Human Reproduction, Vol. 14, No. 4, 1057-1066,
April 1999
© 1999 European Society of Human Reproduction and Embryology
Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern
1 Department of Obstetrics and Gynaecology and 2 Department of Anatomy, University of Goettingen, Robert-Koch-Str. 40, 37075 Goettingen and 3 School of Veterinary Medicine Hannover, Buenteweg 15, 30559 Hannover, Germany
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Abstract |
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Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm–oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.
Key words: human/IVF/oocyte/scanning electron microscopy/zona pellucida
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Introduction |
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The zona pellucida is an extracellular matrix which surrounds the growing as well as the mature oocyte. It is composed of three different glycoproteins (ZPA, ZPB, ZPC) which together build a structure that varies greatly between different oocytes. This heterogeneity becomes particularly clear when examining scanning electron microscope (SEM) photographs. Motta et al. (1991) and later Harris et al. (1994) described the zona of a mature human oocyte as a network with multiple pores and hollows which is created by a three-dimensional arrangement of filaments.
The porous structure might be the result of foot-like cytoplasmic branches from granulosa cells of the surrounding corona radiata, penetrating the zona pellucida to come in close contact to the plasma membrane of the oocyte during oogenesis. Apart from this network-like appearance a more compact and smooth surface has also been described. According to Sundström (1982) this type can be found on non-ovulatory, immature oocytes.
During fertilization spermatozoa have to recognize the zona pellucida and to bind to it (sperm–oocyte interaction). The initial binding of acrosome-intact spermatozoa occurs through terminal 
- and ß-galactose of the ZPC (Litscher et al., 1995
). Other zona proteins are also involved in this primary binding.
The aim of the present investigation was to answer the following questions: (i) Does the morphology of the zona pellucida depend on the state of maturity of the oocyte? (ii) Is there any correlation between the number of spermatozoa as well as their distribution patterns on the zona pellucida and the state of maturity of the oocyte? (iii) Is there any correlation between the number of spermatozoa as well as their distribution patterns on the zona pellucida in fertilized or unfertilized oocytes? (iv) Does the quality of the ejaculate influence the binding patterns of spermatozoa on the zona pellucida?
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Materials and methods |
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Patients
We used SEM to investigate 449 oocytes from 145 patients in the in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programme at the Department of Obstetrics and Gynaecology, University of Goettingen, Germany.
Unfertilized IVF or ICSI oocytes could be used without any restrictions whatsoever, whereas the analysis of fertilized oocytes was extremely limited by the German Embryo Protection Law of 1991. Only oocytes which showed a polyploid fertilization (
2 pronuclei) after IVF were allowed to be investigated. Immature oocytes (with no polar body) from ICSI patients were not injected and were also used for SEM analysis. All oocytes used for SEM came into the investigation purely by chance due to these restrictions.
Scanning electron microscopy
We used a technique which has been previously described (Schwartz et al., 1996). A prerequisite of this technique was the use of coverslips which were previously coated with poly-L-lysin (Phillips and Shalgi, 1980).
Analysis
The zona pellucida structure was classified in mature and immature as well as fertilized and unfertilized IVF and ICSI oocytes. An oocyte was classified as mature if, under the light microscope, one polar body was clearly visible in the perivitelline space, and immature if no polar body could be detected and/or the germinal vesicle was visible inside the cytoplasm.
Besides the analysis of the zona morphology, sperm binding patterns, i.e. the distribution of spermatozoa bound to the zona pellucida, were investigated on IVF oocytes. According to the gold standard, these oocytes had been inseminated after sperm preparation (swim-up technique) with 100 000 spermatozoa from husbands. For the analysis of sperm binding patterns, a three-figure code was used. This code was based on the results of our own investigations made by light microscopy (Michelmann et al., 1995) and gave information about: the number of bound spermatozoa; the distribution of the binding sites on the surface of the zona; and the appearance of cluster-like attachments of spermatozoa on parts of the zona.
The three-figure code consists of the following numbers. First number = number of spermatozoa bound onto the zona; 0 = none; 1 = 1–10; 2 = 11–50; 3 = 51–100; 4 = >100. Second number = distribution patterns of the bound spermatozoa; 0 = no spermatozoa on the zona; 1 = regular; 2 = irregular. Third number = existence of sperm clusters on the zona; 0 = none; 1 = no sperm clusters; 2 = sperm clusters present.
The results referring to zona morphology and sperm binding patterns are presented in histograms according to the percentage distribution. All results were tested for significance with the RxC contingency table. Values of P < 0.05 were considered significant.
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Results |
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Surface structure of the zona pellucida
Structure types
We investigated the zona pellucida surface structures of 359 oocytes from 95 patients by SEM. All oocytes remained unfertilized after IVF (n = 147) or ICSI (n = 212) treatment. According to the SEM pictures four different types of surface structures of the zona pellucida could be distinguished.
Type A represented a distinct net-like structure made out of numerous pores and hollows which were arranged on the surface like windows (Figures 1 and 2). Type B was also a net-like structure which was composed of pores and hollows. In contrast to type A they were flatter and of smaller diameter (Figures 3 and 4). The zonae of type C had an uneven and spongy surface with very few or no porous areas. The net-like structure had nearly disappeared (Figures 5 and 6). Type D was characterized by a relatively smooth exterior of the zona pellucida. Pores and hollows hardly occurred and were scattered only in certain areas. On most of the surface they seemed to have melted into each other to form an even exterior (Figures 7 and 8). Sperm tails were clearly visible.
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The majority (n = 220) of the 359 investigated oocytes had a zona pellucida of type A (n = 57) and type B (n = 163) with many pores of different sizes and depths. A rather large group of 126 oocytes had zona type C with a typically uneven surface. Only a small number of oocytes (n = 13) had a zona pellucida of type D.
When the surface of either zona type A or B was enlarged 10 000 to 20 000 times, an infinite number of small beads (80–130 nm in diameter), lined up like pearls on a string, became visible (Figure 9). In some parts of the zona these pearls formed clearly visible filaments which were also recognizable in the depth of the pores.
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In zona type C these pearl string-like filaments were also clearly visible (Figure 10
). However, their arrangement appeared less organized. If these little beads represent the basic construction material of the zona pellucida then it must be present in all zona types regardless of their appearance. So presumably in types C and D a conversion of the pearl string-like material must have taken place, especially in type D where the beads appeared to have melted into each other and were hardly recognisable (not shown).
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On certain oocytes still surrounded by some attached granulosa cells, cytoplasmic filaments from these cells penetrated the surface of the zona pellucida (Figure 11
).
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Mature and immature oocytes
The evaluation of the zona pellucida surface of unfertilized oocytes was done on mature (n = 229) versus immature (n = 82) oocytes. As the availability of oocytes for analysis was random, the number of mature and immature oocytes differed. Zona types A and B occurred most frequently in mature and immature oocytes without significant differences in numbers. Zona types C and D also appeared in the same numbers in both groups (Figure 12
). Thus, a significant correlation between the state of oocyte maturity and the surface structure of the zona pellucida was not found.
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Fertilized and unfertilized oocytes
Surface structures of fertilized (polyploid) oocytes (n = 21) were compared to unfertilized oocytes (n = 229). In fertilized oocytes four different types (A–D) of zona morphology could be seen (Figure 13
). However, polyploid oocytes had a zona structure of types C and D twice as often as did unfertilized oocytes which, in contrast, had zona type B in a significantly higher number.
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Oocytes after IVF and ICSI treatment
SEM of oocytes after ICSI (n = 178) or IVF treatment (n = 133) showed that ~50% had zona type B regardless of IVF or ICSI treatment (Figure 14
). All other types of zona morphology (A, C and D) appeared in the same numbers, regardless of whether the oocytes were treated by IVF or ICSI. This means that the different treatment of oocytes in the course of IVF or ICSI did not influence their surface morphology.
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Sperm binding patterns
Unfertilized oocytes after IVF
We analysed the binding patterns of sperm on the zona pellucida of 216 unfertilized oocytes from 55 IVF patients (Figure 15
). The number of bound sperm was counted solely on that part of the zona surface which was visible in the SEM pictures.
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Nearly one-quarter of unfertilized oocytes (23%) did not have any spermatozoa attached to the zona pellucida while 44% had only very few (1–10) bound spermatozoa (Figure 16
). On the surface of 22% of the oocytes a moderate number of spermatozoa (11–50) had bound (Figure 17). Only a few unfertilized oocytes had either between 51 and 100 spermatozoa attached to the zona (8%) (Figure 18) or an uncountable number (3%).
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The distribution patterns of spermatozoa on the zona pellucida (even or uneven distribution with or without cluster formation) were not related to the number of bound spermatozoa. In some oocytes, distinct areas on the zona without any spermatozoa were clearly visible. Such areas could be seen even on oocytes with uncountable numbers of attached spermatozoa (Figure 19
). The binding of spermatozoa onto the zona pellucida occurred in an even or uneven distribution. In the latter case sperm clusters could sometimes be seen. A cluster signified the binding of a high number of spermatozoa on a clearly limited area of the zona.
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Fertilized, polyploid oocytes after IVF
SEM pictures of 13 polyploid oocytes after IVF from 12 patients were analysed for their sperm binding patterns. In contrast to unfertilized oocytes, polyploid oocytes always had spermatozoa attached to the zona. On some (15%) <10 spermatozoa had bound. On unfertilized oocytes this low number of bound spermatozoa was much higher (68%). On 32% of the analysed polyploid oocytes >50 spermatozoa had bound with a mostly uneven distribution. In 50% of them cluster formations were clearly visible. In general it must be stated that on polyploid oocytes significantly more spermatozoa had bound compared to unfertilized ones.
Mature and immature oocytes after IVF
To find out if the degree of maturity had some influence on the sperm binding patterns, mature (n = 64) and immature (n = 21) oocytes were analysed after IVF. Only those oocytes were taken for analysis which were inseminated with spermatozoa from ejaculates classified as normospermic. The percentage of oocytes without any bound spermatozoa was the same in both groups (11%:10%), while oocytes with an uncountable number of bound spermatozoa could be found only in the group of mature oocytes (Figure 20). Sperm clusters were recognizable on mature as well as immature ones. There were no statistically significant differences in the number of bound spermatozoa between mature and immature oocytes.
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Oocytes from patients with or without fertilization after IVF
Unfertilized oocytes from two groups of patients after IVF were analysed. In the first group (eight patients; 35 oocytes) no fertilization occurred after IVF whereas in the other group (47 patients; 176 oocytes) each patient had at least one fertilized oocyte. Oocytes from the first group (no fertilization) had sperm binding patterns which were completely different from that of the second group with at least one fertilization (Figure 21
). In 48% of oocytes from the first group no spermatozoa were visible on the zona and 40% of the oocytes had a very small number of bound spermatozoa (1–10) only. However, it has to be taken into account that 75% of these oocytes were inseminated with ejaculates which were classified as below normospermia. In contrast this was the case in only 23% of patients in the second group.
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Unfertilized oocytes inseminated with normal or pathological ejaculates
It was our intention to find out if the quality of ejaculates had any influence on the sperm binding patterns. Therefore 126 unfertilized oocytes from 37 patients whose partner had a normal spermiogram were compared to 86 unfertilized oocytes (18 patients) which were inseminated with ejaculates below WHO criteria.
An extremely high number of oocytes (43%) which were inseminated with pathological ejaculates did not have any sperm attached to the zona and an even higher number (48%) had only a few bound spermatozoa. Oocytes with >50 spermatozoa attached to the zona were not found (Figure 22).
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It is of interest to note that even after insemination with spermatozoa from normal ejaculates there are also oocytes (10%) without attached spermatozoa and that 41% of the oocytes had only up to 10 bound spermatozoa. On the other hand, the remaining 49% of oocytes which were also not fertilized had a high number of bound spermatozoa.
To sum up, it can be said that oocytes inseminated with normal ejaculates had significantly more spermatozoa bound to the zona compared to oocytes inseminated with spermatozoa from ejaculates below WHO criteria.
Oocytes with different zona morphologies
To find out if different zona morphologies led to different sperm binding patterns oocytes were analysed according to their zona morphology. Oocytes (n = 50) with a net-like structure (types A and B) were compared to those of zona types C and D with a smooth and compact structure (n = 36). All oocytes had been inseminated with normospermic ejaculates.
In both groups 54% of oocytes had <10 attached spermatozoa (Figure 23). All other distribution patterns also occurred in nearly the same percentages without significant differences. With regard to the zona morphology it was not clear why certain oocytes had a high number of bound spermatozoa while others had almost none. Distinct areas on all types of zona surfaces were detectable where either no spermatozoa existed or sperm clusters appeared.
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Ultrastructure of sperm–zona pellucida interaction
SEM revealed very heterogeneous courses of gamete interaction and penetration of spermatozoa into the zona. Different phases of sperm fusion became visible. They ranged from an extremely superficial, loose attachment (Figure 24
) to the commencement of penetration (Figure 25) and finally to a total fusion of the sperm head with only the tail remaining visible (Figure 26).
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In most cases a flat, tangential attachment of the sperm head to the surface of the zona appeared (Figure 25
), followed by an intrusion into the zona in exactly this position. However, vertical binding with a penetration by the tip of the head first also occurred (Figure 27). Especially in oocytes where large numbers of bound spermatozoa (with or without clusters) were detectable, the vertical binding and penetration was the most usual way.
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In oocytes with a net-like, porous structure of the zona, sperm heads very often disappeared deeply into the pores so that only the tails were visible from the outside (Figure 26
). The filaments, resembling a string of pearls, surrounded the sperm head as soon as it penetrated the zona (Figure 28). It was not possible to draw conclusions about the acrosomal status of bound spermatozoa by looking at SEM pictures.
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Discussion |
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Surface structure of the zona pellucida
The surface structure of the zona pellucida from different mammals was described by several authors. In mice, hamsters, pigs and cattle two structures could be differentiated. The first was a net-like structure formed out of different layers of a string-like material perforated by numerous pores. The second had a totally different appearance with a smooth and compact structure without any pores.
Both zona types also occur in human oocytes (Familiari et al., 1992). Our own results confirmed those findings. We too found on SEM pictures two distinct zona types which were categorized into types A and B for the more porous and types C and D for the compact and smooth surfaces.
Familiari et al. (1989a,b, 1992) showed that zona filaments from mice and humans were constructed out of little beads which were lined up like a string of pearls. So far it is still unknown if these `pearls' are identical with a single glycoprotein (ZPA, ZPB, ZPC) or if they represent an oligomer made of several glycoproteins. In addition they found that on degenerated oocytes those filaments were no longer detectable and that the pearl string-like beads had been melted into each other. This resembles the structure described by us as zona type D.
But even on A and B type zonae some areas were detectable in which the surface appeared compact and smooth without pores as in types C and D. As described previously, these spots were not the result of mechanical irritations especially during ICSI treatment of these oocytes (Schwartz et al., 1996).
Cells of the corona radiata are in close contact with the egg-plasma membrane of the oocyte through cytoplasmic filaments penetrating the zona (Suzuki et al., 1994). Macchiarelli et al. (1992) speculated that the net-like structure of the zona might have originated from the penetration of those filaments. However, this theory cannot explain why in our results 39% of all analysed oocytes did not have any pores.
Comparison of the surface morphology of mature and immature oocytes
In the literature there are highly contradictory opinions about the surface structure of human oocytes during the final stages of oogenesis. Several papers assumed a correlation between the type of surface morphology and the stage of maturity (Calafell et al., 1992). Familiari et al. (1992) and Motta et al. (1991) described a net-like, porous surface mainly in mature oocytes while immature and degenerated oocytes had a compact type with no pores. These results could not be verified by Suzuki et al. (1994) who, like Sathananthan (1994), detected a porous structure already at the germinal vesicle stage where cytoplasmic filaments from the corona radiata penetrated the zona pellucida surface forming the net-like surface. According to our own data we can confirm the latter results. We also cannot find any correlation between the appearance of the zona surface and the maturity of the oocyte.
Comparison of the zona morphology of fertilized and unfertilized oocytes
After penetration of the spermatozoon into the zona, the so-called zona reaction occurs which leads to a change in the chemical and physical characteristics of the zona. In connection with these biochemical changes, modifications on the zona surface can be expected, which might be visible in SEM pictures. In the literature contradictory results are reported by different authors. Familiari et al. (1992) could not find any changes correlated to fertilization whereas Nikas et al. (1994) and Suzuki et al. (1994) reported a high correlation in the zona morphology between fertilized and unfertilized oocytes. According to their findings, fertilized oocytes had a compact surface (types C and D) in contrast to unfertilized ones with a porous structure (types A and B). These results match our own findings where the majority of fertilized, i.e. polyploid oocytes, had zona types C or D whereas unfertilized oocytes mainly were of zona types A or B. Because our analysis of fertilized oocytes was restricted to polyploid ones only, we could not prove if these structural differences were actually related to fertilization or were the result of other factors.
Comparison of the surface morphology of oocytes after IVF or ICSI treatment
To answer the question if the surface morphology of the zona pellucida might be influenced by different in-vitro techniques we compared oocytes after IVF treatment to those after ICSI treatment. Whereas the cumulus complex of IVF oocytes was dissolved by the enzymatic reaction of spermatozoa, ICSI oocytes were treated with hyaluronidase immediately after follicular puncture to get the same effect. If the handling of oocytes had some influence on the zona surface then different morphologies of ICSI and IVF oocytes must be expected. However, comparison of oocytes from both groups showed no differences. This is certainly evidence that all zona types and their different peculiarities are not exogenous side effects of the treatments related to IVF, ICSI or SEM.
Sperm binding patterns
Fertilized and unfertilized oocytes after IVF
After IVF treatment the sperm binding patterns on unfertilized as well as fertilized (i.e. polyploid) oocytes were analysed. According to our own data obtained previously through the analysis of light microscope pictures all ooyctes have an extremely heterogeneous sperm binding pattern. These patterns did not correlate with oocyte maturity, the occurrence of fertilization or the patients they came from (Michelmann et al., 1995).
The analysis of SEM pictures from 216 unfertilized oocytes also confirmed these results: the number and the distribution patterns of bound spermatozoa on the zona pellucida was highly variable. These findings supported the data of several other investigations (Bedford and Kim, 1993) which assumed that factors such as maturity of the oocytes (Mahadevan et al., 1987), morphology of the zona pellucida (Familiari et al., 1988), or anomalies of the spermatozoa (Liu et al., 1989) were the reasons for this variation.
Liu et al. (1989) reported that from all unfertilized oocytes that they analysed, 23% did not have any bound spermatozoa on the zona pellucida. We also could not detect any spermatozoa on the surface of 23% of these oocytes after SEM analysis or on 25% after investigation by light microscopy (Michelmann et al., 1995).
After IVF, oocytes from the same patient not only had different numbers of bound spermatozoa but also different distribution patterns of spermatozoa.
In oocytes with >10 bound spermatozoa only 50% had an even distribution pattern. On all of the other oocytes spermatozoa bound in extremely heterogeneous ways. Areas totally free of any spermatozoa were close to those which were overloaded, with spermatozoa sometimes forming cluster-like arrangements.
These sperm clusters on the surface of oocytes have been found not only on human oocytes (Michelmann et al., 1995) but also on cattle oocytes (Hyttel et al., 1988). It is because of this heterogeneous binding that some sperm function tests, such as the hemizona assay (Burkman et al., 1988), can no longer be recommended. This test is based on the faulty assumption that sperm binding on oocytes is always evenly distributed.
Like Mahadevan et al. (1987) we found significantly more spermatozoa bound to the zona pellucida of fertilized oocytes than compared to unfertilized ones. These results do not agree with those obtained by Bedford and Kim (1993). Because we used only polyploid cells in a very small number (n = 13) for SEM it is quite possible that, compared to normally fertilized oocytes, polyploid ones had significantly more bound spermatozoa on the zona pellucida.
In this connection, it is of interest to mention that in spite of the high number of motile spermatozoa used for in-vitro fertilization only a relatively small number bound to the zona pellucida. Perhaps Sundström (1982) was correct when he suspected a so-called selection function of the zona. This leads to the conclusion that this `selection function' would be disturbed in all of those oocytes which were overloaded with bound spermatozoa. Furthermore it could explain why polyploid oocytes had a significantly higher number of bound spermatozoa.
Mature and immature oocytes
Several investigators did not find any differences in the number of bound spermatozoa between mature and immature oocytes (Lopata and Leung, 1988; Tesarik et al., 1988; Liu et al., 1989; Bedford and Kim, 1993). We also could not find any such differences. However, only in mature oocytes were >50 bound spermatozoa detected.
In contrast to these findings Oehninger et al. (1991) and Franken et al. (1994) mentioned, in connection with the hemizona assay, that on mature oocytes significantly more spermatozoa bound than on immature ones. They assumed that the meiotic maturity was correlated with increased potential of sperm binding.
Oocytes from patients with or without fertilization
To find out if a total lack of fertilization is related to zona morphology we divided the patients into two groups. Patients in the first group had at least one fertilized oocyte after IVF while there was no fertilization at all in the second group. Unfertilized oocytes from patients in the first group had, on average, more bound spermatozoa than oocytes of the second group. The reason for this difference was male subfertility in most cases. In the group with no fertilization 75% of all male partners had a sperm quality below WHO criteria while in group 1 only 23% of males were subfertile.
The question about a correlation between the number of bound spermatozoa and achieved fertilization is answered inconsistently in the literature. While Mahadevan et al. (1987), Liu et al. (1989), Franken et al. (1989) as well as Liu and Baker (1992) found such a correlation, Bedford and Kim (1993) and Michelmann et al. (1995) refuted it. So our results from 1995 contradict our results in the present study. But as already mentioned light microscopy results cannot be compared to the SEM results as the sample size and criteria of analysis were different. Contrary to the light microscopy analysis fertilized oocytes could not be analysed by SEM because of the destructive effect of this technique.
Unfertilized oocytes inseminated with different ejaculate qualities
The comparison of sperm binding patterns on oocytes inseminated with normal or pathological spermatozoa confirmed that sperm quality had some influence on the binding capacity of spermatozoa. This result agreed with the results of Mahadevan et al. (1987) and Liu et al. (1989). However, even after insemination with good quality ejaculates 10% of the oocytes did not have any bound spermatozoa on the zona pellucida. This is another indication that heterogeneous sperm binding patterns are not purely related to different ejaculate qualities.
Zonae morphologies
Motta et al. (1991) found a significantly higher number of bound spermatozoa on zonae with a porous and net-like structure (types A + B) than on compact zonae (types C and D). They assumed that the porous structure allowed a wider range of sperm binding sites compared to the more compact structure. Henkel et al. (1995) as well as Familiari et al. (1988) also believed that there was a close correlation between zona morphology and sperm binding capacity.
In contrast to those results we were not able to find any correlation between sperm binding and zona morphology. All zona types (A–D) showed a high variety of sperm numbers and sperm binding patterns and no correlation with any specific surface morphology.
Ultrastructure of gamete interaction on SEM pictures
In 1982 Sundström described a tangential binding and penetration of the mammalian spermatozoon on and into the zona pellucida. In most mammals sperm binding starts with the contact of the equatorial segment of the spermatozoa and the outer surface of the zona pellucida (Dobris and Katz, 1991). We were able to confirm this flat position of the spermatozoon on the zona at the beginning of gamete interaction. However, in addition we also found other types of gamete fusion which had already been described by Familiari et al. (1992) and Motta et al. (1991). Sperm heads bind on the zona pellucida in many different positions, even with the tip of the head first. This `head-first' binding type was found especially on zonae with big surface pores by us and other investigators (Tsuiki et al., 1986; Familiari et al., 1988). In a At x10 000 magnification it became clearly visible that the fusing sperm head was covered by pearl string-like filaments as described earlier.
In contrast to the findings of Familiari et al. (1988) we found all different types of sperm penetration on all types of zona (A–D) by us. Familiari et al. (1988) never saw any spermatozoa penetrating a compact and smooth zona (type C and D) but only loose attachments in a flat position on these types.
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4 To whom correspondence should be addressed
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References |
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Submitted on September 4, 1998; accepted on December 31, 1998.
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