Ablation of the sperm-associated antigen 11A (SPAG11A) protein by active immunization promotes epididymal oncogenesis in the rat.

Incidence of cancer in the epididymis is very rare. It is proposed that proteins specific to this organ may contribute to this unique property. We previously demonstrated that siRNA-mediated knockdown of SPAG11A mRNA resulted in increased proliferation of epididymal epithelial cells, whereas overexpression of this gene caused reduced proliferation in immortalized cell lines. In this study, we evaluated the oncogenesis-related anatomical and transcriptome changes in the epididymis of SPAG11A-immunized rats challenged with a low dose of diethyl nitrosamine (DEN). DEN treatment or SPAG11A immunization alone did not cause any histopathological changes in the epididymis. Interestingly, indications of oncogenesis were observed in SPAG11A-immunized + DEN-treated rats. Using high throughput sequencing, we observed that 3549 transcripts that were differentially expressed in the caput epididymis of DEN only-treated rats displayed similar differential expression in the caput epididymis of SPAG11A-immunized rats, indicating that the microenvironment that contributes to oncogenesis sets in when SPAG11A protein is ablated. Differential expression of genes that are involved in 10 major cancer related pathways was also analyzed. Majority of the genes related to these pathways that were differentially expressed in the caput epididymis of DEN only-treated rats also showed similar pattern in the caput epididymis of SPAG11A-immunized rats. For the first time, results of our study demonstrate that ablation of SPAG11A by active immunization renders the epididymis susceptible to oncogenesis and that this protein may be one of the factors that contributes to the rarity of epididymal cancer.

Transgenesis and active immunization mediated reduction of sperm associated antigen 11A mRNA and protein levels affect fecundity in the rat.

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.

siRNA-mediated knockdown of sperm-associated antigen 11a (Spag11a) mRNA in epididymal primary epithelial cells affects proliferation: a transcriptome analyses.

Differential expression of a variety of proteins in the four major regions of the epididymis contributes to maturation of spermatozoa and region-specific cellular functions as well. Proliferation of epithelial cells of the epididymis is highly controlled and thus is one of the major reasons for the nonoccurrence of cancers in this organ system. The molecular mechanisms and the contribution of region-specific genes in epithelial cell proliferation are not yet fully understood. In this study, for the first time, we analyzed the role of sperm-associated antigen 11a (Spag11a), a caput-specific beta-defensin-like antimicrobial gene in governing epididymal cell proliferation and global gene expression. siRNA-mediated knockdown of Spag11a mRNA in epididymal primary epithelial cells resulted in increased cell proliferation. Out of the 68,842 genes analyzed, 4182 genes were differentially expressed (2154 upregulated and 2028 downregulated). A variety of genes that participate in different cellular processes and pathways were differentially regulated. Genes that are important for epithelial cell proliferation were found to be differentially regulated and these changes were confirmed by real-time PCR. Overexpression of Spag11a in immortalized rat caput epididymal cells resulted in decreased proliferation capacity. Results of this study indicate that Spag11a plays a crucial role in governing epididymal epithelial cell proliferation.

Male reproductive tract antimicrobial expression in the extremes of ages of rats.

Alterations in the global gene expression profile are considered to contribute to the various physiological and pathological changes during the course of ageing. Genes that code for the molecular components of the innate system are alter markedly as ageing occurs; and this may define the susceptibility of very young and very old individuals to reproductive tract infections. The expression pattern of genes that code for beta-defensins (effectors of innate immune response) in male reproductive tract tissues of different stages of ageing is not yet reported. Further, the induction of beta-defensins during endotoxin challenge and whether epigenetic modulators can influence the expression of these genes in different stages of ageing are not reported. We analyzed the basal mRNA levels of beta-defensins and defensin-like proteins (Sperm Associated Antigen 11 (SPAG11) family members), their induction during endotoxin challenge and modulation by epigenetic modifiers (Trichostatin A and Azacytidine) in the caput, cauda, testis, prostate and seminal vesicle of rats that represent early stage to late stages of life (20 day to 730 day old). We observed differential basal gene expression pattern in the male reproductive tract tissues and the induction by LPS was not consistent neither among the age groups not the tissues analyzed. Trichostatin A and Azacytidine also influenced antimicrobial gene expression and the pattern was not consistent in different tissues obtained from different age groups. Results of this study demonstrate that antimicrobial gene expression varies to a great extent during ageing and is strongly influenced by endotoxins and epigenetic modulators.

Characterization of isolated rat caput epididymal primary epithelial cells: A molecular biology approach.

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.