Research on fertilization in mammalian species has revealed that Ca2+ is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca2+ is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca2+ plays a pivotal role. Calpain-1 and calpain-2 are Ca2+-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca2+. Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.
Yadira Bastián, Ana L Roa-Espitia, Adela Mújica and Enrique O Hernández-González
Tania Reyes-Miguel, Ana L Roa-Espitia, Rafael Baltiérrez-Hoyos and Enrique O Hernández-González
Mammalian sperm cells acquire fertilizing capacity as a result of a process termed capacitation. Actin polymerization is important for capacitation; inhibiting actin polymerization prevents the adhesion and fusion of the sperm with the ovule. The main function of RHO proteins CDC42 and RHOA is to direct actin polymerization. Although these two RHO proteins are present in mammalian sperm, little is known about their role in capacitation, the acrosome reaction, and the way in which they direct actin polymerization. The purpose of this study was to determine the participation of CDC42 and RHOA in capacitation and the acrosome reaction and their relationship with actin polymerization using guinea pig sperm. Our results show that the inhibition of CDC42 and RHOA alters the kinetics of actin polymerization, capacitation, and the acrosome reaction in different ways. Our results also show that the initiation of actin polymerization and RHOA activation depend on the activation of CDC42 and that RHOA starts its activity and effect on actin polymerization when CDC42 reaches its maximum activity. Given that the inhibition of ROCK1 failed to prevent the acrosomal reaction, the participation of RHOA in capacitation and the acrosomal reaction is independent of its kinase 1 (ROCK1). In general, our results indicate that CDC42 and RHOA have different roles in capacitation and acrosomal reaction processes and that CDC42 plays a preeminent role.
Karina Pastén, Yadira Bastian, Ana L Roa-Espitia, Deneb Maldonado-García, Guillermo Mendoza-Hernández, Cesar I Ortiz-García, Adela Mújica and Enrique O Hernández-González
Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm–egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45 kDa. We find that the size of the mature ADAM15, 45 kDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65 kDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65 kDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm–egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm–egg binding.
Karina Pastén-Hidalgo, Rosaura Hernández-Rivas, Ana Lilia Roa-Espitia, Manuel Sánchez-Gutiérrez, Francisco Martínez-Pérez, Alma Olivia Monrroy, Enrique O Hernández-González and Adela Mújica
Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm–egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell–cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm–oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm–egg binding.
Alberto Darszon, Juan J Acevedo, Blanca E Galindo, Enrique O Hernández-González, Takuya Nishigaki, Claudia L Treviño, Chris Wood and Carmen Beltrán
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm–egg signaling and unveil novel contraception targets.