The morphology and timing of fertilization and early ... · blastocysts of more than thirty cells. Thus, tubal transport of fertilized eggs was completed more than 102 h after ovulation

/ . 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

.


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


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


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


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).


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.


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


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.


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


/. Embryol. exp. Morph., Vol. 15, Part 2 PLATE 3



(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).


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).


(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


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


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.


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.


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{Manuscript received 27 October 1965)

Documents

The morphology and timing of fertilization and early ... · blastocysts of more than thirty cells. Thus, tubal transport of fertilized eggs was completed more than 102 h after ovulation