ABSTRACT
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 & Wrób-lewska, 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Tarkowski’ s observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct.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.
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 vasec-tomized 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 μ,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 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.
Results of transfers of naked eggs and eggs with the zona pellucida (inspection on 11th day)

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 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 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.
Naked 2-cell egg adhering to epithelium of oviduct. 4h after transfer, × 500.
Naked blastomeres adhering to epithelium of oviduct. Contact between blastomeres and epithelium is very close over a large surface. 8 h. × 500.
Naked blastomeres adhering to epithelium of oviduct. Contact between blastomeres and epithelium is very close over a large surface. 8 h. × 500.
Naked 2-cell egg wedged between folds of oviduct. Shape of egg corresponds to surface surrounding it. 24 h. × 500.
Naked blastomere lying in lumen of oviduct. Several leucocytes adhere to its surface. 16 h. × 500.
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 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).
Group of naked and blastomeres and one 2-cell egg in zona pellucida. Two small blastomeres originated from one of the transplanted naked blastomeres. Metaphase spindle can be seen in one of the blastomeres. 24 h. × 350.
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 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 al. (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
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.
1- and 2-cell naked eggs can be recovered from oviducts by flushing only within 2 h of transplantation.
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
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.
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.
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.
Acknowledgements
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’ J. 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.