Papers by Mariana Weigel Muñoz
bioRxiv (Cold Spring Harbor Laboratory), Mar 22, 2024
7th International Conference on the Epididymis: Program, 2019
Sperm cell research in the 21st century: historical discoveries to new horizons, 2012
Fertilization is a complex process that requires the successful completion of a series of orchest... more Fertilization is a complex process that requires the successful completion of a series of orchestrated steps. In most invertebrates and non-mammalian vertebrates, spermatozoa that leave the testes already have the ability to fertilize the egg. In mammals, however, testicular sperm are not yet capable of fertilizing an egg, requiring to undergo several physiological changes during their transit through the male and female reproductive tracts, known as sperm maturation and capacitation, respectively, in order to gain their fertilizing ability. Once in the proximity of the egg, sperm must pass through the cumulus cells that surround the egg, bind to and penetrate the zona pellucida (ZP) and, finally, fuse with the egg plasma membrane. Most of these events involving cell-to-matrix and cell-to-cell interactions are mediated by specific molecules present in both gametes. One of these proteins is rat epididymal protein CRISP1 (formerly known as DE)which was the first identified member of the highly conserved Cysteine-RIch Secretory Protein (CRISP) family, characterized by the presence of sixteen conserved cysteine residues, ten of which are clustered in the C-terminal domain of the molecule. The present manuscript will focus on the results obtained in our laboratory on both the behavior of CRISP1 during capacitation and its role in gamete interaction.Fil: 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; ArgentinaFil: Cohen, Debora Juana. 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: 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: Ernesto, Juan Ignacio. 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: Vasen, Gustavo. 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
Encyclopedia of Reproduction, 2018
Fertilization in mammals is a complex process involving a series of coordinated steps in which sp... more 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.
Frontiers in Cell and Developmental Biology, 2021
Mammalian fertilization is a complex process involving a series of successive sperm-egg interacti... more 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 Ca2+ 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 regu...
Las proteinas CRISP (Proteinas Secretorias Ricas en Cisteinas) de mamiferos se encuentran present... more 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 im...
Andrology, 2019
Background: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing a... more 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.
Scientific Reports, 2018
Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm duri... more 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. Gamete interaction in mammals involves a series of sequential and coordinated steps that culminates in the union of the sperm and egg genomes. Differently from lower vertebrates, the highly differentiated mammalian sperm that leave the testes do not have the ability to fertilize an egg. This requires that sperm first undergo a maturation process while passing through the epididymis where they will acquire progressive motility and the ability to recognize and interact with the egg 1,2. After ejaculation, mature epididymal sperm need to undergo another process known as capacitation that occurs while ascending the female tract and which enables sperm both to undergo the acrosomal reaction, an exocytotic event that takes place in the head, and to develop a vigorous flagellar movement termed hyperactivation 3. Although the mechanisms underlying the acquisition of sperm fertilizing ability are still under investigation 4,5 , it is known that sperm maturation involves numerous changes at the gamete surface level, most of which occur as a result of the association of epididymal proteins with the sperm plasma membrane 6,7. Two such proteins are CRISP1 and CRISP4 8-11 which belong to a highly conserved and widely distributed family among vertebrates known as Cysteine RIch Secretory Proteins (CRISP). In mammals, the CRISP family comprises four members (CRISP1-4) (20-30 kDa) mainly expressed in the male reproductive tract and characterized by the presence of sixteen conserved cysteines, ten of which are located in the C-terminal region or cysteine-rich domain (CRD) connected to the plant pathogenesis-related 1 (PR-1) domain located in
Biology of reproduction, Jan 22, 2018
Epididymal sperm protein CRISP1 has the ability to both regulate murine CatSper, a key sperm calc... more 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...
Sperm Acrosome Biogenesis and Function During Fertilization, 2016
The acrosome reaction (AR) is a universal requisite for sperm-egg fusion. However, whereas throug... more 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.
Journal of Cell Biology, 2015
Ca2+-dependent mechanisms are critical for successful completion of fertilization. Here, we demon... more Ca2+-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 Ca2+ 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 Ca2+ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca2+ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orienta...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015
Lectin-glycan recognition systems play central roles in many physiologic and pathologic processes... more 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 ...
Asian Journal of Andrology, 2015
revealed that the major amount of CRISP1 can be removed from the cells by exposure to high ionic ... more revealed that the major amount of CRISP1 can be removed from the cells by exposure to high ionic strength whereas a minor fraction of the protein remains strongly bound to spermatozoa after this treatment and can only be removed by agents that release integral proteins. 6 Moreover, whereas the amount of the tightly-attached CRISP1 population was similar in spermatozoa recovered from successive regions of the epididymis, the ionically-bound protein increased from caput to cauda, suggesting that CRISP1 associates strongly with sperm cells as soon as they enter the epididymis and then loosely during epididymal transit. 6 Considering that each epididymal segment has its own pattern of gene expression 7,8 and that different secretion pathways have been described for this organ (i.e., merocrine and apocrine), it is possible that the two population of CRISP1 are secreted by different pathways in different epididymal regions. To further characterize the interaction of CRISP1 with spermatozoa, we examined whether the exposure of caput or cauda epididymal spermatozoa to CRISP1 was sufficient to load the protein on the cells. The lack of positive results under all the conditions tested suggested the need of another epididymal fluid component to achieve CRISP1-sperm interaction. Considering the high concentrations of Zn 2+ described in the rat epididymis 9 and the existence of Zn 2+-binding sites in snake CRISP proteins, 10 we next investigated the possible role of Zn 2+ in the association of CRISP1 with spermatozoa. Western blot results showed an increase in CRISP1 only in extracts from caput spermatozoa that had
Molecular Human Reproduction, 2013
Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gam... more 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 proteinprotein 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.
Journal of Andrology, 2011
Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch S... more Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 ''knockout'' animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.
Journal of Andrology, 2012
Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved ... more Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.
Biology of Reproduction, 2011
Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during ma... more 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 Zn 2+ 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 Zn 2+ , and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn 2+ , 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 Zn 2+ 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 Zn 2+ 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 Zn 2+ supported a direct interaction between CRISP1 and Zn 2+. Incubation of either cauda epididymal fluid or purified CRISP1 with Zn 2+ , followed by native-PAGE and Western blot analysis, revealed the presence of highmolecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn 2+ complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.
Biological Research, 2011
Epididymal protein CRISP1 is a member of the CRISP (Cysteine-RIch Secretory proteins) family and ... more Epididymal protein CRISP1 is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-defi cient mice exhibited normal fertility, CRISP1-deffi cient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zonafree eggs in vitro, confi rming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.
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Papers by Mariana Weigel Muñoz