| School of Public Health, Nanjing Medical University, Nanjing 210029Issues of population, resources and environment are the key problems in influencing human survival and the economic development. Effective and safe contraceptive technology or drugs discovery and development are the major scientific issues in population control and life-quality promotion. It is usually considered that contraception is the responsibility of female. With the promotion of social division of labour and social status, male should play more active role in population control. However, in the presence of "reversible drugs are ineffective and effective drugs are irreversible" situation, there is no breakthrough in male contraception drugs development.It is well known that, in order to accommodate the complex physiological changes of female reproductive tract, the spermatozoa in testis, which are premature and lack of motility and fertility, must be experienced the structural modifications in the epididymis, including the further folding and modification of the exterior proteins. Some defects of exterior proteins of spermatozoa will influence the sperm maturation and hyperactivity, which leads to male infertility. During the research of the contraception resulting from immune mechanism, the anti-sperm antibody in many patients indicates that we can develop the sperm plasma-antigen vaccine to achieve immuno-infertility.Eppin (SPINLW1; Epididymal protease inhibitor) was first found in 2001 by Prof. O’Rand. Eppin is a single-copy gene characterized by three splice variants encoding two isoforms Eppin1/3 and Eppin2, the former of which contains a signal peptide (Richardson et al.,2001). These isoforms represent the first member of a family of protease inhibitors characterized by a dual inhibitor consensus sequence on human chromosome 20q12-13.2. Eppin, a cysteine-rich protein found only in the testis and epididymis, contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences (Richardson et al., 2001). It was first reported in The Science that, in 78 percent of the rhesus monkeys, immunization with recombinant Eppin protein caused the high titers of anti-Eppin antibody both in serum and semen, the significant reduction of sperm motility without aberrant morphology of sperm, and the successfully reversible contraception.The titers of anti-eppin antibody in serum and semen determine the efficacy of the immuno-contraception. It has been reported that the anti-eppin antibody results in the obstacles in semen coagulation and decreases the sperm motility, following with the sterility. Our laboratory has used the recombinant Eppin protein produced in vivo to immunize the male monkey and found the reduced motility of the sperm, following with the sterility. However, we have not found the obstacles in semen coagulation. Therefore, we hypothesized that there be another memchanisms underlying the reduced motility of sperm. Futhermore, we do not know wether the anti-eppin antibody influences the function of sertoli cells in testis. Furthermore, no reports have mentioned the different localization and function between the two isoforms of Eppin. It is well-known that the sub-cellular localization of proteins is closely relative to protein function as its proper and accurate locations are a prerequisite for the highly ordered functions in cellular systems. To solve the questions described above, we carried out the following research.Part I. Mechanisms of the anti-eppin antibody decreases transmembrane epithelial electronic resistance(TEER) of the sertoli cells in testis(1) Establish the co-culcure method of the primary sertoli cells with germ cells in vitro. After incubation with the anti-eppin antibody, analyze TEER of the primary sertoli cells.(2) Analysis of tyrosine or serine phosphrylation of occluding, claudin-11, E-Cadherin, F-actin and ZO-1 through co-immunoprecipitation with anti-tyrophosphrylation antibody or with anti-serine phosphrylation antibody respectively. Furthermore, proceed the co-immunoprecipitation to analyze the interaction between the proteins, including occludin with occludin, claudin-11 with claudin-11, E-Cadherin with E-Cadherin, occludin with claudin-11, occludin with ZO-1, claudin-11 with ZO-1, and F-actin with ZO-1.(3) Incubation with or without the anti-eppin antibody, proceed the sertoli cells with immunofluorecence analysis to identify the changes of the localization of occludin and claudin-11.From the results of the measurements described above, the anti-eppin antibody can increase the tyrosine phosphrylation of E-Cadherin, occludin and claudin-11, without influencing the tyrosine phosphrylation of ZO-1 and F-actin. Additionally, the anti-eppin antibody increases the serine phosphrylation of occludin but not influencing other proteins’serine phosphorylation. The subsequent results confirm that the anti-eppin antibody decreases the interaction among claudin-11s, blocks the interaction among E-Cadherins, which results in the reduced TEER of the sertoli cells.PartⅡ. Subcellular localization of two isoforms of Eppin and the characterization of Eppin in the motility of human sperm.(1) To determine the localization of Eppin1/3, Eppin2, the WAP domain and the Kunitz domain, we fused pEGFP-C 1 vector with the four different cDNAs corresponding to each of the protein and transfected each into COS-7 cells. In our study, we show that Eppin1/3 but not Eppin2, is a Golgi protein. The Kunitz domain also shows distinct accumulation in cytoplasmic granules and the WAP domain localizes non-specifically throughout the cell, including the intranuclear region.(2) BFA interfering experiment and immunogold experiment uncover that Eppin2 is actually present as a kind of recycling protein.(3) From the previous study, the motion and functions of the recycling proteins in the cell usually require the association with the cytoskeleton, especially the microtubule. To identify that Eppin could associate with the microtubule, we chose TUBB3, the subunit of beta-tubulin specific expressed in human testis, brain and some kinds of cancer cells to proceed the follow-up investigations. Consequently, we transfected HEK 293 cells both with pEGFP-Eppin1/3 and pHcRedN1/1-TUBB3 or both with pEGFP-Eppin2 and pHcRedN1/1-TUBB3 and then co-stained the two proteins. The co-localization assay and co-immunoprecipitation analysis show that, whether in co-transfected HEK 293cells or mature sperm, Eppin could interact with TUBB3. Furthermore, the interaction between Eppin with TUBB3 is calcium-concentration independent.(4) To confirm that Eppin was localized on the mitochondrial membrane, we isolated mitochondria from sperm and extracted the membrane protein from purified mitochondria. The results of western blotting showed that TUBB3 is the inherent protein on mitochondrial membrane and Eppin is localized on the mitochondrial membrane through the interaction with TUBB3. Furthermore, on the mitochondrial membrane, the interaction between Eppin and TUBB3 was not blocked except when treated with the anti-eppin antibody or (3-ME. It is concluded that the disulfide bonds, which cross-linked Eppin and TUBB3, play the key point for Eppin’s localization on mitochondrial membrane.(5) After incubation with the anti-eppin antibody, the mithchondiral membrane potential decreases, the ATP production of sperm decreases and the activated motility of sperm is significantly inhibited. The images of the electronic microscopy show the loss of the regular helical pattern of mitochondria around the midpiece of sperm and the partial loss of SMR. The results described above have shown that Eppin (whether Eppin1/3 or Eppin2) is interacted with TUBB3, the subunit of beta-tubulin and the interaction between Eppin with TUBB3 is responsible for Eppin’s localization on sperm mitochondrial membrane. It is regretful that we cannot identify Eppin1/3 or Eppin2 is on sperm mitochondrial membrane for the limitation of anti-eppin antibody. The cross-linkage through the disulfide bonds among Eppin links the adjacent mitochondria and maintain the regular helical structure of sperm mitochondria. Once the cross-linkage is blocked by anti-eppin antibody, the regular helical pattern of mitochondria is disrupted, the mitochondrial potential and ATP production are decreased and the motility of sperm is inhibited significantly. Conclusion:It is concluded that our studies uncover the mechanism of the anti-eppin antibody in decreasing TEER of the sertoli cells in testis. Furthermore, our studies uncover that Eppin plays the key role in the motility of sperm, perfecting the mechanism research of the contraception of Eppin. |
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