Influence of reproductive tract obstruction on expression ）f epididymal proteins and their restoration after patency

Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends： a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 （HE2）, fertilization antigen-1 （FA-1）, clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency （P〈O.05）. Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent, Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.

Human ribonuclease 9,a member of ribonuclease A superfamily,specifically expressed in epididymis,is a novel sperm-binding protein

To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.

Molecular cloning and identification of mouse epididymis-specific gene mHongl, the homologue of rat HongrES1

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHongl. The mHongl gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHongl mRNA shows similarity with HongrES1 in the expression patterns： （i） specific expression in epididymal tissue, especially in the cauda region; and （ii） androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHongl with high specificity and sensitivity was raised. Like HongrES1, the mHongl protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHongl protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHongl is localized to the equatorial segment, which is different from that of HongrES 1. The mHongl protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHongl is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHongl.

From the epididymis to the egg： participation of CRISP proteins in mammalian fertilization

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-Rlch Secretory Proteins （CRISP） as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein （hCRISP1） is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis （CRISP2）, epididymis （CRISP3-4） or during ejaculation （CRISP3） are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.

Epididymosomes： transfer of fertility-modulating proteins to the sperm surface

A variety of glycosylphosphatidylinositol （GPI）-linked proteins are acquired on spermatozoa from epididymal luminal fluids （ELF） during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles （epididymosomes, EP） which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane （TM） proteins, including plasma membrane Ca^2＋-ATPase 4 （PMCA4）. Synthesized in the testis, PMCA4 is an essential protein and the major Ca^2＋ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4＇s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca^2＋-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.