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Sperm cells are terminally differentiated cells that are essential for reproduction in sexually reproducing species. Consistent with their highly specialized function, sperm cells harbor a unique proteome containing many proteins not expressed in somatic cells. In contrast, the post-translational landscape of the sperm proteome remains largely unexplored, limiting our understanding of how modifications such as glycosylation impact sperm function and sperm-egg interactions. Here, we used glycopeptide-centric glycoproteomics to comprehensively characterize protein N-glycosylation in sperm from three mammalian species, revealing clear conservation of glycosylation profiles. We find that glycosylation patterns in sperm proteins are distinct from those in plasma, with as clear distinctive features less sialyation and more paucimannosylation in sperm. Moreover, based on their subcellular location, sperm protein glycosylation varies, with paucimannose species enriched in the acrosomal vesicle, oligomannose species in the sperm head membrane, and complex glycan species in the acrosomal membrane.

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air–liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen–progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.A functional equine in vitro oviduct epithelium model was established in Transwell inserts and microfluidic chips using either a direct seeding or a de-differentiation/re-differentiation protocol.

Diabetes is associated with poor oocyte quality and the dysregulation of ovarian function and is thus a leading contributor to the increasing prevalence of female reproductive pathologies. Accordingly, it is well-established that insulin fulfills a key role in the regulation of several facets of female reproduction. What remains less certain is whether proinsulin C-peptide, which has recently been implicated in cellular signaling cascades, holds a functional role in the female germline. In the present study, we examined the expression of insulin, C-peptide, and its purported receptor; GPR146, within the mouse ovary and oocyte. Our data establish the presence of abundant C-peptide within follicular fluid and raise the prospect that this bioactive peptide is internalized by oocytes in a G-protein coupled receptor-dependent manner. Further, our data reveal that internalized C-peptide undergoes pronounced subcellular relocalization from the ooplasm to the pronuclei postfertilization. The application of immunoprecipitation analysis and mass spectrometry identified breast cancer type 2 susceptibility protein (BRCA2), the meiotic resumption/DNA repair protein, as a primary binding partner for C-peptide within the oocyte. Collectively, these findings establish a novel accumulation profile for C-peptide in the female germline and provide the first evidence for an interaction between C-peptide and BRCA2. This interaction is particularly intriguing when considering the propensity for oocytes from diabetic women to experience aberrant meiotic resumption and perturbation of traditional DNA repair processes. This therefore provides a clear imperative for further investigation of the implications of dysregulated C-peptide production in these individuals.

The proteomic composition of extracellular vesicles (epididymosomes) secreted by the mouse epididymis has been determined by applying multiplexed tandem mass tag based quantification coupled with high resolution LC-MS/MS. This analysis confirmed that epididymosomes encapsulate an extremely rich and diverse proteomic cargo, which is commensurate with their putative role in coordinating the post-testicular maturation and storage of spermatozoa.

Quantitative proteomic and phosphoproteomic analyses of Australian saltwater crocodile spermatozoa reveals that these cells respond to capacitation stimuli by mounting a cAMP-mediated signaling cascade that is analogous to that of spermatozoa from the mammalian lineage. Similarly, the phosphorylated proteins responsible for driving the functional maturation of crocodile spermatozoa share substantial evolutionary overlap with those documented in mammalian spermatozoa.

One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the germline. Lipid peroxidation events in the sperm plasma membrane result in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which accentuate the production of reactive oxygen species (ROS) and cause cellular damage. One of the key enzymes involved in the metabolism of polyunsaturated fatty acids to 4HNE in somatic cells is arachidonate 15-lipoxygenase (ALOX15). Although ALOX15 has yet to be characterized in human spermatozoa, our previous studies have revealed a strong link between ALOX15 activity and the levels of oxidative stress and 4HNE in mouse germ cell models. In view of these data, we sought to assess the function of ALOX15 in mature human spermatozoa and determine whether the pharmacological inhibition of this enzyme could influence the level of oxidative stress experienced by these cells. By driving oxidative stress in vitro with exogenous H2O2, our data reveal that 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176; a selective ALOX15 inhibitor) was able to significantly reduce several deleterious, oxidative insults in spermatozoa. Indeed, PD146176 attenuated the production of ROS, as well as membrane lipid peroxidation and 4HNE production in human spermatozoa. Accordingly, ALOX15 inhibition also protected the functional competence of these cells to acrosome react and bind homologous human zonae pellucidae. Together, these results implicate ALOX15 in the propagation of oxidative stress cascades within human spermatozoa and offer insight into potential therapeutic avenues to address male in fertility that arises from oxidative stress.Pharmacological inhibition of ALOX15 reduced the level of oxidative stress experienced by human spermatozoa in response to exogenous ROS challenge.

The reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposing male mice to a mild heat stress. For this purpose, adult male mice were exposed to an elevated ambient temperature of 35°C under two exposure models. The first involved acute exposure for 24 h, followed by recovery periods between 1 day and 6 weeks. The alternative heating regimen involved a daily exposure of 8 h for periods of 1 or 2 weeks. In our acute model, we identified elevated sperm mitochondrial ROS generation (P< 0.05), increased sperm membrane fluidity (P< 0.05), DNA damage in the form of single-strand breaks (P< 0.001), and oxidative DNA damage (P< 0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P< 0.001) and round spermatids (P< 0.001) isolated from testes after 1 day heat recovery. Despite these lesions, the spermatozoa of heat-treated mice exhibited no differences in their ability to achieve hallmarks of capacitation or to fertilize the oocyte and support development of embryos to the blastocyst stage (all P> 0.05). Collectively, our acute heat stress model supports the existence of heat susceptible stages of germ cell development, with the round spermatids being most perturbed and spermatogonial stem cells exhibiting resistance to this insult. Such findings were complemented by our chronic heat stress model, which further supported the vulnerability of the round spermatid population.Environmental heating induces a state of oxidative stress in the male germ line, affecting multiple germ cell types; primarily the round spermatid and pachytene spermatocyte populations.

Lipid peroxidation products, such as 4-hydroxynonenal (4HNE), are causative agents responsible for extensive protein damage within the male and female germlines. Recently, we have demonstrated that 4HNE production can initiate the proteolytic degradation of the molecular chaperone Heat Shock Protein A2 (HSPA2) in male germ cells. These events may be partially responsible for HSPA2 deficiency in the spermatozoa of patients that repeatedly fail in vitro fertilization. Given this, mechanisms that limit the production of 4HNE will be highly advantageous for the preservation of male fertility. The propagation of 4HNE in somatic cells has been linked to the enzymatic actions of arachidonate 15-lipoxygenase (ALOX15), a member of the lipoxygenase family of proteins. In view of this association, this study sought to explore ALOX15 as a physiological target to manipulate the levels of 4HNE produced in the male germline. Herein, we have demonstrated that ALOX15 is markedly upregulated in response to oxidative stress in round spermatids and the GC-2 cell line. Pharmacological inhibition of ALOX15 in GC-2 cells resulted in a significant reduction in both mitochondrial and cytoplasmic reactive oxygen species, as well as a dramatic reduction in 4HNE. Importantly, the reduced bioavailability of this aldehyde appears to confer positive downstream effects to its target proteins such that HSPA2 could be protected from damage by 4HNE. Taken together, these results suggest that the actions of ALOX15 are intimately tied to the production of 4HNE. Thus, the ALOX15 protein may be a promising new target for the mitigation of germline oxidative stress.Cellular oxidative stress and 4HNE generation are ameliorated through the inhibition of ALOX15 in male germ cells of the mouse.

Spermatozoa acquire fertilization potential during passage through a highly specialized region of the extratesticular ductal system known as the epididymis. In the absence of de novogene transcription or protein translation, this functional transformation is extrinsically driven via the exchange of varied macromolecular cargo between spermatozoa and the surrounding luminal plasma. Key among these changes is a substantive remodeling of the sperm proteomic architecture, the scale of which has yet to be fully resolved. Here, we have exploited quantitative mass spectrometry-based proteomics to define the extent of changes associated with the maturation of mouse spermatozoa; reporting the identity of >6,000 proteins, encompassing the selective loss and gain of several hundred proteins. Further, we demonstrate epididymal-driven activation of RHOA-mediated signaling pathways is an important component of sperm maturation. These data contribute molecular insights into the complexity of proteomic changes associated with epididymal sperm maturation.

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated viaimmunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.