J. Embryol. exp. Morph. Vol. 33, 2, pp. 343-353, 1975
Printed in Great Britain
343
Zona pellucida denudation, blastocyst proliferation
and attachment in the rat
By M. A. H. SURANI 1
From the Physiological Laboratory, University of Cambridge
SUMMARY
The mode of zona pellucida denudation and blastocyst proliferation and attachment was
investigated in Wistar rats during day 5 of pregnancy and in ovariectomized pregnant animals
given 50 mg progesterone daily until the 10th day, when 0-2/*g oestradiol was injected
together with progesterone.
During day 5 of pregnancy, when only 5 empty zonae were detected from 32 animals,
zona denudation occurred by lysis, which took 6 h. Zona denudation occurred by shedding
in the ovariectomized pregnant animals; 190 empty zonae were detected from 40 animals.
Oestradiol administration to the experimental animals caused lysis of the shed zonae 24-30 h
after oestradiol treatment.
Air-dried preparations of the embryos showed that the blastomeres proliferated exponentially from a mean of 27-52 ±4-0 cells per embryo at 12.00 h on day 5 of pregnancy to
53-96 ± 2-58 cells per embryo at 22.00 h, with a cell doubling time of 10 h in normal animals.
In the ovariectomized animals, blastocysts were arrested at about the 100-cell stage on the
10th day. Oestradiol administration to the experimental animals did not induce mitosis; the
embryos implanted without further cell division.
The number of free embryos recovered declined from a mean of 12-0 per animal at
12.00 h to 4-25 per animal at 22.00 h on day 5 of pregnancy. The number of embryos recovered from the ovariectomized animals varied between one and 11 per animal
The results show that cell division continued in embryos within ovariectomized pregnant
animals in the absence of oestrogen, until about the 100-cell stage. There was no further cell
division before nidation when oestradiol was administered. The empty zonae were, however,
lysed between 24 and 30 h after oestradiol treatment. The zona lytic factor appears to be
oestradiol dependent and of maternal origin. A causal relationship exists between zona lysis
and embryo attachment, which is independent of the number of cells per blastocyst.
INTRODUCTION
A distinction is usually made between the lysis and the shedding of the zona
pellucida, which is dependent on the physiological state of the mother (Dickraann, 1969; McLaren, 1969; Mintz, 1970). No trace of the membrane can be
found following lysis, whereas an empty zona is seen after shedding. In the rat,
lysis occurs on day 5 of pregnancy and the blastocysts then attach to the uterus.
On the other hand, shedding of the zona in ovariectomized rats given progesterone does not lead to implantation (Yasukawa & Meyer, 1966). Lysis could be
1
Author's address: Physiological Laboratory, Downing Street, Cambridge CB2 3EG,
U.K.
344
M. A. H. SURANI
governed by maternal or trophoblastic factors or a combination of the two.
Information on the precise timing of zona lysis and attachment of blastocysts as
well as the extent and manner of blastomere proliferation is given in this paper.
Use was made of ovariectomized delayed-implantation animals (Cochrane &
Meyer, 1957) to determine the effects of steroids on these parameters and their
relationship to the egg-implantation process.
MATERIALS AND METHODS
Animals
Adult Wistar rats of 180-200 g body weight were maintained at 21 °C on a
lighting schedule of 05.00 h to 19.30 h. One or two virgin females were placed
with each male, and vaginal smears were examined daily. The morning on which
sperm were found in the smear was designated day 1 of pregnancy.
Experimental design
Day 5 pregnant animals were killed at two-hourly intervals between 12.00 h
and 22.00 h. 'Experimental delay' animals were ovariectomized on the morning
of day 3 of pregnancy and maintained thereafter by daily injections of 5-0 mg
progesterone per animal in 0-2 ml arachis oil until the 10th day, when 0-2 /.ig
oestradiol-17/? was injected together with the progesterone. The animals were
killed at regular intervals until 30 h after oestradiol treatment. Preliminary
experiments in which 0-2 ml of 0-2 % pontamine blue was injected intravenously
(Psychoyos, 1966#) showed implantation sites by 30 h after oestradiol treatment.
Injection of colchicine
All animals received an intraperitoneal injection of 0-5 mg colchicine in
0-5 ml physiological saline 2 h prior to being killed. Stathmokinetic agents have
differential effects on embryonic and adult tissues (Williams, 1973). Preliminary
experiments on day 5 pregnant animals in groups with and without colchicine
injections showed no significant difference in any of the parameters measured;
all animals thereafter received colchicine.
Collection of blastocysts and empty zonae
The method was standardized as far as possible. Uterine horns were dissected
out and freed from fat and connective tissue; each horn was then flushed with
0-5 ml of phosphate-buffered saline. Flushings from one animal were collected
in an embryological cavity dish. The total number of blastocysts with and
without a zona pellucida as well as the number of empty zonae was determined
by careful viewing of the contents with the aid of a Wild dissecting microscope.
Blastocysts were separated from the rest of the material.
Preimplantation rat blastocyst
345
Determination of total cell number and number of cells in mitosis
Blastocysts were prepared according to the air-drying technique (Tarkowski,
1966) and stained with Giemsa to determine the total number of blastomeres per
embryo and the number of cells in mitosis. Most of the preparations were completed within half an hour after the animal was killed. Preparations which were
inadequate for counting owing to considerable overlapping of blastomeres were
discarded.
RESULTS
Day 5 of pregnancy
A total of 274 embryos was examined from 32 animals in the fifth day of
pregnancy.
Timing of zona denudation
The total number of blastocysts and the numbers of those with and without a
zona pellucida were recorded for each animal. The total number of naked
embryos in a group was expressed as a percentage of the total recovery, to serve
as an index for the rate of zona lysis. The zona pellucida began to lyse at 14.00 h.;
at 16.00 h, 46 % were lysed and, at 18.00 h, 87 % were lysed (Table 1, Fig. 1).
All embryos were naked by 20.00 h. Very few empty zonae were found; of a total
of five found, three were seen at 12.00 h, one at 14.00 h, and one at 16.00 h.
Two embryos were observed actively 'hatching' out of the zona pellucidaone at 16.00 h and the other at 18.00 h.
Recovery offree embryos
The mean number of embryos recovered from the uterus declined from 14.00 h
onwards; this is during the period when the majority of zonae lysed. The decline
in the mean number of free embryos recovered was from 12-0 at 12.00 h to 8-3 at
16.00 h. Even fewer embryos were recovered in the later groups and the mean
number declined to 4-25 per animal at 22.00 h (Fig. 1). The highest number of
embryos (14 per animal) were recovered from two females at 12.00 h, and the
minimum of four came from one female at 22.00 h. The decline in the rate of
recovery was 64-42%. The 'missing' embryos could be recovered by violent
flushing of the uterus.
Number of cells per embryo
The total number of blastomeres increased from a mean of 27-52 ±4-01 at
12.00 h to 53-69 ± 2-58 at 22.00 h (Fig. 2, Table 1). The line of best fit to the cell
numbers during the afternoon of day 5 of pregnancy has a correlation coefficient
of 0-8367, and it is described by y = 37-59 -2-58x + 0-148x2 where x is the time
on day 5 of pregnancy and y is the mean cell number. Figure 2 illustrates
12.00
14.00
16.00
18.00
20.00
22.00
Total
Time
6
4
7
7
4
4
32
Number
animals
72
48
58
54
25
17
274
Number
embryos
recovered
23
27
21
26
14
8
119
Number
embryos
for air-dried
preparation
12±l-79
12± 1-41
8-29 + 2-06
7-71 + 1-90
6-25 ±1-71
4-25 ±1-73
Mean number
embryos
recovered per
animal ± S.D.
1000
1000
2-6
42
460
870
zona-free
eggs
/o
27-52 ±401
30-99 ±4-54
34-38 ±5-32
38-89 ± 605
44-07 ± 4-46
53-69 ±2-58
Mean number
cells per
embryo + S.D.
Table 1. Recovery of free embryos at various times during day 5 of pregnancy
1-6
2-5
20
2-3
3-3
2-3
0-5
1-7
0-5
0-6
1-6
0-4
>
Number mitotic cells
per embryo
K
,
Mean
Range
as
Preimplantation rat blastocyst
10
12
14
16
18
20
347
22
Time (h), day 5 of pregnancy
Fig. 1. Relationship between zona lysis and the recovery of free embryos during day
5 of pregnancy. •
• , % zona-f ree eggs; •
• , number of free blastocysts
per animal.
blastocyst proliferation on a logarithmic scale. All embryos are represented on
the graph; the line of best fit to the cell numbers has a correlation coefficient of
0-8121 and is described by log y = 2-550 + 0-0621 x where x is the time and y is
the log mean cell number. There is a linear relationship of growth with time;
cell numbers increased exponentially, with a cell doubling time of approximately 10 h. There was no detectable difference in the total cell number between
zona-encased and naked embryos when approximately equal numbers of the
two types of embryos were examined from the same animal. Taken as a group,
the naked blastocysts had a greater number of cells per embryo than the zonaencased blastocysts in the 18.00 h group. Also, blastocysts with about 40 cells
or more were naked, whereas those with 30 cells or less were usually zonaencased.
Number of cells in mitosis per blastocyst
The number of cells in mitosis was variable both within the group and among
blastocysts from the same animal (Table 1).
Delayed implantation in ovariectomized animals
Forty animals were used, from which 208 naked embryos were recovered
together with 190 empty zonae pellucidae (Table 2).
348
M. A. H. SURANI
v
41
40
§>
3-9
/>
3-8
3-7
•
-
*b
P
•o
«o
3-6
60
2
3-5
3-4
3-3
''2
3-2
12
14
16
18
20
Time (h), day 5 of pregnancy
22
Fig. 2. Blastocyst proliferation during day 5 of pregnancy; log mean cell number
versus time (logjv = 2-550 + 0-0621*). • , zona-encased blastocysts; O, zona-free
blastocysts.
The fate of the zona pellucida
In contrast to the total of only 5 empty zonae recovered from 32 animals on
day 5 of pregnancy, 190 were recovered from the ovariectomized pregnant
animals. On the basis of a maximum expected number of 14 empty zonae per
animal, just over 50 % were recovered. Following the treatment of animals with
0-2 jug oestradiol per animal along with 5-0 mg progesterone, the recovery rate
of empty zonae declined to 41-42 % at 18-20 h post oestradiol, 14-29 % at 24 h,
7-14 % at 27 h and none were found in the 30 h group. Denudation of the zona
occurs predominantly by shedding in these animals, followed by lysis when
oestrogen is injected together with progesterone. The pontamine blue reaction
at the implantation sites was detected at 30 h after oestradiol treatment, which
compares with the normal pregnant animals on day 5 of pregnancy, when lysis
of the zona is immediately followed by embryo attachment.
Number
animals
9
6
10
6
4
5
40
Group
1
2
3
4
5
6
Total
0-6
10-16
18-22
24
27
30
Hours after
oestradiol
58
33
59
27
17
14
208
Number
blastocysts
recovered
Number
empty zonae
recovered
( % of maximum
expected)
68 (53-97)
48 (57-14)
58 (41-42)
12 (14-29)
4 (7-14)
0
190
Number
blastocysts
for air-dried
preparation
25
15
28
13
10
7
98
i
Mean
40-121
46-130
56-123
48-121
98-121
66-102
Range
Number cells per
blastocyst
96-55
96-38
100-84
96-23
104-7
100-2
Average 99-14
Table 2. Recovery of blastocysts from ovariectomized pregnant animals
U)
3
B"
350
M. A. H. SURANI
Blastocyst proliferation
The number of cells per embryo was variable but on average there were 100
cells per embryo in all the groups. Of all the blastocysts examined, not a single
blastomere was found in mitosis.
Recovery offree embryos and zonae
The number of free embryos from the animals was variable (between 1 and
11). Repeated flushings did not improve the recovery rate of the embryos or
free zonae.
DISCUSSION
Zona pellucida denudation of the embryos depends upon the physiological
state of the pregnant animal, and is predominantly by lysis on day 5 of pregnancy. During normal pregnancy, lysis occurs over a short time interval of 6 h
between 14.00 h and 20.00 h. In contrast, during ovariectomized delay, zona
denudation occurs at random and is completed only by the 10th day of delay,
shedding being the form of denudation as judged by the large number of empty
zonae recovered. Oestrogen surge occurs in the afternoon of day 4 of pregnancy
(Yoshinaga, Hawkins & Stocker, 1969) and zona lysis is completed by 20.00 h
on day 5 of pregnancy, which compares well with the decline in numbers of
empty zonae to nil, 30 h following oestradiol treatment of the experimental
delay animals. This suggests that the zona lytic process is oestradiol dependent.
Dickmann (1969) suggested that lysis is dependent upon trophoblast
maturation, since late blastocysts lose the zona sooner than early blastocysts or
morulae when transferred to pseudopregnant recipients (Dickmann & Noyes,
1960; Dickmann & De Feo, 1967). Lysis also occurs in blastocysts transferred
to ovariectomized non-treated recipients, although the process requires 24 h and
possibly 72 h after the initiation of lysis (Dickmann, 1968) as opposed to 6 h
for oestrogen-dependent lysis. The shedding of the zona which has been observed in vitro, in the oviduct (AUoiteau & Psychoyos, 1966), in ectopic sites in the
mouse (Fawcett, Wislocki & Waldo, 1947; Runner, 1947; Orsini & McLaren,
1967) and in ovariectomized pregnant animals may occur after trophoblast
maturation, as it has not been observed in stages earlier than the blastocyst; the
precise mode of shedding, which may involve mechanical breakage of the zona
or trophoblastic enzymes, remains unknown. Lysis however appears to be due
to an oestrogen-dependent maternal factor and indeed could be effected prematurely in hypophysectomized animals given small amounts of oestrone
together with progesterone when blastocysts and morulae become denuded
(Wu, Dickmann & Johnson, 1971). Subthreshold levels of oestrogen given to
ovariectomized pregnant animals can cause zona lysis although implantation
does not take place (Psychoyos, 1966 b). An endopeptidase unique to uterine
secretions in the rat uterine fluid is produced in response to exogenous oestrogen
(Joshi & Murray, 1974). Such an enzyme could be responsible for zona lysis.
Preimplantation rat blastocyst
351
Electron microscopy revealed that blastocyst attachment in the rat occurs in
the afternoon of day 5 of pregnancy (Tachi, Tachi & Lindner, 1970). The
pontamine blue reaction for increased capillary permeability is detected at sites
where the eggs are naked, but not in cases where they are still zona-encased
(Ljungkvist & Nilsson, 1974). The decline in the recovery of free embryos concomitant with zona lysis is therefore considered as a very early physiological
event leading to embryo attachment and implantation. A similar correlation
between the times of zona lysis and blastocyst attachment is also found in the
mouse (Mintz, 1970). Lysis of the empty zona in ovariectomized pregnant
animals, commencing at around 24 h after oestradiol treatment, occurs at the
time when attachment is initiated (Psychoyos, 1969) and when the uterus is
maximally sensitive to decidualization stimulus (De Feo, 1967).
Blastocyst cell number increased exponentially, with a cell doubling time of
10 h on day 5 of pregnancy. The mode of proliferation is similar to that of the
mouse preimplantation embryo (Barlow, Owen & Graham, 1972; Graham,
1973). Mitotic figures in blastocysts from the same animal and also within a
group were variable. The number of cells in the zona-encased and naked
embryos from the same animal were similar, although the naked blastocysts in
the 18.00 h group had a greater number of cells than did the zona-encased
embryos. The larger number of cells in the naked blastocysts could be explained
by the fact that some of the animals in the group may have been at a slightly
more advanced stage of pregnancy. The average number of cells in diapausing
embryos was around 100 per embryo, which is twice the number of cells found
in the embryos on day 5 of pregnancy at the time of implantation (a mean of
53-7 per embryo in the 22.00 h group of day 5). Therefore, mitotic activity does
continue in the absence of oestrogen, the embryo ceasing to proliferate by the
10th day of delay when the metabolic rate is very low (Mohla & Prasad, 1971).
Following release from diapause by oestradiol treatment, no mitotic figures were
observed in any of the blastocysts examined and implantation occurred without
further cell proliferation. The embryos appear to be blocked in the Gx phase of
the cell cycle, with resumption of protein, RNA and DNA synthesis following
oestradiol stimulation (Prasad, Dass & Mohla, 1968; Sanyal & Meyer, 1972)
and with further cell division presumably occurring after implantation. Mouse
blastocysts are also blocked in the G t phase of the cell cycle during diapause
(Sherman & Barlow, 1972), have an increased cell number (McLaren, 1968) and
do not show mitotic figures 26 h after release from diapause (Sherman & Barlow,
1972).
Embryos with varying numbers of cells can implant, as judged by cell counts
in the embryos during normal pregnancy and in the ovariectomized animals.
Zona lysis has been observed only in utero, and is oestrogen dependent. The
zona lysis factor appears to be of maternal origin and can be induced prematurely by exogenous oestrogen, causing zona lysis of blastocysts and morulae
(Wu et al. 1971; Dickmann, 1972; Psychoyos, 19666). Shedding of the zona, an
352
M. A. H. SURANI
endogenous process, cannot take place at a stage earlier than the blastocyst
stage; it occurs randomly and is independent of hormonal control. Progesterone,
however, delays shedding and prevents lysis (Dickmann, 1972). A causal
relationship exists between the time of zona lysis and the time of blastocyst
attachment; implantation rapidly follows lysis, with the appearance of increased capillary permeability at sites where the embryos are naked (Ljungkvist
& Nilsson, 1974). The zona lysis factor which induces blastocyst attachment
regardless of the number of cells in mature blastocysts may have an alternative
function in causing changes in the cell surface properties. This is an attractive
concept supported by extensive studies in the mouse (Mintz, 1970; Pinsker &
Mintz, 1973) although the precise mechanism of the effect and the nature of the
zona lysis factor remains unknown.
I thank Dr R. G. Edwards for his help and advice and the Medical Research Council for
support. The work was also supported by a grant to Professor C. R. Austin from the Ford
Foundation.
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(Received 24 May 1974, revised 11 July 1974)