/. Embryol. exp. Morph. Vol. 23, 3, pp. 539-547, 1970
Printed in Great Britain
539
The role of the zona pellucida in the development
of mouse eggs in vivo
By J A C E K A. M O D L l t t S K F
From the Department of Embryology, University of Warsaw
Up to the present time the function and significance of the zona pellucida in
the development of mammalian eggs has not been fully explained. Zona-free
mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres
isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964)
showed that in this species also cleavage can occur when the zona pellucida is
absent, although the blastomeres exhibit a tendency to fall away from each
other.
Tarkowski's observations (unpublished) would appear to show, however, that
naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the
oviduct. A few hours after transplanting the naked eggs none could be recovered
by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered. However, naked morulae and blastocysts transplanted to the oviduct develop and may become implanted in the
uterus (Tarkowski, 1961; Mystkowska & Tarkowski, 1968). Moore, Adams &
Rowson (1968) also transplanted single naked blastomeres from 2- and 4-cell
rabbit eggs to the oviduct and found that no further development occurred.
Contrary to the above findings, Nicholas & Hall (1942) claimed that isolated
'half' blastomeres and pairs of naked 1-cell rat eggs transferred to the uterus
were able to continue development beyond implantation, suggesting that the
presence of zona pellucida is not essential for the development of early cleavage
stages in vivo.
The purpose of the present study was to follow the fate of naked mouse eggs
in vivo after their transplantation to the oviduct.
1
Author's address: Department of Embryology, University of Warsaw, Warsaw 64,
Poland.
35-2
540
J. A. M O D L I N S K I
MATERIALS AND METHODS
Females of the A, CBA-p, CBA-T6T6 inbred strains and of an outbred albino
colony were used as donors and recipients. The recipients usually ovulated
spontaneously, while in donors ovulation was either spontaneous or induced
with PMSG and HCG. The majority of the recipients were mated with vasectomized males; recipients mated with normal males were also used for some
transfers of zona-free eggs.
The embryos were transplanted in stages varying from 1-cell to blastocyst.
All the operations were carried out in Tyrode solution with added bovine
plasma albumin. The zona pellucida was removed enzymatically with 0-5 %
pronase in Tyrode solution (Mintz, 1962). The eggs were kept in the pronase
until the zona pellucida became markedly thin, and the digested membrane was
finally removed by pipetting. The eggs were transplanted by the method devised
by Tarkowski (1959), by incising the ovarian capsule and inserting a micropipette through the infundibulum into the lumen of the oviduct. Transplantations were made on the 1st or 2nd day of pregnancy or pseudopregnancy (the
day when a vaginal plug was found was taken as the first day). The naked and
control eggs remained in vitro for an average of 20-25 min.
There were four groups of experiments :
I. Naked and control eggs from the 1st, 2nd, 3rd and 4th day of development
were transplanted separately to pseudopregnant females. Dissections were
performed on the 11th day to show any implantations.
II. Zona-free and normal 1- and 2-cell eggs were transplanted to pseudopregnant recipients. Oviducts and uteri were flushed on the 4th day of pregnancy.
III. Naked and control 1- and 2-cell eggs were transplanted separately or
together. Oviducts were excised and flushed 15, 30, 45, 60, 75 and 120 min after
transplantation.
IV. 1- and 2-cell naked eggs were transplanted, and the oviducts fixed 1, 2,
3, 4, 6, 8, 12, 24, 28 and 36 h after transfer, in Zenker-Helly or in Susa. The
oviducts were sectioned at 5/*, stained with haematoxylin and eosin, or with
azure II and eosin with or without haematoxylin, and embedded in DPX.
RESULTS
Group I. After transplanting 123 naked 1-, 2- and 4-cell eggs (donors in the
1st and 2nd day of pregnancy) not a single case of implantation was found,
while about 50 % of control eggs in the same stages implanted (Table 1). After
transplanting naked embryos from the 3rd day of pregnancy (8-cell to morula)
implantation was found to occur sporadically. The number of control eggs
which implanted (about 40 %) was lower than that found after transplanting
younger embryos, but was far greater than in the case of eggs without the zona
pellucida (16 %). On the other hand almost no differences were found in
No. of transplantations
5
5
6
7
I
II
III
IV
c
Day of
pregnancy
of the
donors
33
26
30
32
planted eggs
Eggs with zona pellucida
17
16
12
9
No.
51 5
61-5
400
281
(%)
Implantations
•*
9
10
11
10
No. of transplantations
t
63
60
56
52
planted eggs
A
Eggs without zona pellucida
0
0
9
13
0
0
161
250
(%)
s
Implantations
No.
Table 1. Results of transfers of naked eggs and eggs with the zona pellucida (inspection on Wth day)
542
J. A. MODLINSKI
frequency of implantation of blastocysts with or without the zona pellucida.
The fall in the number of implantations after transplanting older stages is
probably significant since it was also observed by Noyes & Dickmann (1961) in
the rat.
Group II. In 11 mice the oviducts and the uteri were flushed 3 days after
transplantation. They had received a total of 35 eggs with the zona pellucida
•3P r
Role of the zona pellucida
543
and 85 naked eggs, all at 1- and 2-cell stages. 12 morulae/blastocysts with the
zona were recovered, but not a single naked one was found.
Group III. In view of the results showing that naked eggs die during their
stay in the oviduct without even attaining the morula stage, attempts were made
to find out how long after transplantation the eggs could survive and what stage
of development they could reach. For this purpose the oviducts into which eggs
had been transplanted (naked eggs and eggs with the zona pellucida as control
eggs) were flushed at different times after transplantation. A total of 54 transplantations were made, in which 162 eggs with the zona pellucida and 419 naked
eggs were transplanted. Comparison of the results obtained from transplanting
the two kinds of eggs revealed significant differences in their behaviour. The
number of control eggs found (with the zona pellucida) did not show, as might
have been expected, any direct dependence on the time spent in the oviduct and
in each time group about 60 % of those transplanted were recovered. Many
fewer naked than control eggs were found after flushing. During the first 45 min
after transplantation about 35 % of the transplanted naked eggs were found.
After 1 h the percentage of naked eggs found dropped rapidly and after 2 h
only one naked egg was found (out of the 46 transplanted). These results agree
with A. K. Tarkowski's observations (unpublished) that after 2-3 h from the
time of transplantation it is impossible to rinse out transplanted naked eggs
from the oviduct.
Group IV. The results of histological studies differ significantly from those
obtained by rinsing the oviducts. Analysis of the preparations revealed that
after 1 h—that is at the time when on flushing the oviducts a sudden decrease
in the number of naked eggs was observed—the naked eggs are present in the
oviduct. The number of naked eggs found during the first 4 h after transplantation was small (about \ of those transplanted) but was maintained at a more or
less stable level. On the other hand it was observed that with increasing length
of time spent by the eggs in the oviduct, there were increasingly frequent cases
Fig. 1. Naked 2-cell egg adhering to epithelium of oviduct. 4 h after transfer,
x 500.
Figs. 2-3. Naked -} blastomeres adhering to epithelium of oviduct. Contact between
blastomeres and epithelium is very close over a large surface. 8 h. x 500.
Fig. 4. Group of naked j- blastomeres adhering to folds of oviduct. 8 h. x 200.
Fig. 5. Naked 2-cell egg wedged between folds of oviduct. Shape of egg corresponds to surface surrounding it. 24 h. x 500.
Fig. 6. Naked \ blastomere lying in lumen of oviduct. Several leucocytes adhere to
its surface. 16 h. x 500.
Fig. 7. Degenerate naked j- blastomere. 28 h. x 500.
Fig. 8. Group of naked \ and i blastomeres and one 2-cell egg in zona pellucida.
Two small -J- blastomeres originated from one of the transplanted naked \ blastomeres. Metaphase spindle can be seen in one of the % blastomeres. 24 h. x 350.
Fig. 9. Group of naked \ blastomeres adhering together in a large group. 4 h. x 350.
544
J. A. MODLINSKI
of eggs adhering to the walls or folds of the oviduct (Figs. 1-4) or becoming
wedged into the narrow and folded parts of the oviduct (the eggs were found
mainly in the ampulla and at the beginning of the isthmus). In some cases the
eggs adhered very firmly to the epithelium over a relatively large area (Figs.
1-3); on some of the cross-sections it is difficult to distinguish the boundary
between the egg cytoplasm and the epithelium of the oviduct. However, there
were no perceptible changes in the appearance of the nucleus and cytoplasm,
but merely changes in shape, usually with elongation and deformation of the
cells. This applied particularly to those eggs which were located in the more
folded parts of the oviduct. They then took on the shape of the surface surrounding the space in which they were located (Fig. 5). 8 h after transplantation the
number of naked eggs found decreased markedly, then there was a gradual
decrease with time, although they were still encountered sporadically after 24 h.
A variable number of leucocytes were also found around some of the naked eggs
remaining after several hours in the oviduct (Fig. 6). After 28 h one degenerate
naked egg was found (Fig. 7), the latest recorded. Not one of the transplanted
naked eggs was found 36 h after transplantation.
However, not all of the naked eggs showed an immediate tendency to adhere
to the walls of the oviduct or to degenerate within the first 24 h. Some of them
lay singly in the wide parts of the oviducts and underwent cleavage, which was
never observed in the adhering eggs. After 12 h, a few single i blastomeres were
found (originating from division of naked \ blastomeres) and two \ blastomeres
with metaphase spindles (Fig. 8). After 24 h one 1-cell naked egg undergoing
division was observed. Naked eggs were also observed adhering to each other
and forming large groups (Fig. 9).
DISCUSSION
Naked eggs transplanted to the oviduct may adhere to its walls, wedge themselves between the numerous folds visible in its lumen or adhere to each other
in groups. It is difficult to explain whether such adhesion—particularly where
contact of the eggs with the epithelium is very close over a large area—is due
only to the great 'stickiness' exhibited by naked eggs or to some other factors.
Unfortunately observation made by means of a light microscope cannot reveal
what kind of contact takes place between the blastomeres and the cells of the
oviducal epithelium. However, whether or not the eggs adhered or became
wedged, between 10 and 20 h after transplantation their appearance was entirely
normal. It is not certain whether the subsequent degeneration of the eggs is a
consequence of their adhesion to the epithelium, or whether it is not in any way
connected with this phenomenon but is caused by some other factors affecting
naked eggs in the oviduct. Degeneration must, however, take place very rapidly
and this process is difficult to demonstrate, since it was only in sporadic cases
that distinctly degenerate naked eggs were found. As divisions were never
observed in adhering eggs it must be assumed that the immobilizing of the eggs
Role of the zona pellucida
545
causes inhibition or disturbance of their first cleavage divisions. The fact that,
in those few cases in which divisions of naked eggs were observed, they were
located in the widest part of the oviduct and were not in contact with its walls,
would appear to confirm this conclusion. This is, however, a rare phenomenon
and presumably the egg sooner or later always adheres to the oviduct walls and
is thus immobilized.
Under such conditions attempts at flushing the oviduct are either ineffectual
or must lead to tearing the eggs from the walls, rupturing the cell membranes of
the blastomeres which then rapidly decompose. In addition, some of the eggs
are so firmly wedged between the folds of the oviducal wall, that flushing is
probably completely ineffective. This would explain why it is almost impossible
to rinse out naked eggs within a relatively short time after transplantation.
All eggs from the 1st and 2nd day and a large proportion of the eggs from the
3rd day of pregnancy, when deprived of their zonae, are immobilized in the
oviduct. Implantation was not found in the recipients' uteri in any case after
transplantation of 1-, 2- and 4-cell naked eggs. Similar results were obtained by
Moore et ah (1968) who transplanted single naked 'half' and 'quarter' blastomeres of rabbit eggs.
In the present study, only eggs of at least 8 blastomeres gave any implantations, the number of implantations increasing with the age of the egg. In studies
on mouse chimeras (Tarkowski, 1961; Mystkowska & Tarkowski, 1968) naked
morulae and blastocysts were transplanted to the oviduct and later became
implanted in the uterus. It may therefore be assumed that from the morula
stage, the external cells undergo some changes protecting the developing embryo
from adhesion and immobilization, and possibly also from the harmful effect of
unidentified factors of the oviducal environment.
The observations thus show that one of the functions of the zona pellucida is
to make it possible for the cleaving eggs to move freely along the oviduct. Up
to the stage of 8 blastomeres, or even slightly longer, the presence of the zona
pellucida is thus a sine qua non condition for development in vivo.
SUMMARY
1. Zona-free eggs and eggs with the zona pellucida intact were transplanted
to the oviduct of female mice on the 1st or 2nd day of pseudopregnancy.
Implantation was not found after transplanting naked eggs from the 1st or 2nd
day of pregnancy (1-, 2- and 4-cell stages). 16% of embryos from the 3rd
day of pregnancy (8-cell-early morulae) and 25 % of embryos from the 4th
day of pregnancy (late morulae and blastocysts) implanted respectively. 51, 61,
40 and 28 % of control eggs (with the zona pellucida) from the 1st, 2nd, 3rd and
4th day of pregnancy respectively became implanted.
2. 1- and 2-cell naked eggs can be recovered from oviducts by flushing only
within 2 h of transplantation.
546
J. A. MODLINSKI
3. Histological examinations revealed the presence of naked 1- and 2-cell
eggs during a period of ca. 24 h; the number of these eggs markedly decreases
with the passage of time. The majority of naked eggs adhere to the epithelium
of the oviduct within a few hours of transplantation. Immobilized eggs do not
undergo cleavage. Degeneration may be the result of the eggs adhering to the
walls of the oviduct, or to the direct action of unidentified factors in the oviducal
environment. It is not until naked eggs have attained at least 8 blastomeres that
they can develop in the oviduct and become implanted in the uterus.
RÉSUMÉ
Le rôle de la membrane pellucide dans le développement d'œufs
de Souris in vivo
1. Des œufs privés de leur zona pellucida ainsi que des œufs à zona intacte
ont été transplantés dans l'oviducte de Souris femelles au cours du premier ou
du deuxième jour de leur pseudogestation. On n'a pu trouver d'implantation
d'œufs nus du premier ou du second jour de gestation (œufs de 1, 2 ou 4 cellules).
Les embryons du troisième jour de gestation (8 cellules—morulas jeunes) et du
quatrième jour de gestation (morulas avancés et blastocystes) se sont implantés
dans 16 et 25 % des cas. 51, 61, 40 et 28 % des œufs témoins (c. à d. avec la
zona pellucida) respectivement du premier, deuxième, troisième et quatrième
jour de la gestation se sont implantés.
2. Des œufs nus à 1 ou 2 blastomères peuvent être récupérés de l'oviducte par
lavage seulement pendant les deux premières heures après la transplantation.
3. Les examens histologiques n'ont pu démontrer la présence des œufs nus à
1 ou 2 cellules que pendant une période approximative de 24 h; le nombre des
œufs diminuait de façon notable avec le temps. La majorité des œufs nus adhèrent
à l'épithélium de l'oviducte après quelques heures de transplantation. Ces œufs
immobilisés ne se segmentent pas. La dégénérescence résulte soit de l'adhérence
des œufs à la paroi de l'oviducte soit de l'action directe de facteurs non identifiés
du milieu tubaire. Ce n'est que lorsque les œufs ont atteint au moins le stade de
8 blastomères qu'ils peuvent se développer dans l'oviducte et s'implanter dans
l'utérus.
I wish to express my sincere thanks to Dr A. K. Tarkowski for his interest and helpful
advice and for critical reading of the manuscript.
Note added in proof. Bronson & McLaren ('Transfer to the mouse oviduct of eggs with
and without the zona pellucida' /. Reprod. Fert. in the press) found a very low implantation rate following transfer of zona-free 8-cell eggs to the oviduct and came to the conclusion, similar to that reported here, that up to this stage the zona pellucida is necessary
for normal development in vivo.
Role of the zona pellucida
547
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(Manuscript received 12 June 1969, revised 29 August 1969)