Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

Distribution of tetraspanins in bovine ovarian tissue and fresh/vitrified oocytes.

Tetraspanin proteins are mostly known as organizers of molecular complexes on cell membranes, widely expressed on the surface of most nucleated cells. Although tetraspanins participate in many physiological processes of mammals, including reproduction, their relevance to the processes of folliculogenesis and oogenesis has not yet been fully elucidated. We bring new information regarding the distribution of tetraspanins CD9, CD81, CD151, CD82, and CD63 at different stages of follicular development in cattle. The found distribution of tetraspanin CD9, CD63, and integrin alpha V in similar areas of ovarian tissue outlined their possible cooperation. We also describe yet-unknown distribution patterns of CD151, CD82, and CD63 on immature and mature bovine oocytes. The unique localization of tetraspanins CD63 and CD82 in the zona pellucida of bovine oocytes suggested their involvement in transzonal projections. Furthermore, we present an unchanged distribution pattern of the studied tetraspanins in vitrified mature bovine oocytes. The immunofluorescent analysis was supplemented by in silico data addressing tetraspanins expression in the ovarian cells and oocytes across several species. The obtained results suggest that in the study of the oocyte development and potentially the fertilization process of cattle, the role of tetraspanins and integrins should also be taken into account.

Expression of αV integrin and its potential partners in bull reproductive tissues, germ cells and spermatozoa.

Integrins are transmembrane receptors expressed in all nucleated mammalian cells, critically involved in cell-matrix adhesion and cell-cell interactions that modulate many signalling cascades. It is assumed that integrins also provide essential functions of the reproductive system. In this study, we describe the detailed localization and distribution of αV integrin in the plasma membrane of bull sperm head and tail. Integrin αV was observed in the area of forming acrosome in developing sperm since the stage of round spermatids and persists in the acrosome during epididymal maturation and ejaculation till the acrosomal exocytosis. We detected CD9 and CD81 tetraspanins as the potential partners of αV integrin. Their similar staining pattern in testicular tissue suggested the involvement of these molecules in the tetraspanin web of "testisomes". Moreover, the complex of αV with ß1 and ß3 integrin subunits cannot be excluded at least in sperm. The presented findings contribute to understanding the mutual action of integrins and tetraspanins during sperm development and maturation.

Tetraspanins, More than Markers of Extracellular Vesicles in Reproduction.

The participation of extracellular vesicles in many cellular processes, including reproduction, is unquestionable. Although currently, the tetraspanin proteins found in extracellular vesicles are mostly applied as markers, increasing evidence points to their role in extracellular vesicle biogenesis, cargo selection, cell targeting, and cell uptake under both physiological and pathological conditions. In this review, we bring other insight into the involvement of tetraspanin proteins in extracellular vesicle physiology in mammalian reproduction. We provide knowledge regarding the involvement of extracellular vesicle tetraspanins in these processes in somatic cells. Furthermore, we discuss the future direction towards an understanding of their functions in the tissues and fluids of the mammalian reproductive system in gamete maturation, fertilization, and embryo development; their involvement in mutual cell contact and communication in their complexity.

Correction to: Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos.

The original article can be found online.

Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos.

It is known that tetraspanin proteins are involved in many physiological somatic cell mechanisms. Additionally, research has indicated they also have a role in various infectious diseases and cancers. This review focuses on the molecular interactions underlying the tetraspanin web formation in gametes. Primarily, tetraspanins act in the reproductive tract as organizers of membrane complexes, which include the proteins involved in the contact and association of sperm and oocyte membranes. In addition, recent data shows that tetraspanins are likely to be involved in these processes in a complex way. In mammalian fertilization, an important role is attributed to CD molecules belonging to the tetraspanin superfamily, particularly CD9, CD81, CD151, and also CD63; mostly as part of extracellular vesicles, the significance of which and their potential in reproduction is being intensively investigated. In this article, we reviewed the existing knowledge regarding the expression of tetraspanins CD9, CD81, CD151, and CD63 in mammalian spermatozoa, oocytes, and embryos and their involvement in reproductive processes, including pathological events.

Missing Information from the Estrogen Receptor Puzzle: Where Are They Localized in Bull Reproductive Tissues and Spermatozoa?

Estrogens are steroid hormones that affect a wide range of physiological functions. The effect of estrogens on male reproductive tissues and sperm cells through specific receptors is essential for sperm development, maturation, and function. Although estrogen receptors (ERs) have been studied in several mammalian species, including humans, they have not yet been described in bull spermatozoa and reproductive tissues. In this study, we analyzed the presence of all types of ERs (ESR1, ESR2, and GPER1) in bull testicular and epididymal tissues and epididymal and ejaculated spermatozoa, and we characterize them here for the first time. We observed different localizations of each type of ER in the sperm head by immunofluorescent microscopy. Additionally, using a selected polyclonal antibody, we found that each type of ER in bull sperm extracts had two isoforms with different molecular masses. The detailed detection of ERs is a prerequisite not only for understanding the effect of estrogen on all reproductive events but also for further studying the negative effect of environmental estrogens (endocrine disruptors) on processes that lead to fertilization.

Detection of CD9 and CD81 tetraspanins in bovine and porcine oocytes and embryos.

Tetraspanins are multifunctional molecules located in specific microdomains on the plasma membrane. Thanks to their ability to form networks with other proteins they can participate in many cellular functions. Tetraspanins are part of the interactive network in gametes; however, their precise role in fertilization is not yet clear. The aim of this study was to compare the localization of CD9 and CD81 tetraspanins during oocyte maturation and early development of the embryos in bovine and porcine model. CD9 was detected on the oocyte plasma membrane and vesicles in the perivitelline space of bovine oocytes and embryos. We suggest that CD9 could be a component involved in transzonal projections. Based on the results of in vitro fertilization assay, CD9 and CD81 seem to be part of a more complex fusion network on the plasma membrane of bovine oocytes. On the other hand, both tetraspanins showed a clustered expression pattern on the plasma membrane and inner margin of zona pellucida (ZP) in porcine oocytes and embryos. We found a new species-specific pattern of CD9 and CD81 distribution in ZP which could reflect their specialized role in processes associated with cell adhesion and intercellular communication upon fertilization.

CD9 and CD81 Interactions and Their Structural Modelling in Sperm Prior to Fertilization.

Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm⁻egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm⁻egg membrane fusion.

Evaluation of protein phosphorylation in bull sperm during their maturation in the epididymis.

Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.