The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus–oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

The capability of human zona pellucida (ZP) to bind selectively to normal functional sperm with normal chromatin has been reported widely in the literature. The aim of this study was to evaluate whether ZP–binding sperm selection may represent a method to retrieve superior spermatozoa for intracytoplasmic sperm injection (ICSI). Patients were divided into two groups: a ZP-ICSI and a conventional ICSI group. In the ZP-ICSI group, spermatozoa for injection were selected after ZP–sperm incubation and spermatozoa that were tightly bound to the ZP were used for ICSI (ZP-ICSI). Clinical outcomes of ZP-ICSI were compared with the outcomes of traditional scientist-selected sperm injection (conventional ICSI). Results did not show any significant difference in fertilization, pregnancy, implantation and take-home-baby rates between conventional ICSI and ZP-ICSI. However, when data relative to patients who received ZP-ICSI were analyzed, an interesting result was observed: higher sperm concentration and morphology correlated with higher ZP–sperm binding. Additionally, patients with higher ZP–sperm binding seem to have improved pregnancy and take-home-baby rates. In conclusion, this study shows that ZP-ICSI is not a superior method compared with conventional ICSI. However, clinical ICSI outcomes were apparently improved in the presence of good ZP–sperm binding. We therefore speculate that sperm competence to ICSI could be reduced when the sperm's ability to bind the ZP is impaired.

That the zona pellucida (ZP) plays a prominent role in the physiology of some human twinning is an attractive, albeit incompletely proven, medical hypothesis. Indeed, an association has been proposed between manipulation of the ZP and/or native ZP microarchitecture and monozygotic (MZ) twins. Ovulation induction also has been theoretically linked to in vivo ZP alterations facilitating MZ twin development. In vitro fertilization (IVF) relies on necessary (and, in some cases extended) embryo culture techniques potentially creating subtle ZP changes and subsequent MZ twinning. With growing experience in the assisted reproductive technologies and particularly IVF, some preliminary reports have noted an increased frequency of MZ twins after procedures that artificially breach the ZP (i.e., intracytoplasmic sperm injection [ICSI], or ‘assisted hatching’). Such ZP manipulations ostensibly enhance oocyte fertilization or facilitate blastocyst hatching, thus improving pregnancy rates for couples undergoing fertility treatment. Evidence exists both to challenge and support the connection between these phenomena and MZ twins. This report outlines the fundamental embryological processes believed responsible for these conflicting observations; the current literature on the subject of human ZP micro-manipulation and MZ twins is also discussed. Twin Research (2000) 3, 217–223.

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

This work evaluated if an in vitro test, with the combined power of the statistical evaluation of spermatozoa and zona pellucida (ZP) competitive binding ability and a rapid method for accessory sperm counts, could predict the bull fertility. Ten Holstein Friesian bulls of known field fertility (five of high and five of low fertility) were selected. An in vitro heterospermic insemination approach, based on differential staining, was tested on 45 possible pairs of bulls (two batches per bull). Motility and quality (abnormalities and membrane status) seminal characteristics and estimated relative conception rates (ERCR) highlighted only one association between membrane integrity and ERCR (p = 0.007). Differences in ZP binding allowed us to rank bulls into two categories based on low and high binding ability. For eight bulls, this classification reflected the ERCR. Differences between batches were reported for two bulls, in which the effect of heterospermic insemination (the number of sperm binding to ZP from different bulls not in a 1:1 ratio) showed a significant bull-related effect (p < 0.001) in the first batch and no effect (p > 0.05) in the second batch for both bulls. Reduction of the number of oocytes per assay from 25 to 5 had no effect (p > 0.5) on the bulls’ ranking. Our results suggest that in vitro competitive binding is a promising approach for estimating bull fertility and support concepts for further implementation, e.g. drastic reduction of oocyte number in a single pair assay and larger scale testing for batches.

Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellcida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

We have investigated whether culture in glycine-containing medium affects subsequent glycine transport by the specific transport system, GLYT1, which is the sole glycine transporter in fertilized mouse ova. When fertilized ova were maintained for 6 h in culture with a physiological level of glycine (1 mM), subsequent transport of radiolabelled glycine was decreased by 40% compared with fertilized ova that had been maintained in glycine-free medium. Kinetic measurements showed that the apparent glycine affinity was decreased after culture with glycine (Km increased from 0.20 to 0.41 mM), but maximal transport rate was unchanged (similar Vmax of 20 and 23 fmol/fertilized ovum/min). These findings could have reflected activation of GLYT1 by prolonged substrate starvation, similar to some other amino acid transport systems. However, our findings were instead consistent with the alteration in glycine transport being due to trapping of glycine within the zona pellucida resulting in competitive transport inhibition even after ova were removed from glycine-containing media. First, even very brief exposures to glycine resulted in decreased subsequent glycine transport rates, with a maximal effect apparent within ~6 min. Second, extensive washing (at least six) reversed the effect. Third, the effect was absent when zona-free fertilized ova were used. Thus, it appears that components of the external environment of preimplantation embryos may continue to affect transport kinetics for a period even after embryos are removed from environments that contain them.

Ultrastructure of the zona pellucida (ZP) of canine and feline oocytes has not been fully investigated. The objective of the study was to evaluate the potential use of the low vacuum scanning electron microscope (LVSEM) with oocytes. This required development of a method to prepare canine and feline cumulus-oocyte complexes (COCs) for LVSEM to provide ultrastructural information on the ZP. COCs were collected from ovaries, and cumulus cells were either partially or completely removed to reveal the ZP. COCs and zona-intact oocytes were fixed at 4°C for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently viewed wet or further processed by critical point drying, and viewed uncoated or sputter coated with gold. Although the spongy surface of the ZP was visible at low vacuum in uncoated oocytes, coated oocytes had more details at high vacuum. The ZP surface of canine and feline oocytes contained numerous various-sized, spherical or elliptical pores that narrowed centripetally splitting into several smaller, deep pores. The round to oblong cumulus cells tightly surrounded the ZP. Each corona radiata cumulus cell tapered into a thin projection that entered the ZP. Our detailed techniques will enable future studies connecting ultrastructural and molecular aspects of oocyte maturation and development in mammals.