The importance of trace minerals copper, manganese, selenium and zinc in bovine sperm-zona pellucida binding.

SummarySperm-zona pellucida (ZP) binding is a necessary event for successful fertilization. The aim of this study was to determine the effect of trace minerals such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) on bovine spermatozoa binding to ZP. Sperm viability, functional membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation (LPO) were also evaluated. For the present study, in vitro fertilization (IVF) medium was supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), all minerals (Cu+Mn+Se+Zn), or tested without supplement (Control). Considerably more sperm bound to ZP when Cu, Se or Zn were added to the IVF medium, but there were no difference compared with the Control, Mn and Cu+Mn+Se+Zn groups. After 1 h of incubation, viability was increased by the addition of Cu, Mn and Se with respect to the Control but, after 2 h, viability was higher only with the addition of Mn to IVF medium. Functional membrane integrity improved in sperm treated with Cu. Acrosome integrity was higher in sperm treated with Zn after 1 h of incubation. LPO was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn. The mean TACs of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were lower than in the Control. In conclusion, the results obtained in the present study determined that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to the ZP, highlighting the importance of these minerals in the fertilization process.

Effects of dietary curcumin supplementation on seminal quality indices and fertility rate in broiler breeder roosters.

1. Decreased semen quality is an underlying contributor to age-related subfertility in broiler breeder roosters. This study investigated the effects of dietary curcumin (derived from turmeric) supplementation as an antioxidant source on semen quality and fertility in broiler breeder roosters. 2. Twenty-eight Ross 308 roosters were randomly allotted to four groups with seven birds in each and were fed a standard diet supplemented with different levels of curcumin at 0 (C0), 10 (C10), 20 (C20) and 30 (C30) mg/bird per day from 48 through to 61 weeks of age. Body weight and semen quality traits were evaluated on a weekly basis and seminal concentrations of malondialdehyde (MDA) as a measure of antioxidation status were quantified at one-week intervals during the first 11 weeks of the trial (48-59 weeks of age). Semen samples from last 2 weeks (60 and 61 weeks of age) were used to artificially inseminate to assess the sperm-egg penetration (SP) in perivitelline membrane and fertility rates. 3. Except for body weight and ejaculate volume, other characteristics, including semen concentration, total sperm production, progressive motility and plasma membrane integrity were linearly improved by the increasing levels of curcumin supplementation (P < 0.01). However, dietary curcumin levels were linearly and quadratically associated with decreased seminal concentration of MDA (P < 0.01 and P < 0.03), percentage of abnormal sperm (P < 0.01 and P < 0.07) and increased plasma membrane functionality (P < 0.01 and P < 0.04), respectively. The SP holes in perivitelline membrane were increased in a linear and quadratic manner in response to increasing levels of curcumin (P < 0.01). Moreover, fertility rate was linearly improved (P < 0.01) as the dosage of curcumin increased, and resulted in 8, 12 and 14% improvements in the birds fed C10, C20 and C30, compared to C0, respectively. 4. In conclusion, the results showed that increasing levels of dietary supplementation of curcumin was associated with beneficial effects on semen quality indices and fertility rate in aged broiler breeder roosters.

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes' zona pellucida.

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 µM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.

Zinc: A Necessary Ion for Mammalian Sperm Fertilization Competency.

The importance of zinc for male fertility only emerged recently, being propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review the properties, biological roles and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss the recently discovered signaling pathways involving zinc in sperm maturation and fertilization. Emphasis is on the zinc-interacting sperm proteome and its involvement in the regulation of sperm structure and function, from spermatogenesis and epididymal sperm maturation to sperm interactions with the female reproductive tract, capacitation, fertilization, and embryo development. Merits of dietary zinc supplementation and zinc inclusion into semen processing media are considered with livestock artificial insemination (AI) and human assisted reproductive therapy (ART) in mind. Collectively, the currently available data underline the importance of zinc ions for male fertility, which could be harnessed to improve human reproductive health and reproductive efficiency in agriculturally important livestock species. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize semen processing procedures for human infertility therapy and livestock AI.

Influence of Glutamine Supplementation on Motility and Fertilization Success of Frozen-Thawed Persian Sturgeon (Acipenser persicus) Sperm.

Amino acids have an important biological role for the prevention of cell damage during cryopreservation. The aim of this study was to investigate the effects of glutamine on post-thaw sperm motility and fertilization success in the Persian sturgeon (Acipenser persicus). Sperm collected from six fish was cryopreserved in extenders containing different glutamine concentrations (2.5, 5 and 10 mm). Sperm samples diluted at the ratio of 1 : 1 using the extenders were subjected to cryopreservation. After dilution, the sperm suspensions were sucked into 250-µl straws; the straws were placed on the tray, frozen in nitrogen vapour and plunged into liquid nitrogen. Then, sperm were thawed in a water bath at 40°C for 5 s and used for analysis. Our results revealed that an increase in the concentration of glutamine caused a significant increase in the motility percentage, curvilinear velocity (VCL) and also fertilization success in the Persian sturgeon (p < 0.05). Comparing all concentrations of glutamine, the best concentration for sperm motility and fertilization rate was 10 mm. In addition, higher post-thaw motility percentage, VCL, and fertilization and hatching rates were obtained with the extender at the concentration of 10 mm (p < 0.05). The findings of this study showed that glutamine was of greater benefit to Persian sturgeon sperm motility during frozen-thawed process.

Plant fertilization: maximizing reproductive success.

Sperm competition does not occur in flowering plants as typically only a single pair of sperm cells is delivered for double fertilization. Two recent reports show that plants are capable of avoiding reproductive failure when defective sperm cells are released.

Adding insult to injury: effects of xenobiotic-induced preantral ovotoxicity on ovarian development and oocyte fusibility.

Mammalian females are born with a finite number of nonrenewing primordial follicles, the majority of which remain in a quiescent state for many years. Because of their nonrenewing nature, these "resting" oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes. In this study, we characterized the mechanisms of ovotoxicity for three ovotoxic agents, 4-vinylcyclohexene diepoxide (VCD), methoxychlor (MXC), and menadione (MEN), all of which target immature follicles. Microarray analysis of neonatal mouse ovaries exposed to these xenobiotics in vitro revealed a more than twofold significant difference in transcript expression (p < 0.05) for a number of genes associated with apoptotic cell death and primordial follicle activation. Histomorphological and immunohistological analysis supported the microarray data, showing signs of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Sperm-oocyte fusion assays on oocytes obtained from adult Swiss mice treated neonatally revealed severely reduced sperm-egg binding and fusion in a dose-dependent manner for all the xenobiotic treatments. Additionally, lipid peroxidation analysis on xenobiotic-cultured oocytes indicated a dose-dependent increase in oocyte lipid peroxidation for all three xenobiotics in vitro. Our results reveal a novel mechanism of preantral ovotoxicity involving the homeostatic recruitment of primordial follicles to maintain the pool of developing follicles destroyed by xenobiotic exposure and to our knowledge provide the first documented evidence of short-term, low- and high-dose (VCD 40-80 mg/kg/day, MXC 50-100 mg/kg/day, MEN 7.5-15 mg/kg/day) neonatal exposure to xenobiotics causing long-term reactive oxygen species-induced oocyte dysfunction.

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations.

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction.

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2). Further experiments analyzing both the ability of other CRISP proteins to bind to the rat egg and the amino acid sequence of their S2 regions show that the amino acid sequence of the S2 is needed for CRISP1 to interact with the egg. CRISP1 appears to be involved in the first step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. The observation that sperm testicular CRISP2 is also able to bind to the egg surface suggests a role for this protein in gamete fusion. Subsequent experiments confirmed the participation of CRISP2 in this step of fertilization and revealed that CRISP1 and CRISP2 interact with common egg surface binding sites. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. These observations contribute to a better understanding of the molecular mechanisms underlying mammalian fertilization.

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs.

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).

Phospholipase Czeta, the trigger of egg activation in mammals, is present in a non-mammalian species.

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCzeta. Since the discovery of PLCzeta, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCzeta is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCzeta orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCzeta cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCzeta cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCzeta. Our findings suggest that PLCzeta may have a universal role in triggering egg activation in vertebrates.

A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.

Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.

Studies on the participation of epididymal sperm protein DE/CRISP-1 in egg activation.

Protein DE (32 kDa) associates with sperm during epididymal maturation and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. In the present work we investigated the participation of DE in two mechanisms probably involved in egg activation: the ability of DE to trigger activation by its interaction with the binding sites on the egg surface (receptor model) and its ability to regulate intracellular calcium channels (sperm factor model). The incubation of eggs with DE did not promote activation parameters such as calcium oscillations or meiosis resumption. Secondly, microinjection of DE into eggs was ineffective in either eliciting calcium release or modifying oscillations induced by an activating sperm extract. Together, these results argue against the participation of DE in egg activation, restricting the activity of this protein and its egg binding sites to the sperm-egg fusion process.

Oviduct-specific glycoprotein modulates sperm-zona binding and improves efficiency of porcine fertilization in vitro.

Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa.

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes.

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.

Primate recombinant zona pellucida proteins expressed in Escherichia coli bind to spermatozoa.

To delineate the role of individual zona pellucida (ZP) glycoproteins during sperm-oocyte interaction, bonnet monkey (bm; Macaca radiata) ZPA (bmZPA), ZPB (bmZPB), and ZPC (bmZPC) have been cloned without native signal sequence and transmembrane-like domain, and expressed in Escherichia coli. Recombinant proteins have been purified from the inclusion bodies in presence of low concentration of chaotropic agent (2 M urea) and high pH (pH 12), and subsequently refolded in presence of oxidized and reduced glutathione. Binding of the recombinant refolded zona proteins to bonnet monkey spermatozoa in an indirect immunofluorescence assay revealed that recombinant bmZPC binds to the head region of the capacitated spermatozoa but does not bind to the acrosome reacted spermatozoa. Recombinant bmZPB binds to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. After induction of acrosome reaction by calcium ionophore A23187, the binding of recombinant bmZPB shifts to the equator, post-acrosome and midpiece of the spermatozoa. bmZPA binds to the principal segment of capacitated spermatozoa but the binding shifts to the equatorial segment, tip of the inner acrosomal membrane and midpiece in acrosome reacted spermatozoa. These studies suggest that polypeptide backbone is sufficient for the binding of ZPA, ZPB and ZPC to spermatozoa in non-human primates. Further studies with recombinant glycosylated zona proteins will help in delineating the role of carbohydrate moieties for higher affinity binding of the ligand to spermatozoa and subsequent signal transduction pathways.

Nuclear reconstitution of plant (Orychophragmus violaceus) demembranated sperm in cell-free extracts from animal (Xenopus laevis) eggs.

Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix--lamina showed the reconstituted nucleus to be filled with a dense network.