Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

Patrat, Catherine; Auer, J.; Fauque, Patricia; Leandri, Roger L; Jouannet, Pierre; Serres, Catherine

doi:10.1186/1471-213x-6-59

Cited by 20 publications

(6 citation statements)

References 52 publications

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“…4G). After 1 hour of incubation, human sperm bound robustly to the native zona matrix and were quantified after fixation and confocal microscopy (avg ± SEM; donor A, 55.2 ± 7.8 sperm per zona; donor B, 43.5 ± 4.4 sperm per zona; donor C, 105.6 ± 15.0 sperm per zona), with results that were comparable to earlier reports (18, 19). From these observations, we conclude that human sperm selection with huZP2 peptide beads is a rapid (35 min) and effective procedure to identify subpopulations of sperm competent for zona pellucida binding and penetration.…”

Section: Resultssupporting

confidence: 83%

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

et al. 2016

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Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2 and huZP2 39-154 peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2Rescue zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2 Rescue eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice.

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Section: Resultssupporting

confidence: 83%

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

et al. 2016

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“…The furin cleavage site is identified at position (350-353), and the propeptide domain at position (351-424). When cleavage takes place the ZP3 undergoes a conformational change that inhibits any further sperm binding to the coat thus preventing polyspermy [ 35 ]. Of the 48 positively selected sites identified, 10 fall within the propeptide domain, with an additional 12 occurring close to the vicinity of the furin cleavage and sperm binding sites, thus suggesting that there is a pressure to improve binding and prevent polyspermy.…”

Section: Resultsmentioning

confidence: 99%

Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

et al. 2010

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BackgroundReproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia.ResultsWe show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality.ConclusionOur in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene.

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“…In contrast to T-type VOCCs inhibitors, L-type VOCCs inhibitors (Nifedipine and Verapamil) failed to inhibit SIZP mediated acrosome reaction. Patrat et al ., [ 26 ] has shown that solubilized zona prepared from unfertilized and fertilized human eggs induces acrosome reaction and increase in [Ca 2+ ] i is mediated by T-type VOCC. However, the ability of SIZP prepared from fertilized eggs to induce acrosome reaction needs further investigation.…”

Section: Discussionmentioning

confidence: 99%

“…The concentrations of the various inhibitors employed in these experiments were based on previously published studies [6,23-26] and inhibitors were procured from Sigma-Aldrich Inc. In addition, effect of prior treatment of capacitated human sperm with Pimozide and Verapamil on the levels of [Ca 2+ ] i in response to SIZP were also determined by fluorimetric assay as described above.…”

Section: Methodsmentioning

confidence: 99%

“…To delineate the involvement of different type of Voltage Operated Calcium Channels (VOCCs) during SIZP mediated induction of acrosome reaction, 1 × 10 6 capacitated sperm were pre-treated with Pimozide (10 and 20 μM) or Mibefradil (5 and 10 μM) as T-Type Ca 2+ channel blocker (CCB); Verapamil (10 and 20 μM) or Nifedipine (10 and 20 μM) as L-Type CCB; for 10 min at 37°C with 5% CO 2 in humidified air prior to the addition of SIZP. The concentrations of the various inhibitors employed in these experiments were based on previously published studies [ 6 , 23 - 26 ] and inhibitors were procured from Sigma-Aldrich Inc. In addition, effect of prior treatment of capacitated human sperm with Pimozide and Verapamil on the levels of [Ca 2+ ] i in response to SIZP were also determined by fluorimetric assay as described above.…”

Section: Methodsmentioning

confidence: 99%

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Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

Bhandari

Bansal

Talwar

et al. 2010

Reprod Biol Endocrinol

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BackgroundHuman egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction.MethodsHeat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP.ResultsSIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction.ConclusionsResults described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

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Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

Cited by 20 publications

References 52 publications

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception

Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

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