Kisspeptin Receptor on the Sperm Surface Reflects Epididymal Maturation in the Dog

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.

SPE-51, a sperm secreted protein with an Immunoglobulin-like domain, is required for sperm-egg fusion in C. elegans

Despite the importance of fertilization, the molecular basis of sperm-egg interaction is not well understood. In a forward genetics screen for fertility mutants in Caenorhabditis elegans we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell-autonomously and localizes to the surface of the spermatozoa. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

Background Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. Results The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

Buffalo Sperm Surface Proteome Profiling Reveals an Intricate Relationship Between Innate Immunity and Reproduction

BackgroundLow conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint which limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. ResultsThe raw mass spectra data searched against in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a GPI-anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst the buffalo sperm surface proteins. The presence of these proteins was confirmed by immunocytochemistry and IVF experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted glycoproteins like BDs and the GPI-anchored proteins. The glycosylation pattern, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BuBDs on the sperm surface strengthens our hypothesis that these BDs assist the buffalo spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD which could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its roles in the regulation of male fertility.

Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.