Julieta Antonella Maldera

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1(-/-) animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1(-/-) mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1(-/-) sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1(+/+) and Crisp1(-/-) sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1(-/-) sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.

A Novel Function for CRISP1 in Rodent Fertilization: Involvement in Sperm-Zona Pellucida Interaction

Abstract Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. In this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. In agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.

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.

Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn2+ present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn2+, and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn2+, but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn2+ concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn2+ exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn2+ supported a direct interaction between CRISP1 and Zn2+. Incubation of either cauda epididymal fluid or purified CRISP1 with Zn2+, followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn2+ complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation. Epididymal CRISP1 weakly associates with rat sperm by a zinc-mediated mechanism that involves the formation of high-molecular-weight complexes between the protein and the cation.

Rat Caltrin Protein Modulates the Acrosomal Exocytosis During Sperm Capacitation

Abstract Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.

Influence of the genetic background on the reproductive phenotype of mice lacking Cysteine-Rich Secretory Protein 1 (CRISP1)

Abstract Epididymal sperm protein CRISP1 has the ability to both regulate murine CatSper, a key sperm calcium channel, and interact with egg-binding sites during fertilization. In spite of its relevance for sperm function, Crisp1-/- mice are fertile. Considering that phenotypes can be influenced by the genetic background, in the present work mice from the original mixed Crisp1-/- colony (129/SvEv*C57BL/6) were backcrossed onto the C57BL/6 strain for subsequent analysis of their reproductive phenotype. Whereas fertility and fertilization rates of C57BL/6 Crisp1-/- males did not differ from those reported for mice from the mixed background, several sperm functional parameters were clearly affected by the genetic background. Crisp1-/- sperm from the homogeneous background exhibited defects in both the progesterone-induced acrosome reaction and motility not observed in the mixed background, and normal rather than reduced protein tyrosine phosphorylation. Additional studies revealed a significant decrease in sperm hyperactivation as well as in cAMP and protein kinase A (PKA) substrate phosphorylation levels in sperm from both colonies. The finding that exposure of mutant sperm to a cAMP analog and phosphodiesterase inhibitor overcame the sperm functional defects observed in each colony indicated that a common cAMPPKA signaling defect led to different phenotypes depending on the genetic background. Altogether, our observations indicate that the phenotype of CRISP1 null males is modulated by the genetic context and reveal new roles for the protein in both the functional events and signaling pathways associated to capacitation. Summary Sentence Analysis of the reproductive phenotype of mice lacking CRISP1 shows the influence of the genetic background on different sperm functional parameters and reveals the involvement of CRISP1 in the cAMP-PKA signaling cascade leading to capacitation.