Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.
Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites.
Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+-dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.
Nowadays, farm animal industries use assisted reproductive technologies (ART) as a tool to manage herds’ reproductive outcomes, for a fast dissemination of genetic improvement as well as to bypass subfertility issues. ART comprise at least one of the following procedures: collection and handling of oocytes, sperm, and embryos in in vitro conditions. Therefore, in these conditions, the interaction with the oviductal environment of gametes and early embryos during fertilization and the first stages of embryo development is lost. As a result, embryos obtained in in vitro fertilization (IVF) have less quality in comparison with those obtained in vivo, and have lower chances to implant and develop into viable offspring. In addition, media currently used for IVF are very similar to those empirically developed more than five decades ago. Recently, the importance of extracellular vesicles (EVs) in the fertility process has flourished. EVs are recognized as effective intercellular vehicles for communication as they deliver their cargo of proteins, lipids, and genetic material. Thus, during their transit through the female reproductive tract both gametes, oocyte and spermatozoa (that previously encountered EVs produced by male reproductive tract) interact with EVs produced by the female reproductive tract, passing them important information that contributes to a successful fertilization and embryo development. This fact highlights that the reproductive tract EVs cargo has an important role in reproductive events, which is missing in current ART media. This review aims to recapitulate recent advances in EVs functions on the fertilization process, highlighting the latest proposals with an applied approach to enhance ART outcome through EV utilization as an additive to the media of current ART procedures.
Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.
Abstract Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.
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