Immunological response and ovarian histology of squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida ZP3 (Mr = 55,000) macromolecules

ZP3 (M, = 55,000) is the major electrophoretic component of the porcine zona pellucida (ZP). In a continuing assessment of ZP3 as a candidate antigen for contraceptive vaccine development, female squirrel monkeys were immunized with 200 μg ZP3 using either Freund's adjuvant (FA) or muramyl dipeptide (MDP) and the effect of such immunization on ovarian histology examined. Two experimental and three control groups were immunized: Group 1 (n = 4), ZP3 plus FA; Group 2 (n = 4),ZP3 plus MDP; and controls—Group 3 (n = 2), ZP3 alone; Group 4 (n = 4), FA alone; and Group 5 (n = 4), saline. High antibody response to ZP3 was detected in the ZP3/FA and ZP3/MDP groups, and a very low response, in the ZP3-alone group. Immune profiles for the ZP3iFA and ZP3/MDP groups were comparable, but titers in the MDP group were consistently lower and decreased more rapidly after 300 days post-immunization (PI) than in the FA group. At 6 months PI, all ovaries from the ZP3/FA group revealed a deficiency of zona-encased oocytes and a reduction in secondary and tertiary follicles compared to controls. At 18–24 months PI, normal ovarian histology in one ZP3/FA injected monkey and the presence of zona-encased oocytes in a second monkey suggested ovarian recovery. Normal ovarian histology was present in all monkeys in the ZP3/MDP group as well as in all controls. These findings indicate that while immunization with ZP3/FA does initially perturb normal ovarian histology, such adverse effects appear to be reversible. Furthermore, immunization using ZP3 with MDP has no adverse effect on the ovary, indicating the importance of proper adjuvant selection in immunocontraceptive (IC) studies. These data encourage continued investigation of the zona IC approach using well-characterized zona immunogens with non-Freund's adjuvants.     

Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida.

To study zona pellucida antigens involved in human fertilization, five monoclonal antibodies (MAbs)--2A1, 2G3, 4A2, 4E12, and 5H4--were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zonae pellucidae. Each MAb reacted not only with solubilized porcine zona glycoproteins but also with the glycoproteins deglycosylated by trifluoromethanesulfonic acid treatment. They also reacted with intact zonae pellucidae of porcine and human oocytes. Three (4A2, 4E12, and 5H4) of the five MAbs showed a significant blocking effect on human sperm binding and penetration of human zonae pellucidae. The 5H4 MAb showed a strong reaction with ZP4 and ZP1 glycoprotein families of porcine zonae pellucidae, and four other MAbs reacted more strongly with ZP3 than with ZP4. The reactivity of 5H4 with porcine zona glycoproteins was destroyed by chymotrypsin digestion, but the antigen epitope was resistant to proteolysis by trypsin and endoproteinase Lys-C. A peptide fragment reactive to 5H4 was isolated by reverse-phase HPLC from endoproteinase Lys-C-treated ZP4 glycoproteins, and its molecular mass was determined to be 7 kDa by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 is a good candidate for development of a contraceptive vaccine.     

Ovaries remain functional in squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida 55,000 macromolecule

Fifty female squirrel monkeys were each immunized with 200 micrograms of a purified preparation of the 55,000 macromolecule (ZP3) from porcine zona pellucida. The fertility status of these immunized monkeys, as well as the effect of ZP3 antibodies on ovarian function, was monitored. High anti-ZP3 titers were achieved (greater than 75% binding levels as determined by radioimmunoassay) and remained high (approximately 67% binding level) for the duration of this study. Hormonal evaluations indicated initial disturbances in normal ovarian steroid secretion and function that were confirmed by laparoscopic observation and oocyte production data. Histological examination of ovaries at 6-7 mo post-injection suggested an interference in folliculogenesis. No pregnancies were observed in the immunized monkeys during this period. By 10-15 mo post-immunization, hormonal and laparoscopic data indicated that ovarian function was recovering in injected monkeys despite the continued presence of high titers to ZP3. Collectively, these results demonstrate that although immunization with ZP3 initially produces disturbances in normal ovarian function that inhibit fertility, these effects are reversible. Such findings encourage the continued intensive investigation of purified porcine zona macromolecules for immunocontraceptive purposes.     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice

The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.     

Inhibition of pig oocyte in vitro fertilization by the action of components of the zona pellucida

The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 mug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner.     

Immunocontraception of captive exotic species. I. Przewalski's horses (Equus przewalskii) and banteng (Bos javanicus)

A contraceptive vaccine made of porcine zonae pellucidae (PZP) was tested in three Przewalski's mares and five banteng cows. The vaccine antigen consisted of the complete family of glycoproteins of the porcine zona pellucida, including the sperm receptor ZP3. All mares and three of five banteng were inoculated with 2 or 3 i.m. injections of approximately 65 μg of antigen given over a 6 week period. Two other banteng received inoculations of only 35 μg of antigen on the same schedule. Two of the three mares and three of five banteng cows were pregnant at the time of inoculation. No new pregnancies, as a result of postinoculation breedings, occurred among either the mares 36 months after 65 μg antigen inoculations or among the banteng for 24 months after 65 μg inoculations. One postinoculation pregnancy resulted among the two banteng receiving only 35 μg of antigen. Differences in fertility between treated and control mares and between preinoculation and postinoculation reproductive performance of the banteng were significant (P < 0.05). Urinary ovarian steroid metabolites and behavioral observations indicated follicular development and ovulations were occurring among treated mares during the year following PZP inoculations. PZP immunization produced progressively elevated anti-PZP antibodies in both species, which provided contraceptive protection. PZP immunization appears to be an effective form of contraception in both species. © 1995 Wiley-Liss, Inc.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits

Rabbits (Oryctolagus cuniculus) and two strains of mice (Mus musculus, one inbred and one outbred) were immunized against porcine zona pellucida (PZP) antigen. Alginate microspheres or cholera toxin B were used alone or in combination when mucosal immunization routes were used. Serum antibody responses and fertility were assessed. Neither rabbit or mouse groups immunized by mucosal routes generated significant antibody responses to PZP as compared to parenteral immunization (ANOVA, P > 0.05). The study shows that porcine zona pellucida is not an effective mucosal antigen in small mammals.     

卵母细胞透明带异常及其对助孕结局影响的研究进展

人类透明带(zona pellucida,ZP)是在卵泡发生过程中由卵母细胞和颗粒细胞共同分泌的由ZP1-ZP4四种糖蛋白分子组成的高度有序结构,它与卵母细胞的成熟、受精、胚胎发育及妊娠结局等预后紧密关联。许多生殖中心实验室发现有些患者的卵出现全部或部分的透明带异常,而且不同实验室发现的透明带异常类型各异。研究发现这些透明带异常与卵母细胞受精、胚胎发育及临床结局有一定的相关性。本文综述了关于透明带异常及其对卵母细胞受精、胚胎发育潜能和临床结局的影响的研究进展。     

Survey of zona pellucida antigens for immunocontraception of cats

The purpose of this study was to screen a panel of native zona pellucida (ZP) antigens isolated from five mammalian species for immunocontraceptive activity in the cat (Felis catus). Native soluble-isolated ZP (SIZP) was prepared from the ovaries of cows (bZP), cats (fZP), ferrets (feZP), dogs (cZP), and mink (mZP). Vaccines were constructed using SIZP from each of the above species encapsulated in liposomes suspended in saline and emulsified with Freund's complete adjuvant (SpayVac). Female cats were immunized once (n = 3 cats per group). Serum was collected for determination of antibody titers against SIZP and for binding of antibodies to feline ovaries. All cats responded to immunization by producing anti-SIZP antibodies. The most immunogenic SIZP in cats was from mink, followed by feZP, cZP, and fZP in descending order. Antibodies had low reactivity for fZP, and no reactivity against feline ovaries was detected by immunohistochemistry. A breeding trial was commenced 20 weeks after immunization. All cats became pregnant, averaging 4.1 +/- 0.7 viable kittens per litter. We have previously shown that porcine SIZP is not an effective antigen for immunocontraception of cats. In this study, SIZP from five other mammalian species were immunogenic in the cat, but ZP antibodies failed to bind to fZP in situ, and fertility was not impeded.     

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.     

Endocrine response in rabbits immunized with native versus deglycosylated porcine zona pellucida antigens

Previous studies evaluating porcine zona pellucida antigens for immunocontraceptive purposes have in some cases revealed altered ovarian function in association with antibody response. This study was undertaken in an attempt to identify zona immunogens that do not cause adverse endocrine effects. To this end, we investigated the effects of highly purified preparations of native and deglycosylated pig zona pellucida antigens on ovarian function and immune response in the rabbit. Thirty female rabbits were immunized, 5 per group, with 100 micrograms each of either 1) SIZP, solubilized isolated zonae pellucidae; 2) ZP3, a purified porcine zona preparation containing the two principle glycoproteins, ZP3 alpha and ZP3 beta, endo-beta-galactosidase-digested ZP3 glycoproteins (approximately 30% deglycosylated) termed 3) ZP3 alpha/EBGD and 4) ZP3 beta/EBGD; and chemically deglycosylated ZP3 alpha and ZP3 beta (greater than or equal to 92% deglycosylated), termed 5) ZP3 alpha/DG and 6) ZP3 beta/DG. Rabbits injected with saline (n = 2) or Freund's adjuvant alone (n = 3) served as controls. Serum LH, FSH, estradiol, and progesterone were measured at 5-day intervals during seven 20-day cycles of hCG-induced pseudopregnancy over 42 wk. Anti-ZP3 titers, determined by RIA, developed in all treatment groups and correlated directly with carbohydrate content. Animals immunized with SIZP, ZP3, and ZP3 beta/EBGD showed a significant elevation of LH and FSH and a significant decline of peak progesterone levels by the fourth pseudopregnancy cycle. In contrast, animals immunized with ZP3 alpha/EBGD, ZP3 alpha/DG, and ZP3 beta/DG showed no significant elevations of gonadotropins and continued to display cyclic progesterone secretion in response to hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.     

Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes

The effect of lectins with different saccharide specificity (ConA, LCA, DBA, WGA and PNA) on enzymatic digestion of the zona pellucida (ZP) of mouse oocytes was studied. All lectins tested, except PNA, induced ZP hardening with different degrees of efficiency. Moreover, extensive ZP digestion with mixed exoglycosidase prevented "spontaneous" ZP hardening. These observations suggest that changes of the carbohydrate moieties can be involved in the hardening of the zona pellucida of mouse oocytes.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.     

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa

IVF of porcine oocytes has been carried out in many laboratories. However, polyspermic fertilization is still a major issue to be solved. It is well known that besides the nucleus, oocyte organelles and the cytoplasm have to undergo a final maturation process before they become fully competent for fertilization. Until now, it is still uncertain whether the zona pellucida (ZP) must also undergo a maturation process and what impact the maturation status may have on sperm recognition and monospermic fertilization. Our data show that the ZP undergoes biochemical changes in the final maturation phase of the oocyte prior to fertilization. During zona maturation, the induction of the acrosome reaction in spermatozoa bound to the zona pellucida shows a different time pattern. Additionally, it was shown by 2D gel electrophoresis that after maturation, ZPA moved 0.8 pI units and ZPB/ZPC 1.3 pI units in the direction of the anode, indicating increased acidity. These preliminary studies indicate that the maturation processes of the oocyte involves biochemical and functional alterations in the zona pellucida. In addition, the morphology of the porcine ZP was investigated before and after maturation at the GVI and metaphase II stage as well as 1h after onset of IVF. No significant consistent structural changes were seen between immature oocytes and those matured in vitro for 48 h. However, at 24 h, the zona structures were more similar to those in in vivo matured oocytes. This phenomenon needs to be elucidated. So far, the only way to avoid polyspermic penetration is to reduce the number of spermatozoa per oocyte used for IVF. The amount depends on the treatment of the sperm and has to be set for each individual boar.     

N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes

The objective was to determine whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation of porcine oocytes, and whether this had a role in fertilization. In the first of three experiments, carbohydrate residues in the ZP of in vitro matured porcine oocytes were blocked with various lectins and the influence of such blocking on sperm-ZP interactions was studied. The second experiment used a lectin-binding assay to determine whether the number of GlcNAc residues in ZP was changed by N-glycosylation during in vitro maturation (IVM) of porcine oocytes. The last experiment determined the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM, on sperm-ZP interactions in porcine oocytes. The primary findings were that: 1) N-glycosylation of GlcNAc residues in porcine ZP occurred during the first 24 h of IVM; and 2) such glycosylation was indispensible for sperm-ZP interactions, e.g., number of sperm bound to ZP, acrosome-reacted sperm, sperm penetration rate, and level of polyspermy (P < 0.05). However, blocking N-glycosylation by tunicamycin treatment during IVM did not adversely influence the progression of oocytes to meiotic metaphase II and male pronucleus formation, indicating that this glycosylation was involved only in the initial stages of fertilization. We inferred that the increase in terminal GlcNAc residues in ZP glycoprotein through new N-glycosylation during the first 24 h of meiotic maturation played a critical role in porcine ZP acquiring the capacity to accept sperm.