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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Key words: human/IVF/oocyte/scanning electron microscopy/zona pellucida
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Introduction |
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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 (spermoocyte 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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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 = 110; 2 = 1150; 3 = 51100; 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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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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When the surface of either zona type A or B was enlarged 10 000 to 20 000 times, an infinite number of small beads (80130 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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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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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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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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Discussion |
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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 (AD) 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 (AD) 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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Notes |
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References |
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Submitted on September 4, 1998; accepted on December 31, 1998.