Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls

There is still debate in the literature on whether or not endurance athletes tend to have low iron stores. In this article, we propose that endurance athletes really are at risk of becoming iron deficient due to an imbalance between absorption of dietary iron and exercise-induced iron loss. The purpose of this article is to present a critical review of the literature on iron supplementation in sport. The effect of iron deficiency on performance, its diagnosis and suggestions for treatment are also discussed. Studies of the nutritional status of athletes in various disciplines have shown that male, but not female, athletes clearly achieve the recommended dietary intake of iron (10 to 15 mg/day). This reflects the situation in the general population, with menstruating women being the main risk group for mild iron deficiency, even in developed countries. Whereas the benefit of iron supplementation in athletes with iron deficiency anaemia is well established, this is apparently not true for non-anaemic athletes who have exhausted iron stores alone (prelatent iron deficiency); most of the studies in the literature show no significant changes due to supplementation in the physical capacity of athletes with prelatent iron deficiency. However, the treatment protocols used in some of these studies do not meet the general recommendations for the optimal clinical management of iron deficiency, that is, with respect to adequate daily dosage, mode of administration and treatment period. For future studies, we recommend a prolonged treatment period (> or = 3 months) with standardised conditions of administration (use of a pharmaceutical iron preparation with known high bioavailability and a dosage of ferrous (Fe++) iron 100 mg/day, taken on an empty stomach). Currently, decisions regarding iron supplementation are best made on the basis of taking care of individual athletes. We believe that there are sufficient arguments to support controlled iron supplementation in all athletes with low serum ferritin levels. Firstly, the development of iron deficiency is prevented. Secondly, the nonspecific upregulation of intestinal metal ion absorption is reverted to normal, thus limiting the hyperabsorption of potentially toxic lead and cadmium even in individuals with mild iron deficiency.     

Pentoxifylline improves sperm binding to the zona pellucida in the hemizona assay

OBJECTIVE: To evaluate the effect of pentoxifylline on sperm binding capacity to zona pellucida (ZP) using the hemizona assay (HZA). DESIGN: The fertility potential of 82 men was evaluated by routine semen analysis. Each ejaculate was incubated with or without pentoxifylline (3 mM) in Ham's F-10 medium (Flow Laboratories, Irvine, Scotland) before the HZA. The effect of the pentoxifylline treatment on sperm-binding capacity to ZP was assessed by the hemizona index. RESULTS: The mean hemizona indexes with medium or pentoxifylline treatment were 23% +/- 2.1% (mean +/- SE) and 41% +/- 3.4%, respectively. Taking into consideration a significant change of the hemizona index on rising above the intra-assay coefficient of variation (+/- 8%) after pentoxifylline treatment, 73.1% of specimens improved, 19.5% deteriorated, and 7.4% remained unchanged. Using a threshold hemizona index of 23% as a discriminator between fertile and infertile specimens, 43.5% of the "pentoxifylline-improved" samples ascended to the fertile zone (> 23%). No correlations were found between sperm variables in the raw semen and the effect of pentoxifylline on sperm binding capacity. CONCLUSION: Pentoxifylline may improve the binding capacity of human spermatozoa. However, this effect is confined to a selected group of patients and cannot be predicted by the basic sperm variables. Thus, to avoid uncertain or damaging effects of pentoxifylline while preparing sperm suspension for assisted reproductive techniques, it is recommended that its effect be tested by the HZA system before its use.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.     

Gamete recognition in mammals: sperm and zona pellucida interactions

Association of sperm with the acellular protective envelope of the oocyte, the zona pellucida, and their penetration is a determinant step in fertilization process. It is at this stage that species barriers take place to prevent cross fertilizations. This association is dependent upon binding of ZP3 oligosaccharides to specific sperm receptors. Their activation triggers the acrosome reaction via transduction pathways and release of proteolytic enzymes that dissociate the zona pellucida network. Then, secondary binding to zona pellucida components allows sperm to penetrate and cross the zona pellucida barrier. Several sperm surface proteins are putative receptors for zona pellucida partners. Repercussion of these studies on human fertility are discussed.     

Human zona pellucida glycoproteins: characterization using antibodies against recombinant non-human primate ZP1, ZP2 and ZP3

Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.     

The paradoxical effects of pentoxifylline on the binding of spermatozoa to the human zona pellucida

In the presence of pentoxifylline, human spermatozoa are induced to increase certain motion characteristics; however, the role of this drug in fertilization remains equivocal. In this study, the influence of pentoxifylline on one aspect of fertilization, that is sperm-zona binding, has been examined. Results from a fluorescence label competitive zona binding (CZB) test showed that spermatozoa exposed to a pentoxifylline challenge of between 0.1 and 5 mM, which was curtailed after 1 h by washing, had a decreased (P < 0.01) ability to bind to intact zona compared with control spermatozoa. The washing procedures also removed (decrease P < 0.01 compared with peak values) some of the enhanced motion characteristics induced by pentoxifylline. These results were in contrast with those obtained using experimental conditions that maintained an increased curvilinear velocity (VCL) and lateral head displacement (ALH) (increase P < 0.001 above baseline controls) in the continued presence of pentoxifylline. Using a hemizona binding (HZB) assay, 3 mM pentoxifylline increased (P < 0.001) sperm-zona binding almost 20% above zona binding with unexposed control spermatozoa. It was concluded that, in the presence of pentoxifylline, there is increased sperm binding to the zona pellucida; however, if the drug is removed by washing, the sperm binding to the zona is decreased in concert with the removal of the enhanced motion characteristics. The application of zona solubilization by acidic conditions in a microchamber enabled the precise determination of sperm numbers in both of the sperm-zona binding assays, and the results demonstrated that a wide variation in sperm numbers was observed in each test, with 63-580 spermatozoa bound in the CZB assay and 56-1340 spermatozoa bound on a hemizona.     

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).     

The molecular genetics of the zona pellucida: mouse mutations and infertility

The zona pellucida is an extracellular matrix surrounding growing oocytes, ovulated eggs and the preimplantation embryo. After mediating the relatively species-specific events of fertilization, the zona pellucida provides a post-fertilization block to polyspermy and protects the growing embryo as it passes down the oviduct. The genes that encode the three zona pellucida proteins (ZP1, ZP2, ZP3) have been characterized in mouse and human. The ability to genetically manipulate the zona pellucida genes in mouse models has enhanced our knowledge of zona pellucida structure and function in vivo and may translate into a better understanding of human fertility.     

The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells

Oocyte development within avian ovarian follicles is an intricate process involving yolk deposition and the formation of extraoocytic matrices. Of these, the perivitelline membrane (pvm) not only plays a role in sperm binding but also provides mechanical support for the large oocyte's journey through the oviduct after ovulation. To date we have focused on the mechanisms for uptake of yolk precursors into oocytes of the chicken; now we extend our studies to a detailed analysis of the pvm. In the course of characterization of its major components, we obtained partial protein sequences; comparison with the GenBank database revealed that one of the pvm proteins is the homologue of mammalian zona pellucida glycoprotein 3 (ZP3), a key component in sperm binding. Following a nomenclature based on gene structure, the protein is referred to as chicken ZPC (chZPC). The chicken protein (444 residues) and murine ZP3 (424 residues) are highly conserved, with 41% of the amino acids identical. As shown by Northern blot analysis, the avian ZPC gene is expressed exclusively in the granulosa cells surrounding the oocyte, in contrast to murine ZP3, which is synthesized by the oocyte. Upon reaching a size larger than 1.5 mm in diameter, follicles accumulate chZPC in highly polarized fashion, i.e., in the space intercalated between the oocyte and the granulosa cells, as revealed by immunohistochemistry of follicle sections. ChZPC synthesis and secretion by granulosa cells was demonstrated directly by metabolic labeling and immunoprecipitation from the culture medium of granulosa cell sheets isolated ex vivo from follicles. Immunoblot analysis and glycosidase treatment of chZPC from preovulatory and freshly ovulated oocytes, as well as laid eggs, revealed that the primary product undergoes a two-step decrease in size from follicle to laid egg that is unlikely to be due to modification of the carbohydrate moiety.     

Expression of mannose-ligand receptors on human spermatozoa: effect of lecithin and association with sperm binding to the zona pellucida

OBJECTIVE: To evaluate the change in the expression of mannose-ligand receptors and sperm binding capacity after the incubation of sperm cells with lecithin liposomes. DESIGN: A randomized, blinded-controlled experiment. SETTING: Andrology laboratory at the Lis Maternity Hospital. PATIENT(S): Fifteen fertile sperm donors and 10 subfertile men. INTERVENTION(S): Incubation of sperm samples with either control medium or 1 mg/mL of liposomal lecithin for 2 hours. MAIN OUTCOME MEASURE(S): Expression of mannose-ligand receptors as evaluated by mannosylated bovine serum albumin-fluorescein isothiocyanate and sperm binding to the zona pellucida as evaluated by the hemizona assay. RESULT(S): The mean +/- SE percentages of spermatozoa with patterns I, II, and III were 86% +/- 4.8%, 11% +/- 3.4%, and 3% +/- 1.6%, respectively, after treatment with control medium and 71% +/- 5.7%, 22% +/- 3.5%, and 7% +/- 2.5%, respectively, after treatment with lecithin. The same effect of lecithin was observed in the 10 sperm samples from subfertile men. The mean +/- SE numbers of sperm that bound to hemizonae after treatment with control medium or lecithin were 116 +/- 32.4 and 176 +/- 29.6, respectively. Statistically significant correlations were observed between the shift in patterns II and III and the enhancement of sperm binding after lecithin treatment (r = 0.44 and 0.6, respectively). CONCLUSION(S): Lecithin shifts the expression of mannose-ligand receptors to the capacitated and acrosoine-reacted patterns and enhances the binding capacity of the sperm cells.     

Species-specific binding of sperm proteins to the extracellular matrix (zona pellucida) of the egg

The zona pellucida is an extracellular matrix surrounding the mammalian egg where species-specific gamete recognition and signaling occur. Pig zona pellucida were isolated in large amounts and used as an affinity matrix for detergent-solubilized, biotinylated membrane proteins of pig spermatozoa. On non-reducing SDS-polyacrylamide gel electrophoresis, specifically bound sperm proteins migrated with M(r) 170,000, 150,000, 130,000, 56,000, and 50,000 (p50). Disulfide bond reduction separated each of the M(r) 130,000-170,000 proteins into M(r) 105,000 (p105) and M(r) 45,000 (p45) subunits, indicating that these high M(r) proteins are related. Based on two-dimensional electrophoresis, the M(r) 56,000 band was composed of three to four proteins that migrated with M(r) 56,000-62,000 (p56-62) in the second (reducing) dimension. p50 bound to heterologous zona pellucida (murine, bovine) and to Xenopus laevis oocyte envelopes, demonstrating a lack of species specificity to its binding and was identified as proacrosin/acrosin based on amino acid sequences of two tryptic peptides and its interaction with monospecific antibodies to proacrosin. In contrast, p105/p45 and one or more of the p56-62 proteins bound to pig zona pellucida but not to the egg extracellular matrices of the other species; these proteins therefore exhibited the species-specific binding to the zona pellucida expected for molecules involved in specific gamete adhesion. Amino acid sequences of nine tryptic peptides derived from p105/p45 did not match peptide sequences in existing databases, establishing it as a unique protein. These (p105/p45 and at least one p56-62 protein) are the first sperm membrane proteins to be identified that bind in a species-specific manner to the egg extracellular matrix.     

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.     

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.     

Systemic lupus erythematosus in women and serum interleukin-6, sperm and zona pellucida antibodies

Systemic lupus erthematosus (SLE) has been very often associated with complication in reproduction. 29 SLE women divided into two groups A (10 patients aged 18-36 years) and B (19 patients aged 42-67 years) were queried with regard to general, obstetric and SLE history by the use of a questionnaire. Serum of each patient was tested for interleukin-6 (by ELISA), sperm antibodies (by mixed antiglobulin reaction and tray agglutination test) and zona pellucida antibodies (passive haemagglutination test and ELISA methods). Neuropathic, cutaneous and nephrotic symptoms prevailed in the SLE women. The group of younger women showed significant problems in fertility (only one woman became a happy mother) while 15 women in the elderly group were successfully pregnant before SLE diagnosis. Low serum II-6 levels were detectable only in 3 cases as a possible consequence of corticoid treatment. Levels of zona pellucida antibodies were higher in the elderly group B, levels of sperm antibodies were higher in contrast to younger SLE women (group A). The laboratory findings may be related to the severity of autoimmune disease and to menopause onset (group B) or good sexual activity (group A).     

Polarized light microscopy and digital image processing identify a multilaminar structure of the hamster zona pellucida

The zona pellucida (zona) is a glycoprotein coat that envelopes the oocyte and embryo, binds sperm during fertilization and facilitates transfer of the embryo through the Fallopian tube. Before implantation can occur, the blastocyst must hatch from the zona. Several lines of evidence suggest that the zona is multilaminar. We hypothesized that the multilaminar structure of the zona filaments could be imaged non-destructively with the polarized light microscope. A recent modification of the polarized light microscope (pol-scope), which combines innovations in polarization optics with novel image processing software, allows measurement of birefringence at all points of the image. Hamster metaphase II oocytes were placed on glass coverslips which replaced the bottom of culture dishes, imaged under differential interference contrast (DIC) and pol-scope optics, then digitized and processed to measure birefringence magnitude and orientation. The pol-scope revealed the zona to be divided into outer and inner layers separated by a zone of low retardance. This finding is consistent with filaments in the outer layer oriented tangentially and in the inner layer oriented radially. The multilaminar structure of the mammalian zona suggested by differential lectin binding and by scanning electron microscopy could be imaged non-destructively with the pol-scope. Because the pol-scope provides a non-destructive method to identify macro-molecular organization of the zona, it may prove useful in developmental studies of hatching and to direct resection of the zona.     

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.     

Compromised sperm protein phosphorylation after capacitation, swim-up, and zona pellucida exposure in teratospermic domestic cats

BACKGROUND: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation). Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection. This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down-regulate the action of host complement. RESULTS: Transgenesis with a yeast artificial chromosome (YAC) was used to produce transgenic mice with the complete genomic gene of the human complement regulator membrane cofactor protein (MCP). Transgenic mice were obtained that exhibit full regulation of MCP as normally observed in humans. Hearts from these mice were shown to be significantly protected from HAR caused by human serum in an in vivo experimental procedure. CONCLUSIONS: We conclude that MCP can protect discordant xenografts from HAR caused by human serum and that transgenic mice can be used effectively as in vivo models for the study of the role of human complement regulatory molecules in xenotransplantation.     

Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.     

Timing of zona pellucida formation in the tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula)

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. The timetable of ZP formation was examined in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula) using conventional histological methods, immunofluorescence and electron microscopy. Ovaries from tammar wallaby pouch young less than 80 days of age contained only primordial follicles with a single layer of flattened granulosa cells. There was no evidence of ZP formation until 98 days, when a small number of eggs surrounded by a single layer of cuboidal granulosa cells had a ZP detectable by periodic-acid-schiff staining and rabbit anti-pig ZP polyclonal antibody labelling. Possum ovaries at 108 and 114 days also contained a small number of eggs with a ZP and a single layer of cuboidal granulosa cells. The antibody also labelled the peripheral cytoplasm of oocytes at this stage and, occasionally, the granulosa cells. Antral follicles were first detected at 144 days in the wallaby and 125 days in the possum, and always contained an egg surrounded by a ZP. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies contained a range of follicle types from primordial through early antrum formation. Electron microscopy confirmed observations made at the light microscope level. The ZP was first detectable in small primary follicles with a single layer of cuboidal granulosa cells in areas where microvilli had begun to form on the egg plasma membrane. Immunogold labelling indicated the egg cytoplasm as the origin of the ZP proteins. The ZP completely filled the space between the egg and the adjacent granulosa cells in preantral follicles, so that there was no perivitelline space.     

Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization

The physiological function of mammalian sperm acrosin has long been believed to be involved in the limited proteolysis of the oocyte zona pellucida, thus enabling the sperm to penetrate this extracellular matrix and to gain access to the oocyte plasma membrane. Here we show that male mice homozygous for a targeted mutation in the mouse acrosin gene are still fertile in spite of the complete absence of acrosin protease activity in the sperm. In vitro fertilization assays verified that sperm from the homozygous mutant mice penetrate the zona pellucida and effect fertilization. Therefore, acrosin is not essential for both sperm penetration of the zona pellucida and fertilization.