a, 1982b) were carried out to identify flagellar components and evaluate

the morphogenesis of spermatozoa 3~-profine and 3~-cystine, radiolabeled amino acids,

were injected in vivo into the rat at various time intervals of development of the principal

piece. Microscopy revealed that the FS of spermatids did in fact incorporate this

radiolabel however these studies had technical limitations. The site of synthesis of the FS

proteins could not be detected due to the nonspecific nature of the labeled precursors.

u) Antibody Loalhrtion

Most natural antigens contain multiple epitopes, or antigenic determinants, which

stimulates the formation of several different antibodies when exposed in animals. When

an antibody recognizes a specific yet different epitope on the same antigen, they are said

to be polyclonal. When an antigen activates the formation of a specific antibody directed

against a specific determinant on that antigen molecule, the antibodies produced are said

to be monoclonal and are more specific than polyclonal antibodies.

In past studies, antibodies that have been generated against the FS to identify

constituent proteins. Monoclonal antibody K32 of Sakai and colleagues (1986),

recognizes the FS of sperm in mice, musk, shm, boar, and human, but its cognate

antigen has not been identified biochemically. Monoclonal anttibody ATC of

Fenderson et al. (1988) recognizes a 67-kDa FS antigen in rat and mouse, but not in

human sperm. Two antibodies synthesized by Jassim (1990. 1991) recognizes FS

antigens in humans, one such antigen is a 97 kDa protein, this was the first human FS

protein whose apparent mass was revealed.

B) Immunocytocbemical and Immuaofluomeent Loerllvtioa of FS proteins

Immunocytochernistry and immunofluorescence have used both monoclonal and

polyclcmal antibodies to identify the site of synthesis of a protein or cellular component.

These techniques simultaneously reveal the distribution of these cellular components.

Limitations using this technique depend upon the specificity of the antibody to the

antigen or antigenic detenniaant as well as biochemical analysis. For example, a

monoclonal antibody reacting with the FS in rat spermatozoa was reported by Jones et al.

(1983), but knmunocytochemical studies were not carried out to determine when it first

appeared during spermatogenesis or it's molecular weight.

Fenderson et al. (1984; 1988) used a monoclonal antibody, ATC, obtained by

hyperimmunizing male BALB/c mice, immunocytochemistry and indirect

immunofluorescence to recognize a 67 kDa protein integral to the FS in rat and mouse.

, Oko (1988) also obtained antibodies specific to individual FS polypeptides in rat. After

isolating the FS and verifying its' purity using EM, anti-FS serum was raised in rabbits

against the FS and used to Smity purify antibodies. Immunocytochernistry and EM

localized a 14.4 kDa polypeptide to the FS. Interestingly, the complete protein

composition of the FS was carried out by mmhg a sodium dodecyl sulfide-

polyacry h i d e gel electrophoresis (SDS-PAGE). This revealed several protein bands

(18 bands) whose molecular masses were then deduced.

C) Molecular Studies

Better understanding of the biological role of the FS, its various structural

components and the regulatory mechanisms involved in their synthesis and assembly in

normal spermatozoa require use of molecular studies. Molecular biological studies are

especially important when dealing with flagellar anomalies associated with infertility

(Chemes et al., 1987; Jassim and Festensteb, 1988; h s i m et al., 1990; Jassim et al.,

1991). A sensitive and general technique has been devised for the dual purposes of

cloning genes by using antibodies as probes and isolating unknown proteins encoded by

cloned DNA (Young and Davis, 1983). The method uses an expression vector, hgtll

(lac5 ninS c 1857 S NO), that permits insertion of foreign DNA into the P-galactosidase

structural gene lac2 and promotes synthesis of hybrid proteins. Efficient screening of

antigen-producing clones in lgt l l recombinant cDNA libraries is achieved through

lysogeny of the phage Library in high-hquency mutant cells of Escherichia coli. These

iysogens produce detectable quantities of antigen on induction. The kgtll has been

exploited to enhance the sensitivity and efficacy of antigen screening. This method may

also isolate proteins specified by previously cloned gene sequences. Therefore,

antibodies to FS proteins raises the prospect of their use to screen libraries.

7. Objective of the Present Research The objectives of this study were to clone, deduce and analyze the amino acid

sequence of the cDNA encoding the entire rat FS 75 protein and to examine the

development of both the transcript mRNA coding for this protein and its tramlation into

DNA. This study includes a complete examination of the expression and regulation of

the rat FS 75 mRNA during spermatogenesis.

III. FIGURES

Figure 1. Diagram of the Mammalian Spermatozoon

Figure 2. Diagram of the Axoneme

Figure 3. Cross section of the middle piece of the spermatozoon

Figure 4.. Cross section of the principal piece of the spermatozoon

Figure 4b. Tridimensional representation of the principal piece of the spermatozoon

Figure 5. Diagram of the Comecting Piece

Figure 6. Longitudinal section of the middle and principal piece of the spermatozoon

Figure 7a. Freeze-hcture of the principal piece of the guinea pig spermatozoon

Figu re 7b. Cross section of the principal piece of the guinea pig spermatozoon

Figure 8. Stages of spermatogenesis ffi the rat

Figure 9. Stages of spenniogenesis in the rat

Figure 10 a-d. Main ultrastructural changes seen in the developing rat sperm

Figure 11 a, b. Step 2 of development of the distal end of the fibrous sheath seen in longitudinal and cross section

Figure 12. Step 1 1 of development of the fibrous sheath

Figure 13. Step 15 of development of the distal end of the fibrous sheath

Figure 14 a, b. Step 15 of development of the proximal end of the fibmus sheath seen in longitudinal and cross section

Figure IS. Topology of the anchored type I1 PKA

Figure 1: Diagram of the Mamrnaliaa Spermatozoon Schematic diagram of a typical mammalian spermatozoon. (From Fawcen, 1975.)

Acrosomal

Connecting piece I

Fiun 2: Diagram of the Axoneme Diagrammatic representation of the axoneme as seen after aldehyde and tannic acid fmation. Numbers 1 - 9 identify the nine evenly spaced peripheral microtubule doublets. Letters A and B identify the two components that make up the peripheral doublets. Subunit A composes a complete circular microtubule. Subunit B is C-shaped. (From Oko and Clermont, i990.)

Peripheral doublet

adial spokes - \ 1 Outer dynein arm

ein arm

2 I

Central singlets

Figure 3: Cross section of the middk piece of the rat spermatozoon Electron microscope photograph showing a cross section of the middle piece of a rat spenn tail. The axoneme (As), which is centrally located, is composed of a "9 + 2" arrangement of rnicrotubules. Associated to and surrounding the axoneme (As) are nine outer dense fibers (ODF). A sheath of mitochondria (m) surround the outer dense fibers (ODF) and the axoneme (Ax) and the entire cell is enclosed in a plasma membrane (p). ~40,000. (From Oko and Clermont, 1990.)

Figure 4.: Cross section o f the principal piece of the rat spermatozoon Electron microscope photograph showing a cross section of the principal piece of a rat sperm tail. The fibrous sheath is composed of two components: the longitudinal columns (LC) and transverse ribs (R). These surround a set circumferentially oriented elements known as the outer dense fibers (ODF) which are numbered 1, 2, 4, 5, 6, 7, 9. The longitudinal columns (LC) of the fibrous sheath has replaced numbers 3 and 8 of the outer dense fibers (ODF). The outer dense fibers (ODF) surround the centrally located axoneme (Ax). Scale bar 0.1 p.

Figure 4b: Tridimensional representation of the principal piece o f the spermatozoon Diagrammatic tridimensional representation of the fibrous sheath from the proximal part of the principal piece of a rat sperm tail. The fibrous sheath is composed of two longitudinal columns facing doublets 3 and 8 of the axoneme and bridging ribs. The fibrous sheath surrounds the outer dense fibers facing microtubule doublets 1,2,4,5,6,7 and 9 of the axoneme. The plasma membrane is not shown on this diagram. (From Oko and Clermont, 1990.)

Longitudinal columns

Figure 5: Diagram of the Connecting Piece Di-tic representation of the general structure of the connecting piece of rat spermatozoon. On the left (a), the diagram represents the connecting piece as seen in side view and in three dimensions. On the right (b), the drawing represents a thin section going through the long axis of the comecting piece. Numerous bridging filaments attach the connecting piece to the basal plate of the head which is located at the surfbce of the nucleus. The striated collar of the comecting piece is characterized by dark bands alternating with narrower light bands (not all of the 9 columns of the collar are depicted). The striated collar is continuous with the outer dense fibers (ODF) of the middle piece and mitochondria are helically arranged around the outer dense fibers (ODF). In immature spermatozoa, a transversely oriented centriole is embedded in the co~ecting piece, distal to the capitulum, but disappears before maturation is complete leaving an empty compartment called a vault (V). (From Oko and Clermont, 1990).

Figure 6: Longitudinal section of the middle and principal piece of the rat sperm tail Electron microscope photograph going through the long axis of two spermatozoa at the junction of the middle and principal piece. The middle piece is identified by the mitochondria1 sheath (m) that surrounds the outer dense fibers (ODF) and axoaeme (Ax). The principal piece is identified by the fibrous sheath (FS) that surrounds the outer dense fibers (ODF) and axoneme (Ax). The annulus (An), associated with a small infolding of the plasma membrane, is visible in each tail. The markedly condensed mitochondria (m) of the middle piece are visible as small squarish profiles, a sharp contrast to the fibrous sheath (FS) which replaces the mitochondria1 structure in the principal piece. The microtubules of the axoaeme (Ax) as well as the outer dense fibers (ODF) can also be identified. ~30,000. (From Clermont et d., 1993).

Figure 7s: Freeze-fracture o f the principal piece of the guinea pig spcrmatomn Freeze-fracture of the principal piece in the guinea pig sperm tail. The longitudinal double row of large particles, referred to as a zipper (arrow), is contained in the plasma membrane of the sperm tail. It corresponds to the point of attachment opposite outer dense fiber #1 seen in thin section on the right (7b). Identically located in the rat is a single strand of trammembrane particles. ~76.000. (From Friend and Fawcett, L974).

Fiprc 7b: Cross section of the principal piece of the guinea pig spermatozoon Thin cross section of the principal piece in the guinea pig sperm tail. In thin cross section, a punctate density (arrowhead) appears to fasten the plasma membrane to the fibrous sheath opposite outer dense fiber #l. Attachment between the longitudinal columns of the fibrous sheath and outer doublets #3 and #8 of the axoneme can be seen. ~76,000. (From Friend and Fawcen, 1974).

Figure 8: Stages of spermatogenesis in the rat Schematic diagram of the 14 cellular associations that characterize the 14 stages of the cycle of the seminiferous epithelium of the rat. The stages of the cycle are indicated by Roman numerals (I-ZZIV) at the base of each column, and the duration of each stage in hours, based on the data of Clermont et al. (1959), is shown. The gem cells present within the seminiferous epithelium include various ciasses of spermatogonia (types Al- 4; Intermediate, In; type B-B"'), spermatocytes (preleptotene, PI; leptotene, L; zygotene, Z; pachytene, P and secondary, II), and spermatids in the 19 steps of spermiogenesis (1-19) (From Dym and Clermont, 1 970).

Figure 9: Stages of spermiogenesis in the rat

Drawings of the steps of spermiogenesis of the rat as seen in semithin sections of glutaraldehyde-fixed testes stained with toluidine blue or iron hematoxylin. The numbers given to the steps of spermiogenesis correspond to those proposed in the classification of Leblond and Clermont (1 W2a).

During the Golgi and Cap phases (steps 1-'I), the periauclear Golgi apparatus elaborates the cap-like acrosomic system. At the opposite pole, the centrioles give rise to the growing flagellum, and attach to the nuclear surface. The mitochondria are seen as small do^ along the plasma membrane.

During the Acrosome phase (steps 8-14), the nucleus elongates and progressively assumes a characteristic sickle shape. The bulk of the cytoplasm accumulates as a lobe around the flagellum which is attached to the base of the nucleus. The caudal tube, inserted around the base of the nucleus in step 7, appears and is seen as thin lines on either side of the flagellum. It elongates and is present throughout this phase. The Golgi apparatus leaves the nuclear region in step 8 and is subsequently located in the cytoplasmic lobule.

hning the Maturu~on phuse (steps 15-19) the spermatid completes its transformation into a spermatozoon (step 19). Following displacement of the annulus, late in step 15, Grom the neck region to the extremity of the cytoplasmic lobe, the mitochondria form the mitochondria1 sheath around the flagellum. The caudal tube disappears in step 15. The Golgi apparatus undergoes dissolution (step 17 or 18) and clusters of lipid droplets appear in the cytoplasm. The bulk of the periflagellar cytoplasm is displaced toward the head of the spermatid and ultimately is detached to form the residual body (RB) (steps 1&, 19, RB ). A small droplet of cytoplasm remains associated with the neck region (step 19) of the spermatozoon. (From Clermont et al., 1993).

Figure 10 r-d: The main altrutructural changes seen in the developing rat sperm Diagram showing the main ultrsstructural changes taking place in the developing tail of the rat spermatid. Four steps of spenniogenesis are illustrated i.e. step 7.9, 14, and 18. Cmss sectional outlines of the proximal and distal regions of the tail are shown with various components such as: the outer dense fibers (ODF) and their anlagen (AODF); and the fibrous sheath (FS) with its longitudinal column (LC), its anlagen (ALC) and its ribs (R). The development of the principal piece with its fibrous sheath (FS) is illustrated in the four cross sectional profiles above, while the development of the middle piece with the outer dense fibers (ODF) and the anlagen of the outer dense fiben (AODF) are illustrated in the thm cross sectional profiles below. Note that until the end of step 14 the tail structures are enclosed within a periaxonemal compartment delimited in its proximal portion by a double plasma membrane @p) which extends down to the annulus (An) located near the connecting piece (CP). Early in step 15, the annulus (An) slides along the axoneme (Ax) and concomitantly the outer dense fibers (ODF) increase in diameter and mitochondria (m) migrate toward the tail.

Lettering: ALC, anlagen of the longitudinal columns of the fibrous sheath; AODF, anlagen of the outer dense fibers; AS, acrosomic system; Ax, axoneme; Ca, capitulum of the connecting piece; CB, chromatoid body; CL, cytoplasmic lobule; CP, comecting piece; Dp, double plasma membrane; FS, fibrous sheath; G, Golgi apparatus; LC, longitudinal columns of the fibrous sheath; m, mitochondria; N, nucleus; ODF, outer dense fibers; R, ribs of the fibrous sheath; SC, striated collar of the connecting piece. (From Oko and Clermont, 1990).

Figure 11 - Figure 14: kvelopmenhl stages of the fibrous sheath in rat sperm Several electron microscope photographs reveal both the longitudinal and cross section appearance of the fibrous sheath through the distal and proximal regions of the principal piece of rat spermatids. The components forming the fibrous sheath are identified during the different steps of spedogenesis. ALC, anfagen of the longitudinal columns; LC, longitudinal columns; RA, rib anlagen; R, ribs;. ~33,000. (From Irons and Clermont, 1 !Ma).

ALC ALC

1 l a Step 2 Distal

12 step 11

13 Step 15 Distal

Step 15 P r ~ i m a l

Figure 15: Topology of the anchored type II PKA This model illustrates the essential features of A-Kinase b c h o ~ g Protein (AKAPs): (a), is a conserved RII-binding site; (b), is a unique targeting domain for subcellular localization i.e. the axoneme; and (c), shows that AKAPs are Likely substrates for PKA activity. (Scott and McCartney, 1994).

IV. MATERIAL AND METHODS

1. Fibrous Sheath Extraction md Antibody Production A) Isolation of the Fibrous Sheatb

Spermatozoa were obtained from the epididymides of S prague-Dawle y rats. The

epididymides were minced in several drops of 0.02 M phosphate buffered saline (PBS),

pH 7, and suspended in 15 rnl of this buffer at 20°C, then stirred and filtered through 150-

pn Nitex netting (Thompson, Montreal, Quebec). The filtrate was centrifbged at 4 K for

10 minutes at 4°C. Then, 0.2 m M phenylmethylsulfonyl fluoride was added to the buffer

and the sperm pellet was washed twice in 15 rnl of PBS. AAer the last centrifugation, the

pellet was resuspended in 5 ml of PBS.

The spermatozoa suspension was then sonicated at 4OC with a Bronwill Biosonik

IV, VWR sonicator (Scientific, San Franciso. CA) set at 100% output for four 15 second

bursts at 30 second intervals. This treatment assured 95% decapitation, verified by phase

contrast microscopy. After sonication, LO ml of PBS was added to the suspension which

was centrifbgated at 4 K for 10 minutes. The pellet was resuspended in 8 ml of 65%

sucrose containing 0.02 M PBS, layered over a sucrose step gradient composed of 8 ml

fhctions of 65%, 70%, and 75% sucrose in 0.02 M PBS (Calvin, 1976), and spun in a

Beckman SW 28 swinging bucket rotor at 1000 K for 70 minutes. The sperm tails that

were removed &om the 65-70% interfiace had 5% head contamination. Therefore, they

were resuspended in PBS to 12 ml and layered and centrifuged at 1000 K through a

sucrose step gradient composed of 12 ml hctions of 65% and 75% sucrose in 0.02 M

PBS. This procedure usually assured tails practically fiee of heads. Each of two of the

twelve tail fractions collected h m the 65075% interf'kces were p l e d and diluted to 36

ml with PBS and pelleted at 100 K for 15 minutes.

Disuffide bonds have been found to stabilize the proteins of the FS (Calvin aad

Bedford, 1971). This property, along with its relative resistance to solubiIization in urea,

has allowed for successful isolation of the FS (Olson et al., 1976; Oko, 1988). Each tail

pellet was e m t e d twice in a buffer containing 2% Triton X-100, 5 mM dithiothreitol

@"IT) and 50 mM Tris-HCI @ydn,xymethylaminornehe-hydrochtotic acid), pH 9.0,

with shaking at 4OC to solubilize all membrane components including the mitochondria1

sheath. The peflet obtained after centrifugation at 10 K for 10 minutes was then

suspended in a buffer containing 4.5-6 M urea and 25 mM DTT and incubated for 3-5

hours under phase-contrast microscope observation until all cytoskeletal structures,

except for the FS, dissolved. This procedure was also used to prepare sperm for

immunofluorescence probing.

B) Preparation of Immune S e n against the FS 75 kD. Protein

The purified FS fkaction resulting fiom the extraction process was solubilized in

2% sodium dodecyl sulfate (SDS) - 5% P-mercaptoethanol @-ME) for 5 minutes at

100°C. The denatured FS was then emulsified in an equal volume (0.5 ml) of Freund's

complete adjuvant and 100 pl (1 pg protein/@) was injected per popliteal lymph node

(Newbould, 1965) in New Zealand white rabbits. The remainder was injected at 8 sites

along the neck and back. The rabbits were boosted with antigen at 3 week intervals and

bled 7-10 days after each boost. The immune sera was stored frozen at -70°C with

protease inhibitors.

C) Affinity Purification of Anti-FS 75 Antibody

To obtain antibodies to the 75 kDa FS polypeptide, immune sera against the FS

was adsorbed onto portions of preparative Western blots containing the major 75 kDa FS

polypeptide. The major 75 kDa FS polypeptide band was detected by staining the

Western blots with 0.2% Ponceau in 3% trichloracetic acid (TCA) and by staining vertical

strips on each side of the blot with a colloidal gold solution adjusted to pH 3. The band

was then washed and destained in three 15 minutes changes of Tris-base buffered saline

containing 0.5% Tween-20 (TWBS), chopped up into 2-3 mm pieces, inserted into a 10

ml syringe, and saturated for 2 hours in 5 ml of Tris-base buffered saline (TBS)

containing 10% normal goat senun (NGS). Following incubation with antiserum for 2

hours the blot pieces were washed 5 times with 5 changes of TWBS and with continual

shaking to remove non-adsorbed or non-specific antibodies. Elution of the adsorbed or

specific antries was similar to a procedure descnid by Talian et al. (1983). After

discarding the solution h m the last wash, the blot pieces were incubated and stirred for 3

minutes in 5 ml of 0.2 M glycine-HCl, pH 2.8. The acid solution containing the eluted

antibodies was then immediately extruded out of the syringe, neutralized with a

predetermined amount of sodium hydroxide (NaOH) and mixed with a 5 ml buffer

containing 40 mM Tris-HCl, 1.8% sodium chloride (NaCl) and 0.1% bovine serum

albumin (BSA), pH 7.4. The elutes were concentrated to as low as SO p1, by the

combined use of Centriprep 30 and Centricon 10 concentrators (Amicon, Danven, MA),

and utilized directly for immunoc ytochemistry . Control solutions were obtained by

replacing immune sera with preimmune sera and by following the same procedures of

adsorption and elution of immune sera on Western blots of low molecular weight

standards (Pharmacia Electrophoresis Calibration Kits, Piscataway , NJ).

D) Peptide Sequencing

The 75 kDa FS polypeptide was immobilized on immobilon-P for amino terminal

peptide sequence analysis (Matsudaira, 1987). Automated gas phase sequencing (Hewick

et d., 1981) was performed in an on-line Porton Model 2090E sequenator according to

manufacturer's instructionst

2. Immuaocytochemistry

A) Tissue Preparation for Light Microscopy

Adult male Sprague-Dawley rats obtained fiom Charles River Canada Inc. were

anaesthetized with sodium pentobarbital (0.1 cd100 g) and perfused through the heart

with Bouin's fixative. An incision was made fkom the diaphragm of the animal up to the

levei of the sternum, this enabled access to the ni cage. The heart was lifted fiom the

thoracic cavity and a perfirsion needle was inserted into the left ventricle. After the blood

was cleared from all vessels with a ringer solution, Bouin's fixative followed for 10

minutes. The testes were dissected fkm the rat and immersed in Bouin's fixative for 24

hours then removed and dehydrated in graded ethanol solutions. The testes and were then

embedded in paratfia, sectioned and mounted onto slides for LM immunopxidase

staining.

B) Light Microscope Immunocytochemistry

Paraffin sections (5 p thick) of the testes were incubated with anti-FS 75

antibody according to Oko and Clermont (1989). The sections wen deparaffinized in

xylene and then hydrated in a series of 5 minute graded ethanol solutions, starting &om

100% and moving to 50% ethanol. During hydration, residual picric acid was neutralized

in 70% ethanol containing 1% lithium carbonate. In addition, endogenous peroxidase

activity was abolished in 70% ethanol containing 1% hydrogen peroxide (Hz@). Once

hydrated, the tissues were washed for 5 minutes in distilled water (dH20) containing 300

m M glycine in order to block free aldehyde groups.

Prior to immunolabeling, non-specific binding sites were blocked for 15 minutes

in TBS containing 0.1 % BSA and 10% NGS, pH 7.4. This was accomplished by placing

40 jd of the solution onto a coverslip and then overturning the tissue face of the slide onto

the drop, thus ensuring that the entire tissue was treated with minimal fluid (Oko, 1988).

The coverslips were then removed by rinsing the slides with TBS, allowing the coverslip

to slide off the tissue. Sections were then incubated at 37°C with the primary antiserum,

(anti-FS 75 antibody diluted 1 : 1 00 in TBS) for 1.5 hours.

Following primary antibody incubation, the sections were washed 5 times for two

minutes in TWBS in order to remove any unbound antibody. The sections were blocked

with 10% NGS diluted in TBS for 5 minutes before incubation with the secondary

antibody. Then, the sections were incubated for 30 minutes at 37OC with a goat anti-

rabbit IgG secondary antibody conjugated to peroxidase, diluted 1:250 in TBS. M e r

incubation with the secondary a n t l i y , the sections were washed 5 times in TWBS and

reincubated for 10 minutes in 200 ml of TBS containing 0.1 M imidazok, 0.03% H2@

and 0.05% d i m h o b e ~ d i n e tetrachloride (DAB, Sigma Chemical Co., St. Louis,

Missouri). The DAB was polymerized in the presence of the peroxidase and H2&

molecules to form an insoluble brown polymer which was deposited at the site of the

antigen-antibody complex (Oko. 1988). The slides were washed with dH2O to remove

any excess DAB, and counterstained with 0.1% rnethylene blue for 8 minutes. The

tissues were then washed in tap water, followed by dH20, and subsequently dehydrated

by passing the slides through graded concentrations of ethanol (50% to 100%)).

Following dehydration, the sections were placed in xykne for 5 minutes, cleaned,

mounted with permounting media, examined aud photographed using a Carl Zeiss light

microscope. A negative control was performed for every section consisting of a section

of rat testes immunostained with normal rabbit serum.

C) T'wue Preparation for Electron Microscopy

Adult male Sprague-Dawley rats fiom Charles River Canada Inc. were

anesthetized and the testes were fixed by perfusion for 20 minutes through the abdominal

aorta with 0.5% glu&taldehyde and 4% paraformaldehyde in 0.1 M PBS containing 1 5

mM lysine (Erdos et al., l986), pH 7.4. The tissues were immersed in the same fixative

for 2 hours at 4OC, then washed three times in 0.15 M PBS containing 4% sucrose, pH

7.4, at 4OC. Tissues were subsequently washed in PBS, dehydrated in graded methanol

up to 90%, and infiltrated and embedded in Lowicryl K4M (Grant et al., 1985). Thin

sections were mounted on Formvar nickel coated grids.

D) Elechon Microscope Inmunocytochemisty

Lowicryl sections on Formvar coated grids were saturated for 30 minutes with

10% NGS in order to block non-specific binding sites. The tissue was then incubated for

1 hour with primary anti-FS 75 antibody, washed 4 times for five minutes in TWBS in

order to remove excess unbound primary antibody. The tissue was then saturated again

with 10% NGS for 15 minutes before being incubated for 30 minutes with secondary

antibody. Colloidal gold-conjugated goat anti-rabbit IgG (laussen Pharmaceutica, Olen,

Belgium) secondary antibodies were used to label the antigenic sites on the FS previously

exposed to primary antibodies. The immunomarking protocol was based on information

given by Janssen Life Science Products (1 985).

Isolated spermatozoa intact or denuded of membranes and mitochondria, were

pelleted through a 65% sucrose gradient and subsequently washed and centrifuged in 0.02

M PBS, pH 7.4. The redtirig pellet was resuspended to obtain 2 X 10' spermatozodml,

and 100 pl aliquots of this suspension was cytocentrifbged and dried onto glass slides

precoa3ed with 3% BSA. Af&r fixation in methanol at 4OC, the slides were riased four

times with ice-cold PBS and incubated in PBS with 5% NGS for 20 minutes. The

spermatozoa were then incubated for 30 minutes with antisem, diluted 1:250 in PBS,

pH 7.0, containing 0.1% BSA and 1% NGS. After four rinses in buffer and a 15 minute

incubation in PBS + 15% NGS, the slides were incubated for 1 hour with rhociamine-

conjugated (~ab,)' goat anti-rabbit IgG (Bio/Can Scientific Inc., Mississauga, Ontario)

diluted 1:25 in PBS containing 0.1% BSA and 1% NGS. The slides were then rimed

thoroughly with PBS and coverslips were mounted with a medium for fluorescence.

Controls consisted of incubating sperm with 1) antibodies that had been exposed to an

excess of 75 kDa protein, 2) preimmune serum, and 3) antibodies to rabbit IgG alone.

4. Isolation of cDNA Clones

Antibodies flmity purified bin the 75 kDa FS protein were used to

immunoscreen a rat testicular Zap (ligtl 1) phage cDNA library (Strategene, La Jolla, CA)

according to the method of Young and Davis (1 983). Several positive clones were

obtained and the cDNA inserts contained within the enriched pBluescript plasmids were

excised using the Ex-Assist helper phase/SOLR system (Stratagene) for sequencing and

hybridization. This particular vector system avoids routine subcloning.

DNA sequence determination of the cDNA clones contained in the vector

pBluescript was accomplished by automated fluorescent-sequencing using the Standard

Sequenasa Version 2.0 Sequencing System kit and protocol (United States biochemical,

Cleveland, OH). The plasmid MCS promoter primers T7 and SP6 were used for the first

round of sequencing. Data b r n this initial step allowed for the generation of specific

primers, made by United States Biochemical, in order to continue sequence

determination. Both cDNAs were sequenced on both strands. DNA sequences were

determined using the dedeoxy chain termination procedure (Sanger et al., 1977).

5. Polymerase Chain Reaction Technique A) Designing Primers

Imm~oscreening a rat testicular Zap (MI 1) phage cDNA library produced two

partid downstream cDNA fagments. In order to isolate and amplify the upstream cDNA

firagment containing the initiation codon for translation, the Polymerase Chain Reaction

(PCR) technique was used. Two oligonucleotide primers (Table 1) were designed using

an oligonucleotide Software Primer Program and sent for synthesis at the Sheldon

Biotechnology Center at McGill University. These primers were designed to anneal to

specific areas of the cDNA encoding for the FS 75 kDa polypeptide. Primer 1 was a

sense oligonucleotide designed according to the mouse cDNA sequence obtained by

Fulcher et al. (1995). Primer 2 was an antisense oligonucleotide designed according to

the partial FS 75 cDNA sequence obtained by immunoscreening (see Figure 22 for primer

position). Primer I was 20 nucleotides in length and Primer 2 was 21 nucleotides long.

Both were verified using the DNAsis computer program to have; an appropriate G/C

content of 50%, no significant complementarity to one another, similar melting

temperatures, and good secondary structures containing a fiee 3' end. Both primers were

diluted to a &a1 working concentration of 10 pM.

TABLE 1

( Oligonucleotide I Base Pair Length I Melting Temp I GC Content I Sequence (5'- 3') 1

1 Sense strand I 20 nucleotides I 5S°C I 50% I AGAGTCATCGCAGCATCCAA 1

B) PCR Technique

2 p1 of the cDNA from a rat testicular Zap (hgtll) phage library was used as a

template for PCR. To this template was added, 2 p1 of dNTP (final concentration of

0.2mM), 10 of 10X PCR buffer (final concenaation of IX), 1 pi of Primer 1 and

Rimer 2 (final concentration of 0.1 pM), double distilled sterile water (ddH20), and 0.5

pl of Taq DNA Polymerase (final concentration 1-5 unitdl00 pl Pharmacia Biotech).

The reaction mixture was brought to a fmal volume of 100 pi. The PCR sample was then

placed in an automated Programmable Thermal Controller (PTC-100, MJ Research Inc.

Fisher, Montreal, Qc), and to the top of the mixture, an overlay of 3060 pl mineral oil

was added to prevent evaporation of the solution.

Primer 2 Antisense strand 2 1 nucleotides 57OC 47% GAAACGAAGTCTGAGTCTGTC

Different temperature cycles were used in order to amplify the cDNA. The

sample, devoid of the Taq enzyme, was denatured for 3 minutes at 94OC. The

temperature was then lowered to 80°C and the Taq enzyme was added to the PCR sample.

Following the addition of the Taq e v e , the PCR samples were again denatured for 45

seconds at 94°C. The primers were allowed to anneal to the cDNA for 40 seconds at

54OC, this temperature was previously determined according to the lowest theoretical

annealing temperam of the primers. The primers were subsequently extended for 2

minutes at 72°C. These three steps were then repeated 30 times for amplification. Lastly

there was a fmal extension for 7 minutes at 7Z°C which enabled the Taq DNA

Polymerase to add on the poly A tail. The resulting PCR product (1" Run Product) was

then kept at 4OC.

Depending on the primers used and the type of DNA being amplified, each of

these steps may be varied. Before performing any additional rounds of PCR to further

amplify the product (in Run Product), it was recommended to optimize the PCR

conditions to determine what results in the most specific and abundant product.

Optimizing PCR conditions involves changing annealing temperatures, increasing or

decreasing the amount of cycles, varying the concentration of DNA template, and

changing the amount of enzyme added. Once the proper conditions have been attained,

PCR is repeated using the previous PCR product (ln Run Roduct) as a template. This

M e r amplifies the desired DNA segment so that an appropriate amount of the new

product (znd Run Product) is visible on an agarose gel. The DNA can then easily be

subcloned into a vector and subsequently sequenced.

C) Analysis of PCR Products

i) A g a m Gel Elechophoresis

A 1% agarose gel was prepaid by dissolving 0.5 g of low electroendosmosis

agarose in 50 ml TAE buffer (121 g of Tris base, 28.55 ml acetic acid and 50 ml of 0.5 M

ethyienediaminaetetraacetic acid, pH 8.0, final concentration EX). After the agarose and

TAE buffer were heated, 2.5 fl of ethidium bromide (0.5 Wml) was added as a marker

to allow us to visualize the DNA witbin the gel using UV illumination. This solution was

then poured into a container containing a comb and was left to hardzn. A 6X loading

buffer (0.25% bromo phenol, 0.25% xylene cyanol, and 30% glycerol at a fmal

concentration of 1X) was added to the PCR products (1 0 pl- L 00 p 1) and each sample was

subsequently loaded into a separate well on the agarose gel. The container was filled with

1X TAE buffer, and the gel was run for 1-3 hours at 35-65 volts. A standard 100 base

pair (bp) ladder (Phannacia Biotech) was used as a marker to estimate the size of the PCR

products, which corresponds to the size of the cDNA merit of interest. The PCR

products were then visualized under UV illumination and photographed (Polaroid-57

film).

ii) DNA isolation

A specific cDNA band was excised from this 1% agarose gel. The cDNA

hgment was extracted and purified fiom the gel using the Agarose Gel DNA Extraction

Kit from Boehringer Mannheim. This kit consisted of a silica matrix, nucleic acid

binding and washing buffers. It functioned in separating the cDNA from the other

constituents by having the cDNA bind to the matrix in the presence of chaotmpic salts.

This separates the DNA from impurities that interfere with subcloning and sequencing.

The eluted cDNA, diluted in ddH20, was subsequently measured on a DU-64 Beckman

spectrophotorneter.

6. Cloning of the PCR Products

A) Ligation

In order to obtain optimal ligation, a 1 3 molar ratio of the p ~ ~ m 2 . 1 TA

cloninga Vector (Invitrogen, San Diego, CA) to PCR product was used (in a 10 pl h a 1

reaction rnixnue). The p ~ ~ T M 2 . 1 TA cloningQ Vector is about 4 kilobases (kb) in length

and is supplied linearized with single 3' T-overhangs to enable direct ligation of the PCR

product at high efficiencies. The p ~ ~ T % l TA cloninga Vector is supplied at a

concentration of 2Sng/p.l, from lnvitrogeno. The ligation reaction consisted of 1 pl of T4

ligase 10X buffer, 2 pl of the p ~ ~ T M 2 . 1 TA cloningQ Vector, 1pl of T4 DNA Ligase, 2.6

pl of PCR product (for a hal ratio of 1:3 for p ~ ~ 9 . 1 TA cloningQ VectorPCR

product), and d m to a final volume of 10 pl. This mixture was incubated at 14OC

overnight. A control vial was also prepared to evaluate the efficacy of ligation. The

ligase was inactivated in a thermal cycler the next day to avoid ditticulties during the next

step.

B) Transformation

Transformation was performed using One Shot INVaF' Competent Cells ( 1 ~ 1 0 '

cWpg DNA Invitrogen). LB/ampicillin plates were prepared by autoclaving I1 g of

NZCYM Broth from GIBCO BRL and 7.5 g of agar in 500 ml of dH20. Then, 1 ml of

ampicillin (50 pg/ml) was added to the mixture at 4540°C and poured onto 10 crn petri

dishes. After the plates cooled, 40 pl of XGal(40 mg/ml) was evenly spread onto each

plate and allowed to soak in for 15 minutes prior to use.

The ligated PCR product:p~~TM2.1 TA cloninga Vector reaction mixture was

centrifiged, and 2 pl of this was added to a vial containing 2 jd of P-ME and 50 pl of

One Shot INVaF' Competent Cells (WaF' E. Coli bacteria). These cells are extremely

fragile and required carefbl handling. The mixture was kept on ice for 30 minutes, and

then the reaction was heat shocked for 30 seconds at 42°C exactly. The sample was put

back on ice for 2 minutes, and 250 pi of S.0.C media was then added to this 50 p1

mixture, mixed gently, and incubated on a shaker for 1 hour at 37OC. The transformation

culture was then spread onto 2-3 separate LBIX-GaVampicillin plates, inverted, and

incubated overnight at 37°C. The following day, the amount of colonies present on the

plates were compared to the control plate to ensure that plating results were satisfactory.

White colonies were expected to contain inserts.

C) Identification of the Positive Clones

In order to determine which colonies actually contained the insert of interest, PCR

was performed on 7 white colonies. This saved time in having to grow all 7 colonies and

extract ali the plasmid DNA from each sample. Thus, each white colony acted as a

template for PCR and each was picked up using a pipette tip and placed in a PCR mixture

containing; 2.5 pl of 10X PCR buffer, 1 pl of each nucleoticte, 0.75 pl of Rimer 1 and

Primer 2, and sterile water for a fiaal volume of 20 pl. Each bacterial colony was mixed

well with the solution and then denatured for 5 minutes at 94OC. The temperature was

subsequently decreased to 80°C, and 5 pl of diluted Taq DNA Polymerase (find

concentration 1-5 units/100 pi Pharmacia Biotech) was added. PCR was then performed

on the bacterial colonies using the same protocol for PCR as described above. Ail 7

colonies were also re-incubated on a fkesh LBK-GaVampicillin plate at 37OC for 3-4

hours in preparation for fbture experiments, mainly the isolation of recombinant plasmid

DNA experiment.

A 1% agarose gel was run on the 7 PCR products in order to determine which

colonies positively contained the insert of interest. A standard 100 basepair ladder

(Pharmacia Biotech) was used as a marker to estimate the size of the PCR products.

Based on these PCR results, one colony was chosen to be sequenced.

D) Isolation of Recombinant Plasmid DNA

One white colony, that was positively confmed using PCR and that had been

previously incubated on a fiesh LB plate, was picked up and amplified further by growing

in 50 mi of LB broth that contained 100 p1 of ampiciilin. This was incubated overnight

on a shaker at 37OC. A control was used to verify that only the plasmid with the insert of

interest was grown. The ampMed rexombinant DNA was then extracted fiom the

bacterial DNA, using the PLASMID Mini Kit fiom QIAGEN, Montreal. The vector

containing the insert of interest was then treated with appropriate restriction enzymes to

cut the insert cDNA fragment from the vector hgment. The product was run on a 1%

agarose gel to verify that the insert was in fact present before sending it for sequencing.

The recombinant DNA was sent to the Sheldon Biotechnology Center at McGill

University for sequencing, on both strands, by automated fluorescent-sequencing utilizing

the Thermo Sequenase fluorescent labeled primer cycle sequencing kit (Amersham Life

Science, Buckinghmshire, England).

7. Northern Blot Analysis A) Total Testicalar RNA Extraction

i ) Preparation of Solutions

This technique required all solutions and equipment to be treated with

diethylpyrocarbonate @EPC, 50 p250 ml), in order to prevent the degradation of RNA.

RNA is very sensitive to RNases therefore gloves were worn during the course of the

experiments. Some solutions needed to be prepared prior to the day of RNA extraction

and are described below.

The lysis buffer was made by dissolving 59 g of Guanidine monothiocyanate in 83

mi DEPC H20 at 50°C adding 10 mM of ethylenediaminetetraacetic acid (EDTA), 50

m M Tris-HCI pH 7.5, and just before use, 8% P-ME was added. A 4 M lithium chloride

(LiCI) solution was prepared by dissolving 16.96 g of LiCl in 100 ml DEPC HzO. The

homogenization buffer consisted of 12 ml of a 5 M NaCi solution, 0.5% SDS, 5 rnM

EDTA, and 20 ml of a 1 M Tris-HCl solution, pH 7.5.

ii) Total RNA Isolation and Purification

Total testicular RNA was isolated from the testes of 10 to 65 day-old Sprague-

Dawley rats after they were anaesthetized with sodium pentobarbital (0.1 mVlOO g body

weight). The testes were placed in a cortex tube with 7 ml of lysis buffer and 570 p1 of P- ME. This was then homogenized with a Polytron homogenizer, and 7 mi of chloroform

was added. The sample was centrifbged at 5 K for 7 minutes in a rotor centrifuge, and the

supernatant was carefully removed and placed into a new tube. L C 1 (7 volumes) was

then added to the tissue homogenate, and the tube was left on ice For 15-20 hours in the

fkidge. The following day, the sample was centrifbged at 11 K for 30 minutes after

balancing tubes with LiCI. The pellet was then resuspended in 15 ml of LiCI, and the

sample was centrifuged again for 30 minutes at 4OC. This step eliminated any excess P ME. The pellet was then resuspended in homogenization buffer (1-2 ml), treated with

proteinase K (1 00 pg/ml) and left at 45°C for 30-45 minutes. Equal volume (2-3 ml) of

chloroform was added to the sample, and then centrifbged at 5 K for 7 minutes. The

supernatant was subsequently removed with a disposable pipette, and placed in a new

tube containing another 2-3 mi of chloroform. This was centrifuged at 5 K For 7 minutes,

and the supernatant was removed, placed in a new tube containing phenoUchloroform (2-

3 ml), and centrifiged at 5 K for 7 minutes. The top layer was then carefblly removed

and transferred to a k s h tube. Then, 2.5 volumes of ethanol was added to the sample,

and this was kept at -70°C for 24 hours. This step allowed the RNA to precipitate. The

tube was ceneifUged for 20 minutes at I 1 K the next day, and the pellet was resuspended

with 300 p1 DEPC water and h a a s f e d to a sterile eppendod tube. 9 pl of a 5 M NaCl

solution aad 700 pl of ethanol were added to the sample, and this was placed at -70°C for

1 hour and then centrifuged. The supernatant was poured out and the pellet was dissolved

in DEPC-H20.

B) Analysis of RNA

To prepare the gel solution, 1.2 g of agarose was dissolved in LO ml of 10X

Hepes-EDTA (200 mM Hepes and 10 mM EDTA, pH 7.8) and 73.8 ml sterile water. The

solution was heated and stirred until it appeared clear and it was then cooled to 60°C in a

water bath. Then, 16.2 ml of 37% formaldehyde was added, and the solution was poured

into a gel container. Total RNA sample previously isolated from rat was prepared by

mixing with 100 pl I OX Hepes-EDTA, 500 pi fonnamide, and 160 p1 formaldehyde to

make a 4X sample buffer. 5 p1 of total RNA (8 lg) was denatured by adding 15 pl of this

sample buffer. The sample was warmed to 60°C for 5 minutes, and 5 pl of bromo phenol

blue was added to the sample which was loaded onto the 1.2% gel containing a running

buffer that consisted of 100 ml 10X Hepes EDTA and 162 ml of a 35% formaldehyde

solution. The gel electrophoresis was run for 24 h o w at 35 mV.

C) Transfer of RNA from Gel to Membmne

The gel was placed in an RNase-fiee glass dish and rinsed with several changes of

distilled water. This step removed the formaldehyde which would have reduced the

retention of RNA by nylon membranes. The gel was then soaked in 20X SSC (87.6 g

NaCl and 88.2 g sodium citrate, pH 7.0) for 45 minutes. The transfer apparatus was set

up and consisted of a vacuum blotting unit &om Pharmacia LKB 2016 vacugene. The

wet membrane (Hybond-N Membrane fkom Amersham, Life Science) was placed on the

surface of the gel and care was taken to prevent gening air bubbles under the membrane.

The surface of the membrane was covered with 400 ml of 20X SSC. The transfer was

carried out for 1 hour and the vacuum unit was set to 50 units of pressure. The gel was

removed and discarded while the nylon was soaked in a water bath for 5- 10 minutes, and

then was placed on a filter paper and Left to dry for 30 minutes. Once dry, the RNA was

cross linked onto the membrane with W light.

D) Hybridization Analysis

The membrane was placed, RNA side up, in the hybridization tube for pre-

hybridization by incubating with 10 ml of hybridization buffer (100 rnl forrnamide. 80 ml

of 1 M NaH2P04,800 p1 of 250 rnM EDTA, 10 g SDS and 10 ml of 2% BSA) overnight

at SS°C in the rotating hybridization oven. The membrane was then re-incubated

overnight at 5S°C with [)*PI-UTP- labeled antisense RNA transcribed fkom the linearized

pBluescript plasmid containing the FS 75 cDNA insert (specific activity 10' cpmlpg).

Following hybridization, the membrane was washed three times in 2X SSC/O.I% SDS for

30 minutes at 68OC. These washes ensured that non-specific binding was eliminated.

The membrane was placed into a radioautographic cassette which contained KODAK

film, and it was stored at -70°C. The film was exposed to the membrane for 24 hours and

was then developed.

8. In situ hybridization

In situ hybridization was performed as described by Morales et al. (1991) using

[ 3 ~ - ~ ~ ~ - l a b e l e d antisense and sense RNA probes. These were transcribed in vitro fkom

the pi3luescript plasmid containing the FS 75 cDNA (specific activity 10' cpdpg) by

using SP6 or T7 RNA plymerases according to the protocol of the supplier (Promega).

Prestained hybridized testicular sections ( 1 p thick) were coated with Kodak NTB-2

nuclear emulsion (Kopriwa and Leblond, 1962) and after 10 days of exposure were

developed with Kodak D- 1 70.

V. RESULTS

I. Light Microscope Immunocytochemistry Immunostaiaiag with the anti-FS 75 affinity purified antibody showed stage-

specific variations in the staining of cross sections of the seminiferous tubules (Figs. 16a-

h). Immunostaining was exclusive to the cytoplasm and flagellum of elongating

spematids, no reactivity was found in testicular interstitial cells, spcrmatogonia,

spermatocytes or round spematids. A weak but gradually increasing reactivity was

found in the cytoplasm and tail of early elongated spermatids (steps 9- 14) (Figs. 16a-c).

Strong immunostaining was visualized in late elongated spermatids (steps 15-17)

exclusive to the cytoplasm and tail (Figs. 16d-f). Peak cytoplasmic reactivity was

reached in step 15 (Fig. 16d) and remained elevated throughout step 16 to early step 17

spennatids (Figs. 16e-f). This coincided with an intensification of the imrnunostaining

along the tail. Between steps 17- 19 a diminution of irnrnunostaining was observed in the

cytoplasm of these cells (Figs. 16g-h). The residual cytoplasmic immunostaining was

incorporated into the cytoplasmic droplets of mature spermatids at the end of step 19 (Fig.

16h).

2. Electron Microscope lmmunocytochemistry The specificity of the anti-75 kDa antibody utilized to imrnunoscreen a Zap rat

testicular library was tested by immunogold labeling. Electron microscopy localized the

mibody against the 75 kDa FS polypeptide exclusively to the longitudinal columns and

nibs of the FS (Figs. 17% 1 7b). It was also noticeable that the anlagen of FS, which is

closely associated with the plasma membrane of steps 2- 17 spermatids and assembles

along the tail in a distal to proximal direction (Irons and Clermont, 1982a), is not

immunoreactive to the FS anti'bodies (not shown). One reason for this may be that these

rudimentary structures ate too small to be detected by the immunocytochemical technique

employed. Controls showed no immunoreactivity.

3. Immunofluorescence

The affinity-purified antibodies raised against the major 75 kDa FS polypeptide

utilized to irnmunoscreen the cDNA rat testicular library was specific for the FS of the

tail. Fluorescent microscopy of membrane-, acrosome-, and mitochondrial-sheath

denuded rat spermatozoa showed a strong reaction between the anti-FS 75 affinity

purified antibody and the principal piece of the sperm tail (Fig. 18). Membrane intact

sperm as well as controls showed no irnmunofluorescence reactivity (results not shown).

4. Isolation of cDNA Clones Several positive cDNA clones were obtained by immunoscreening a rat testicular

Zap (hgtll) phage cDNA !ibrary with the anti-75 kDa FS antibody. Of these, two

positive cDNA clones were selected for sequencing. The f i t was a 1.5 kb hgment

(clone I ) and the second a 2.4 kb hgment (clone 2) (Fig. 19). Sequence analysis

revealed that both were partial cDNAs representing downstream hgments of the FS 75

mRNA that overlapped one another (Fig. 19). The overlapping region between these two

clones share identical nucleotides. The upstream fragment containing the initiation codon

(start site) for translation was missing in both cDNAs. Due to this fact, the Polymerase

Chain Reaction technique was employed to identify the complete open reading frame of

the cDNA encoding the 75 kDa FS polypeptide.

5. PCR Cloning A) Analysis of the PCR Products using Agarose Gel Electrophoresis

Two partial cDNAs representing the downstream fhgments of the FS 75 mRNA

were cloned by immunoscmning a rat testicular Zap (kgtll) phage cDNA library with

Wty purified anti-FS 75 antibody. The upstream hgment containing the initiation

codon for translation was missing in both therefore two oligonucleotide primers were

designed for use with the Polymerase Chain Reaction technique. Rimer 1, designed from

the 5' sense region of a recently cloned mouse FS cDNA (Fulcher et al., 1995), and

Primer 2 designed fiom the 3' antisense end of the partial FS 75 cDNA designated clone 1

(Fig. 19). PCR amplification generated a 1.2 kb DNA hgment (Fig. 20). This hgment

was then cloned into a pCRm2.1 TA cloningQ Vector. This 1.2 kb hgment was

expected according to recent data published on the mouse FS polypeptide (Fulcher et al.,

1995).

B) Ligation and Transformation

DNA purified from the agarose gel was ligated into the pCRN2. 1 TA cloningm

Vector. The ligation products were subsequently transformed into One Shot INVaF'

Competent Cells, incubated in S.O.C. media, plated onto LBK-Gal/ampicillin plates, and

incubated overnight at 37T. The results showed a good distribution of white colonies,

each expected to contain the 1.2 kb pair insert, compared to the distriiution of the blue

colonies devoid of any insert (data not shown).

C) Identification of the Positive Clones

To verify the presence of the DNA insert, these positive white colonies were

analyzed using the PCR method already described. This was done in order to save time,

by not having to grow all the white colonies, and not having to extract all the plasmid

DNA before being able to sequence the samples. Thus, PCR was performed on 7 white

colonies using Primer I and Rimer 2. The predicted size of the PCR product containing

the insert was approximately 1.2 kb. AAer PCR amplification, the 7 samples were

resolved on a 1% agarose gel electrophoresis using a standard 100 basepau ladder, which

identified 4 positive PCR products containing the insert of interest (Fig. 2 1).

D) Sequencing of the PCR Product

Based on the PCR results seen in Figure 21, one colony was chosen for

sequencing. Following isolation of the recombinant plasmid DNA, the plasmid DNA was

separated fiom the bacterial DNA using a plasmid DNA extraction kit &om QIAGEN.

The purified plasmid DNA was then sequenced using a Thenno Sequenase fluorescent

labeled primer cycle sequencing kit (Amersham Life Science, Buckinghamshire,

England).

The nucleotide sequence of the PCR product overlapped and matched the cloned

cDNAs and included the remaining open reading b e of the FS 75 kDa protein (Fig.

19). Linking the overlapping segments created a 2786 nucleotide (nt) sequence (Fig. 22).

Sequence analysis using the DNAsis program showed the longest possible initiation of

translation codon at nt 55, a TAA stop codon at nt 2596, and a polyadenylation signal

sequence AATAAA at nt 2767 (Fig. 22). The complete cDNA encoded a predicted

protein of 847 amino acids containing 32 cysteine residues, 64 serine residues, 37

threonine residues and 20 tyrosine residues (Fig. 22). The amino terminal sequence,

Q,S,P,S,N,P,A,T,K,S of the isolated 75 kDa FS protein as determined fiom peptide

sequencing, was found to be 188 amino acids downstream fkom the beginning of the

predicted translation initiation site indicating post-translational processing of the original

translational product (Fig. 22). Computer analysis of the cDNA sequence encoding the

rat FS 75 kDa protein using the Prosite database (Bairoch A, 1993) revealed; one

potential CAMP- and a cGMP-dependent protein kinase phosphorylation site; twelve

potential cAMP-dependent protein kinase C phosphorylation sites; thirteen casein kinase

I1 phosphorylation sites; two potential tyrosine kinase phosphorylation sites and one N-

myristoylation sites. Computer analysis of the rat FS 75 using the National Center for

Biotechnology Information (NCBI) database employing the BLAST network service

(Altschul et ai., 1990) did not detect any significant homologies over an extended region

to other known somatic or germ cell-specific protein. It did reveal that the nucleotide

sequence of the open reading frame of the rat FS 75 was found to be 93% identical with

the previously cloned mouse FS cDNAs (Carrera et al., 1994, accession #U07423;

Fulcher et al., 1995, accession #U10341) and two regions within the rat FS 75 cDNA

were homologous to intracellular anchoring domains within AKAP 75 and 79 (Fig. 22).

6. Northern Blot analysis Developmental Northern Blot analysis of testicular RNA isolated fkom rats aged

10-65 days (Fig. 23) and probed with the FS 75 antisense RNA showed the first detection

of a 3.0 kb mRNA on day 30 postpartum. This corresponds to the initiation of

spermiogenesis (haploid development of germ cells). On day 35 postpartum, at the tiw

the developing testis is enriched with more mature spematids, the intensity of the

message gradually increased and remained high through to adulthood. When total RNA

was hybridized with a sense probe, this failed to reveal a signal (results not shown).

Northern analysis was also performed using total RNA on liver, kidney, brain, spleen,

muscle, heart and lung (results not shown). No signal was detected, this confirmed

testicular specificity as previously documented (Fulcher et al., 1 995).

7. In situ hybridization In situ hybridization, petformed with pet forme labeled antisense RNA probe in

adult testis and visualized by radioautography, demonstrated that the mRNA encoding the

FS 75 kDa protein was detected at very low levels in steps 1-2 spermatids. The transcript

became increasingly abundant between steps 3-7 (Figs. 24% 24b) and reached a

maximum level by steps 8-9. Maximum levels of mRNA corresponded to the same time

haascription ceased (step 9). From steps 11 to 13, corresponding to the time the

spennatids are undergoing nuclear condensation and elongation, the radioautographic

reaction remained moderately intense. At steps 14- 16, the radioautographic reaction

showed a sharp drop in intensity and by step 17-19 the transcript gradually disappeared

(Fig. 24b). No radioautographic reaction was observed in testicular interstitial cells such

as: spennatogonia, spermatocytes or Sertoli cells (Figs. 24% 24b). A sense control probe

did not yield any radioautographic reaction (results not shown).

Figure 16 a-h. Light microscope sections immunostained with anti-FS 75 antibody

Figure 17 a, b. Electron microscope sections immunostained with anti-FS 75 antibody

Figure 18. Immunofluorescent microscopy using the anti-FS 75 antibody

Figure 19. Diagrammatic representation of three isolated cDNA clones

Figure 20. Analysis of the amplified PCR product using agarose gel electrophoresis

Figure 21. Identification of positive clones using PCR and gel electrophoresis

Figure 22. Nucleotide and deduced amino acid sequence of the FS 75 cDNA

Figure 23. Developmental Northern Blot analysis

Figure 248. In situ hybridization

Figure 24b. In situ hybridization

Figure 16 a-h: Light microscope sections immunostaiaed with anti-FS 75 antibody Immunoperoxidase demonstration on rat testicular sections of the time and site of FS protein expression in the cytoplasm and flagellum of elongated spermatids with antibodies generated against the major FS kDa polypeptide. The micrographs of this plate are representing cross sections of tubules at different stages of the cycle of the seminiferous epithelium. (A) Elongated spermatids at step 1 I (stagex) are unreactive, the background staining is normal. (B) Elongated spermatids at step 13 (stage Xm) are generally unreactive but show a weak reaction in the distal part of their tail due to the FS. (C) Elongated spermatids at step 14 (stage XIV) exhibit a moderate reaction in their cytoplasm and tail. Elongated spermatids at step 15 (D) and step 16 (E) (stages I and 11 respectively) show a strong cytoplasmic and tail reaction. Elongated spermatids at step 17 (F) (stage III) and step 18 (G) (stage IV) exhibit a less stronger reaction in their cytoplasm than in the preceding stages but the reaction on the tail remains intense. (H) At the moment of spermiation (step19 spermatids, stage W) a punctate reaction is observed at the level of the cytoplasmic droplets (brown) but not in the residual bodies (blue) however a filter was not used to illuminate the color difference. The tails of these spermatids are weakly stained. x600.

Figure 17 a, b: EM sections immunostained with anKFS 75 antibody Electron microscope sections through Lowicryl embedded tissue immunostained with antibody-amity purified against the 75 kDa FS polypeptide. The 10 nm gold particles show the sites of immunoreactivity. (A) Cross sections through the tails of step 17 spermatids. The fibrous sheath shows a strong immunoreactivity (arrow) while the outer dense fibers and axoneme show no reactivity. ~55,000. (B) Longitudinal section through the junction of the middle and principal piece of the tail of step 16 spermatid shows the distinct immunoreactivity of affinity-purified 75 kDa FS antibody with the fibrous sheath ( a m ) , the mitochondria and axoneme are clearly not immunoreactive. ~55,000.

Figure 18: ImmunoOuorescent microscopy using the an ti-FS 75 antibody Immunofluorescent microscopy of rat spermatozoa denuded of plasma membrane, mitochondrial sheath and acrosome. The antibody-affmity purified against the 75 kDa FS reacted specifically with the principal piece of the tail as seen using a fluorescent label. The fluorescent label specifically identifies the fibrous sheath. x800.

Figure 19: Diagrammatic repmentation of three isolated cDNA clones Schematic drawing depicting the size and overlapping portion of two cDNAs isolated by immunoscreening. Clone 1 is a 1.5 kb hgment, clone 2 is 2.4 kb hgment. A 1.2 kb PCR insert, containing the upstream fiagment of the FS 75 mRNA, was amplified using two primers. Below is the restriction map of the entire open reading M e of the FS 75 cDNA. The largest possible open reading b e for the rat FS 75 cDNA is 2786 nucleotides.

Figure 20: Analysis of the amplified PCR products using agarose gel electrophoresis PCR products amplified with primers 1 and 2 were run on an agarose gel electrophoresis counter stained with ethidium bromide. A major 1 .Z kb band was visualized under W light (arrowhead). Lane 1, first run PCR reaction. Lane 2, second run PCR reaction. Lane 3, 100 bp ladder (Pharmacia, Montreal, QC).

Figure 21: Ideotification of positive clones using PCR and gel electrophoresis A 1% agarose gel electrophoresis is shown representing the amplification of 7 positively cloned white bacterial colonies. PCR was performed on these colonies using Primer I and Primer 2. Each colony was used as a template from which amplification of out target insert was performed. Four of these colonies positively revealed the presence of the DNA fkagment of interest which is approximately 1.2 kb in length (arrowhead). These four colonies confirmed the presence of the insert within the p ~ ~ T M 2 . 1 TA cloning@ Vector. Lane 1-7, amplification product of 7 positive colonies. Lanes 1, 4, 6, and 7 contain the 1.2 kb insert of interest. Lane 8, 100 bp ladder (Pharmacia, Montreal, Qc).

Figure 2 2: Nucleotide and deduced amino acid sequence of the FS 75 cDNA A combined nucleotide sequence and deduced amino acid sequence of the testis-specific haploid expressed FS 75 cDNA is shown. A single underline represents the AKAP-like domains. There are two such regions located at amino acid residue 361-374 and 423-430. The double underlined segment represents the N-terminal region determined by Edman degradation located at amino acid residue 1 88- : 97. The N-Myristoy lation site is identified in bold lettering and is located at amino acid residue 241-246. There are two potential tyrosine b a s e phosphorylation sites, these are dot underlined and located at amino acid residues 148-155 and 803-809. There is one potential cAMP/cGMP- dependent protein b a s e phosphorylation site located at amino acid residue 436439. Then are twelve potential protein kinase C phosphorylation sites located at amino acid residues 7-9, 19-2 1,84086, 134-1 36,260-262, 304-306,349-35 1,448-450,592-594,597- 599,620-622, and 80 1-803. There are thirteen potential casein b a s e II phosphorylation sites located at amino acid residue 64-67,2 12-2 15,252-255, 28 1-284,397-400,480-483, 490493, 525-528, 642-645, 648-65 1, 678-68 1, 743-746, and 8 10-8 13. The location of two primers used for PCR amplification is identified using a broken line with an arrow pointing in the 5' to 3' direction. Primer 1 is 20 nucleotides long and is located at nucleotide 1-20, it was designed using information fkom the mouse major FS protein. Primer 2 is 2 1 nucleotides long and is located at nucleotide 124 1 - 1262. This primer was designed &om information gathered by screening of the rat testicular Library.

49 TCC CAA ATG ATT GCT TAC TGT GGC ACT ACA AAG ATG TCT GAT GAC ATT 96 1 M I A Y C G T T K M S D D I 1 4

9 7 GAC TOG TTA CAC AGC CGC AGA GGC GTG TGC M G GTA GAT CTT TAC AGC 1 4 4 1 5 O W L S R R G V C K V D L Y S 3 0

145 CCA GAA GGA CAA CAA GAT CAG GAC CGA AAA GTG ATA TGC TTT GTG GAT 1 9 2 3 1 P E G Q Q D Q D R K V I C F V D 46

193 G!CG TCC ACC TTG M T GTG OAA GAT GAT TCC M G GGT GCT GCT GGT CCC 2 4 0 4 7 V S T L N V E D D S K G A A G P 62

241 MiG TCA GAC GGC GAA TTA AAC CTG GAG M T CTG GAA GhA AAA GAG ATT 288 6 3 R S D G E L N L E N L E E K E I 70

289 ATC GTG ATC AM3 GAC ACA GAA AAA CAA GAC CAG CCT AAG ACG GAG GGA 336 7 9 V I K D T E K Q D Q P K T E G 94

3 3 7 TCT GTATGC CTT T T C A A A C A A G C T CCC TCTGACCCC ATAAGT GTC CTC 3 8 4 9 S S V C L F K Q A P S D P I S V - L 1 1 0

3 8 5 AAC TGG CTC CTC AAT GAT CTC CAG M G TAC GCC TTG GGT TTC CAA CAT 4 3 2 N W L L N D L Q K Y A L G F Q H 126

433 GCA TTA AGT CCC TCA GCC TCT AGC TGT AAA CAT AAA GTA GGA GAC CTA 4 8 0 1 2 7 A L S P S A S S C K H R V G D L 1 4 2

481 GAG GGT GAT TAT CAC AAA A!FT CCC TCA GAG AAC TGC TAC AGT OTG TAT 520 1 4 3 E G D Y H K I P S E N C Y S V Y 158 ........... o*......@*m.a.a.~a**.~.e. 529 GCT GAT C M GTA M C TTA GAT TAT Tn; AM3 AAk GGA CCT CAA M C CTT 5 7 6 1 5 9 A D Q V N L D P L N K G P Q N L 174

673 CCT GAT GGA GAA TQT TCG An] GM3 GAC CTC TCC TAC TAT GTC M C CGA 720 2 0 7 P D G E S M D D L S Y Y V N R 222

769 TTG GAA GOT GGA M C AAA TGC CTC CAT CAT TCC ATG TAT ACA TCA GGA 816 2 3 9 t E O Q I K C L H H S M Y T S G 254

8 1 7 G M A M GGG AAA ACC AGC CCC CGG AGT GCT GTC AGC M A T T GCT TCT 8 6 4 2 5 S E G T S P R S A V S I A S 270

865 GAG ATG GCC CAT GAA GCT GTT GAA CTG ACC 2 7 1 E M A H E A V E L T

913 AAT GGA GAA GAA GGC AGG GAT GGC CGG AAA 2 8 7 N G E E G R D G R K

961 TTA TCT M T AAG M C M G TGT GGA GAG M G 3 0 3 L S N K N K C G E K

1009 GAC AGC AAA GAA TTT GCA GAT TCC A X AGC 3 1 9 D S K E F A D S I S

1057 GCA AAT CAG GTA GCA TCT GAC ATG ATG GTC 3 3 S A N Q V A S D M M V

1105 AZlA GTG CAC AGC TGT GGG MG CCA ATT CCA 3 5 1 K V H S C G R P I P

1153 AGG GTA CTA TTA M G CAC ACC M G GAA ATT 367 B v L L K H 'l' g E I

1201 TCA TGC ATG AAG AAC TTG CAT AAT ATA ACA 3 8 3 S C M K N L H N I T

= w I m m ~ I - a - C I I I I ~ I I

1249 TCA GAC TTC GTT TCT GCT GTC M G AGG M T 3 9 9 S D F V S A V K R N

1297 CAA M T GCA GCA GAC ATC ATG GAG GCC ATG

TCA TCA GAA ATG CGT GGC S S E M R G

ACC T!CT TTG TAT AGT GAG T F L Y S E

CAG CAG ATG TGC CCA AAA Q Q M C P K

AAG GGG CTT ATG GTT TAT K G L M V Y

TCT GTT ATG AAA ACC RYS S V M K T L

GCT TGT GTG GTC CTG AAG w

GTA TCT GAT CTG ATT GAC V S D L X D

CTT TTC AAT CAT GGT AAA L F N H G K

CTA AAG CGT CTG GTC AGT 4 1 5 Q N A A D M E B M L K R L V S 436

1393 G C A M X TTAAMGCT GGA ACT CIIT G E CCG AIU TGC AAG M T CAG AGC 1440 4 4 7 A T L K A G T f I D P K C K N Q S 462

1441 CTT GAG TIC TCA GCT A N AAA GCT GAA ATG AAA GGA AM3 GAC AAA GGC 1488 4 6 3 L E F S A M K A E M K G K D K G 4 7 8

1489 AAA ACT AAA GOA GAT CCA TGC TGT AAA TCA CTG ACA AGT GCT GAG AGA 1536 4 7 9 K T K G D P C C R S L T S A E R 494

1537 GTC AGC GAACAT ATC CTCrUrrrGM;AGC CTT ACT An; TGGMC AAC CAG 1584 1 9 S V S E H I L K E S L T M W N N Q 510

1585 AM3 C M GGA ACC CAA GGC AGG GTG CCT AAC A M GTA TGC CCT AGT AAA 1632 S l l K Q G T Q G R V P N V C P S K 526

1633 GAT GAIL M G AGA GkG JUG ATC AGT CCT ?KC ACA GAC TCA CTG GCC AM; 1680 5 2 7 D E K R E K I S P S T D S L A R 542

1681 GAT CTA ATT GTC TCT GCC CTT ATG CTC ATT CAA TAC CAT CrO ACC C M 1 7 2 8 5 4 D L X V S A L L I Q Y B L T Q 558

CAA GCG AAG GGC AAA GAT CCA TGT GAA GAG GAG TGC CCT GGT TCC TCC Q A K G K D P C E E E C P G S S

An; GGC TAT ATG TCG CAA AGT GCA CAA TAT GAA M G AGT GGA GGT GGC M G Y M S Q S A Q Y E K S G G G

CAA AGT TCT AAA TCA CTT TCA ATG AAG CAT TTT GAA TCT CGT GGA GCT Q S S K S L S M K H F E S R G A

CCT GGA CCA TCT ACA TGT GCG AAG GAA AAT CAA CTT GAG TCC CAG AAG P G P S T C A K E N Q L E S Q K

ATG GAT ATG TCA AAC ATG GTT CTG TCC CTG ATT CAG AAA CTT CT'G AGT M D M S N M V L S L I Q K L L S

GAG AGC CCT TTC AGC TGT GAT GAA CTA TCT G M AGT GAG M T AAG CGT E S P F S C D E S E S E N K R

TGT TGT GAT TCT CGA TCA M G CAA GCA GCT CCC GTG GCC AAG AGA CCT C C D S R S K Q A A P V A K R P

GAA GAT CAA AGC CAA GAC AGC ACA GAA ATG GAC TTC ATC AGC GGG ATG E D Q S Q O S T E M D F I S G M

M G C M ATG M C CGG CAG TTT ATA GAC C M CTA GTA GAA TCG GTG ATG K Q M N R Q F I D Q L V E S V M

AAA CTG TGC C!FG ATC ATG GCT AAG TAC AGC AAC M T GGA GCA GCA CTA K L C L I M A K Y S N N G A A L

OCT GAG CTA GAA GAG CAA GCA GCC CTA GCA AGC AAT GGC CCC AGA TGT A E L E E Q A A L A S N G P R C

GGC CGT GAG GCT Gn; ATG TCA CAA AGT TAT CTG clu ACT CCT GGC CCT G R E A V M S Q S P L E T P G P

GAA GTT ATC GTC AAC AAT CAG TGC TCT ACA AGC M C TTG CAG AAG CM; E V I V N N Q C S T S N L Q K Q

ATG CTC TAC 'ITC ATG GGA GAT GAT GAT GGA CAA CTA GAG M G CTC CCT M L Y . F M G D D D G Q L E K L P

G M GTT TCA GCT M G GCA CCA GAG AAA GGT TAC AGT GTA GGA GAT CTT E V S A K A A E K G Y S V G D L

...e...e...*. eo..e...~.*e..eeo*

CTT CAG GAG GTC ATG AM; TTT GCC AAA GAA CGA CAA CTG GAT GAA GCC L Q E V M K F A K E R Q L D E A

GTG GGC M C ATG GCT AG24 AAG C M CTG CTG GAC TGG CTT CTC GCT AAC V G N M A R K Q L L D W L L A N

2593 CTG T M GCT GAG M T TCC TCT GAC TCC CTC CAT CCA TCC TCT CCC COO 2640 847 L 847

2641 CAG CAA TTC CIU: CCA GCT OOA GCC ACC CTC ACC ATC AGG CTG GTA M C 2608

2689 TGC ACA ATT GTG A X ACA TTT ACC M T ACA TCT GAG CAG !l'TG M C TGT 2736

2737 OM M T ACG Goo TGC CCT CCT GGG CAA CAT OM T M AM AAA TK: AM 2784

2785 M 2786

Figure 23: Developmental Northern Blot analysb Developmental Northern Blot analysis of 8 pg samples of total testicular RNA from 10 to 65 day-old Sprague Dawley rats probed with [32~] -labeled antisense RNA transcribed from our FS cDNA. On day 30 postpartum (Iane 5), corresponding to the time in development when round spermatids first appeared, a 3.0 kb transcript is visualized. With time this transcript gradually increases in intensity and remains highly expressed into adulthood (Jane 9,65 day old rat).

I I I I I 10 ' 15 '20 25 30 35'40 45 65 days

23

Figure 24.: In situ hybridization In situ hybridization of rat testicular cross section with [3~-labeled coding region probe for FS 75 mRNA and visualized by radioautography. Silver grains overlie step 5 round spermatids (RS). Step 18 elongated spermatids (SP), pachytene spermatocytes (P), spermatogonia (G) and Sertoli cells (S) are seemingly devoid of radioautographic reaction* x 1 000.

Figure 24b: In situ hybridization In situ hybridization of rat testicular cross section with [3~-labeled coding region probe for FS 75 mRNA and visualized by radioautography. Most of the silver p i n s overlie step 7 round spermatids (RS). Step 19 elongated spermatids (SP), pachytene spermatocytes (P), sperrnatogonia (G) and Sertoli cells (S) are devoid of radioautographic reaction. x 1000.

VII. DISCUSSION

The major fibrous sheath 75 kDa polypeptide (FS 75) of the rat bas been cloned

(accession #AF008 1 14) during the course of this investigation and found to be unique in

this species. The rat, an ideal model organism for experimental study on the testis, is

physiologically similar to humans and has created a genetic reservoir for human disease.

The cloning of this and other rat sperm-specific proteins is compelling for several

reasons: (1) to predict the structure and function of these proteins, (2) to develop

immunochemical and genetic assays, and (3) to understand the pathogenesis of infertility

related to defective and deficient haploid-specific proteins.

Sequence comparison of the cloned rat FS 75 cDNA and two previously cloned

mouse FS cDNAs (Carrera et al., 1994; Fulcher et al.. 1995) reveals that these proteins

are 93% identical. Also, the rat FS 75 is not homologous to any other known somatic and

germ cell-specific protein. The cloned and characterized mouse cDNA, designated Fscl

by Fulcher et al. (1995), encodes a FS component that matches the cDNA of Carrera et al.

(1994) and extends the sequence of the precursor protein by 147 nucleotides at the 5' end

predicting a protein 849 amino acids long. In the rat, a combination of PCR and

immunoscreening produced a FS 75 cDNA sequence that was 2786 nucleotides in length,

identifying it as the longest possible complete open reading frame. Interestingly, our rat

nucleotide sequence is shorter than that of mouse and predicts a precursor protein of 847

amino acids. This difference appears to be species specific.

Carrera et al. (1994) showed that the mouse FS polypeptide @82) is synthesized

as a precursor protein of approximately 92 kDa (840 amino acids) which is proteolitically

processed into a mature form approximately 72 kDa (661 amino acids). The amino acid

length of the mature protein was established by determining the N-terminal sequence

fiom the precursor protein using Edman's degradation. The N-terminal end of the mouse

p82 polypeptide is Q,S,P,S,NP,A,TJC,S. The N-terminal end of our purified rat FS 75

LDa polypeptide is Q,S,P,S,N9PA,TJC,S which matches that of the mouse. la the rat, the

Kterminal end begins at amino acid residue 188 (corresponding to nucleotide 616) and

produces a 660 amino acid protein. The upstream merit, composed of 187 amino

acids, contains a potential initiation of translation codon beginning at nucleotide 55. The

resemblance between the major FS protein of mouse and rat suggest a high degree of

conservation in the FS protein between these two species.

Carrera et al. (1994) found that the major mouse FS polypeptide @82) has 2

regional domains similar to the A-base &choring Proteins (AKAPs) which are

responsible for anchoring CAMP-dependent protein kinases (PKA). This activity was

confirmed by an in v i m ligand blotting assay and led Carrera et al. (1994) to conclude

that the mouse FS could potentially act as a scafblding protein for the subcellular

localization of regulatory proteins. Sequence analysis of the rat FS 75 cDNA revealed

identical regional domains suggesting that the rat FS 75 localizes PKA. This fmding

corroborates documentation by Horowitz et al. (1988) which states that the rat FS binds

the RD regulatory subunit of PICA. At the time of his study, the specific rat protein

responsible for binding PKA was unknown. The AKAP-like domains of the newly

sequenced rat FS 75 cDNA potentially provides the missing link and suggests that the FS

75 behaves as an AKAP. Studies have shown that activation of PKA is dependent upon CAMP and

increased levels of CAMP increases the frequency of the flagellar beat due to activation of

protein phosphorylation (Lindemann, 1978; Garbers and Kopf, 1980; Brokaw, 1987).

Two CAMP molecules are required to bind to each of the two regulatory subunits of PKA

in order to release two catalytic subunits which then phosphorylave s e ~ e , threonine and

tyrosine residues on target substrate proteins. If AKAPs function to bind PKA which

phosphorylate target proteins and the FS 75 protein behaves as an AKAP, then bound and

activated PKA can phosphorylate near by target proteins such as the axoneme, a

cytoskeletal structure known to play a major role in sperm motility (Kamiya and

Okarnoto, 1985; Mitchell and Rosenbaum, 1985; Huang et al., 1979; Brokaw et al.,

1982). The essential structure of the inner dynein and outer dynein arms of the axoneme

that aid to control the force and speed of axmemal bending or sliding may be regulated

by PKA bound to FS 75 in addition to or exclusive of other regulatory mechanisms.

AKAPs themselves have also been shown to contain phosphorylation sites for PKA

activity, suggesting that AKAPs are likely substrates for PKA (Glantz et af., 1993).

Analysis of the rat FS 75 cDNA reveals several potential phosphorylation sites for PKA,

conoborathg the hypothesis that FS 75 proteins function as AKAPs. The preferential

phosphorylation activity of PKA directed towards the FS 75 protein potentially promotes

motility by providing mechanical suppa.

The effect of CAMP-dependent phosphorylation is to activate regulatory

mechanisms that control the kequency of flagellar oscillation in addition to the effects

caused by changes in sliding velocity (Okuno and Brokaw, 1979; Brokaw, 1987). Brito

et al. (1989) previously showed that the major 80 kDa protein in h e rat FS is

phosphorylated on serine residues. Evidence by Carrera et al. (1994) suggests the

presence of phosphoserine and/or phosphothreonine in the mouse p82 however Carrera et

al. (1994) did not fmd evidence of phosphotyrosine. Computer analysis of the rat FS 75

cDNA revealed two potential tyrosine kinase phosphorylation sites. Several other

interesting potential phosphorylation sites which may promote fbrther mechanical and

regulatory mechanisms include 13 casein kinase I1 phosphorylation sites and 12 protein

kinase-C phosphorylation sites. Numerous cysteine residues, 32 in all, predicts extensive

cross-linking by disulfide bond formation which may account for the elastic rigidity of

the FS promoting streamline motility.

Further sequence analysis identified a potential N-myristoylation site close to the

amino or 5' end of the rat FS 75 cDNA. This suggests that the rat FS 75 kDa protein may

covalently anchor to the inner leaflet of the plasma membrane via myristic acid, a 14

carbon saturated fatty acid. Developmentally, ultrastruc~ual studies have shown that the

anlagen and mature FS develop adjacent to the plasma membrane (PM) (Irons and

Clermont, 1982a; Clennont et al., 1990a; Clermont et al., 1990b; Oko and Clermont,

1991). Several freeze-fiacture studies have permitted direct visualization of structural

differentiation in spermatozoon membranes to investigate functional uniqueness (Friend

and Fawcett, 1974; Friend and Rudolf, 1974; Fawcett, 1975; Friend, 1977). Friend and

Fawcett, (1 974) showed that a linear array of particles opposite fiber I, called a zipper, is

the dominant keze-hcture characteristic of the principal piece of rat, guinea pig and

other species. This suggests that the fimction of this zipper is to attach the plasma

membrane to the underlying fibrous sheath. We believe that the N-myristoylation site on

the FS 75 may be the attachment site which binds the P M and the FS together. The

importance of such a finding is twofold. First, it is crucial for assembly of the FS.

Examination of the FS during spermiogenesis reveals that the stability of the developing

FS may result from the aachoring of the FS to the plasma membrane. This stabilization

assures the progressive assembly of the FS. Second, it plays an important role in

streamlining during motility. The whole flagellum acquires greater efficiency in moving

through points of fixation between the axoaemal complex, the fibrous sheath, and the

plasma membrane. Otherwise, the axonemal complex and cytoskeletal components

would be wavering in a relatively loose sleeve, not l l l y utilizing its motile power for

movement of the tail (the whole tail must move in unison for sperm to swim effectively).

Recently, the chromosomal location of the p82 mouse FS protein was mapped

using an interspecific backcross panel. The p82 was mapped to the very proximal end of

chromosome X (Moss et al., 1996). Its presence on chromosome X was surprising since

heterochromatization and presumptive inactivation of the X and Y chromosomes (Moss et

al., 1996) characterize spermatogenesis. The results of these investigators and the fact

that the mRNA and the translation product of this gene are expressed during

spermiogenesis, indicate that the p82 gene is not subject to X chrornosorne inactivation.

Based on the developmental Northern Blot analysis and in situ hybridization

studies, the mRNA encoding the FS 75 protein is first transcribed in steps 1-2 round

spermatids and is abundant in steps 7-8. The mRNA remains highly expressed in steps 9-

12 but declines dramatically in steps 14-1 6 concomitantly with a dramatic increase in

protein synthesis as revealed by immunocytochemistry. The appearance of the FS 75

protein after transcriptional activity terminates in the developing spermatid indicates that

FS 75 mRNA is translationally regulated.

Present research has shown that the FS 75 has to self-associate in order to form

both the longitudinal columns and the circumferentiai ribs. The seif-association of FS

components and the process by which this may occur is still unclear. For example, the

ODF proteins have been shown to self-associate by leucine zippers or by particular motif

repeats like PCX (Shoa et al., 1997). Studying the mechanism of how FS proteins

self-associate is one of the challenges we face today.

In conclusion, cloning the major rat FS 75 kDa protein and the subsequent cDNA

analysis provides a better understanding of the role this flagellar protein plays in sperm

motility. Dysplasia of the fibrous sheath, an inherited sperm tail defect observed

occasionally in iofertile patients, has been associated with sperm immofility. Thus, it will

be of great importance to determine the genetic status of the FS 75 kDa protein in infertile

men suffering from dysplasia of the sperm fibrous sheath. Further analysis to locate and

characterize components involved in and responsible for efficient sperm motility will help

in understanding the pathogenesis of infertility.

XI. CONCLUSIONS

In summary, the fibrous sheath of the rat is composed of several proteins of which

the 75 kDa is a major polypeptide. Structurally, the whole fibrous sheath, located

immediately beneath the plasma membrane, forms two longitudinal columns that attach

opposite microtubule doublet three and eight. The columns are connected by ribs

oriented circderentially (Clermont et al., 1 99Oa). The structural complexity and

location of the fibrous sheath suggest that it is a cytoskeletal component of the

spermatozoon's tail and that it may play a structural, mechanical and regulatory role

during axonerne bending and sliding. The potential MAP-like activity of the FS 75

protein suggests it hctions to tether type I1 PKA to specific subcellular structures in

order to permit and regulate the preferential phosphorylation of specific target proteins.

The FS 75 protein may also be a likely substrate for PKA activity and as such, the FS 75

protein may provide the mechanical support required for motility. The presence of

several potential kinase phosphorylation sites such as: cAMP/cGMP-dependent; tyrosine;

protein4 and casein 11 supports the crucial role that the major rat FS 75 protein may play

in sperm motility. The hypothesis that the FS 75 protein is anchored to the cytoplasmic

face of the plasma membrane during early development and into maturation is supported

by the fmding that there is a potential N-myristoyiation binding site for myristic acid. In

addition to this, the inactive C subunits of PKA may also promote binding to the

cytoplasmic face of the plasma membrane, in conjunction with or independent of both the

regulatory subunit and other anchoring proteins (Clegg et al., 1989). Developmental

Northern Blot analysis and in situ hybridization documents the FS 75 mRNA is mainly

haploid expressed and the appearance of the FS 75 protein after transcriptional activity

terminates in the developing spermatid indicates that the FS 75 mRNA is translationally

regulated.

MI. ORIGINAL CONTRIBUTIONS

1) We cloned and sequenced the major fibrous sheath 75 kDa protein in the rat.

2) Sequence analysis provided information on the potential function of this protein. As

an A-Kinase A n c h o ~ g Protein, it tethers type U PKA which regulates

phosphorylation of target proteins. As a heavily phosphorylated protein, it may

mechanically support sperm motility.

3) The N-myristoylation site found by sequence analysis explains what has been

observed morphologically for years. That is, how the fibrous sheath and the plasma

membrane are associated. Myristic acid, a 14 carbon saturated fatty acid, binds to the

N-myristoylation site on the fibrous sheath 75 kDa protein and covalently anchors the

inner leaflet of the plasma membrane to the fibrous sheath.

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