Study of CRISP proteins as regulators of fertility and the immune system

Las proteínas de la familia CRISP1-4 (Cysteine-RichSecretoryProteins) se expresan tanto en el tracto reproductor de mamíferos donde actúan como mediadores del proceso de fertilización, como en órganos de importancia inmunológica. En base a ello, el objetivo general de esta Tesis Doctoral ha sido investigar la relevancia de las CRISP como reguladores tanto de la fertilidad como del sistema inmune a través del empleo de animales simples y múltiples knockout (KO) para las CRISP. Teniendo en cuenta resultados previos de nuestro laboratorio indicando que la proteína CRISP1 exhibe la capacidad de inhibir a CatSper, el principal canal de calcio del espermatozoide esencial para la fertilidad masculina, como primer objetivo nos planteamos continuar investigando la actividad de CRISP1 como regulador de CatSper y explorar su posible empleo para el desarrollo de un método anticonceptivo masculino. Los resultados revelaron que la exposición de espermatozoides a HC-056456 (HC), un compuesto que inhibe la actividad de CatSper tal como CRISP1, fue capaz de inhibir la capacidad fertilizante de los espermatozoides tanto in vitro como in vivo, apoyando la posibilidad de desarrollar un método anticonceptivo masculino basado en la actividad inhibitoria de CRISP1 sobre CatSper. La normal fertilidad de los machos simples KO para las proteínas CRISP1, CRISP2 y CRISP4 generados en nuestro laboratorio, junto a la observada subfertilidad de los machos carentes de dos proteínas CRISP (CRISP1 y CRISP4) simultáneamente, apoyan la existencia de mecanismos de compensación entre los diferentes miembros de la familia CRISP. En base a ello, y considerando la no disponibilidad del ratón simple KO para la proteína CRISP3, el segundo objetivo de esta Tesis consistió en el desarrollo y caracterización tanto del animal simple KO para CRISP3 como de animales múltiples KO para diferentes CRISP a través del empleo de la técnica CRISPR/Cas9. Los resultados revelaron que la presencia de la mutación en el gen Crisp3 afectó la expresión de CRISP1, llevando a la generación de un animal doble KO (DKO) para CRISP1 y CRISP3. La caracterización de estos animales a nivel reproductivo mostró una bajada significativa de la fertilidad tanto en los machos como en las hembras, representando ésta la primera evidencia de la relevancia de las CRISP para la fertilidad femenina. Más aun, observamos que la subfertilidad de esta colonia se debería, principalmente, a defectos en el desarrollo temprano de los embriones, revelando por primera vez el rol de las CRISP en eventos posteriores a la fertilización. Los animales carentes de más de dos proteínas CRISP simultáneamente se generaron empleando una construcción capaz de interferir con los genes Crisp1 y Crisp3 en animales fértiles DKO CRISP2/CRISP4 disponibles en el laboratorio. El estudio de la fertilidad de los animales triples (TKO) y cuádruples (CKO) KO generados reveló que los machos eran prácticamente infértiles a juzgar por un promedio de crías por camada inferior a 1. El análisis de los mecanismos subyacentes a este fenotipo reveló defectos en la migración por el tracto femenino y capacidad fertilizante de los espermatozoides como así también en el desarrollo embrionario temprano, indicando que la severa bajada de la fertilidad sería la resultante de una combinación de defectos a diferentes niveles. Finalmente, en base a observaciones previas de nuestro grupo indicando la presencia de CRISP1 y CRISP3 en células dendríticas (CD), investigamos el posible rol inmunoregulador de las CRISP utilizando animales simples y DKO para estas proteínas. Los resultados mostraron que la ausencia de CRISP1 y CRISP3 modifico la expresión de citoquinas de las CD y produjo un aumento en el porcentaje de Linfocitos T regulatorios en un co-cultivo de ambas células, favoreciendo una respuesta inmune de perfil tolerogénico. Consistente con ello, la inyección de células tumorales MA-10 en los animales simple KO y DKO condujo a un mayor crecimiento tumoral in vivo comparado con los controles, apoyando el rol de las CRISP en la regulación del sistema inmune. En conjunto, consideramos que estos resultados contribuirán a una mayor comprensión de los mecanismos involucrados en la regulación de la fertilidad y del sistema inmune, abriendo la posibilidad de que las CRISP sean empleadas en el futuro como agentes diagnósticos y/o targets terapéuticos.The Cysteine-Rich Secretory Proteins (CRISP1-4) are mainly expressed in the mammalian male reproductive tract where they act as mediators of the fertilization process as well as in organs of immunological importance. Based on this, the main aim of this Thesis has been to study the relevance of CRISP as possible regulators of fertility and the immune system through the use of simple and multiple knockout (KO) animals for these proteins. Based on previous results from our laboratory indicating that mouse CRISP1 protein exhibits the ability to inhibit CatSper, the main sperm calcium channel essential for male fertility, the first aim was to continue studying the role of CRISP1 as a CatSper regulator and to explore the possibility of its use for male contraceptive development. Results revealed that exposure of sperm to HC-056456 (HC), a drug that inhibits CatSper activity as CRISP1, was capable of inhibiting the sperm fertilizing ability in vitro as well as in vivo, supporting the possibility of developing a male contraceptive method based on the inhibitory activity of CRISP1 on CatSper channel. The normal fertility of single KO males for CRISP1, CRISP2 and CRISP4 generated by our group together with the subfertility observed in males lacking two CRISP proteins (CRISP1 and CRISP4) simultaneously, supports the existence of compensatory mechanisms among different CRISP family members. Based on this, and considering the unavailability of the single KO animal for CRISP3, the second objective of this work has been the generation and characterization of the single KO for CRISP3 as well as of multiple KO for different CRISP using the CRISPR/Cas9 technique. Results revealed that the mutation in Crisp3 affected the expression of CRISP1, leading to the generation of a double KO animal (DKO) for CRISP1 and CRISP3. The reproductive characterization of these DKO mice showed a significant decrease in fertility of both males and females, representing the first evidence of the relevance of CRISP proteins for female fertility. Moreover, we observed that the subfertility of this colony could be mainly due to defects in early embryo development, revealing for the first time the role of CRISP proteins in post-fertilization events. The animals lacking more than two CRISP simultaneously were generated through the introduction of a construct capable of interfering with Crisp1 and Crisp3 genes in fertile DKO CRISP2/CRISP4 animals already available in our laboratory. Evaluation of fertility of the generated triple (TKO) and quadruple (QKO) KO mice revealed that the males were almost infertile as judged by an average litter size of less than one. Analysis of the mechanisms underlying this phenotype revealed severe defects in sperm migration through the female tract and sperm fertilizing ability as well as on early embryonic development, indicating that the severe decrease in male fertility would be the result of a combination of defects at different levels. Finally, based on previous observations of our group indicating the presence of CRISP1 and CRISP3 in dendritic cells (CD), we investigated the possible immunoregulatory role of CRISP using both single KO and DKO mice for these proteins. Results revealed that the absence of CRISP1 and CRISP3 modified the cytokine expression profile of the CD and produced an increase in the percentage of regulatory T lymphocytes in a co-culture of both cells, favoring a tolerogenic type immune response. Consistent with this, injection of MA-10 tumor cells in KO and DKO animals led to a greater in vivo tumor growth compared to controls, supporting the role of CRISP as regulators of the immune system. Altogether, we believe these results will contribute to a better understanding of the mechanisms involved in the regulation of fertility and the immune system, opening the possibility of using CRISP in the future as diagnostic agents and / or therapeutic targets.Fil: Curci, Ludmila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Pharmacological inactivation of catsper blocks sperm fertilizing ability independently of the capacitation status of the cells: implications for non-hormonal contraception

Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1–20 μM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm’s ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm–egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.Fil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Carvajal, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sulzyk, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gonzalez, Soledad Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Functional redundancy and compensation: deletion of multiple murine Crisp genes reveals their essential role for male fertility

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca2+ 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 multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2−/−.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 Ca2+ 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 functionalredundancy/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: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Brukman, Nicolás Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rojo, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Carvajal, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sulzyk, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gonzalez, Soledad Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Relevance of cystein-rich secretory proteins for male fertility

Cysteine-Rich Secretory Protein (CRISP) 1, 2, 3 and 4 are mainly expressed in thereproductive tract and have key roles in mammalian fertilization. In spite of this,knockout (KO) mice for each individual protein are fertile whereas double KO(DKO) CRISP1/CRISP4 are subfertile, suggesting the existence of compensatorymechanisms between homologous CRISP family members. Recent results fromour lab revealed that DKO CRISP1/CRISP3 are also subfertile.Fil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sulzyk, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaReunion Anual de Sociedades de BiocienciaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de Ciencia y Tecnología de Animales de LaboratorioAsociación Argentina de NanomedicinasThe Histochemical Societ

Relevance of CRISP proteins for epididymal physiology, fertilization, and fertility

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.Fil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Carvajal, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gonzalez, Soledad Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Relevance of cystein-rich secretory proteins for fertilization, fertility and early embryo development

CRISP1 and CRISP3 are two members of the Cysteine Rich Secretory Protein family expressed in the male and female mammalian reproductive tracts. In spite of the important roles of CRISP1 in fertilization, CRISP1 knockout mice are fertile, suggesting compensatory mechanisms among CRISP proteins. As the functional role of CRISP3 in reproduction still remains unclear, in this work we investigated the relevance of CRISP3 for both fertilization and fertility by generating single Crisp3 and double Crisp1/Crisp3 knockout mice. Methods: Crisp3-/- and Crisp1-/-/Crisp3-/- (DKO) mice were generated by using the novel CRISPR/Cas9 technique. DNA sequencing was used to confirm the null-allele mutations and Western blot for evaluating protein expression. Fertility was evaluated by mating males with one females and in vivo fertilization by analyzing the percentage of fertilized eggs recovered from the ampulla of mated females. Finally, early development was evaluated as the percentage of fertilized eggs reaching the blastocysts stage 5 days after in vitro incubationResults: Male and female fertility were significantly reduced in both Crisp3-/- and DKO animals. Protein expression analysis revealed the lack of CRISP1 protein not only in DKO but also in Crisp3-/- mice. Subsequent experiments were carried out using only those mice exhibiting mutations in both genes. Examination of in vivo fertilization to elucidate the mechanisms underlying fertility defects showed significantly lower fertilization rates in DKO females than in controls but normal fertilization rates when control females were mated by DKO males. Interestingly, the eggs normally fertilized by DKO sperm exhibited a significantly lower ability to reach the blastocyst stage compared to controls, supporting an additional role for CRISP1 and CRISP3 in early embryo development.Conclusion: Our results confirm the relevance of CRISP1 and CRISP3 for both fertilization and fertility and may contribute to a better understanding of how paternal factors could impact on embryo development.Fil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Brukman, Nicolás Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rojo, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaInternational Federation of Placenta Associations 2019 (IFPA2019), 8th Latin American Symposium on Maternal-Fetal Interaction and Placenta (VIII SLIMP)Ciudad Autónoma de Buenos AiresArgentinaSociedad Argentina de BiologíaSociedad de Obstetricia y Ginecología de Buenos AiresSociedad Argentina de DiabetesSociedad Argentina de Endocrinología Ginecológica y ReproductivaSociedad Argentina de Investigación Bioquímica y Biología MolecularSociedad Argentina de Investigación Clínic

From the epididymis to the egg: participation of CRISP proteins in mammalian 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 by using CRISP (Cysteine-RIch Secretory Proteins) as model molecules. Substantial evidence obtained using 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 homologue 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 testes (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 homologue proteins to ensure the success of fertilization. Together, our observations indicate that CRISP proteins escort sperm 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.Fil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Brukman, Nicolás Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Carvajal, Guillermo. Fundacion de Instituto de Biologia y Medicina Experimental; ArgentinaFil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Gómez Elías, Matías Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

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

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, ourobservations 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.Fil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Carvajal, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Maldera, Julieta Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Curci, Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres, Pablo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin