HELGOL,~NDER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 51, 125-135 (1997)

Ultrastructural invest igat ion of spermatogenes is in the nemertine worm Procephalothrix sp.

(Palaeonemertini , Anopla)

A. A. R e u n o v 1 & W. K l e p a l 2

tInstitute of Marine Biology, Far Eastern Branch of the Russian A c a d e m y of Sciences; 17 Palchevsky st., 690041 Vladivostok, Russia

2Institute for Zoology, University of Vienna, Biozentrum; I4 Althanstrasse, A-I090 Vienna, Austria

ABSTRACT: Spermatogenesis and sperm structure of the nemertine worm Procephalothrix sp. were studied by transmission electron microscopy. It is shown that a flagellum and proacrosomal vesicles are common in spermatogonia and spermatocytes as in spermatogenesis of a number of marine invertebrates with external fertilization. Originally, the animals were collected as Procephalothrix Sloiralis but they were found to have a type of spermatozoon different from that of P. spiralis as de- scribed by Turbeville & Ruppert (1985). The re-identification of the material collected in the Japan Sea has shown that the features are characteristic of P. spiralis (Coe, 1930). This finding suggests that P spiralis shows variations in different parts of the world.

INTRODUCTION

The ultrastructure of the sperm cells of nemer t ines has been the subject of several in- vest igations (Afzelius, t971; Franz~n, 1983; Turbeville & Ruppert, 1985; Stricker & Cavey,

1986; Franz6n & Sensenbaugh, 1988; Reunov & Chernyshev, 1992; Jespersen, 1994). These studies have shown that some nemer t eans have so-called primitive spermatozoa

or aquasperm (Franz~n, 1983; Turbeville & Ruppert, 1985). In other nemer t ines the head of the sperm may be modified, showing an e longa ted nucleus (Afzelius, 1971; Stricker &

Cavey, 1986; Franz6n & Sensenbaugh, 1988; Reunov & Chernyshev, 1992). Also aberrant

spermatozoa without flagella have been found in nemer t ines by light microscopy (Ger- her, 1969). Ultrastructural invest igat ions of all cell stages of spermatogenes is were car-

ried out on the hoplonemer t ines Tetrastemma phyllospadicola (Stricker & Cavey, 1986) and T. nigrifrons Reunov & Chernyshev, 1992) possessing modified spermatozoa. The

data on the spermatogenes is of the most pr imit ive nemer t ines (Palaeonemertea) are ba-

sed on the description of spermiogenesis and sperm structure of Cephalothrix rufifrons (Jespersen, 1984). In our opinion it would be interest ing to study the ultrastructure of all

spermatogenic stages in pa laeonemer t ines in more detail. It is known that in spermato- genesis of many marine invertebrates wi th externa l fertilization a f lagel lum and/or pro-

acrosomal vesicles are common in spermatogonia and spermatocytes (Hodgson & Reu-

nov, 1994; Reunov & Hodgson, 1994). This differentiat ion of f lagel lum and/or proacroso-

�9 Biologische Anstalt Helgoland, Hamburg

126 A .A . Reunov & W. Klepal

mal vesicles in ear ly spe rmatogenes i s appea r s to be an in teres t ing p r o b l e m of cell bio- logy that has not yet been c leared up. In the nemer t in i with modif ied spe rms descr ibed so far, Tetrastemma phyllospadicola (Stricker & Cavey, 1986) and T nigrifrons (Reunov & Chernyshev, 1992), these characters were not de tec ted . The product ion of the acrosomal mate r ia l in both species is s tar ted in the spermat ids . The tail formation in T. nigrifrons be- gins dur ing spermiogenes is . On the other hand, the secondary spe rma tocy te s of T. phyl- lospadicota can p roduce f lagel la which are cons idered a typical (Stricker & Cavey, I986). The aim of this s tudy is to descr ibe all s tages of spe rmatogenes i s in Procephalothrix sp. and to check whe the r it is poss ible to observe the ear ly ar is ing of tails and p roacrosomal vesic les d u n n g spe rmatogenes i s of this pa l aeonemer t ine .

MATERIAL AND METHODS

The samples of Procephalothrix sp. (Anopla: Palaeonemer t in i ) were col lec ted from F e b r u a r y to May 1992 in the inter t idal zone of the Ussur ian bay (Japan Sea, Russia), and ident i f ied by Dr. A. V. Chernyshev. The addi t ional re- ident i f ica t ion of this mate r ia l by an analysis of histological sections of the worms from the gullet areas was m a d e by Dr. W. Senz in the Zoological Insti tute at the Universi ty of Vienna. For the u l t ras t ructura l inve- s t igat ions the spec imens were f ixed in 2.5 % g lu t a r a ldehyde in 0.1 M sod ium cacodyta te buffer (pH 7,4 fixative osmolari ty = 1090 mOsm) and in 2 % osmium te t roxide . Fol lowing dehydra t ion in a g r aded series of e thanol and p ropy lene oxide, the mater ia l was embed- d e d in Epon. Sections were cut on an Ultracut-E (Reichert) u l t ramicro tome us ing glass and d iamond knives, s ta ined with uranyl ace ta te and l ead citrate, and e x a m i n e d with a Zeiss EM 9S-2 t ransmission e lec t ron microscope. Five samples of ma ture males were stu- died. For scanning e lect ron microscopical invest igat ions, samples of Procephalothrix sp. were fixed as above. The mater ia l was pa s sed th rough a g r a d e d e thanol series, critical- point dried, coa ted with gold and p h o t o g r a p h e d with a JSM-35 CF scann ing e lect ron microscope. Most of the p repara t ion for and the examina t ion of the spec imens with the e lec t ron microscopes was carr ied out in the Zoological Insti tute at the Univers i ty of Vienna.

RESULTS

S p e r m a t o g o n i a

In Februa ry and March the testes of Procephalothrix sp. are full of spe rma togon ia (Fig. 1). Usually these cells are e longa te (8-9 pm long and 3-4 pm wide) and they have a nucleus (about 4-5 p m long and 2 g m wide) with small pa tches of chromat in sca t te red th roughout the nucleoplasm. In the nuc leop lasm there are one or two nucleol i about 0.8 p m in diameter . The spe rmatogon ia have f lagel la which e m e r g e from the cent r io le just be low the cell m e m b r a n e (Fig. 3). The cy top lasm contains Golgi bodies, e l o n g a t e d mit- ochondr ia and numerous m e m b r a n e - b o u n d e lec t ron-dense vesicles which are a s sumed to be proacrosomal vesicles (Fig. 4). In terce l lu lar b r idges connect the t ight ly p a c k e d sper- ma togon ia (Fig. 5). The wall of the testis is l ined by e longa ted pe r i tonea l cells wi th an ovoid nucleus (Fig. 2). In the cy top lasm of these cells there is a cons ide rab le n u m b e r of vacuoles .

Spermatogenes i s in Procephalothnx sp. 127

S p e r m a t o c y t e s

In Apri l the gonads contain spermatocy tes and spermat ids (Fig. 6). The pr imary sper- matocytes are round to ovoid (about 6 x 3 pm) and they have a f lagel lum. The d iamete r of the sphero ida l nucleus of the p r imary spermatocy tes is about 3 pm. The chromat in within the nucleus is now highly condensed . In the z y g o t e n e / p a c h y t e n e s tages there are typical synap tonemal complexes within the chromat in (Fig. 7). The in terce l lu lar b r idges connect the spermatocy tes (Fig. 8). The cytoplasm contains ovoid mitochondria , Golgi bo- dies and numerous e lec t ron-dense proacrosomal vesicles which are r andomly distr ibu- ted (Fig. 8). Spermatocy tes also have one f lagel lum each (Fig. 9). It was not poss ib le to inves t iga te the second spermatocytes . Usually, the pr imary spermatocytes , spermat ids and dividing cells ( supposedly the meiot ica l ma tura t ion stages) may be seen (Fig. 6). It should be s t ressed that more than two nuclei can be observed in these d ividing cells (Fig. 10).

S p e r m a t i d s

The early spermat ids are about 4 pm long and 1.5 pm Wide. Each cell has one round or ovoid nucleus about 1.3 lJm in diameter . Within the cytoplasm the p roacrosomal ve- sicles are s i tuated at the p resumpt ive poster ior pole of the cell (Fig. 11) and fuse to form one large acrosomal vesicle near the centr ioles and the tail. This vesicle even tua l ly has a d i ame te r of 0.6 pm and is full of e lec t ron-dense mater ia l inside of which g lobules of even h igher electron dens i ty may be seen (Fig. 12). Dur ing the course of d e v e l o p m e n t the acro- somal vesicle migra tes to the p resumpt ive anter ior pole of the late spe rma t id (Fig. 13) and becomes concave (Figs 14, 16). Some per iacrosomal mater ia l appea r s in the cy toplasm within the inner curvature of the concave vesicle (Figs 16, 17). In the late spe rmat id the acrosomal vesicle is loca ted la teral to the apical pole of the nucleus (Fig. 17). Dur ing sper- miogenes i s the mi tochondr ia congrega te a round the base of the nucleus (Fig. 13). The nuc leus changes its shape from round to e longated . The condensa t ion of chromat in con- t inues and is almost comple te in the late spermat ids . Throughout spe rmiogenes i s the de- ve lop ing spermat ids are connec ted by in terce l lu lar b r idges (Fig. 15).

Spermatozoon

In May the gonads contain spermatozoa only. The head of the spermatozoon is about 4 p m long (Figs t9-23). The spe rmatozoon has a cyl indr ical nucleus wi th an acrosome at its apical pole (Figs 18, 19). The c u p - s h a p e d acrosomal vesicle consists of some homoge- nous e lec t ron-dense substance with a l ight centre. The granular pe r iac rosomal mater ia l contains central l ight subs tances and pe r iphe ra l d a r k substances . The acrosome remains in a la tera l posi t ion relat ive to the nucleus (Figs 18, 19). The nucleus is r o u n d e d at its api- cal pole and it has a centr iolar fossa on its basa l pole (Figs 19, 21). The mid -p i ece of the spe rmatozoon comprises one r i ng - shaped mi tochondr ium (Fig. 20) which sur rounds the basa l par t of the nucleus and the p rox imal and distal centrioles. An e l ec t ron -dense ve- sicle which is thought to represen t a s torage vesicle is p resen t in connect ion with the cen- tr ioles (Fig. 21) The distal centriole has an anchor ing f iber appara tus that consists of n ine radia l ly or iented forked e lements (Fig. 22). It is the basal body of the tai l (flagellum), which has the typical a r r angemen t of the axonema l microtubuIes (9+2).

128 A.A. Reunov & W. Klepal

Spermatogenesis in Procephalothrix sp. 129

130 A.A. Reunov & W. Klepal

Spermatogenesis in Procephalothrix sp. 131

132 A . A . R e u n o v & W. K l e p a l

Figs 1-5. Procephalothnx sp. Transmiss ion electron microscopy (TEM). 1: S p e r m a t o g o n i a (sg) in contact with the gonad wall. n - nucleus, nu - nucleolus, bl - basal lamina. Scale bar - 2 ~lm. 2: Somatic per i toneal cell (sc) lining the wall of the testis, sg - spermatogonia , bl - basal lamina. Scale bar - 3 pm. 3: S p e r m a t o g o n i u m with flagella (f). Scale bar - 1 pm. 4: Proacrosomal vesicles (pay) in spermatogonia . Scale bar - 0.5 pro. 5: Intercel lular br idge (Ib) be twee n spe rmatogon ia . Scale

b a r - 1 ]am

Figs 6-10. Procephalothrix sp. TEM. 6: Spe rma tocy tes (sc) and spermat ids (st) in g o n a d of Proce- phalothrix sp. mc - meiotically dividing cells. Scale bar - 3 tam. 7: Primary spermatocy te , s - synap- tonemat complex. Scale bar - 1 pro. 8: Primary sper rna tocytes connec ted by intercel lular br idges (Ib). m - mitochondria , p a v - proacrosomal vesicles. Scale bar - 2 ]~m. 9: Primary spe rma tocy t e with fla- gella (f). m - mi tochondr ium, n - nucleus. Scale bar - 1 pm. 10: Meiotically dividing spermatocy tes .

n - nucleus. Scale bar - 1 pm

Figs 11-17. Procephalothrix sp. TEM. 11: Proacrosomal vesicles (pay) s i tuated at the p re sumpt ive poster ior pole of the spermatid, t - tail. Scale bar - 0.5 ]Jm. 12: Acrosomal vesicle (av) in basal part of the spermatid, m - mitochondrion, dc - distal centriole, pc - proximal centriole, t - tail. Scale bar - 0.5 ]am. 13: Acrosomal vesicle (av) migra t ing to lie at the p re sumpt ive anterior po le of the sper- matid, n - nucleus, m - mitochondrion. Scale bar - 1 ]am. 14: Acrosomal vesicle (av) c h a n g i n g form. n - nucleus, m - mitochondrion. Scale bar - 1 ]am. 15: Spermat ids connec ted by intercel lular br idges (Ib). Scale bar - 1 pm. 16: Concave acrosomal vesicle (av) in spermatid, p - pe r i ac rosomal material . Scale bar - 0.5 ]am. 17: Acrosomal vesicle s i tuated lateral to the nucleus of spermatid. Arrow shows

the per iacrosomal material . Scale bar - 0.5 ~m

Figs 18-23. Procephalothrix sp. TEM. 18: Acrosome of spermatozoon, av - ac rosomal vesicle, lc - light centre of the acrosomal vesicle, ls - light subs t ance of per iacrosomal material , ds - dark subs t ance of the per iacrosomal material, n - nucleus . Scale bar - 0.5 ]am. 19: S p e r m a t o z o o n of Pro- cephalothrix sp. a - acrosome, n - nucleus, cf - centr iolar fossa, m - mitochondria , t - tail. Scale bar - 1 pro. 20: R ingshaped mi tochondr ium (m) in the mid - p iece of spermatozoon, n - nuc leus . Scale bar - 0.5 ]am. 21: Mid-piece of spermatozoon, cf - centr iolar fossa, dv - dark vesicle, pc - proximal centriole, dc - distal centriole. Scale bar - 0.3 ]am. 22: Pericentriolar complex of distal centriole. Scale bar - 0.5 ~lm. 23: Scanning electron microscopy. Spe rma tozoon of Procephalothrix sp. a - acro-

some, n - nucleus, m - mitochondria , t - tail. Scale bar - 1 ]am

D I S C U S S I O N

T h e g o n a d w a l l in Procephalothnx sp . c o n s i s t s of p e r i t o n e a l cel ls w i t h t h e i r b a s a l la-

m i n a . P e r i t o n e a l ce l l s f o r m i n g t h e g o n a d w a l l a r e t y p i c a l fo r n e m e r t i n e s ( S t A c k e r & C a -

vey , 1986; T u r b e v i l l e & R u p p e r t , 1985; R e u n o v & C h e r n y s h e v , 1992; J e s p e r s e n , 1994) a n d

m a n y o t h e r i n v e r t e b r a t e s . T h e e x c e p t i o n w a s d e s c r i b e d for t h e n e m e r t i n e Tubulanus rhabdotus w h e n t h e s e ce l l s w e r e a b s e n t in t h e m a l e g o n a d s ( T u r b e v i l l e & R u p p e r t , I 985 ) .

T h e d e v e l o p i n g s p e r m ce l l s in t h e g o n a d s of Procephalothrixsp. a r e c o n n e c t e d b y in -

t e r c e l l u l a r b r i d g e s b u t t h e y a r e n o t a g g r e g a t e d in c l u s t e r s a s is c h a r a c t e r i s t i c fo r Tetra- stemma sp . ( S t r i c k e r & C a v e y , 1986; R e u n o v & C h e r n y s h e v , 1992). It is l i k e l y t h a t t h e or-

g a n i z a t i o n in d u s t e r s is a m o r e a d v a n c e d c h a r a c t e r a m o n g t h e n e m e r t i n e s .

It w a s s h o w n t h a t t h e f l a g e l l u m a n d / o r p r o a c r o s o m a l v e s i c l e s a r e c o m m o n in p r e -

s p e r m i o g e n i c ce l l s of Procephalothrix sp . a s in o t h e r m a r i n e i n v e r t e b r a t e s s u c h as s p o n -

g e s ( P a u l u s , 1989), c n i d a r i a n s ( D e w e l & C l a r k , 1972; S c h m i d t & H o l t k e n , 1980; L a r k m a n ,

1984), p o l y c h a e t e s ( E c k e l b a r g e r , 1984), e c h i n o d e r m s {Longo & A n d e r s o n , 1969; K a t o &

I s h i k a w a , 1982; Y a m a s h i t a , 1983), c h i t o n s ( H o d g s o n e t aL, 1988), b i v a l v e s ( R e u n o v &

Spermatogenesis in Procephatothrix sp. 133

Hodgson, 1994), brachiopods (Hodgson & Reunov, 1994), sipunculids (Klepal, 1993; Reu- nov & Rice, 1993), priapulids (Adrianov et al., 1991).Therefore, it may be assumed that this early development of a flagellum and proacrosomal vesicles is characteristic of the deve- lopment of a primitive sperm. The possible reasons for early arising flagella and proacro- somal vesicles were recently discussed (Hodgson & Reunov, 1994; Reunov & Hodgson, 1994). Yamashita (1983) suggested that the early development of the acrosome in the Ophiuroidea is correlated with its relatively large size and the short durat ion of sperma- togenesis. Pre-spermiogenic development of the proacrosomal vesicles was also observed in holothurians the sperm of which has a large acrosome (Atwood, 1974). The duration of spermatogenesis of the Holothurioidea is not particularly short (Reunov et al., 1984). It is also doubtful that the early formation of the acrosomal material in the spermatogenesis of priapulids, sipunculids, polychaetes, brachiopods, nemer t ines and bivalves could be ex- p la ined by a short spermatogenests. However, it might be that there Is some correlation be tween the early arising of proacrosomal vesicles and the size of the acrosome. For ex- ample, in the brachiopod Discinisca tenuis the spermatozoon has a large acrosome and the proacrosomal vesicles are formed in the spermatogonium whilst in the brachiopod Kraussina rubra the sperm has a very small acrosome, formed during spermiogenesis (Hodgson & Reunov, 1994). Unfortunately, this correlation does not appear to exist in the spermatogenesis of nemert ines. The average diameter of the acrosome of the modified sperm (development of the acrosome during spermiogenesis) of Tetrastemma phyllospa- dicola is about 1.1 ~m and that of Tetrastemma nigritrons is about 0.5 l~m (see Stricker & Cavey, 1986; Reunov & Chernyshev, 1992). The average diameter of the acrosome of the Procephalothrix sp sperm (pre-spermlogenic deve lopment of the acrosome) is about 0.8 gin, so that the correlation be tween the sizes of the acrosomes and the beg inn ing of the formation of the proacrosomal vesicles is not conclusive in this case. As an alternative it may be assumed that the pre-spermiogenic development of acrosomal e lements is an ancestral feature characteristic for the development of the primitive spermatozoa.

Supposedly the flagella might be a character not only of early spermatogenical cells but also of early ovogenical cells. As was shown in the actinia Actinia fragacea (Larkman, 1984), the early germinat ive cells of the female may have flagella and centriolar comple- xes identical to those found in the germinat ive cells of the male. It could be suggested that the flagella in early germinative cells may be interpreted as pleslomorphic or rudi- mental . This may provide evidence for the possibility that the origin of Metazoan game- tes is to be found in monociliated ancestral ceils (Reunov & Hodgson, 1994). The possibi- lity that flagella may be rudimenta l was discussed for muscle cells of brachiopods, phoro- nids, echinodermates, polychaetes and vertebrates (see Gardiner & R~eger, 1980). These authors support Sorokin's idea (Sorokin, 1962) that rudimentary cilia in muscle cells re- present vestiges inher i ted from a primitive ancestor.

It was not possible to observe the second spermatocytes. The dividing cells be tween the spermatocytes I and the spermatids present the meiotical maturat ion stages. So it may be assumed that dur ing the spermatogenesis of Procephalothrix sp. the first and second divisions of matura t ion occur within the spermatocyte I.

It is surpris ing that Procephalothrlx spiralis (Coe, 1930) as identif ied by Dr. A. V. Chernyshev, collected from the intertidal zone of the Ussurian Bay (Russia), has a diffe- rent type of sperm compared with that of P, spiralis described by Turbeville & Ruppert (1985). Unfortunately, these authors did not ment ion where they got their animals. The

134 A . A . R e u n o v & W. Klepa l

s p e r m of P. spiralisdescribed by Turbev i l l e & Ruppe r t (1985) has a typical p r i m i t i v e s truc-

tu re wi th the e x c e p t i o n of a m i t o c h o n d r i a l col lar a r o u n d the cen t r io les w h i c h is usua l ly

m o r e charac te r i s t i c of m o d i f i e d sperms . T h e s p e r m a t o z o o n of Procephalothrix sp. descr i -

b e d in this p a p e r has s o m e p r imi t ive f ea tu re s l ike fo rked pe r i cen t r io l a r p r o j e c t i o n s (Fer-

ragut i , 1984) a n d the o rgan i za t i on of t he tai l (9 + 2). T h e r e are also m o d i f i e d e l e m e n t s l ike

an e l o n g a t e d n u c l e u s a n d a m i t o c h o n d r i a l col lar a r o u n d the cen t r io la r c o m p l e x . T h e

s i d e w a y pos i t ion of the a c r o s o m e re l a t ive to t he axis of t he s p e r m a t o z o o n is mos t l ike ly an a b e r r a n t fea tu re . T h e compos i t ion of the m i d d l e p i e c e of the Procephalothrix sp. s p e r m

cou ld be c o m p a r e d wi th that in s p e r m a t o z o a of Amphiporus cruentatus (Turbevi l le &

Rupper t , 1985) and Gononemertes parasita (Franzdn & S e n s e n b a u q h , 1988). In t h e s e ca-

ses the cen t r io la r a p p a r a t u s is also s u r r o u n d e d by one c i rcular m i t o c h o n d r i o n . In M a l a - cobdella grossa (Afzelius, 1971), Tetrastemma nigrifrons (Reunov & C h e r n y s h e v , 1992)

a n d Cerebratulus lacteus (Longo et al., 1988) the n e c k r e g i o n i nc ludes s o m e s e p a r a t e

mi tochondr i a . T h e spe rms of Tetrastemma phyllospadicola (Str icker & Cavey , 1986) a n d Cephalothrix ruff[tons ( Jespersen , 1994) h a v e one e l o n g a t e d m i t o c h o n d r i u m w h i c h is

s i t ua t ed la te ra l ly in the m i d d l e par t of the s p e r m a t o z o o n . It shou ld be s t r e s s e d tha t the

s to rage ves i c l e b e i n g in con tac t wi th t he cen t r io les of the Procephalothrix sp. s p e r m was no t d e s c r i b e d in s p e r m a t o z o a of o the r n e m e r t i n e s . In re la t ion to the a s y m m e t r i c a l posi-

t ion of the a c r o s o m e the s p e r m of this n e m e r t i n e is s imi lar to the s p e r m a t o z o a of Cepha- lothrix rufifrons ( Jespersen , 1994) and Tetrastemma phyllospadicola (S t r icker & Cavey , 1986). A c o m p a r i s o n of the u l t r a s t ruc tu re of the s p e r m of Procephalothrix spirafis f rom the

J a p a n Sea wi th that d e s c r i b e d or ig ina l ly by Turbev i l l e & Ruppe r t (1985) l e a d s to the con-

c lus ion tha t t he d i f f e rences m a y be d u e to d i f fe ren t g e o g r a p h i c a l d i s t r ibu t ion of t he spe- cies. N e v e r t h e l e s s , this f ind ing ra i sed the i n t e r e s t i ng q u e s t i o n c o n c e r n i n g t h e n e c e s s i t y

for c o m p a r a t i v e analys is of t he se two P. spiralis spec ies f rom d i f fe ren t par ts of t he wor ld .

Acknowledgements. We would like to thank Dr. A. V. Chernyshev for collection and identification of Procephalothrix sp. and Dr. W. Senz for re-identification of this material. Many thanks to both of them and to Prof. Salvini-Plawen for the discussion of the manuscript. Thanks to Dr. V. V. Isaeva for useful discussions on some aspects of this exploration. We are grateful to Mag. D. Gruber for her technical assistance. Financial support for one of us (A.A.R.) was given by the organization ,,Fbrde- rung von Auslandsbeziehungen" and a grant from the Russian Fund of Basic Research: RFBR No 96-04-49702.

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