/. Embryol exp. Morph., Vol. 15, 2, pp. 169-191, April 1966
With 3 plates
Printed in Great Britain
169
The morphology and timing of fertilization and
early cleavage in the Mongolian gerbil
and Deer mouse
By J. H. MARSTON 1 AND M. C. CHANG 2
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 few
myomorph rodents and in only two members of the family Cricetidae. The
Golden hamster (subfamily Cricetinae: Mesocricetus auratus) was studied by
Graves (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) examined
the 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 the
Mongolian gerbil (subfamily Gerbillinae: Meriones unguiculatus) and the Deer
mouse (subfamily Cricetinae: Peromyscus maniculatus bairdii).
METHODS
Animals
Mongolian gerbils were bred in a controlled-light environment (day from
7 a.m. to 7 p.m.) under constant conditions of management (Marston & Chang,
1965). Mature females (55-80 g) were monogamously paired with vigorous
males and examined twice daily, at 9 a.m. and 5 p.m., for vaginal spermatozoa
and a copulation plug. The animals were also under regular surveillance between
these times. They were sacrificed or unilaterally ovariectomized under pentobarbital sodium anaesthesia at various times after mating. Day 0 of pregnancy
was 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 pursued
by the male. On day 1 only immotile and broken spermatozoa were found in the
9 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 under
conditions resembling those used for commercial mouse breeding. Mature
females (14-24 g) were caged polygamously with vigorous males and examined
once each day, at 9 a.m., for the presence of vaginal spermatozoa. Mating
usually occurred in the late evening, and thus day 1 of pregnancy was the day
when spermatozoa were first detected in the 9 a.m. vaginal smear. The animals
were sacrificed or unilaterally ovariectomized under ether anaesthesia at
various times after mating.
Induced ovulation
Aged virgin and several multiparous Mongolian gerbils, at unselected stages
of 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.). The
hormone preparations ('Equinex' and 'A.P.L.', Ayerst Laboratories Inc.)
were injected subcutaneously in not more than 0-10 ml of sterile saline. Ten
gerbils were inseminated surgically, under pentobarbital sodium anaesthesia,
from 12 to 18 h after H.C.G. administration by injecting 0-10 ml of an epididymal
spermatozoa suspension into each uterine horn. The caudae epididymides of a
mature male were macerated in 1-0 ml of Ringer's solution at body temperature
to provide this suspension of spermatozoa.
Immature Deer mice, aged approximately 6 weeks, were injected intraperitoneally with 10-30 i.u. P.M.S. followed after 56 h by 10 i.u. H.C.G. The
mature animals were aged 3-5 months, and in some cases had recently been
caged with mature males. At unselected stages of their oestrous cycles they
received the same hormone treatments, and one additional group received a
single injection of 10 i.u. H.C.G. All the animals were inseminated with a
suspension 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 recently
ruptured follicles on each ovary was noted. The ovarian capsule, Fallopian tube
and uterine horn were carefully and separately examined for the presence of
eggs. Eggs were mounted for phase-contrast microscopy, fixed, and stained
according to Marston & Chang (1964), Chang (1952) and Yanagimachi &
Chang (1961).
Examination of spermatozoa
Fresh suspensions of epididymal spermatozoa were examined under a phasecontrast 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 detailed
study. Additional smears were prepared and stained with Giemsa (Hancock,
1952), haematoxylin and by the Feulgen technique. Fluorescence microscopy
using 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 the
remainder had immotile, broken spermatozoa at 9 a.m. and had presumably
mated the prevous evening at some time after 5 p.m. Mating was observed in
five females as early as 1 p.m., but in most cases it was not observed sooner than
3 p.m.
(2) Time of ovulations. The majority of animals had begun to ovulate by
9-10 p.m. on day 0 (Table la) and had completed their ovulation by 1 a.m. on
day 1. One of the twenty-four animals ovulating before 4 a.m. on day 1 had
eggs in the ovarian capsule, the others yielded eggs from the slightly swollen
ampulla of the Fallopian tube. Thus, transfer of freshly ovulated eggs to the
ampulla 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 had
definitely occurred. Table 1 a shows that for the majority of animals and eggs
penetration occurred between midnight and 4 a.m. on day 1 at about 2-3 h
after 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 the
pronuclear 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. For
the majority of animals and eggs the first cleavage was completed between
midnight 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 timing
of the second to fifth cleavages of the fertilized Mongolian gerbil egg. As
93-3 % of the eggs examined at 9-10 a.m. on day 3 were still two-celled, the
second cleavage seemed to occur more than 30 h after the first cleavage. The
intervals 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. Four
of these eggs actually had seven blastomeres which each contained a formed
nucleus (Plate 1, fig. 25). It seemed that one blastomere from the four-cell stage
had 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 5
but otherwise the eggs were in the caudal or intra-mural isthmus of the Fallopian
1, 3-4 p.m.
1, 9-10 p.m.
2, midnight-1 a.m.
2, 3-4 a.m.
2, 9-10 a.m.
Total
fertilized
eggs
examined
Number
of
animals
Prophase
10
9
100
92
43
23
Pro-metaphase
4-cell
Early
fertilization*
24
14
Metaphase
Telophase
7t
53
21
8-cell
50
4th cleavage
25
16-cell
Early
two-cell
4*
86
Blastocyst
100
8
7
41
Two-cell
—
12
50
70
Pronuclear
t Probably abnormal and about to degenerate.
3rd cleavage
Stage of development ( % total fertilized eggs)
Table \c
15
93
41
17
6
24
14
7
blastocyst, and still in the Fallopian tube.
2-cell
Unpenetrated
Stage of development (% total fertilized eggs)
Pronuclear
Table lb
Damaged
Stage of development (% total eggs)
16(18)
—
69
31
25 (27)
—
64
24
42(42)
2
29
19
34(35)
3
18
9
t All of these animals were mating at 5 p.m. on day 0.
Total
number
of eggs
recovered
(ovulated)
Five animals each provided two Fallopian tubes.
21
12
70
22
19
Total
fertilized
eggs
examined
5
5
15*
6
5
Number
of
animals
Day 3, 9-10 a.m.
5
Day 4, 9-10 a.m.
5
Day 5, 9-10 a.m.
10
Day 6, 9-10 a.m.
5
* Nineteen-celled morula—not yet a
Examination
(time of day)
Day
Day
Day
Day
Day
Number of
animals
with
penetrated
eggs
0, 9-10 p.m.
10f
6(3)
2
1, midnight-1 a.m.
101
8 (8)
3
1,3-4 a.m.
10f
10(10)
7
1, 9-10 a.m.
10t
10(10)t
9
* Sperm head has entered vitellus and started its transformation.
% 1 animal had a cystic ovary and yielded four unpenetrated eggs.
Examination
(time of day)
Day
Day
Day
Day
Examination
(time of day)
Number
of
animals
Number of
animals
ovulating
(completed
ovulation)
Table \a
Table 1 a-c. Time of ovulation, fertilization and cleavage in the naturally mated Mongolian gerbil
(each animal providing one Fallopian tube or uterine horn)
O
X
>
O
co
H
O
>
X
Fertilization and early cleavage in mice
173
tube. On day 6 eggs were only recovered from the uterus, and 85-8 % were
blastocysts of more than thirty cells. Thus, tubal transport of fertilized eggs
was 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 ovulation
rate was 4-6 (± 0-3, range 3-6) with twenty-seven ovulations from the right
ovary and nineteen from the left.
At 9-10 a.m. on day 1, eight animals all had motile spermatozoa in their
uteri and had completed their ovulation. Five of these animals yielded recently
penetrated eggs and three had unpenetrated eggs. At re-examination 6-24 h. later
all the animals yielded penetrated eggs.
At midnight to 1 a.m. on day 2, two Deer mice yielded four pronuclear eggs
from two Fallopian tubes. Three hours later a single Fallopian tube from another
animal contained a two-celled egg and one in the cleavage anaphase. At 9-10
a.m. on day 2 five two-celled eggs were recovered from three Fallopian tubes
of 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 ovulation
rates 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 the
mature Mongolian gerbil and immature Deer mouse. With the exception of one
animal, which was approximately 8 days pregnant and had ovulated sixteen
normal eggs, the mature Deer mouse ovulated a normal number of eggs. The
mature Deer mice were possibly only responding to the H.C.G. with an induced
ovulation from mature, cyclic, ovarian follicles (cf. Chitty & Austin, 1957).
The morphology of the eggs and their surrounding cumulus clot suggested that
in both species ovulation had been induced at 10-14 h after the injection of
H.C.G.
Penetrated eggs were recovered from three Mongolian gerbils inseminated
at 12 h after H.C.G. injection. Out of seventy-two one-celled eggs recovered
from these animals at 12-14 h after insemination, twelve (16-6 %) were normal
but unpenetrated; three eggs (4-2 %) had an intact spermatozoon in the perivitelline space but not in the vitellus, and fifty-seven (79-2 %) were normal
pronuclear eggs. Approximately 240 unpenetrated eggs were recovered from the
remaining animals at 22-36 h after H.C.G. injection. Seventeen of these eggs were
morphologically abnormal; six eggs had degenerate second metaphase spindles;
eight were grossly degenerate, and in three the second metaphase chromosomes
had apparently formed a single pronucleus with one or more nucleoli, but without the abstriction of a second polar body. First polar bodies could be identified
in 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 following
treatment with P.M.S. and H.C.G.
Dose
Dose
of
P.M.S.
of
H.C.G.
(i.u.)
(i.u.)
group
Number
animals
with
recently
ovulated
eggs
40
30
20
10
10
10
10
10
4
4
4
4
3
3
4
4
Immature Deer mice
30
20
10
0
10
10
10
10
5
5
5
4
5
5
5
4
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)*
Mature Deer mice
30
20
0
10
40
10
10
10
10
10
4
5
4
4
3
4
5
4
3
3
6-5 (2-16)t
3-8 (1-5)
5-5 (4-7)
4-3 (3-5)*
8-7 (8-10)*
Mature Mongolian gerbils
No. of
animals
per
Mean
number
of recently
ovulated
eggs
(range)
* Additional observations from Dept. of Anatomy, University of Birmingham. (These
animals were not inseminated.)
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 all
had well-organized second metaphase spindles.
In the Deer mouse 75 % of the animals examined more than 3 h after artificial
insemination yielded penetrated eggs (Table 3). If the rate of development of
eggs following induced ovulation is not markedly different from that after
natural ovulation, these results demonstrate the cleavage of Deer mouse eggs.
All the Deer mice examined 2-5 h after insemination had motile spermatozoa
in their uteri. Only three penetrated eggs were recovered at 3-4 h after insemination; whereas at 4-5 h the eggs from three immature mice were in early
stages 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 after
penetration. The two-celled stage lasted for approximately 30 h as judged by the
recovery of two-celled eggs at 52-53 h after insemination. The timing of subsequent cleavages was variable and they occurred at intervals of 10-20 h. The
third cleavage was complete in sixteen (76 %) of the eggs examined 72-74 h
after 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 uterus
as morulae.
Immature
Immature
Immature
Immature
24-25
52-53
72-74
96
Immature
4-5
Mature
Mature
3^
20-22
Mature
Group
2-3
Examination
(hours after
insemination)
30
15
26
21
2
1
3
2
34
31
26
26
3
3
3
3
41
3
28
9
2
5
—
—
28
41.
19
17
Total
eggs
recovered
5
3
4
4
No. of
animals
Total eggs
recovered
from animals
with
fertilized
eggs
No. of
animals
with
fertilized
eggs
I
8
!
I
a.
All normal, unpenetrated and at second
maturation metaphase
3 recently fertilized with enlarging sperm
head. One has second polar body abstricted •5"
41 all early pronuclei or enlarging sperm
head. 35 have abstricted second polar body
7 late pronuclei, 3 prometaphase,
7 metaphase, 11 two-cell
1 metaphase, 13 two-cell
8 two-cell, 3 second cleavage, 1 four-cell
1 two-cell, 4 four-cell, 10 eight-cell,
4 fourth-cleavage, 2 16-cell
4 fourth cleavage, 2 16-cell, 5 fifth cleavage
Details of fertilized eggs
Table 3. Morphology of Deer mouse eggs at various times after artificial insemination
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 spermatozoon
head measured 4-5 fi long by 2-9 ju, at its widest in the Mongolian gerbil (mean
often 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 acrosome of both species stained pink with Giemsa and seemed to overlie the anterior portion of the nucleus; it fluoresced bright red with acridine orange,
merging to yellow orange where it overlay the nucleus. The nucleus fluoresced
bright apple-green and the mid-piece was pale green. In the Mongolian gerbil
a cytoplasmic droplet, fluorescing pale green without any red, was usually
carried at various levels on the mid-piece of the epididymal spermatozoon, but
in the Deer mouse it was smaller and generally at the end of the mid-piece. The
spermatozoon mid-piece and main-piece measured 40-45 ii and 85-100 ju, in the
Mongolian 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 Mongolian
gerbils 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 completely
absent by 9-10 a.m. at about 9-12 h after ovulation. In naturally mated Deer
mice the cumulus clot was dense up to 5 p.m. on day 1 but the eggs were naked
by midnight on day 1.
The dimensions of the penetrated and unpenetrated eggs of both species are
summarized in Table 4.
In both species the egg cytoplasm was finely granular with some coarse
inclusions. However, when recently ovulated unpenetrated Deer mouse eggs were
carefully compressed and examined under the oil-immersion objective, fine
'cortical granules' could be identified in the cortex close to the surface of the
vitellus and the cytoplasmic membrane (Plate 2, figs. 28, 29). The cortical
granules resembled those described in unpenetrated eggs of the Golden hamster
in 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 Mongolian
gerbil eggs.
More than 80 % of the recently ovulated eggs of the Mongolian gerbil had a
distinct first polar body, in which the chromatin was variously arranged as bars,
strands or granules: the first polar body often appeared lobulated or fragmented
into two or three parts. In Deer mouse eggs the first polar body could only be
identified as a faint cytoplasmic globule in recently ovulated eggs. After staining,
Unpenetrated (ca. 4 h from
induced ovulations)
Early penetrated or pronuclear
(natural matings day 1, 9 a.m.10 a.m.)
Deer mouse (Peromyscus
maniculatus bairdii)
105-8
103-2
30
16
102-6
32
1131
99-6
25
18
100-9
25
Mean
overall
diameter (/*)
71-5
74-4
68-9
72-6
69-2
69-4
Mean
vitelline
diameter (/*)
* Variability of measurements was such that, within species, differences were not significant.
Unpenetrated (9 p.m. day 0,
3 a.m. day 1)
Early penetrated (9 p.m. day 0,
3 a.m. day 1)
Pronuclear (midnight day 0,
9 p.m. day 1)
Pronuclear or first cleavage
(midnight day 1, 3 a.m. day 2)
Group
Mongolian gerbil
(Meriones unguiculatus)
Species
No. of
eggs
measured
Table 4. Measurements of living Mongolian gerbil and Deer mouse single-celled eggs*
9-6
101
7-8
7-8
7-8
8-4
Mean
thickness
of zona
pellucida (/*)
1
178
J. H. MARSTON & M. C. CHANG
it was difficult to identify and usually did not contain discrete masses of
chromatin.
In both species, all the recently ovulated, unpenetrated eggs contained a
second metaphase spindle, usually lying paratangentially to the surface of the
vitellus. Under the present method of fixation and staining the spindles had
relatively broad ends without any indication of centriolar structures: they were
difficult to observe in the living egg, but showed a similar structure.
Eggs at early stages of fertilization were only recovered from the ampulla of
the Fallopian tube, and penetration occurred while the cumulus clot about the
eggs was still very dense. The number of spermatozoa in the ampulla appeared
low, and usually not more than three spermatozoa were identified in the cumulus
clot surrounding recently penetrated eggs.
(3) Spermatozoa penetration of the zona pellucida
Actual penetration of the zona pellucida was not observed. However, at
3-4 a.m. on day 1 one Mongolian gerbil egg had the filamentous tip of the
spermatozoan main piece extending obliquely through the zona pellucida in
the form of a 'penetration curve' (Dickmann, 1964). The greater portion of the
main piece had entered the vitellus and pronuclei were forming. At 4-5 h after
artificial insemination a Deer mouse egg was observed with approximately half
of 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 space
although the tip of the mid-piece had entered the vitellus and the spermatozoan
head was enlarged. Single 'penetration slits' (Austin, 1951a) in the form of
'penetration curves' were also identified in nine Mongolian gerbil eggs and one
Deer mouse egg. They were especially prominent when a portion of the vitellus
protruded 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; yet
the occurrence of intact, 'supplementary spermatozoa' in the perivitelline space
of pronuclear eggs was 13-3 % after superovulation (see above), 15-8 % for 114
pronuclear eggs and 11-8 % for 102 cleaving or two-celled eggs from natural
matings. Of these thirty-eight eggs, thirty-three had one supplementary spermatozoon, four had two, and one egg had four spermatozoa. Supplementary
spermatozoa were not observed in the perivitelline space of eight- to sixteencelled eggs, and only three eggs in the stages from the second to third cleavages
had a supplementary spermatozoon. All the supplementary spermatozoa
examined in detail had lost the filamentous tip of the spermatozoan head, including 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 penetrated by two spermatozoa. One pronuclear egg from a naturally mated animal
had a supplementary spermatozoon. The other two eggs were recovered from
artificially inseminated animals and each had two distinct spermatozoan tails
in the perivitelline space: in one the spermatozoan heads were just entering
the vitellus and in the other two male pronuclei were forming. These eggs
were lost during fixation, so the probable occurrence of dispermic fertilization
could not be proved.
(5) Spermatozoan penetration into the vitellus
In fertilized Mongolian gerbil eggs the spermatozoan tail had completely
entered the vitellus, and well-stained preparations showed that it usually had
separated into two or more filaments. These filaments remained united at their
proximal ends, but separated along the mid-piece and the whole of the main
piece. Few Deer mouse eggs were examined at late stages of pronuclear development, and in these it was either impossible to identify the spermatozoan tail
or else its location in the vitellus or perivitelline space was questionable. Sixty
recently penetrated or early pronuclear eggs were examined in their fresh condition, and forty-seven (78 %) had virtually all of the fertilizing spermatozoan
tail lying in the perivitelline space (Plate 2, fig. 30). After staining, all of thirtyseven eggs appeared to have the entire main-piece and a posterior portion of the
mid-piece in the perivitelline space: the tip of the mid-piece had an irregular
outline and had probably entered the vitellus.
(6) The second maturation division, second polar body emission and transformation
of the fertilizing spermatozoon
Twenty-one Mongolian gerbil and eleven Deer mouse eggs were examined in
detail during these stages. They were probably examined within 2 h of penetration and their morphology agreed with the observations on rat, mouse and
Golden 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 of
the second maturation spindle (Plate 2, fig. 41) and coalesced to form a distinct
mid-body which underwent condensation (Plate 2, fig. 43) during emission of
the polar body. Constriction of the cytoplasmic membrane seemed to occur on
either side of the mid-body, so that remnants of the spindle fibres extended
between 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 to
the first cleavage and sometimes beyond this time. In about twenty Mongolian
gerbil eggs remnants of a similar mid-body were also found with the first polar
body. The chromatin of the second polar body did not form a vesicular nucleus
at any stage of development in either species.
180
J. H. MARSTON & M. C. CHANG
Stages in the transformation of the fertilizing spermatozoon's head are
illustrated by Plate 1, figs. 8, 9, and Plate 2, figs. 34-36. In both species the head
showed marked enlargement and rounding out soon after entering the vitellus.
Its staining intensity was reduced and a distinct nuclear membrane could not be
identified 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 specimens : all other figures are of fixed and stained preparations. The photographs were taken on a
phase-contrast microscope. 1st = First polar body; 2nd = second polar body; m.b. = midbody 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 polar
body ( 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 the
early 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 the
larger (xca. 1000).
Fig. 16. Detail of pronuclear egg at about 10 h after penetration to show (a) female and (b) male
pronuclei (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
J. H. MARSTON & M. C. CHANG
PLATE 1
facing p. 180
Fertilization and early cleavage in mice
181
mouse eggs a small 'rod' also appeared to separate from the spermatozoon's
head 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 identified
as originating from the spermatozoon's head. No such structure was found in
Mongolian gerbil eggs.
(7) Pronuclear development
In both species pronuclear development followed the pattern described by
Austin (19516, 1956a) and Odor & Blandau (1951) for the rat and Golden
hamster (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 and
remnants of the second maturation spindle. In Mongolian gerbil eggs the male
pronucleus 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 growth
and development of the male and female pronuclei seemed to be coordinated.
In Deer mouse eggs the pronuclei generally had one nucleolus with a large
central inclusion of bright contrast (Plate 2, fig. 49): a few pronuclei had one
large nucleolus and up to four small nucleoli. The number of nucleoli in
Mongolian gerbil pronuclei varied with the development of the egg. However,
the female pronucleus tended to have one nucleolus, and rarely more than three
nucleoli. The male pronucleus tended to have one to three large nucleoli, but
these were frequently associated with up to ten or more small nucleoli. In both
species maximal development of the pronuclei was reached at about 3 h before
the onset of cleavage, and in the Mongolian gerbil it appeared that the male
pronucleus entered prophase of the first cleavage division shortly before the
female pronucleus. Pronuclei at maximal development tended to lie centrally
in the vitellus: they did not make contact with one another and showed no
evidence of pronuclear fusion.
(8) Syngamy, first cleavage and the two-celled stage
Seven Mongolian gerbil eggs were examined during prophase of the first
cleavage division. They showed progressive dissolution of the nucleoli, disappearance of pronuclear membrane, and loss of pronuclear contrast within the
vitellus. This was especially obvious in living eggs under the phase-contrast
microscope; and eventually the pronuclei were invisible (Plate 3, figs. 56-58).
In the stained preparations two separate groups of loosely interwoven chromatin
strands could be identified; they lay centrally in the vitellus and were presumably
the pronuclear chromosomes. In slightly earlier stages, chromatin filaments
could 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 stage
each showed two, separate, fairly compact groups of chromosomes which were
not oriented in any one direction. The chromosome groups appeared to be
haploid and the chromosomes did not show distinct chromatids (Plate 2,
figs. 59, 60, 64; Plate 3, figs. 71, 72, 73): the chromosomes gave the impression
of 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 metaphase
spindle and no intermediate stages between pro-metaphase and metaphase were
identified. The first cleavage spindle was fully formed in twenty Mongolian
gerbil and eight Deer mouse eggs. A single group of chromosomes lay centrally
in 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 the
equivalent structures of the second maturation division. However, the spindle
structure was not so clearly defined and intensely stained as the second maturation spindle (cf. Odor & Blandau, 1951). In good preparations the separation
of 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 in
the 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 male
pronucleus (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. exp. Morph., Vol. 15, Part 2
PLATE 2
27
35 UM 36 "T- 37 r *
J. H. MARSTON & M. C. CHANG
38
facing p. 182
/. Embryol. exp. Morph., Vol. 15, Part 2
J. H. MARSTON & M. C. CHANG
PLATE 3
facing p. 183
Fertilization and early cleavage in mice
183
(Plate 3, fig. 65), but it was not possible to identify any centriolar structures at
the poles of the spindle.
One Deer mouse egg was examined in early anaphase and the chromosomes
were seen as two groups of distinctly oriented fibres separating along the axis
of the spindle. Three Mongolian gerbil eggs in early telophase showed a distinct
mid-body at the equator of the spindle. It seemed to consist of an aggregation
of 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 telophase were recovered following the completion of cytokinesis but before formation of the blastomere nuclei. The greatly condensed mid-body of the telophase
spindle 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 phasePLATE 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 and
fertilizing 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 the
preparation 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 the
preparation 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 pronuclei could be identified in the living eggs. Cytoplasmic cleavage seemed to be
equal in both species, and there was probably some reduction in cytoplasmic
volume at cleavage.
Following the completion of cytokinesis, formation of the blastomere nuclei
was first indicated by the appearance of primary nucleoli within the chromatin of
late telophase (Plate 3, fig. 78). Expansion of the nucleus, formation of a distinct
nuclear membrane and development of large nucleoli occurred very rapidly,
for no intermediate stages were identified. Two-celled eggs are illustrated by
Plate 1, figs. 18, 19, and Plate 2, figs. 52 and 53.
Remnants of the fertilizing spermatozoan tail were not identified in twocelled Deer mouse eggs, but 52-6 % of thirty-eight eggs had a distinct second
polar 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 % the
remnants of the fertilizing spermatozoon's tail were closely associated with the
mid-body as they extended across the cleavage furrow and lay within both
blastomeres. Six additional ova (9-8 %) had remnants of the tail lying in only
one 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 blastomeres distinctly larger than the others (Plate 2, fig. 51). However, four of these
unequally cleaved eggs were recovered 72-74 h after artificial insemination, and
they could be abnormal. The blastomeres of the eight- and sixteen-celled stages
appeared relatively equal in both species, and were usually in a compact, almost
spherical, group (Plate 1, figs. 23, 26; Plate 2, figs. 54, 55). Usually each blastomere 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 most
well-stained eight- to sixteen-celled Mongolian gerbil eggs (Plate 1, fig. 25).
These remnants appeared to extend from one blastomere to another, lying
within as many as four blastomeres, and nodular, darkly straining bodies could
occasionally be identified along their length. The bodies were thought to be the
mid-bodies of the most recent cleavage spindles, for their number in any one
egg tallied with the number of completed cleavages. Similar mid-bodies were
identified in Deer mouse eggs.
Blastocysts were not examined in the Deer mouse, but in the Mongolian
gerbil on day 6 they contained more than thirty cells, had an intact, undistended
zona pellucida and generally resembled early blastocysts of the rat, mouse and
Golden hamster (Plate 1, fig. 26).
Fertilization and early cleavage in mice
185
(10) Abnormalities offertilization and cleavage
At 3 to 4 a.m. on day 1 one Mongolian gerbil yielded five pronuclear eggs
with second polar bodies, normal male pronuclei and vitelline sperm tails: three
of these eggs had normal female pronuclei, one had six micro-nuclei beside a
small female pronucleus, and in the other a subnucleus lay beside the female
pronucleus. At 9-10 a.m. on day 1 another animal yielded three normal pronuclear eggs and one egg with a subnucleus close to the female pronucleus. Two
animals were found with abnormal eggs at midnight to 1 a.m. on day 2. In the
first case, one out of six two-celled eggs had a single subnucleus associated with
the blastomere nucleus (Plate 1, fig. 20). The other animal yielded one normal
and four abnormal pronuclear eggs. The abnormal eggs had second polar
bodies, vitelline sperm tails and apparently normal male pronuclei. The female
pronuclei were represented by ten or more micro-nuclei scattered through the
vitellus in a manner which suggested that they had originated from fragments of
the female chromatin (Plate 1, fig. 4). In two of the four cases the animals had
received surgical anaesthesia when their eggs were at early stages of fertilization.
In the Deer mouse two probable cases of dispermic fertilization have already
been noted. Three of twenty-four pronuclear eggs from naturally mated Deer
mice contained a small subnucleus lying close to the female pronucleus in the
presence 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 Deer
mouse eggs showed degeneration of their cytoplasm. After staining, the second
maturation spindle seemed broader and more loosely organized, the fibres were
less densely stained, and individual fragments of chromatin were often widely
displaced from the metaphase plate. Approximately ten Mongolian gerbil and
eight Deer mouse unpenetrated eggs were found with very degenerate cytoplasm and ten or more micro-nuclei scattered through the vitellus. These micronuclei had one or two nucleoli (Plate 1, fig. 17). In both species unfertilized eggs
traversed the Fallopian tube at an apparently normal rate and could be recovered from the uterus as grossly degenerate forms.
DISCUSSION
In the present study the timing of the critical events of ovulation, penetration
and cleavage could not be precisely defined. As a result, it is difficult to establish
the need for spermatozoan capacitation (Austin, 1951a; Chang, 1951) and
'maturation' of the ovulated egg (Austin & Braden, 1954) as essential preliminaries to penetration in the Mongolian gerbil and Deer mouse.
In the Mongolian gerbil, an interval of 8-12 h was observed between mating
and spermatozoan penetration, at which time the egg was aged about 4 h
after 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 penetration
would cover the time required for 'maturation' and penetration of the cumulus
and zona pellucida. The observations in the Deer mouse were not strictly
physiological, because induced ovulation and artificial insemination were used.
However, an interval of about 3 h was observed between insemination and
spermatozoan penetration; and as insemination was probably performed soon
after induced ovulation, this interval would include the time for transportation,
penetration of the cumulus and zona pellucida, and would cover any period
necessary for 'capacitation' and egg 'maturation'.
Austin & Braden (1954) suggested that during the 2-4 h interval between
ovulation and penetration in the naturally mated rat, some change occurred
in the egg membranes which had to be completed before the spermatozoon could
penetrate the egg; this change might represent a final stage for the 'maturation'
of the egg. The interval between ovulation and penetration following natural
mating was about 5 h in the mouse (Braden & Austin, 1954) and 2 h in the
Golden hamster (Austin, 1956 c; Strauss, 1956), but it was insignificant in the
rabbit (Austin & Braden, 1954) and sheep (Braden, 1959). The present observations support Braden's suggestion (1959) that 'maturation' may be a process
peculiar to rodent species, for there was a sufficient time for 'maturation' to
occur in the Deer mouse and Mongolian gerbil eggs.
The interval between ovulation and penetration was reduced following delayed
mating in the mouse (Braden & Austin, 1954), following induced ovulation in
the rat (Austin, 1951a) and mouse (Braden, 1959), and also differed between
two inbred strains of mouse (Braden, 1958). These differences could be related
to variation in the density of the cumulus clot about the eggs, thus suggesting
that an essential change in state of the cumulus clot might occur during maturation (Braden, 1959): such a change would be expected to be most subtle,
possibly occurring within the intercellular matrix of the cumulus. In the present
study we have not detected any gross differences between the cumulus surrounding recently ovulated and recently penetrated eggs. It has not yet been
clearly established whether 'maturation' coincides with a complete failure of
spermatozoa to enter the cumulus, or whether it represents the time for them to
penetrate the cumulus. If 'maturation' is indeed related to a change in the
cumulus, it could be independent of the condition of the eggs and related to the
effects of post-ovulatory ageing interacting with the environment of the Fallopian
tube.
Our observations on the rates of cleavage have been summarized in Table 5
and compared with the available data for other Cricetidae. The duration of the
two- to four-cell stage was approximately 30 h in the Golden hamster, Deer
mouse and Mongolian gerbil. It was considerably longer than the intervals
between subsequent cleavages and between fertilization and the first cleavage.
The rate of tubal transport was slower in the Mongolian gerbil and the eggs
were thus in a more advanced state of cleavage when they entered the uterus.
16-18
2-cell
Induced
18-20
ovulation
with artificial
insemination
at time of
ovulation
Natural
< 24
Field vole
mating
(Micro tus
(possibly
(ovulation
agrestis:
ca. 12)
probably
Microtinae)
induced by
coitus)
Mongolian
Natural
22-24
mating
gerbil
(evening),
(Meriones
unguiculatus: spontaneous
ovulation
Gerbillinae)
Deer mouse
(Peromyscus
maniculatus
bairdii:
Cricetinae)
Method
Golden hamster Natural
(Mesocricetus mating
auratus:
(evening),
Cricetinae)
spontaneous
ovulation
Species
(subfamily)
20-24
16-32 cells
20-24
> 102 h
post
ovulation
> 30
Variable but
usually
> 8 cells
Variable but
usually
> 70 h post
coitum
Variable:
minimal
estimated
ca. 12
Variable:
minimal
estimate
ca. 12
Variable:
minimal
estimate
ca. 12
Present study
Austin (1957),
Chitty &
Austin (1957)
Source
Third cleavage Graves (1945),
Venable (1946a),
probably
Ward (1948),
initiated in
Austin (1956),
Fallopian
Strauss(1956)
tube and
completed in Hamilton &
Samuel (1956)
the uterus
Harvey et al.
(1961)
Present study
8-16 cells
Stage
ca. 72 h after
insemination
and induced
ovulation
60-66 h after
ovulation.
< 72 h post
coitum
Time
Variable,
10-20
12-16
8-16
cell
Variable,
10-20
12-16
4-8
cell
Entry to uterus
ca. 30
24-26
2-4
cell
Estimated duration of each stage (h)
Table 5. Estimated rates of cleavage in Cricetidae
oo
§
s
188
J. H. MARSTON & M. C. CHANG
The morphology of the Mongolian gerbil spermatozoon resembled that of the
Libyan gerbil (subfamily Gerbillinae: Meriones libycus) in having a finely
tapered apex, whereas the Deer mouse spermatozoon was similar to that of
the Field vole and Cotton rat (subfamily Cricetinae: Sigmodon hispidus) and had
a sharply recurved apex (cf. illustrations of Austin, 1957; Bishop & Walton,
1960). We have not observed any change in the spermatozoon before penetration of the zona pellucida, but from the morphology of supplementary spermatozoa in Mongolian gerbil eggs it seemed that the acrosome had been lost during
penetration. 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 Deer
mouse there was some evidence that the fertilizing spermatozoon's tail did not
enter the vitellus, a situation similar to that in the Field vole (Austin, 1957) and
unlike that in other rodents. However, more information is required on this
point.
The unpenetrated Deer mouse egg had distinct cortical granules, which
were visible under the phase-contrast microscope. Such cortical granules
have only been recorded for the Golden hamster (Austin, 1956Z?; Yanagimachi
& Chang, 1961), although Szollozi (1962) has suggested that they are present
at the ultra-microscopic level in most mammalian eggs. The cortical granules
underwent dissolution during sperm penetration. The Deer mouse and Golden
hamster both belong to the subfamily Cricetinae, and their eggs exhibit a strong
'zona block' and a weak 'vitelline block' to polyspermic penetration. This
property is shared with the Field vole egg and completely reversed in Mongolian
gerbil eggs (cf. Austin, 1956a, 1957). Dispermic fertilization was not observed in Mongolian gerbil eggs, although penetration by more than one
spermatozoon occurred in 10-15 % of all cases, whereas dispermic fertilization
usually followed dispermic penetration in the Deer mouse, Golden hamster, and
Field vole. It would be of interest to know whether the presence of microscopic
cortical granules is characteristic for unpenetrated eggs from members of the
subfamily Cricetinae and also to define the function of these granules in establishing the 'zona block' to polyspermy.
In both species, a distinct mid-body was formed on the second maturation
and first cleavage spindles, and probably on subsequent cleavage spindles up to
the fourth cleavage. In the Mongolian gerbil egg a mid-body was probably
formed on the first maturation spindle, as evidenced by the remnants associated
with the first polar body. This confirms observations (Marston, Yanagimachi,
Chang & Hunt, 1964) that mid-body formation occurs on all maturation and
early cleavage spindles of the mouse, rat and Golden hamster. The work of
Buck (1963) suggests that the mid-body may play some part in the definition of
the future plane of cleavage or polar body emission, or even be actively involved in the process of cleavage and emission.
Fertilization and early cleavage in mice
189
Organized division of the second maturation spindle was not observed in
unpenetrated Mongolian gerbil and Deer mouse eggs, although it does occur
frequently in the Golden hamster (Austin, 1956a; Yanagimachi & Chang, 1961).
The presence of micro-nuclei and scattered chromatin granules in unpenetrated
aged eggs suggests that fragmentation of the spindle had occurred. It follows
that the presence of micro-nuclei in penetrated eggs might result from partial
fragmentation of the female chromatin at some time during, or subsequent to,
the completion of the second maturation telophase. These eggs may have
developed after the penetration of abnormal eggs, possibly damaged by the
effects of ageing or experimental anaesthesia.
SUMMARY
1. The timing of ovulation, penetration of spermatozoa and cleavage has
been studied in naturally mated Mongolian gerbils maintained in a controlled
environment.
2. The timing of sperm penetration and cleavage was studied in Deer mice
following artificial insemination of mature and immature animals close to the
time of gonadotrophin-induced ovulation. A few naturally mated animals
were also studied.
3. The morphology of fertilization and cleavage in the Mongolian gerbil
and Deer mouse is described and illustrated.
RESUME
Morphologie et chronologie de la fecondation et des premiers clivages chez deux
rongeurs: la gerbille de Mongolie (Meriones unguiculatus, Gerbillidae)
et Peromyscus maniculatus (Cricetidae)
1. La chronologie de l'ovulation, de la penetration des spermatozoides et de
la segmentation a ete etudiee chez des gerbilles de Mongolie naturellement
accouplees et maintenues dans un milieu controle.
2. La chronologie de la penetration des spermatozoides et de la segmentation
a ete etudiee chez des Peromyscus apres insemination artificielle d'animaux
matures et immatures, peu de temps apres l'ovulation induite par les gonadotrophines. Quelques animaux accouples naturellement ont egalement ete
etudies.
3. On decrit et on illustre la morphologie de la fecondation et de la segmentation 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 of
Veterinary Surgeons for providing a travel grant. Dr R. Yanagimachi gave considerable
help in the examination of cortical granules in Deer mouse eggs, and Dr L. T. Turbyfill
co-operated most valuably in the studies on induced ovulation. Messrs J. Zucker and
T. Luuko were responsible for the care of the animals. Part of the cost of preparing this
manuscript was provided by a grant from the Ford Foundation.
190
J. H. MARSTON & M. C. CHANG
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