Fertilization in mammals is a complex process involving a series of coordinated steps in which spermatozoa need to penetrate the coats that surround the ovulated egg (i.e., cumulus oophorus and zona pellucida) to then fuse with the egg plasma membrane. This article will describe the unique features of the eutherian mammalian gametes as well as the strategies and molecular mechanisms involved in each of the stages of the fertilization process.

Numerous reports showed that the epididymis plays key roles in the acquisition of sperm fertilizing ability but its contribution to embryo development remains less understood. Female mice mated with males with simultaneous mutations in Crisp1 and Crisp3 genes exhibited normal in vivo fertilization but impaired embryo development. In this work, we found that this phenotype was not due to delayed fertilization, and it was observed in eggs fertilized by epididymal sperm either in vivo or in vitro . Of note, eggs fertilized in vitro by mutant sperm displayed impaired meiotic resumption unrelated to Ca 2+ oscillations defects during egg activation, supporting potential sperm DNA defects. Interestingly, cauda but not caput epididymal mutant sperm exhibited increased DNA fragmentation, revealing that DNA integrity defects appear during epididymal transit. Moreover, exposing control sperm to mutant epididymal fluid or to Ca 2+ -supplemented control fluid significantly increased DNA fragmentation. This, together with the higher intracellular Ca 2+ levels detected in mutant sperm, supports a dysregulation in Ca 2+ homeostasis within the epididymis and sperm as the main factor responsible for embryo development failure. These findings highlight the contribution of the epididymis beyond fertilization and identify CRISP1 and CRISP3 as novel factors essential for sperm DNA integrity and early embryo development.

In mammals, sperm that leave the testes are nonfunctional and require a complex post-testicular maturation process to acquire their ability to recognize and fertilize the egg. The crucial maturation changes that provide sperm their fertilizing capability occur while passing through the epididymis. Due to the widespread use of assisted reproductive technologies to address male infertility, there has been a significant decrease in research focusing on the mechanisms underlying the maturation process over the past decades. Considering that up to 40% of male infertility is idiopathic and could be reflecting sperm maturation defects, the study of post-testicular sperm maturation will clearly contribute to a better understanding of the causes of male infertility and to the development of both new approaches to maturing sperm in vitro and safer male contraceptive methods. Based on this, the present review focuses on the physiopathology of the epididymis as well as on current approaches under investigation to improve research in sperm maturation and as potential therapeutic options for male infertility.

Mammalian fertilization is a complex process involving a series of successive sperm-egg interaction steps mediated by different molecules and mechanisms. Studies carried out during the past 30 years, using a group of proteins named CRISP (Cysteine-RIch Secretory Proteins), have significantly contributed to elucidating the molecular mechanisms underlying mammalian gamete interaction. The CRISP family is composed of four members (i.e., CRISP1-4) in mammals, mainly expressed in the male tract, present in spermatozoa and exhibiting Ca 2+ channel regulatory abilities. Biochemical, molecular and genetic approaches show that each CRISP protein participates in more than one stage of gamete interaction (i.e., cumulus penetration, sperm-ZP binding, ZP penetration, gamete fusion) by either ligand-receptor interactions or the regulation of several capacitation-associated events (i.e., protein tyrosine phosphorylation, acrosome reaction, hyperactivation, etc.) likely through their ability to regulate different sperm ion channels. Moreover, deletion of different numbers and combination of Crisp genes leading to the generation of single, double, triple and quadruple knockout mice showed that CRISP proteins are essential for male fertility and are involved not only in gamete interaction but also in previous and subsequent steps such as sperm transport within the female tract and early embryo development. Collectively, these observations reveal that CRISP have evolved to perform redundant as well as specialized functions and are organized in functional modules within the family that work through independent pathways and contribute distinctly to fertility success. Redundancy and compensation mechanisms within protein families are particularly important for spermatozoa which are transcriptionally and translationally inactive cells carrying numerous protein families, emphasizing the importance of generating multiple knockout models to unmask the true functional relevance of family proteins. Considering the high sequence and functional homology between rodent and human CRISP proteins, these observations will contribute to a better understanding and diagnosis of human infertility as well as the development of new contraceptive options.

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca(2+)channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multipleCrispgene mutants by CRISPR/Cas9 technology. Whereas targeting ofCrisp1andCrisp3yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertileCrisp2(-/-).Crisp4(-/-)mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca(2+)dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

Fil: Carvajal, Guillermo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Biologia y Medicina Experimental. Fundacion de Instituto de Biologia y Medicina Experimental. Instituto de Biologia y Medicina Experimental; Argentina

BACKGROUND:The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process.OBJECTIVES:In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1-4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility.MATERIALS AND METHODS:We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team.RESULTS:Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects.DISCUSSION AND CONCLUSION:Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology.

The Caenorhabditis elegans spe-9 class genes, which show specific or predominant expression in the male germline, are indispensable for fertilization [1, 2]. However, due to the rapid evolution of genes involved in reproduction, we do not currently know if there are spe-9 class genes in mammals that play similar roles during fertilization to those found in C. elegans. In mice, the Izumo1 gene encodes a sperm-specific transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain that is absolutely required for gamete fusion [3, 4]. In this study, we hypothesized that C. elegans has a new member of the spe-9 class genes coding for an IZUMO1-like protein. We screened C. elegans microarray data [5, 6] to identify male germline-enriched genes that encode membrane proteins with Ig-like domains. A deletion (tm3715) in one such gene (F28D1.8) caused hermaphrodites to show a male germline-dependent self-sterility, so we have named it spe-45. Mutant spe-45 worms seemed to normally undergo spermatogenesis (spermatid production by meiosis) and spermiogenesis (spermatid activation into actively motile spermatozoa). spe-45 mutant spermatozoa, however, could not complete gamete fusion, which is a characteristic of all spe-9 class mutants [1, 2]. Moreover, spe-45 self-sterile worms were rescued by a transgene expressing chimeric SPE-45 protein in which its Ig-like domain was replaced by the Ig-like domain from mouse IZUMO1. Hence, C. elegans SPE-45 and mouse IZUMO1 appear to have retained a common function(s) that is required during fertilization.

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 cAMP-PKA 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.

Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology.

The acrosome reaction (AR) is a universal requisite for sperm-egg fusion. However, whereas through the animal kingdom fusion of spermatozoa with the egg plasma membrane occurs via the inner acrosomal membrane exposed after the AR, in eutherian mammals, gamete fusion takes place through a specialized region of the acrosome known as the equatorial segment (ES) which becomes fusogenic only after the AR is completed. This chapter focuses on the different molecular mechanisms involved in the acquisition of the fusogenicity of the ES after the AR. We provide an update of the knowledge about the proteins proposed to have a role in this process either by modifying cytoskeletal and/or membrane molecules or by relocalizing to the ES after the AR to subsequently participate in gamete fusion.

Lectin-glycan recognition systems play central roles in many physiologic and pathologic processes. We identified a role for galectin-1 (Gal-1), a highly conserved glycan-binding protein, in the control of sperm function. We found that Gal-1 is expressed in the epididymis and associates with sperm during epididymal maturation. Exposure of sperm to Gal-1 resulted in glycan-dependent modulation of the acrosome reaction (AR), a key event in the fertilization process. Gal-1-deficient (Lgals1(-/-)) mice revealed the essential contribution of this lectin for full sperm fertilizing ability both in vitro and in vivo. Mechanistically, Lgals1(-/-) sperm exhibited defects in their ability to develop hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, Lgals1(-/-) sperm showed a decreased ability to control cell volume and to undergo progesterone-induced AR, phenotypes that were rescued by exposure of the cells to recombinant Gal-1. Interestingly, the AR defect was associated with a deficiency in sperm membrane potential hyperpolarization. Our study highlights the relevance of the Gal-1-glycan axis in sperm function with critical implications in mammalian reproductive biology.

Ca(2+)-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca(2+) channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus-oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca(2+) channel involved in hyperactivation and essential for fertility. Given the critical role of Ca(2+) for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.

Alopecia areata (AA), a cell mediated autoimmune disease, is the second most common form of hair loss in humans. While the autoimmune disease is responsible for the underlying pathogenesis, the alopecia phenotype is ultimately due to hair shaft fragility and breakage associated with structural deficits. Quantitative trait genetic analyses using the C3H/HeJ mouse AA model identified cysteine-rich secretory protein 1 (Crisp1), a hair shaft structural protein, as a candidate gene within the major AA locus. Crisp1 transcripts in the skin at various times during disease development were barely detectable. In situ hybridization identified Crisp1 expression within the medulla of hair shafts from clinically normal strains of mice but not C3H/HeJ mice with AA. Follow-up work with 5-day-old C3H/HeJ mice with normal hair also had essentially no expression of Crisp1. Other non-inflammatory based follicular dystrophy mouse models with similar hair shaft abnormalities also have little or no Crisp1 expression. Shotgun proteomics, used to determine strain difference in hair proteins, confirmed that there was very little CRISP1 within normal C3H/HeJ mouse hair in comparison to 11 other strains. However, mutant mice with hair medulla defects also had undetectable levels of CRISP1 in their hair. Crisp1 null mice had normal skin, hair follicles, and hair shafts indicating that the lack of the CRISP1 protein does not translate directly into defects in the hair shaft or hair follicle. These results suggest that CRISP1 may be an important structural component of mouse hair and that its strain-specific dysregulation may indicate a predisposition to hair shaft disease such as AA.

Alopecia areata (AA), a cell mediated autoimmune disease, is the second most common form of hair loss in humans. While the autoimmune disease is responsible for the underlying pathogenesis, the alopecia phenotype is ultimately due to hair shaft fragility and breakage associated with structural deficits. Quantitative trait genetic analyses using the C3H/HeJ mouse AA model identified cysteine-rich secretory protein 1 (Crisp1), a hair shaft structural protein, as a candidate gene within the major AA locus. Crisp1 transcripts in the skin at various times during disease development were barely detectable. In situ hybridization identified Crisp1 expression within the medulla of hair shafts from clinically normal strains of mice but not C3H/HeJ mice with AA. Follow-up work with 5-day-old C3H/HeJ mice with normal hair also had essentially no expression of Crisp1. Other non-inflammatory based follicular dystrophy mouse models with similar hair shaft abnormalities also have little or no Crisp1 expression. Shotgun proteomics, used to determine strain difference in hair proteins, confirmed that there was very little CRISP1 within normal C3H/HeJ mouse hair in comparison to 11 other strains. However, mutant mice with hair medulla defects also had undetectable levels of CRISP1 in their hair. Crisp1 null mice had normal skin, hair follicles, and hair shafts indicating that the lack of the CRISP1 protein does not translate directly into defects in the hair shaft or hair follicle. These results suggest that CRISP1 may be an important structural component of mouse hair and that its strain-specific dysregulation may indicate a predisposition to hair shaft disease such as AA.

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Las proteinas CRISP (Proteinas Secretorias Ricas en Cisteinas) de mamiferos se encuentran presentes en el tracto reproductor masculino y han sido propuestas como mediadores del proceso de fertilizacion. En base a ello, y a su alta homologia con las proteinas PR1 y Ag5 involucradas en el sistema de defensa, el objetivo de esta Tesis ha sido investigar la relevancia de las CRISP tanto para la fertilidad como para el sistema inmune. Previos resultados de nuestro grupo indican que los ratones knockout para la proteina epididimaria CRISP1 (Crisp1-/-) son fertiles pese a presentar espermatozoides con una menor capacidad fertilizante. Dado que una mutacion puede producir fenotipos diferentes segun su fondo genetico, evaluamos el fenotipo de los animales Crisp1-/- generados en un fondo genetico homogeneo. Los resultados revelaron que los machos presentaron no solo una desventaja en la fertilidad sino tambien nuevos defectos en parametros funcionales de los espermatozoides, confirmando la importancia de realizar estudios en animales con diversos fondos geneticos. La proteina testicular CRISP2 participa en el proceso de fertilizacion y ha sido propuesta como un auto-antigeno capaz de generar orquitis. Con el fin de investigar su relevancia para la fertilidad, ratas macho y hembra fueron inmunizadas con CRISP2, observando que esta proteina es capaz de generar una respuesta inmune especifica en animales de ambos sexos, que no genera orquitis ni compromete la fertilidad. Estos resultados sugieren que, a diferencia de CRISP1, CRISP2 no seria un blanco anticonceptivo ni un antigeno responsable de la inmunoifertilidad. En cuanto a CRISP3, observamos que si bien existen dos isoformas en el espermatozoide humano, una de las cuales se libera durante la capacitacion y otra que permanece en el espermatozoide capacitado, esta proteina no actuaria como factor decapacitante ni seria relevante para la fertilizacion. Finalmente, en lo que respecta al posible rol de las CRISP en el sistema inmune, observamos que tanto CRISP3 como su homologo CRISP1 se expresan en celulas dendriticas inmaduras y maduras. Mas aun, el empleo de animales knockout para CRISP1 revelo que esta proteina modularia el perfil secretorio de citoquinas de las celulas dendritricas. En conjunto, los resultados obtenidos indican la relevancia de las CRISP para la regulacion tanto de la fertilidad como del sistema inmune.

Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Biologia y Medicina Experimental. Fundacion de Instituto de Biologia y Medicina Experimental. Instituto de Biologia y Medicina Experimental; Argentina