Expression and function of RNase9 protein in male reproductive system

Abstract

Objective To investigate the expression, localization and function of Ribonuclease 9 (RNase9) in the male reproductive system, so as to provide a theoretical basis for the diagnosis and treatment of infertility caused by abnormal RNase9 expression. Methods Bioinformatic techniques were used to analyze the structure and thereby predict the function of human RNase9. Recombinant human RNase9 protein was obtained using a prokaryotic expression vector constructed by recombinant DNA technology. RT-PCR was performed to evaluate the difference in RNase9 protein expression in various tissues. The RNase9 protein expression in epididymis was compared among elderly [aged (76.3±8.1) years], adults [aged (31.2± 5.6) years] and fetuses [ (0.8±0.2) years]. Fluorescence immunohistochemistry was used to localize RNase9 proteins in the testis, epididymis and sperm. Ribonuclease activity of RNase9 was measured with yeast tRNA substrate assay. Sperm suspensions were added with pre-immune serum (Group A) or mouse anti-RNase9 antiserum (Group B) , and then subjected to antibody-blocked sperm motility assay to determine the effect of RNase9 protein on indicators of sperm motility [mean curvilinear velocity (VCL) , mean straight line velocity (VSL) , mean path velocity (VAP) ]. From 8 to 12 week-old Golden hamsters intramuscularly injected with progesterone and human chorionic gonadotropin, ova were harvested and incubated with the sperms from Groups A and B (the sperms in Group A had been blocked with pre-immune serum and those in Group B with anti-RNase9 antiserum for 5 h; there were 60 ova used in group A and 62 in Group B). The effect of RNase9 on ovum penetration ability of the anti-body blocked sperms was evaluated by hamster ovum penetration assay. Results Bioinformatic analysis indicated that RNase9 gene is a new member of RNase A superfamily, with a 23-aminoacid signal peptide sequence at the N-terminal, a relative molecular weight of about 20 000 and an isoelectric point (PI) of 5. 92. RNase9 was shown to contain five N-glycosylation sites, two casein kinase Ⅱ phosphorylation sites, one tyrosine kinase phosphorylation site, one NDUFC2 site and two labeling sites of pancreatic -ribonuclease functional domain. RNase9 protein was ubiquitously expressed in human epididymis, testis, heart, lung, liver, spleen, kidney, and stomach tissues, whereas the expression level was higher in the epididymis. Expression of RNase9 in epididymis was significantly higher in adults than that in the epididymis of fetuses and elderly (all P 0.05). Conclusion Expression and localization of RNase9 protein in the epididymis and sperm suggest that the protein may play an important role in sperm maturation and development, but may not affect the motility and ovum-penetrating ability of mature sperms rats. Key words: RNase9; Testis; Epididymis; Sperm; Sperm motility; Sperm-ovum interactions