Regulation Of Androgen On Gene Expression Profile Of Mouse Epididymis

Epididymis is a part of male reproductive system, and the place where sperm maturation and a series of functions are gradually acquired, such as formation of sperm motility, improvement of acrosome function, changes of metabolic types, recognition and fusion of sperm and egg.Based on current knowledge, the structure and function of epididymis is dependent on androgen levels to a large extent. Experimental results showed that sperm quality and fertility of animals were significantly decreased after application of androgen antagonists, but the molecular mechanisms remained unclear.Gene expression profile is the most widely used gene chip now, which can screen and analyze differentially expressed genes according to their tissue specific, developmental stage specific and differentiation stage specific. In addtion, microarray results can comprehensively reflect the mechanism of specific physiological processes, role of targets and specific ways by clustering analysis and gene annotation features. In this study, whole genome expression profile was used to investigate the function and molecular mechanism of androgen on mouse epididymis and sperm maturation. ObjectivesTo investigate the effects of androgen antagonists (Dutasteride) on mouse sperm maturation, genes that were regulated by androgen and related to sperm maturation were screened by analyzing gene expression profile. The study provided theoretical basis for molecular biology of sperm maturation and male infertility treatment.Materials and MethodsMale SPF mice were divided into 3 groups, control group, low and high dose groups,12 in each. Mice were treated with continuous oral administration for 8 weeks. Low and high dose groups were treated with 1.5 mg/kg/d and 7.5 mg/kg/d dutasteride respectively, control group was treated with same volume corn oil. After treatment, testes and epididymis were removed and weighted to evaluate the effect of dutasteride on mouse reproductive organs. Computer assisted sperm analysis system (CASA) was used to analyze density, sperm progressive motility and sperm motility to evaluate the quality of mouse epididymal sperm. Enzyme linked immunosorbent assay (ELISA) was used to detect the testosterone and dihydrotestosterone in serum to evaluate the levels of androgen in mouse.NimbleGen whole genome expression profile was used to screen the differentially expressed genes between high dose group and control group. RNA samples were extracted from tissues using trizol method, then purified and tested by UV quantitative and electrophoresis before cDNA synthesis, purification and label, then hybridization was processed on NimbleGen hybridization system 4. After 16 hours, chips were put into GenePix 4000B for scanning and images were processed and analyzed by GenePix pro V6.0 software. After that, genes were screened according to signal strength after probe signals were normalizated. Finally, Genespring and David software were used for further analysis.PSMB8, GSTA1, AQP1 and IGFBP4 genes were validated by real time fluorescence quantitative PCR (qPCR) method using both epididymal epithelial cell line PC1 and epididymal tissues as templates. Finally, possible biological functions of these genes were analyzed based on experimental results and literatures.ResultsDHT levels in serum were significantly decreased when compared control group with low and high dose groups (P<0.01). The average weight of epididymis was decreased significantly, while that of testes did not differ significantly (P>0.05). CASA analysis indicated that density, sperm progressive motility and sperm motility were decreased in low and high dose groups.Compared with control group,1227 genes showed significantly changes in high dose group using gene expression profile, while 635 genes were down regulated and 592 genes were up regulated. Differentially expressed genes were mainly involved in physiological processes, such as metabolism and transport of macromolecules, cell communication, apoptosis, androgen metabolism and transfer activity.Gene expression profile of mouse epididymis was analyzed and some of these genes were validated by qPCR method, and the results were consistent with the chips.ConclusionsDHT level was related to the quality of epididymal sperm, and when concentration of inhibitor was high, the DHT level was low, then the quality of epididymal sperm was also low.The differentially expressed genes in epididymis between high dose group and control group were screened using gene expression profile. The study initially established profile database of sperm maturation related genes in epididymis and provided reference material for molecular biology of sperm maturation.