Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.

Spermatozoa acquire motility and fertilizing ability during their transit through the epididymis. A wide variety of proteins secreted into the epididymal lumen are added on to the sperm surface to allow morphological and molecular changes involved in sperm maturation. Proteins of the Sperm Associated Antigen 11 (SPAG11) family are known to be localized on the sperm surface. The rat SPAG11A protein was implicated in sperm maturation during epididymal transit in vitro. However, systematic analyses on the significance of SPAG11A in fertility and sperm function is not yet reported in vivo. In this study, using testicular electroporation, we generated transgenic rats that express shRNA to ablate endogenous Spag11a mRNA. Genotyping revealed the integration of the plasmid that expresses shRNA against Spag11a mRNA. Significant decrease in the mRNA levels of Spag11a and its encoded protein was observed in the caput epididymis of transgenic rats. We also generated an active immunization rat model to ablate endogenous SPAG11A protein by administering recombinant SPAG11A protein. Immunized rats had a high antibody titer in the serum and the tissue fluids of caput, cauda and testis. In both these model systems, the litter size and sperm count was significantly reduced. However, spermatozoa obtained from the transgenic or immunized rats underwent capacitation and acrosome reaction and the associated calcium release. Results of this study indicate the role of SPAG11A in fecundity and sperm production and not in sperm function, especially capacitation and acrosome reaction.

Beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.

Spermatogenesis and sperm maturation in the male reproductive tract is dictated by a variety of proteins secreted in the testis and epididymis. Though the proteome of these tissues is known, the functional role of many of these proteins remains uncharacterized. In this study, we characterize the rat Lysozyme-like (Lyzl) genes and proteins.

Sperm maturation in the epididymis is a tightly regulated process which involves secretion and addition of a variety of proteins onto the sperm surface. The molecular mechanisms governing these processes has gained interest in the last decade. In vitro model systems to study the role of epididymal proteins in sperm maturation and other physiological process are very important. Isolation of epididymal cells, culture of epididymal explants and generation of immortalized cells were standardized to be used as in vitro models to study epididymal function. However, isolation and maintenance of primary cultures of epididymal epithelial cells seems to be the best option because of its closeness to the in vivo conditions. Though structural and morphological characterization of primary cultures of epididymal epithelial cells were carried out, the same were not conducted at the molecular level. In this study, we isolated adult rat epididymal primary epithelial cells (EPECs) and characterized them for their purity and cell specific expression of molecular markers. Isolated EPECs exhibited normal cell morphology and were sub cultured and maintained up to 3 weeks. EPECs expressed the epithelial marker, E-cadherin and their purity was estimated to be 73% using flow cytometry. EPECs abundantly expressed CRISP1, Urp1a, Pate-F, Crisp1, Ar and Spag11e, markers of epididymal cells and were negative for Urp1b and Pate, markers negative for epididymis. Results of our study provide a systematic characterization of EPECs at the molecular level and thus a refinement to the previously reported characterization methods.

Uroplakins (UPKs) play an important role in the normal and pathophysiology of the urothelium. They protect the urothelium and play a crucial role during urothelial infections by Uropathogenic E. coli. However, their functions beyond this organ system remain unexplored. A wide variety of proteins secreted in the male reproductive tract tissues contribute to spermatogenesis, sperm maturation, fertilization and innate immunity. However, the presence of UPKs and their possible contribution to the male reproductive tract physiology is not yet reported. Hence, in this study, we characterized UPKs in the male reproductive tract of rats. To the best of our knowledge, for the first time, we report the expression of UPKs in the male reproductive system. Upk1a, Upk1b, Upk2 and Upk3b mRNA and their corresponding proteins were abundantly expressed in the caput, cauda, testis, seminal vesicles and the prostate. Their expression was not developmentally regulated. UPK protein expression was also localized on the spermatozoa, suggesting a role for these proteins in sperm function. To study the role of UPKs in innate immunity, Upk mRNA expression in response to endotoxin challenge was evaluated in vitro and in vivo. In the rat testicular and epididymal cell lines, Upk mRNA levels increased in response to lipopolysaccharide challenge. However, in the caput, cauda, testes, seminal vesicle and prostate obtained from LPS treated rats, Upk mRNA expression was significantly reduced. Results of this study indicate a role for UPKs in male reproductive physiology and innate immune responses.