The morphology and timing of fertilization and early ... · blastocysts of more than thirty cells....
Transcript of The morphology and timing of fertilization and early ... · blastocysts of more than thirty cells....
/ . Embryol exp. Morph., Vol. 15, 2, pp. 169-191, April 1966 1 6 9With 3 plates
Printed in Great Britain
The morphology and timing of fertilization andearly cleavage in the Mongolian gerbil
and Deer mouse
By J. H. MARSTON1 AND M. C. CHANG2
From the Worcester Foundation for Experimental Biology,Shrewsbury, Massachusetts, U.S.A. and the Department of
Biology, Boston University
Ovulation, fertilization and early cleavage have been investigated in fewmyomorph rodents and in only two members of the family Cricetidae. TheGolden hamster (subfamily Cricetinae: Mesocricetus auratus) was studied byGraves (1945), Venable (1946a, b), Ward (1948), Strauss (1956) and Harvey,Yanagimachi & Chang (1961), using fixed and stained materials; and Austin(1956a), Samuel & Hamilton (1942) and Hamilton & Samuel (1956) examinedthe living eggs. The effects of delayed fertilization were investigated by Chang &Fernandez-Cano (1958) and Yanagimachi & Chang (1961). The Field vole(subfamily Microtinae: Microtus agrestis) was studied by Austin (1957).
The present paper deals with the processes of fertilization and cleavage in theMongolian gerbil (subfamily Gerbillinae: Meriones unguiculatus) and the Deermouse (subfamily Cricetinae: Peromyscus maniculatus bairdii).
METHODS
Animals
Mongolian gerbils were bred in a controlled-light environment (day from7 a.m. to 7 p.m.) under constant conditions of management (Marston & Chang,1965). Mature females (55-80 g) were monogamously paired with vigorousmales and examined twice daily, at 9 a.m. and 5 p.m., for vaginal spermatozoaand a copulation plug. The animals were also under regular surveillance betweenthese times. They were sacrificed or unilaterally ovariectomized under pento-barbital sodium anaesthesia at various times after mating. Day 0 of pregnancywas defined as the day when motile spermatozoa and/or a copulation plug were
1 Author's address: Department of Anatomy, Medical School, University of Birmingham,Birmingham 15, England.
2 Author's address: Worcester Foundation for Experimental Biology, Shrewsbury, Mass.,U.S.A.
170 J. H. MARSTON & M. C. CHANG
found in the vagina at 5 p.m. and the female was still being vigorously pursuedby the male. On day 1 only immotile and broken spermatozoa were found in the9 a.m. vaginal smear.
Deer mice were obtained from Dr F. W. Bronson, The Jackson Laboratory,Bar Harbour, Maine, and allowed to breed in the same light environment underconditions resembling those used for commercial mouse breeding. Maturefemales (14-24 g) were caged polygamously with vigorous males and examinedonce each day, at 9 a.m., for the presence of vaginal spermatozoa. Matingusually occurred in the late evening, and thus day 1 of pregnancy was the daywhen spermatozoa were first detected in the 9 a.m. vaginal smear. The animalswere sacrificed or unilaterally ovariectomized under ether anaesthesia atvarious times after mating.
Induced ovulation
Aged virgin and several multiparous Mongolian gerbils, at unselected stagesof their oestrous cycle, received 10-40 i.u. of pregnant mare serum (P.M.S.)
followed after 52 h by 10 i.u. human chorionic gonadotrophin (H.C.G.). Thehormone preparations ('Equinex' and 'A.P.L.', Ayerst Laboratories Inc.)were injected subcutaneously in not more than 0-10 ml of sterile saline. Tengerbils were inseminated surgically, under pentobarbital sodium anaesthesia,from 12 to 18 h after H.C.G. administration by injecting 0-10 ml of an epididymalspermatozoa suspension into each uterine horn. The caudae epididymides of amature male were macerated in 1-0 ml of Ringer's solution at body temperatureto provide this suspension of spermatozoa.
Immature Deer mice, aged approximately 6 weeks, were injected intra-peritoneally with 10-30 i.u. P.M.S. followed after 56 h by 10 i.u. H.C.G. Themature animals were aged 3-5 months, and in some cases had recently beencaged with mature males. At unselected stages of their oestrous cycles theyreceived the same hormone treatments, and one additional group received asingle injection of 10 i.u. H.C.G. All the animals were inseminated with asuspension of epididymal spermatozoa (as above) at 14-15 h after the H.C.G.
injection, according to the technique of Dzuik & Runner (1960).
Examination of ovaries and eggs
After ovariectomy or autopsy the number of corpora lutea or recentlyruptured follicles on each ovary was noted. The ovarian capsule, Fallopian tubeand uterine horn were carefully and separately examined for the presence ofeggs. Eggs were mounted for phase-contrast microscopy, fixed, and stainedaccording to Marston & Chang (1964), Chang (1952) and Yanagimachi &Chang (1961).
Examination of spermatozoa
Fresh suspensions of epididymal spermatozoa were examined under a phase-contrast microscope. Specimens were mixed with an equal volume of 10%
Fertilization and early cleavage in mice 171
buffered neutral formol, and then prepared as thin smears for more detailedstudy. Additional smears were prepared and stained with Giemsa (Hancock,1952), haematoxylin and by the Feulgen technique. Fluorescence microscopyusing acridine orange was performed according to Bishop & Smiles (1957).
RESULTS
A. Observations from natural matings in the Mongolian gerbil
(1) Time of mating. Seventy-three females were used in this study: 53 (72-6 %)were mating and had motile spermatozoa in the vagina at 5 p.m., while theremainder had immotile, broken spermatozoa at 9 a.m. and had presumablymated the prevous evening at some time after 5 p.m. Mating was observed infive females as early as 1 p.m., but in most cases it was not observed sooner than3 p.m.
(2) Time of ovulations. The majority of animals had begun to ovulate by9-10 p.m. on day 0 (Table la) and had completed their ovulation by 1 a.m. onday 1. One of the twenty-four animals ovulating before 4 a.m. on day 1 hadeggs in the ovarian capsule, the others yielded eggs from the slightly swollenampulla of the Fallopian tube. Thus, transfer of freshly ovulated eggs to theampulla seemed to occur rapidly.
(3) Time of spermatozoon penetration. All the animals examined up to 4 a.m.on day 1 had their uteri distended by a mass of spermatozoa, so mating haddefinitely occurred. Table 1 a shows that for the majority of animals and eggspenetration occurred between midnight and 4 a.m. on day 1 at about 2-3 hafter the completion of ovulation.
(4) Time of cleavage. Fifteen animals were examined from midnight to 1 a.m.on day 2 and nine yielded eggs at various stages of the first cleavage (Table 1 b),although 3 h earlier only one of five animals had an egg advanced beyond thepronuclear stage. At 3-4 a.m. on day 2 one animal had only pronuclear eggs,two had eggs in the first cleavage, and three yielded only two-celled eggs. Forthe majority of animals and eggs the first cleavage was completed betweenmidnight and 4 a.m. on day 2, at about 22-24 h after penetration.
From the available data (Table 1 c) it was only possible to estimate the timingof the second to fifth cleavages of the fertilized Mongolian gerbil egg. As93-3 % of the eggs examined at 9-10 a.m. on day 3 were still two-celled, thesecond cleavage seemed to occur more than 30 h after the first cleavage. Theintervals between subsequent cleavages were estimated to be 20-24 h.
At 9-10 a.m. on day 4 seven eggs were undergoing the third cleavage. Fourof these eggs actually had seven blastomeres which each contained a formednucleus (Plate 1, fig. 25). It seemed that one blastomere from the four-cell stagehad not yet cleaved and was associated with six recently cleaved blastomeres.
One sixteen-celled egg was recovered from the uterus at 9-10 a.m. on day 5but otherwise the eggs were in the caudal or intra-mural isthmus of the Fallopian
Tab
le
1 a-
c.
Tim
e of
ov
ulat
ion,
fer
tili
zati
on
and
clea
vage
in
the
natu
rall
y m
ated
Mon
goli
an g
erbi
l(e
ach
anim
al p
rovi
ding
on
e F
allo
pian
tu
be o
r ut
erin
e ho
rn)
Tab
le
\a
Exa
min
atio
n(t
ime
of d
ay)
Num
ber
ofan
imal
s
Num
ber
ofan
imal
sov
ulat
ing
(com
plet
edov
ulat
ion)
Num
ber
ofan
imal
sw
ithpe
netr
ated
eggs
Tot
alnu
mbe
rof
egg
sre
cove
red
(ovu
late
d)
Stag
e of
dev
elop
men
t (%
tot
al e
ggs)
Dam
aged
Unp
ene-
trat
edE
arly
fert
iliza
tion*
Pr
onuc
lear
Day
0,
9-10
p.m
. 10
f 6(
3)
2D
ay 1
, m
idni
ght-
1 a.
m.
101
8 (8
) 3
Day
1,3
-4
a.m
. 10
f 10
(10)
7
Day
1,
9-10
a.m
. 10
t 10
(10)
t 9
* Sp
erm
hea
d ha
s en
tere
d vi
tellu
s an
d st
arte
d it
s tr
ansf
orm
atio
n.%
1 a
nim
al h
ad a
cys
tic o
vary
and
yie
lded
fou
r un
pene
trat
ed
eggs
.
16(1
8)
—
69
31
—25
(27)
—
64
24
12
42(4
2)
2 29
19
50
34(3
5)
3 18
9
70t
All
of t
hese
ani
mal
s w
ere
mat
ing
at 5
p.m
. on
day
0.
Exa
min
atio
n(t
ime
of d
ay)
Num
ber
ofan
imal
s
Tot
alfe
rtili
zed
eggs
exam
ined
Tab
le
lb
Stag
e of
dev
elop
men
t (%
tot
al
Pro
-met
a-P
ronu
clea
r P
roph
ase
phas
e M
etap
hase
fert
ilize
d eg
gs)
Tel
opha
seE
arly
two-
cell
Tw
o-ce
ll
Day
1,
3-4
p.m
. 5
21D
ay 1
, 9-
10 p
.m.
5 12
Day
2,
mid
nigh
t-1
a.m
. 15
* 70
Day
2,
3-4
a.m
. 6
22D
ay 2
, 9-
10 a
.m.
5 19
100 92 43 23
10 924 14
Fiv
e an
imal
s ea
ch p
rovi
ded
two
Fal
lopi
an t
ubes
.
8 7 41 100
X > co H O O O X >
Tab
le
\c
Exa
min
atio
n(t
ime
of d
ay)
Tot
alN
umbe
r fe
rtili
zed
of
eggs
anim
als
exam
ined
Stag
e of
dev
elop
men
t ( %
tot
al f
ertil
ized
egg
s)
2-ce
ll4-
cell
3rd
clea
vage
8-
cell
4th
clea
vage
16
-cel
lB
last
ocys
t
Day
3, 9
-10
a.m
.D
ay 4
, 9-
10 a
.m.
Day
5,
9-10
a.m
.D
ay 6
, 9-
10 a
.m.
5 5 10 5
15 17 24 14
93 6 7
4153 21 7t
5025
4* 86
* N
inet
een-
cell
ed m
orul
a—no
t ye
t a
blas
tocy
st,
and
still
in
the
Fal
lopi
an t
ube.
t Pr
obab
ly a
bnor
mal
and
abo
ut t
o de
gene
rate
.
Fertilization and early cleavage in mice 173
tube. On day 6 eggs were only recovered from the uterus, and 85-8 % wereblastocysts of more than thirty cells. Thus, tubal transport of fertilized eggswas completed more than 102 h after ovulation.
B. Observations on natural mating in the Deer mouse
Ten naturally mated, mature animals were examined. The mean ovulationrate was 4-6 (± 0-3, range 3-6) with twenty-seven ovulations from the rightovary and nineteen from the left.
At 9-10 a.m. on day 1, eight animals all had motile spermatozoa in theiruteri and had completed their ovulation. Five of these animals yielded recentlypenetrated eggs and three had unpenetrated eggs. At re-examination 6-24 h. laterall the animals yielded penetrated eggs.
At midnight to 1 a.m. on day 2, two Deer mice yielded four pronuclear eggsfrom two Fallopian tubes. Three hours later a single Fallopian tube from anotheranimal contained a two-celled egg and one in the cleavage anaphase. At 9-10a.m. on day 2 five two-celled eggs were recovered from three Fallopian tubesof three animals.
C. Observations from induced ovulations
The results of the P.M.S. and H.C.G. treatments are summarized in Table 2.Naturally mated, mature Mongolian gerbils and Deer mice have mean ovulationrates of 6-6 (± 0-05) and 4-6 (± 0-3) respectively (cf. above and Marston &Chang, 1965). Thus, the hormonal treatments induced super-ovulation in themature Mongolian gerbil and immature Deer mouse. With the exception of oneanimal, which was approximately 8 days pregnant and had ovulated sixteennormal eggs, the mature Deer mouse ovulated a normal number of eggs. Themature Deer mice were possibly only responding to the H.C.G. with an inducedovulation from mature, cyclic, ovarian follicles (cf. Chitty & Austin, 1957).The morphology of the eggs and their surrounding cumulus clot suggested thatin both species ovulation had been induced at 10-14 h after the injection ofH.C.G.
Penetrated eggs were recovered from three Mongolian gerbils inseminatedat 12 h after H.C.G. injection. Out of seventy-two one-celled eggs recoveredfrom these animals at 12-14 h after insemination, twelve (16-6 %) were normalbut unpenetrated; three eggs (4-2 %) had an intact spermatozoon in the peri-vitelline space but not in the vitellus, and fifty-seven (79-2 %) were normalpronuclear eggs. Approximately 240 unpenetrated eggs were recovered from theremaining animals at 22-36 h after H.C.G. injection. Seventeen of these eggs weremorphologically abnormal; six eggs had degenerate second metaphase spindles;eight were grossly degenerate, and in three the second metaphase chromosomeshad apparently formed a single pronucleus with one or more nucleoli, but with-out the abstriction of a second polar body. First polar bodies could be identifiedin this last group, so they were not ovulated primary oocytes. Approximately
174 J. H. MARSTON & M. C. CHANG
Table 2. Induced ovulation in the Mongolian gerbil and Deer mouse followingtreatment with P.M.S. and H.C.G.
Doseof
P.M.S.(i.u.)
Mature Mongolian gerbils 40
Immature Deer mice
Mature Deer mice
302010
302010
0
3020
0
1040
* Additional observations fromanimals were not inseminated.)
Doseof
H.C.G.(i.u.)
10101010
10101010
101010
1010
No. ofanimals
pergroup
4444
5554
454
43
Numberanimals
withrecentlyovulated
eggs
3344
5554
454
33
Meannumber
of recentlyovulated
eggs(range)
390 (10-79)21 3 (16-25)18-3 (8-27)200 (9-25)
8-8 (2-20)12-6 (4-29)10-2 (3-19)
3-5 (2-5)*
6-5 (2-16)t3-8 (1-5)5-5 (4-7)4-3 (3-5)*8-7 (8-10)*
Dept. of Anatomy, University of Birmingham. (These
t One animal, found to be pregnant at autopsy, yielded sixteen normal eggs.
80 % of the unpenetrated eggs had first polar bodies, and the normal eggs allhad well-organized second metaphase spindles.
In the Deer mouse 75 % of the animals examined more than 3 h after artificialinsemination yielded penetrated eggs (Table 3). If the rate of development ofeggs following induced ovulation is not markedly different from that afternatural ovulation, these results demonstrate the cleavage of Deer mouse eggs.
All the Deer mice examined 2-5 h after insemination had motile spermatozoain their uteri. Only three penetrated eggs were recovered at 3-4 h after insemi-nation; whereas at 4-5 h the eggs from three immature mice were in earlystages of fertilization, and thirty-five (92 %) had emitted the second polar body.The first cleavage was completed within 24 h of insemination, or 18-20 h afterpenetration. The two-celled stage lasted for approximately 30 h as judged by therecovery of two-celled eggs at 52-53 h after insemination. The timing of sub-sequent cleavages was variable and they occurred at intervals of 10-20 h. Thethird cleavage was complete in sixteen (76 %) of the eggs examined 72-74 hafter insemination; and two animals yielded fertilized eggs from their uteri.At 96 h the majority of eggs had completed the fourth cleavage and five (45 %)had commenced the fifth cleavage: these eggs were recovered from the uterusas morulae.
Tab
le 3
. Mor
phol
ogy
of D
eer
mou
se e
ggs
at v
ario
us ti
mes
aft
er a
rtif
icia
l ins
emin
atio
n
Exa
min
atio
n(h
ours
aft
erin
sem
inat
ion)
2-3 3^
4-5
20-2
2
24-2
552
-53
72-7
4
96
Gro
up
Mat
ure
Mat
ure
Imm
atur
e
Mat
ure
Imm
atur
eIm
mat
ure
Imm
atur
e
Imm
atur
e
No.
of
anim
als
4 4 3 5 3 3 3 3
Tot
aleg
gsre
cove
red
17 19 41.
28 34 31 26 26
No.
of
anim
als
wit
hfe
rtili
zed
eggs
— 2 3 5 2 1 3 2
Tot
al e
ggs
reco
vere
dfr
om a
nim
als
wit
hfe
rtil
ized
eggs
— 9 41 28 30 15 26 21
Det
ails
of
fert
ilize
d eg
gsI a. I •5
" 8 !
All
nor
mal
, unp
enet
rate
d an
d at
sec
ond
mat
urat
ion
met
apha
se3
rece
ntly
fer
tiliz
ed w
ith
enla
rgin
g sp
erm
head
. One
has
sec
ond
pola
r bo
dy a
bstr
icte
d41
all
ear
ly p
ronu
clei
or
enla
rgin
g sp
erm
head
. 35
hav
e ab
stri
cted
sec
ond
pola
r bo
dy7
late
pro
nucl
ei,
3 pr
omet
apha
se,
7 m
etap
hase
, 11
tw
o-ce
ll1
met
apha
se,
13 t
wo-
cell
8 tw
o-ce
ll,
3 se
cond
cle
avag
e, 1
four
-cel
l1
two-
cell
, 4
four
-cel
l, 10
eig
ht-c
ell,
4 fo
urth
-cle
avag
e, 2
16-
cell
4 fo
urth
cle
avag
e, 2
16-
cell,
5 f
ifth
clea
vage
176 J. H. MARSTON & M. C. CHANG
D. Morphological observations(1) Spermatozoon
The Mongolian gerbil and Deer mouse spermatozoa are illustrated by Plate 1,fig. 6 and Plate 2, fig. 37. In profile the nuclear portion of the spermatozoonhead measured 4-5 fi long by 2-9 ju, at its widest in the Mongolian gerbil (meanoften measurements on stained specimens), and 5-6/tby 3-7 ju, in the Deer mouse:the apical portion of the head measured 5-0 fi and 2-7 /i, respectively. The acro-some of both species stained pink with Giemsa and seemed to overlie the an-terior portion of the nucleus; it fluoresced bright red with acridine orange,merging to yellow orange where it overlay the nucleus. The nucleus fluorescedbright apple-green and the mid-piece was pale green. In the Mongolian gerbila cytoplasmic droplet, fluorescing pale green without any red, was usuallycarried at various levels on the mid-piece of the epididymal spermatozoon, butin the Deer mouse it was smaller and generally at the end of the mid-piece. Thespermatozoon mid-piece and main-piece measured 40-45 ii and 85-100 ju, in theMongolian gerbil, respectively, and 15-8 ju, and 59-5 pi in the Deer mouse.
(2) Secondary oocyte
The eggs of both species were ovulated as secondary oocytes (Plate 1, fig. 1;Plate 2, fig. 27) surrounded by a dense cumulus clot. In unmated Mongoliangerbils the clot was still present up to 12 h after the estimated time of ovulation;but in mated animals the clot was dense at 3-4 a.m. on day 1 and completelyabsent by 9-10 a.m. at about 9-12 h after ovulation. In naturally mated Deermice the cumulus clot was dense up to 5 p.m. on day 1 but the eggs were nakedby midnight on day 1.
The dimensions of the penetrated and unpenetrated eggs of both species aresummarized in Table 4.
In both species the egg cytoplasm was finely granular with some coarseinclusions. However, when recently ovulated unpenetrated Deer mouse eggs werecarefully compressed and examined under the oil-immersion objective, fine'cortical granules' could be identified in the cortex close to the surface of thevitellus and the cytoplasmic membrane (Plate 2, figs. 28, 29). The corticalgranules resembled those described in unpenetrated eggs of the Golden hamsterin size, distribution and general appearance (Austin, 19566; Yanagimachi &Chang, 1961). They were not observed in penetrated Deer mouse eggs; and,despite careful examination, cortical granules were not identified in Mongoliangerbil eggs.
More than 80 % of the recently ovulated eggs of the Mongolian gerbil had adistinct first polar body, in which the chromatin was variously arranged as bars,strands or granules: the first polar body often appeared lobulated or fragmentedinto two or three parts. In Deer mouse eggs the first polar body could only beidentified as a faint cytoplasmic globule in recently ovulated eggs. After staining,
Tab
le 4
. Mea
sure
men
ts
of l
ivin
g M
ongo
lian
ger
bil
and
Dee
r m
ouse
sin
gle-
cell
ed e
ggs*
Spe
cies
Gro
up
No.
of
eggs
mea
sure
d
Mea
nov
eral
ldi
amet
er (
/*)
Mea
nvi
tell
ine
diam
eter
(/*
)
Mea
nth
ickn
ess
of z
ona
pell
ucid
a (/*
)
Mon
goli
an g
erbi
l(M
erio
nes
ungu
icul
atus
)
Dee
r m
ouse
(P
erom
yscu
sm
anic
ulat
us b
aird
ii)
Un
pen
etra
ted
(9 p
.m.
day
0,3
a.m
. da
y 1)
Ear
ly p
enet
rate
d (9
p.m
. da
y 0,
3 a.
m.
day
1)P
ronu
clea
r (m
idni
ght
day
0,9
p.m
. da
y 1)
Pro
nu
clea
r or
fir
st c
leav
age
(mid
nigh
t da
y 1,
3 a
.m.
day
2)
Un
pen
etra
ted
(ca.
4 h
fro
min
duce
d ov
ulat
ions
)E
arly
pen
etra
ted
or p
ronu
clea
r(n
atu
ral
mat
ings
day
1,
9 a.
m.-
10 a
.m.)
25 25 32 30 18 16
100-
9
99-6
102-
6
103-
2
1131
105-
8
69-4
69-2
72-6
68-9
74-4
71-5
8-4
7-8
7-8
7-8
10
1
9-6
* V
aria
bili
ty o
f m
easu
rem
ents
was
suc
h th
at,
wit
hin
spec
ies,
dif
fere
nces
wer
e no
t si
gnif
ican
t.
1
178 J. H. MARSTON & M. C. CHANG
it was difficult to identify and usually did not contain discrete masses ofchromatin.
In both species, all the recently ovulated, unpenetrated eggs contained asecond metaphase spindle, usually lying paratangentially to the surface of thevitellus. Under the present method of fixation and staining the spindles hadrelatively broad ends without any indication of centriolar structures: they weredifficult to observe in the living egg, but showed a similar structure.
Eggs at early stages of fertilization were only recovered from the ampulla ofthe Fallopian tube, and penetration occurred while the cumulus clot about theeggs was still very dense. The number of spermatozoa in the ampulla appearedlow, and usually not more than three spermatozoa were identified in the cumulusclot surrounding recently penetrated eggs.
(3) Spermatozoa penetration of the zona pellucida
Actual penetration of the zona pellucida was not observed. However, at3-4 a.m. on day 1 one Mongolian gerbil egg had the filamentous tip of thespermatozoan main piece extending obliquely through the zona pellucida inthe form of a 'penetration curve' (Dickmann, 1964). The greater portion of themain piece had entered the vitellus and pronuclei were forming. At 4-5 h afterartificial insemination a Deer mouse egg was observed with approximately halfof the main piece extending through the zona pellucida in a 'penetration curve'.The rest of the main piece and most of the mid-piece lay in the perivitelline spacealthough the tip of the mid-piece had entered the vitellus and the spermatozoanhead was enlarged. Single 'penetration slits' (Austin, 1951a) in the form of'penetration curves' were also identified in nine Mongolian gerbil eggs and oneDeer mouse egg. They were especially prominent when a portion of the vitellusprotruded through the slit during compression of the egg (Plate 1, fig. 5;Plate 2, fig. 33). 'Penetration slits' were not seen in unpenetrated eggs.
(4) Spermatozoa in the perivitelline space
Polyspermic fertilization was not recognized in Mongolian gerbil eggs; yetthe occurrence of intact, 'supplementary spermatozoa' in the perivitelline spaceof pronuclear eggs was 13-3 % after superovulation (see above), 15-8 % for 114pronuclear eggs and 11-8 % for 102 cleaving or two-celled eggs from naturalmatings. Of these thirty-eight eggs, thirty-three had one supplementary sper-matozoon, four had two, and one egg had four spermatozoa. Supplementaryspermatozoa were not observed in the perivitelline space of eight- to sixteen-celled eggs, and only three eggs in the stages from the second to third cleavageshad a supplementary spermatozoon. All the supplementary spermatozoaexamined in detail had lost the filamentous tip of the spermatozoan head, in-cluding the acrosome, but otherwise their structure appeared intact (Plate 1,fig. 7).
Fertilization and early cleavage in mice 179
Three of 135 one-celled or early two-celled Deer mouse eggs had been pene-trated by two spermatozoa. One pronuclear egg from a naturally mated animalhad a supplementary spermatozoon. The other two eggs were recovered fromartificially inseminated animals and each had two distinct spermatozoan tailsin the perivitelline space: in one the spermatozoan heads were just enteringthe vitellus and in the other two male pronuclei were forming. These eggswere lost during fixation, so the probable occurrence of dispermic fertilizationcould not be proved.
(5) Spermatozoan penetration into the vitellus
In fertilized Mongolian gerbil eggs the spermatozoan tail had completelyentered the vitellus, and well-stained preparations showed that it usually hadseparated into two or more filaments. These filaments remained united at theirproximal ends, but separated along the mid-piece and the whole of the mainpiece. Few Deer mouse eggs were examined at late stages of pronuclear develop-ment, and in these it was either impossible to identify the spermatozoan tailor else its location in the vitellus or perivitelline space was questionable. Sixtyrecently penetrated or early pronuclear eggs were examined in their fresh con-dition, and forty-seven (78 %) had virtually all of the fertilizing spermatozoantail lying in the perivitelline space (Plate 2, fig. 30). After staining, all of thirty-seven eggs appeared to have the entire main-piece and a posterior portion of themid-piece in the perivitelline space: the tip of the mid-piece had an irregularoutline and had probably entered the vitellus.
(6) The second maturation division, second polar body emission and transformationof the fertilizing spermatozoon
Twenty-one Mongolian gerbil and eleven Deer mouse eggs were examined indetail during these stages. They were probably examined within 2 h of pene-tration and their morphology agreed with the observations on rat, mouse andGolden hamster eggs recorded by Austin (1951 b, 1956a), Odor & Blandau(1951) and Chang & Hunt (1962).
During the early telophase, granular elements aggregated at the equator ofthe second maturation spindle (Plate 2, fig. 41) and coalesced to form a distinctmid-body which underwent condensation (Plate 2, fig. 43) during emission ofthe polar body. Constriction of the cytoplasmic membrane seemed to occur oneither side of the mid-body, so that remnants of the spindle fibres extendedbetween the vitellus and polar body through the mid-body (Plate 1, figs. 13,14, 15). The mid-body could be identified close to the second polar body up tothe first cleavage and sometimes beyond this time. In about twenty Mongoliangerbil eggs remnants of a similar mid-body were also found with the first polarbody. The chromatin of the second polar body did not form a vesicular nucleusat any stage of development in either species.
180 J. H. MARSTON & M. C. CHANG
Stages in the transformation of the fertilizing spermatozoon's head areillustrated by Plate 1, figs. 8, 9, and Plate 2, figs. 34-36. In both species the headshowed marked enlargement and rounding out soon after entering the vitellus.Its staining intensity was reduced and a distinct nuclear membrane could not beidentified even though its demarcation from the cytoplasm was clearly defined.The mid-piece usually separated from the head during transformation. In Deer
EXPLANATION OF PLATES
Figs. 1, 3, 5, 16-20, 22-24, 27-31, 49, 51, 52, 54 and 56-58 are photographs of fresh speci-mens : all other figures are of fixed and stained preparations. The photographs were taken on aphase-contrast microscope. 1st = First polar body; 2nd = second polar body; m.b. = mid-body of second maturation division; r. = rod; sp. = spermatozoan tail.
PLATE 1
Mongolian gerbil: fertilization and cleavage stages
Fig. 1. Unpenetrated egg (9-10 a.m. on day 1) slightly compressed (x ca. 250).
Fig. 2. Same: to show metaphase of second maturation division (polar view) and first polarbody ( x ca. 250).
Fig. 3. Pronuclear egg (9-10 a.m. on day 1) (xra. 250).
Fig. 4. Pronuclear egg with micro-nuclei (midnight, day 2) ( x ca. 250).
Fig. 5. Spermatozoan penetration slit with protrusion from vitellus (x ca. 1000).
Fig. 6. Epididymal spermatozoon (xca. 1000).
Fig. 7. Supplementary spermatozoa in perivitelline space (x ca. 1000).
Figs. 8-12. Sequence to show transformation of fertilizing spermatozoon's head into theearly male pronucleus (x ca. 1000).
Fig. 13. Female pronucleus and emission of second polar body (x ca. 1000).
Fig. 14. Second polar body, mid-body and remnants of second maturation spindle(x ca. 1000).
Fig. 15. Detail of pronuclear egg at about 4 h after penetration. The male pronucleus is thelarger (xca. 1000).
Fig. 16. Detail of pronuclear egg at about 10 h after penetration to show (a) female and (b) malepronuclei (x ca. 1000).
Fig. 17. Degenerate, unpenetrated egg with micro-nuclei (24h after ovulation) (xca. 250).
Fig. 18. Two-cell eggs (9-10 a.m. on day 3) (xca. 135).
Fig. 19. Two-cell egg (9-10 a.m. on day 3) (x ca. 250).
Fig. 20. Two-cell egg with sub-nucleus in one blastomere (midnight, day 2) (x ca. 250).
Fig. 21. Four-cell egg (9-10 a.m. on day 3) (x ca. 250).
Fig. 22. Four-cell eggs (9-10a.m. on day 3) (xca. 135).
Fig. 23. Eight-cell egg (9-10 a.m. on day 4) (x ca. 135).
Fig. 24. Blastocyst (9-10 a.m. on day 6) (x ca. 250).
Fig. 25. Seven-cell egg (9-10 a.m. on day 4) (x ca. 250).
Fig. 26. Eight-cell egg (9-10 a.m. on day 4) (x ca. 250).
J. Embryol. exp. Morph., Vol. 15, Part 2 PLATE 1
J. H. MARSTON & M. C. CHANG facing p. 180
Fertilization and early cleavage in mice 181
mouse eggs a small ' rod' also appeared to separate from the spermatozoon'shead in four out of eleven eggs (Plate 2, fig. 37). It appeared to have two or three'prongs', but was not highly refractile and could not be positively identifiedas originating from the spermatozoon's head. No such structure was found inMongolian gerbil eggs.
(7) Pronuclear development
In both species pronuclear development followed the pattern described byAustin (19516, 1956a) and Odor & Blandau (1951) for the rat and Goldenhamster (Plate 1, figs. 11, 12, 15, 16; Plate 2, figs. 38-40, 44-49).
During development it was usually possible to identify the female pronucleus,which, in the early stages, was closely associated with the second polar body andremnants of the second maturation spindle. In Mongolian gerbil eggs the malepronucleus was often close to remnants of the fertilizing spermatozoon's tail.The male pronucleus was larger than the female (Plate 1, figs. 3, 15, 16; Plate 2,figs. 30, 49, 56) in both species at all stages; but the actual rate of growthand development of the male and female pronuclei seemed to be co-ordinated.
In Deer mouse eggs the pronuclei generally had one nucleolus with a largecentral inclusion of bright contrast (Plate 2, fig. 49): a few pronuclei had onelarge nucleolus and up to four small nucleoli. The number of nucleoli inMongolian gerbil pronuclei varied with the development of the egg. However,the female pronucleus tended to have one nucleolus, and rarely more than threenucleoli. The male pronucleus tended to have one to three large nucleoli, butthese were frequently associated with up to ten or more small nucleoli. In bothspecies maximal development of the pronuclei was reached at about 3 h beforethe onset of cleavage, and in the Mongolian gerbil it appeared that the malepronucleus entered prophase of the first cleavage division shortly before thefemale pronucleus. Pronuclei at maximal development tended to lie centrallyin the vitellus: they did not make contact with one another and showed noevidence of pronuclear fusion.
(8) Syngamy, first cleavage and the two-celled stage
Seven Mongolian gerbil eggs were examined during prophase of the firstcleavage division. They showed progressive dissolution of the nucleoli, disap-pearance of pronuclear membrane, and loss of pronuclear contrast within thevitellus. This was especially obvious in living eggs under the phase-contrastmicroscope; and eventually the pronuclei were invisible (Plate 3, figs. 56-58).In the stained preparations two separate groups of loosely interwoven chromatinstrands could be identified; they lay centrally in the vitellus and were presumablythe pronuclear chromosomes. In slightly earlier stages, chromatin filamentscould be identified lying close to the pronuclear membrane, or, in its absence,close to the margin of the pronuclear material.
182 J. H. MARSTON & M. C. CHANG
Eight Mongolian gerbil and three Deer mouse eggs in the pro-metaphase stageeach showed two, separate, fairly compact groups of chromosomes which werenot oriented in any one direction. The chromosome groups appeared to behaploid and the chromosomes did not show distinct chromatids (Plate 2,figs. 59, 60, 64; Plate 3, figs. 71, 72, 73): the chromosomes gave the impressionof moving towards one another as if during the completion of syngamy (Plate 3,fig. 74). No indication was obtained as to the mechanism of this movement.
Syngamy was completed at the formation of the first cleavage metaphasespindle and no intermediate stages between pro-metaphase and metaphase wereidentified. The first cleavage spindle was fully formed in twenty Mongoliangerbil and eight Deer mouse eggs. A single group of chromosomes lay centrallyin the vitellus, loosely arranged at the equator of the spindle (Plate, 3, figs. 61,65,66). The metaphase plate and spindle were approximately twice as large as theequivalent structures of the second maturation division. However, the spindlestructure was not so clearly defined and intensely stained as the second matura-tion spindle (cf. Odor & Blandau, 1951). In good preparations the separationof the metaphase chromosomes into two chromatids could be recognized
PLATE 2
Deer mouse: fertilization and cleavage stages
Fig. 27. Impenetrated egg (9-10 a.m. on day 1) (x ca. 250).
Fig. 28. Cortical granules in an impenetrated egg (x ca. 1000).Fig. 29. Recently penetrated egg without cortical granules. Fertilizing spermatozoan tail inthe perivitelline space (x ca. 1000).Fig. 30. Pronuclear egg. The male pronucleus is the larger (9-10 a.m. on day 1) (x ca. 250).Fig. 31. Pronuclear egg with subnucleus. (9-10 a.m. on day 1) (x ca. 250).Fig. 32. Epididymal spermatozoon (xca. 1000).Fig. 33. Spermatozoan penetration slit with protrusion from vitellus (x ca. 1000).Figs. 34-40. Sequence to show transformation of fertilizing spermatozoon's head into malepronucleus (x ca. 1000).Fig. 41. Early telophase of second maturation division with swollen spermatozoon head(cf. fig. 35) (xca. 250).Fig. 42. Metaphase of second maturation division (x ca. 1000).Fig. 43. Late telophase of second maturation division. The preparation is slightly distorted(xca. 1000).Figs. 44-48. Sequence to show transformation of female chromatin into female pronucleus(xca. 1000).Fig. 49. Pronuclei (9-10 a.m. on day 1). The male pronucleus is the larger (x ca. 1000).Fig. 50. Early second polar body and mid-body (x ca. 1000).Fig. 51. Four- and eight-cell eggs (72-74 h after insemination) (x ca. 250).Fig. 52. Two-cell egg (24 h after insemination) (x ca. 250).Fig. 53. Two-cell egg (9-10 a.m. on day 2) (x ca. 250).Fig. 54. Eight-cell egg (74 h after insemination) (x ca. 250).Fig. 55. Morula (96 h after insemination) (x ca. 250).
J. Embryol. ex p. Morph., Vol. 15, Part 2 PLATE 2
27
35 UM 36 "T- 37 r * 38
J. H. MARSTON & M. C. CHANG facing p. 182
/. Embryol. exp. Morph., Vol. 15, Part 2 PLATE 3
J. H. MARSTON & M. C. CHANG facing p. 183
Fertilization and early cleavage in mice 183
(Plate 3, fig. 65), but it was not possible to identify any centriolar structures atthe poles of the spindle.
One Deer mouse egg was examined in early anaphase and the chromosomeswere seen as two groups of distinctly oriented fibres separating along the axisof the spindle. Three Mongolian gerbil eggs in early telophase showed a distinctmid-body at the equator of the spindle. It seemed to consist of an aggregationof granules in the form of an annulus about the equator of the spindle (Plate 3,figs. 62, 67, 68). Three Mongolian gerbil and two Deer mouse eggs in late telo-phase were recovered following the completion of cytokinesis but before forma-tion of the blastomere nuclei. The greatly condensed mid-body of the telophasespindle could be identified lying centrally in the cleavage furrow (Plate 3, figs.69, 70, 75, 76).
In living eggs the vitellus was usually spherical during early stages of mitosis,and appeared to become elongated or irregular in shape during telophase.Actual formation of the cleavage furrow was not observed, and under the phase-
PLATE 3
The first cleavage division: Mongolian gerbil
Figs. 56-58. Transformation of pronuclei during prophase (midnight-1 a.m. on day 2)(x ca. 250).
Fig. 59. Egg from fig. 58 after fixation and staining. Prometaphase (x ca. 250).
Fig. 60. Prometaphase (x ca. 250).
Fig. 61. Metaphase (xca. 250).
Fig. 62. Telophase (x ca. 250).
Fig. 63. Two-cell egg showing mid-body (day 2) (x ca. 250).
Fig. 64. Detail of fig. 59 (x ca. 500).Figs. 65, 66. Metaphase plate (equatorial view) (x ca. 500).Figs. 67, 68. Early telophase to show the mid-body (x ca. 500).Figs. 69, 70. Late teleophase after completion of cytokinesis to show the mid-body andfertilizing spermatozoon's tail (xca. 500).
First cleavage division: Deer mouse
Figs. 71, 72. Late pro-metaphase (x ca. 250 and 500).Fig. 73. Early metaphase (x ca. 250).Fig. 74. Metaphase plate (polar view) ( x ca. 500).Fig. 75. Late telophase after completion of cytokinesis (x ca. 250).Fig. 76. Detail of fig. 75 to show mid-body (x ca. 500).Fig. 77. Early two-cell egg (x ca. 250).
Fig. 78. Detail of fig. 77 with montage to show mid-body in the cleavage furrow; in thepreparation it lay below the focal plane (x ca. 500).Fig. 79. Early two-cell egg (x ca. 250).Fig. 80. Detail of fig. 79 with montage to show mid-body in the cleavage furrow; in thepreparation it lay below the focal plane (x ca. 500).
184 J. H. MARSTON & M. C. CHANG
contrast microscope no stages of mitosis beyond the disappearance of the pro-nuclei could be identified in the living eggs. Cytoplasmic cleavage seemed to beequal in both species, and there was probably some reduction in cytoplasmicvolume at cleavage.
Following the completion of cytokinesis, formation of the blastomere nucleiwas first indicated by the appearance of primary nucleoli within the chromatin oflate telophase (Plate 3, fig. 78). Expansion of the nucleus, formation of a distinctnuclear membrane and development of large nucleoli occurred very rapidly,for no intermediate stages were identified. Two-celled eggs are illustrated byPlate 1, figs. 18, 19, and Plate 2, figs. 52 and 53.
Remnants of the fertilizing spermatozoan tail were not identified in two-celled Deer mouse eggs, but 52-6 % of thirty-eight eggs had a distinct secondpolar body and the mid-body of the cleavage spindle lay in the cleavage furrow.In sixty-one two-celled Mongolian gerbil eggs, 18 % had a second polar body,65-5 % had a distinct mid-body in the cleavage furrow, and in 55-8 % theremnants of the fertilizing spermatozoon's tail were closely associated with themid-body as they extended across the cleavage furrow and lay within bothblastomeres. Six additional ova (9-8 %) had remnants of the tail lying in onlyone blastomere.
(9) Second to fifth cleavage stages
Four-celled Mongolian gerbil eggs showed almost equal cytoplasmic cleavage(Plate 1, figs. 20, 22), but all five Deer mouse eggs at this stage had two blasto-meres distinctly larger than the others (Plate 2, fig. 51). However, four of theseunequally cleaved eggs were recovered 72-74 h after artificial insemination, andthey could be abnormal. The blastomeres of the eight- and sixteen-celled stagesappeared relatively equal in both species, and were usually in a compact, almostspherical, group (Plate 1, figs. 23, 26; Plate 2, figs. 54, 55). Usually each blasto-mere nucleus had three to five nucleoli up to the sixteen-celled stage, thereafter,the number of nucleoli tended to be reduced to one or two per nucleus.
Remnants of the fertilizing spermatozoan's tail could be identified in mostwell-stained eight- to sixteen-celled Mongolian gerbil eggs (Plate 1, fig. 25).These remnants appeared to extend from one blastomere to another, lyingwithin as many as four blastomeres, and nodular, darkly straining bodies couldoccasionally be identified along their length. The bodies were thought to be themid-bodies of the most recent cleavage spindles, for their number in any oneegg tallied with the number of completed cleavages. Similar mid-bodies wereidentified in Deer mouse eggs.
Blastocysts were not examined in the Deer mouse, but in the Mongoliangerbil on day 6 they contained more than thirty cells, had an intact, undistendedzona pellucida and generally resembled early blastocysts of the rat, mouse andGolden hamster (Plate 1, fig. 26).
Fertilization and early cleavage in mice 185
(10) Abnormalities of fertilization and cleavage
At 3 to 4 a.m. on day 1 one Mongolian gerbil yielded five pronuclear eggswith second polar bodies, normal male pronuclei and vitelline sperm tails: threeof these eggs had normal female pronuclei, one had six micro-nuclei beside asmall female pronucleus, and in the other a subnucleus lay beside the femalepronucleus. At 9-10 a.m. on day 1 another animal yielded three normal pro-nuclear eggs and one egg with a subnucleus close to the female pronucleus. Twoanimals were found with abnormal eggs at midnight to 1 a.m. on day 2. In thefirst case, one out of six two-celled eggs had a single subnucleus associated withthe blastomere nucleus (Plate 1, fig. 20). The other animal yielded one normaland four abnormal pronuclear eggs. The abnormal eggs had second polarbodies, vitelline sperm tails and apparently normal male pronuclei. The femalepronuclei were represented by ten or more micro-nuclei scattered through thevitellus in a manner which suggested that they had originated from fragments ofthe female chromatin (Plate 1, fig. 4). In two of the four cases the animals hadreceived surgical anaesthesia when their eggs were at early stages of fertilization.
In the Deer mouse two probable cases of dispermic fertilization have alreadybeen noted. Three of twenty-four pronuclear eggs from naturally mated Deermice contained a small subnucleus lying close to the female pronucleus in thepresence of a normal second polar body and male pronucleus (Plate 2, fig. 31).
(11) Fate of unfertilized eggs
More than 24 h after ovulation unpenetrated Mongolian gerbil and Deermouse eggs showed degeneration of their cytoplasm. After staining, the secondmaturation spindle seemed broader and more loosely organized, the fibres wereless densely stained, and individual fragments of chromatin were often widelydisplaced from the metaphase plate. Approximately ten Mongolian gerbil andeight Deer mouse unpenetrated eggs were found with very degenerate cyto-plasm and ten or more micro-nuclei scattered through the vitellus. These micro-nuclei had one or two nucleoli (Plate 1, fig. 17). In both species unfertilized eggstraversed the Fallopian tube at an apparently normal rate and could be re-covered from the uterus as grossly degenerate forms.
DISCUSSION
In the present study the timing of the critical events of ovulation, penetrationand cleavage could not be precisely defined. As a result, it is difficult to establishthe need for spermatozoan capacitation (Austin, 1951a; Chang, 1951) and'maturation' of the ovulated egg (Austin & Braden, 1954) as essential pre-liminaries to penetration in the Mongolian gerbil and Deer mouse.
In the Mongolian gerbil, an interval of 8-12 h was observed between matingand spermatozoan penetration, at which time the egg was aged about 4hafter ovulation. The time between mating and ovulation was probably sufficient
186 J. H. MARSTON & M. C. CHANG
for spermatozoan transportation and capacitation; thus, the delay in penetrationwould cover the time required for 'maturation' and penetration of the cumulusand zona pellucida. The observations in the Deer mouse were not strictlyphysiological, because induced ovulation and artificial insemination were used.However, an interval of about 3 h was observed between insemination andspermatozoan penetration; and as insemination was probably performed soonafter induced ovulation, this interval would include the time for transportation,penetration of the cumulus and zona pellucida, and would cover any periodnecessary for 'capacitation' and egg 'maturation'.
Austin & Braden (1954) suggested that during the 2-4 h interval betweenovulation and penetration in the naturally mated rat, some change occurredin the egg membranes which had to be completed before the spermatozoon couldpenetrate the egg; this change might represent a final stage for the 'maturation'of the egg. The interval between ovulation and penetration following naturalmating was about 5 h in the mouse (Braden & Austin, 1954) and 2 h in theGolden hamster (Austin, 1956 c; Strauss, 1956), but it was insignificant in therabbit (Austin & Braden, 1954) and sheep (Braden, 1959). The present obser-vations support Braden's suggestion (1959) that 'maturation' may be a processpeculiar to rodent species, for there was a sufficient time for 'maturation' tooccur in the Deer mouse and Mongolian gerbil eggs.
The interval between ovulation and penetration was reduced following delayedmating in the mouse (Braden & Austin, 1954), following induced ovulation inthe rat (Austin, 1951a) and mouse (Braden, 1959), and also differed betweentwo inbred strains of mouse (Braden, 1958). These differences could be relatedto variation in the density of the cumulus clot about the eggs, thus suggestingthat an essential change in state of the cumulus clot might occur during matura-tion (Braden, 1959): such a change would be expected to be most subtle,possibly occurring within the intercellular matrix of the cumulus. In the presentstudy we have not detected any gross differences between the cumulus sur-rounding recently ovulated and recently penetrated eggs. It has not yet beenclearly established whether 'maturation' coincides with a complete failure ofspermatozoa to enter the cumulus, or whether it represents the time for them topenetrate the cumulus. If 'maturation' is indeed related to a change in thecumulus, it could be independent of the condition of the eggs and related to theeffects of post-ovulatory ageing interacting with the environment of the Fallopiantube.
Our observations on the rates of cleavage have been summarized in Table 5and compared with the available data for other Cricetidae. The duration of thetwo- to four-cell stage was approximately 30 h in the Golden hamster, Deermouse and Mongolian gerbil. It was considerably longer than the intervalsbetween subsequent cleavages and between fertilization and the first cleavage.The rate of tubal transport was slower in the Mongolian gerbil and the eggswere thus in a more advanced state of cleavage when they entered the uterus.
Tab
le 5
. Est
imat
ed r
ates
of
clea
vage
in
Cri
ceti
dae
Est
imat
ed d
urat
ion
of e
ach
stag
e (h
)E
ntry
to
uter
us
Spec
ies
(sub
fam
ily)
Met
hod
2-ce
ll2-
4ce
ll4-
8ce
ll8-
16ce
llT
ime
Stag
eSo
urce
Gol
den
ham
ster
N
atur
al(M
esoc
rice
tus
mat
ing
aura
tus:
(e
veni
ng),
Cri
cetin
ae)
spon
tane
ous
ovul
atio
n
16-1
824
-26
Dee
r m
ouse
(Per
omys
cus
man
icul
atus
bair
dii:
Cri
cetin
ae)
Fiel
d vo
le(M
icro
tus
agre
stis
:M
icro
tinae
)
Mon
golia
nge
rbil
(Mer
ione
sun
guic
ulat
us:
Ger
billi
nae)
Indu
ced
ovul
atio
nw
ith a
rtif
icia
lin
sem
inat
ion
at t
ime
ofov
ulat
ion
Nat
ural
mat
ing
(ovu
latio
npr
obab
lyin
duce
d by
coitu
s)N
atur
alm
atin
g(e
veni
ng),
spon
tane
ous
ovul
atio
n
18-2
0
< 2
4(p
ossi
bly
ca.
12)
22-2
4
ca.
30
Var
iabl
e:m
inim
ales
timat
eca
. 12
> 3
0
12-1
6
Var
iabl
e,10
-20
Var
iabl
e:m
inim
ales
timat
eca
. 12
20-2
4
12-1
6
Var
iabl
e,10
-20
Var
iabl
e:m
inim
ales
timat
edca
. 12
20-2
4
60-6
6 h
afte
rov
ulat
ion.
< 7
2 h
post
coitu
m
ca. 7
2 h
afte
rin
sem
inat
ion
and
indu
ced
ovul
atio
n
Var
iabl
e bu
tus
ually
> 7
0 h
post
coitu
m
>
102
hpo
stov
ulat
ion
Thi
rd c
leav
age
prob
ably
initi
ated
in
Fallo
pian
tube
and
com
plet
ed i
nth
e ut
erus
8-16
cel
ls
Var
iabl
e bu
tus
ually
> 8
cel
ls
Gra
ves (
1945
),V
enab
le (1
946a
),W
ard
(194
8),
Aus
tin (1
956)
,St
raus
s(19
56)
Ham
ilton
&Sa
mue
l (1
956)
Har
vey
et a
l.(1
961)
Pres
ent
stud
y
Aus
tin (1
957)
,C
hitty
&A
ustin
(195
7)
16-3
2 ce
lls
Pres
ent
stud
y
s § oo
188 J. H. MARSTON & M. C. CHANG
The morphology of the Mongolian gerbil spermatozoon resembled that of theLibyan gerbil (subfamily Gerbillinae: Meriones libycus) in having a finelytapered apex, whereas the Deer mouse spermatozoon was similar to that ofthe Field vole and Cotton rat (subfamily Cricetinae: Sigmodon hispidus) and hada sharply recurved apex (cf. illustrations of Austin, 1957; Bishop & Walton,1960). We have not observed any change in the spermatozoon before penetra-tion of the zona pellucida, but from the morphology of supplementary sperma-tozoa in Mongolian gerbil eggs it seemed that the acrosome had been lost duringpenetration. The presence of 'penetration slits' and 'penetration curves'through the zona pellucida agrees with previous observations in other species(Austin, 1951 a; Austin & Bishop, 1958; Dickmann, 1964; Dickmann &Dzuik, 1964; Dzuik & Dickmann, 1965; Yanagimachi, 1964). In the Deermouse there was some evidence that the fertilizing spermatozoon's tail did notenter the vitellus, a situation similar to that in the Field vole (Austin, 1957) andunlike that in other rodents. However, more information is required on thispoint.
The unpenetrated Deer mouse egg had distinct cortical granules, whichwere visible under the phase-contrast microscope. Such cortical granuleshave only been recorded for the Golden hamster (Austin, 1956Z?; Yanagimachi& Chang, 1961), although Szollozi (1962) has suggested that they are presentat the ultra-microscopic level in most mammalian eggs. The cortical granulesunderwent dissolution during sperm penetration. The Deer mouse and Goldenhamster both belong to the subfamily Cricetinae, and their eggs exhibit a strong'zona block' and a weak 'vitelline block' to polyspermic penetration. Thisproperty is shared with the Field vole egg and completely reversed in Mongoliangerbil eggs (cf. Austin, 1956a, 1957). Dispermic fertilization was not ob-served in Mongolian gerbil eggs, although penetration by more than onespermatozoon occurred in 10-15 % of all cases, whereas dispermic fertilizationusually followed dispermic penetration in the Deer mouse, Golden hamster, andField vole. It would be of interest to know whether the presence of microscopiccortical granules is characteristic for unpenetrated eggs from members of thesubfamily Cricetinae and also to define the function of these granules in estab-lishing the 'zona block' to polyspermy.
In both species, a distinct mid-body was formed on the second maturationand first cleavage spindles, and probably on subsequent cleavage spindles up tothe fourth cleavage. In the Mongolian gerbil egg a mid-body was probablyformed on the first maturation spindle, as evidenced by the remnants associatedwith the first polar body. This confirms observations (Marston, Yanagimachi,Chang & Hunt, 1964) that mid-body formation occurs on all maturation andearly cleavage spindles of the mouse, rat and Golden hamster. The work ofBuck (1963) suggests that the mid-body may play some part in the definition ofthe future plane of cleavage or polar body emission, or even be actively in-volved in the process of cleavage and emission.
Fertilization and early cleavage in mice 189
Organized division of the second maturation spindle was not observed inunpenetrated Mongolian gerbil and Deer mouse eggs, although it does occurfrequently in the Golden hamster (Austin, 1956a; Yanagimachi & Chang, 1961).The presence of micro-nuclei and scattered chromatin granules in unpenetratedaged eggs suggests that fragmentation of the spindle had occurred. It followsthat the presence of micro-nuclei in penetrated eggs might result from partialfragmentation of the female chromatin at some time during, or subsequent to,the completion of the second maturation telophase. These eggs may havedeveloped after the penetration of abnormal eggs, possibly damaged by theeffects of ageing or experimental anaesthesia.
SUMMARY
1. The timing of ovulation, penetration of spermatozoa and cleavage hasbeen studied in naturally mated Mongolian gerbils maintained in a controlledenvironment.
2. The timing of sperm penetration and cleavage was studied in Deer micefollowing artificial insemination of mature and immature animals close to thetime of gonadotrophin-induced ovulation. A few naturally mated animalswere also studied.
3. The morphology of fertilization and cleavage in the Mongolian gerbiland Deer mouse is described and illustrated.
RESUME
Morphologie et chronologie de la fecondation et des premiers clivages chez deuxrongeurs: la gerbille de Mongolie (Meriones unguiculatus, Gerbillidae)
et Peromyscus maniculatus (Cricetidae)
1. La chronologie de l'ovulation, de la penetration des spermatozoides et dela segmentation a ete etudiee chez des gerbilles de Mongolie naturellementaccouplees et maintenues dans un milieu controle.
2. La chronologie de la penetration des spermatozoides et de la segmentationa ete etudiee chez des Peromyscus apres insemination artificielle d'animauxmatures et immatures, peu de temps apres l'ovulation induite par les gonado-trophines. Quelques animaux accouples naturellement ont egalement eteetudies.
3. On decrit et on illustre la morphologie de la fecondation et de la segmen-tation chez la gerbille de Mongolie et les Peromyscus.
This work was supported by a grant from the National Institute of Health (GM 10529-01)and the Population Council Inc. One of us (J. H. M.) is grateful to the Royal College ofVeterinary Surgeons for providing a travel grant. Dr R. Yanagimachi gave considerablehelp in the examination of cortical granules in Deer mouse eggs, and Dr L. T. Turbyfillco-operated most valuably in the studies on induced ovulation. Messrs J. Zucker andT. Luuko were responsible for the care of the animals. Part of the cost of preparing thismanuscript was provided by a grant from the Ford Foundation.
190 J. H. MARSTON & M. C. CHANG
REFERENCES
AUSTIN, C. R. (1951a). Observations on the penetration of sperm into the mammalian egg.Austr. J. scient. Res. B, 4, 581-96.
AUSTIN, C. R. (19516). The formation growth and conjugation of pronuclei in the rat egg./ . Rl microsc. Soc. 71, 295-306.
AUSTIN, C. R. (1956 a). Ovulation, fertilization and early cleavage in the hamster (Meso-cricetus auratus). J. Rl microsc. Soc. 75, 141-54.
AUSTIN, C. R. (19566). Cortical granules in hamster eggs. Expl Cell Res. 10, 533-40.AUSTIN, C. R. (1957). Fertilization, early cleavage and associated phenomena in the Field
vole (Microtus agrestis). J. Anat. 91, 1-11.AUSTIN, C. R. & BISHOP, M. W. H. (1958). Role of the rodent acrosome and perforatorium
in fertilization. Proc. R. Soc. B, 149, 241-8.AUSTIN, C. R. & BRADEN, A. W. H. (1954). Time relations and their significance in ovulation
and penetration of eggs in rats and rabbits. Aust. J. biol. Sci. 7, 179-94.BISHOP, M. W. H. & SMILES, J. (1957). Induced fluorescence in mammalian gametes with
acridine orange. Nature, Lond., 179, 307-8.BISHOP, M. W. H. & WALTON, A. (1960). Chapter 7 of Marshall's Physiology of Reproduction,
3rd ed. Ed. A. S. Parkes. London: Longmans.BRADEN, A. W. H. (1958). Variation between strains of mice in phenomena associated with
sperm penetration and fertilization. / . Genet. 56, 37-47.BRADEN, A. W. H. (1959). Spermatozoon penetration and fertilization in the mouse. Proc.
Int. Symp. Expl Biol. Spallanzani (Pavia).BRADEN, A. W. H. & AUSTIN, C. R. (1954). Fertilization of the mouse egg and the effect of
delayed coitus and of hot shock treatment. Austr. J. biol. Sci. 7, 522-65.BUCK, R. C. (1963). The central spindle and the cleavage furrow. In The Cell in Mitosis,
pp. 55-65. New York: Academic Press.CHANG, M. C. (1951). Fertilizing capacity of spermatozoa deposited in Fallopian tubes.
Nature, Lond., 168, 697.CHANG, M. C. (1952). Fertilizability of rabbit ova and the effects of temperature in vitro on
their subsequent fertilization and activation in vivo. J. exp. Zool. 121, 351-81.CHANG, M. C. & FERNANDEZ-CANO, L. (1958). Effects of delayed fertilization on the develop-
ment of pronucleus and the segmentation of hamster ova. Anat. Rec. 132, 307-17.CHANG, M. C. & HUNT, D. M. (1962). Morphological changes of sperm head in the ooplasm
of mouse, rat, hamster and rabbit. Anat. Rec. 148, 417-26.CHITTY, H. & AUSTIN, C. R. (1957). Environmental modification of oestrus in the vole.
Nature, Lond., 179, 592-3.DICKMANN, Z. (1964). The passage of spermatozoa through and into the zona pellucida of
the rabbit egg. / . exp. Biol. 41, 177-82.DICKMANN, Z. & DZUIK, P. J. (1964). Sperm penetration of the zona pellucida of the pig egg.
/ . exp. Biol. 41, 603-8.DZUIK, P. J. & DICKMANN, Z. (1965). Sperm penetration through the zona pellucida of the
sheep egg. / . exp. Zool. 158, 237-8.DZUIK, P. J. & RUNNER, M. N. (1960). Recovery of blastocysts and induction of implanta-
tion following artificial insemination of immature mice. / . Reprod. Fert. 1, 321-31.GRAVES, A. P. (1945). Development of the Golden hamster during the first nine days. Am. J.
Anat. 77, 219-52.HAMILTON, W. J. & SAMUEL, D. M. (1956). The early development of the Golden Hamster
(Cricetus auratus). J. Anat., Lond., 90, 395-414.HANCOCK, J. L. (1952). The morphology of bull spermatozoa. / . exp. Biol. 29, 445-53.HARVEY, E. B., YANAGIMACHI, R. & CHANG, M. C. (1961). Onset of estrus and ovulation in
the Golden Hamster. / . exp. Zool. 146, 231-6.MARSTON, J. H. & CHANG, M. C. (1964). The fertilizable life of ova and their morphology
following delayed insemination in mature and immature mice. / . exp. Zool. 155,237-52.
Fertilization and early cleavage in mice 191MARSTON, J. H. & CHANG, M. C. (1965). The breeding, management, and reproductive
physiology of the Mongolian Gerbil {Meriones unguiculatus). J. Lab. Anim. Care, 15,34-48.
MARSTON, J. H., YANAGIMACHI, R., CHANG, M. C. & HUNT, D. M. (1964). The morphologyof the first cleavage division in the Mouse, Mongolian Gerbil, Golden Hamster andRabbit. Anat. Rec. 148, 417.
ODOR, P. L. & BLANDAU, R. J. (1951). Observations on fertilization and the first segmentationdivision in rat ova. Am. J. Anat. 89, 29-61.
SAMUEL, D. M. & HAMILTON, W. J. (1942). Living eggs of the Golden Hamster. / . Anat.,Lond., 76, 204-8.
STRAUSS, F. (1956). The time and place of fertilization of the Golden Hamster egg. / . Embryol.exp. Morph. 4, 42-56.
SZOLLOZI, D. (1962). Cortical granules. A general feature of mammalian eggs? / . Reprod.Fert. 4, 223-4.
VENABLE, J. H. (1946a). Pre-implantation stages in the Golden Hamster (Cricetus auratus).Anat. Rec. 94, 105-24.
VENABLE, J. H. (19466). Volume changes in the early development of the Golden Hamster.Anat. Rec. 94, 139-62.
WARD, M. C. (1948). The early development and implantation of the Golden Hamster{Cricetus auratus), and associated endometrial changes. Am. J. Anat. 82, 231-76.
YANAGIMACHI, R. (1964). Sperm penetration into Hamster egg in vitro. 5th Int. Cong. Anim.Reprod. A.I. (Trento), 3, 321-4.
YANAGIMACHI, R. & CHANG, M. C. (1961). Fertilizable life of Golden Hamster ova and theirmorphological changes at the time of losing fertilizability. / . exp. Zool. 148, 185-204.
{Manuscript received 27 October 1965)