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The epididymis is a complex organ where spermatozoa acquire motility and ability to fertilize the egg. Epididymal maturation lasts 1 or 2 weeks and exposes the immature spermatozoa to a sequentially modified milieu, promoting intense interactions with secretions by the epididymal epithelium. Sperm surface modifications in response to interactions with epididymal secretions are key steps to achieve fertility ability. However, the precise molecular mechanisms that convert an immotile and infertile gamete into a highly motile cell capable of fusion with an oocyte are still unknown. Recent data on proteomics and transcriptomics of epididymal fluid and epididymosomes brought new ideas of the physiological roles of proteins and miRNAs in epididymal maturation in spermatozoa. This review focuses on the recent discoveries on epididymal fluid composition and its role on sperm maturation and preservation, linking to their survival and fertility potential.

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The present study was conducted to decipher the proteome of in vivo‐produced pre‐implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC‐MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre‐implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa‐mir‐106b‐5p; hsa‐mir‐30‐5p; hsa‐mir‐103a‐5p and hsa‐mir‐106a‐5p) with potential interactions with embryo‐expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF‐beta, p53 and Toll‐like receptor. The current study describes the first major proteomic profile of 6‐day‐old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Despite the development of efficient boar semen extenders, there is still room for improvement of new formulas using new molecules that could increase fertilisation outcomes and substitute cryoprotectants and antibiotics. The goal of this work was to evaluate if the essential oils from the leaves of Myrrhinium atropurpureum and Cymbopogon citratus are suitable as additives in boar semen extender. The major compounds found in the essential oils from M. atropurpureum were 1,8-cineole (37.37%) and terpinolene (19.18%); and geranial (49.8%) and neral (33.24%) in essential oil of C. citratus. The addition of 1% and 0.1% of both essential oils to extended semen had immediate spermicidal effects (p < 0.05). Lower concentrations were tested and no cytotoxic effect was observed when M. atropurpureum essential oil was added at 0.001%. Differently, essential oil from C. citratus reduced sperm motility, membrane functionality and integrity and mitochondrial membrane potential even in concentrations as low as 0.001%. Also, addition of essential oils in low concentrations had no inhibitory effect against Escherichia coli and Pseudomonas aeruginosa growth. We conclude that the essential oils from C. citratus and M. atropurpureum, rich in monoterpenes, are cytotoxic to swine spermatozoa, therefore unsuitable as semen extender additives.

SUMMARYHypogonadism is defined as the inadequate gonadal production of testosterone. Low serum testosterone leads to infertility by impairing spermatogenesis and reducing sperm count, however, the impact of hypogonadism in epididymal sperm maturation is poorly understood. From the testis, spermatozoa are transported into the epididymis where they find a specific microenvironment composed of a complex mixture of proteins that facilitate sperm storage and maturation. Inside the epididymal ductule, spermatozoa undergo several changes, resulting in their becoming capable of fertilizing eggs. Protein disulfide isomerases (PDIs) are known to participate in the folding and assembly of secreted proteins in the endoplasmic reticulum. However, little is known about the control and function of PDIs in the testis and epididymis, particularly during male development. The aim of this work was to compare the expression and distribution of PDI and PDIA3 (ERp57) in the testis and epididymis of healthy and GnRH-immunized boars. We detected higher amounts of PDIA3 and PDI in sperm preparations and fluid from the proximal regions of the epididymis of healthy boars. However, we observed an increase in PDIA3 expression in the testis and cauda epididymis in the immunocastrated group. GnRH-immunized boars showed a marked increase in PDI content in cauda spermatozoa and fluid, indicating a possible endocrine dysregulation of PDI. The results of our study suggest that PDIs are associated with epididymal sperm maturation and may be attractive candidates for monitoring male fertility.

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