Fadd Phosphorylation Study On The Effects On Spermatogenesis In Male Mice

FADD(Fas-associated death domain),was first recognized as an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as cell development, cell cycle progression and cell proliferation. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region. To understand the significance of Ser191 phosphorylation of FADD on male reproduction, our laboratory collaborated with State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, and chose the model of FADD^-/-tgFADD (S191D) mouse and their littermates as control to carry out our research.In the process of breeding mice, we found that FADD(S191D) male mice were infertile. We can obtain FADD(S191D) mice only by mating mice with the gene type of FADD^+/-tgD and FADD^+/-. The most obvious feature of FADD(S191D)mouse was leanness. The testes were obviously smaller than control, and the testis index was significantly decreased compared to control. The pathological section of the testis showed that the spermatogenesis was impaired. Few mature sperm were found in the seminiferous lumen, and a large number of strong eosinophilic staining of spermatogenetic cells were observed in the lumen. We counted the numbers of testicular germ cells and found that spermatogonia, spermatocytes and round spermatids in FADD(S191D) testis were reduced to varying degrees, in which round spermatids decreased the most obviously. The sperm number in epididymal decreased, and the sperm deformity rate increased significantly when compared to control.To find the mechanism of spermatogenesis disorder in FADD (S191D) mouse, TUNEL staining was used to compare the apoptosis rate of FADD (S191D) testis and littermate control, and we found FADD (S191D) had more apoptosis cells in the seminiferous lumen than control, primarily in spermatocytes. Combined with the decrease of the germ cells, especially haploid sperm cells, we speculated that the phosphorylation of FADD may affect all stage during spermatogenesis, especially meiosis of spermatocytes.To study the meiotic abnormalities during prophase I in FADD(S191D) mouse, synaptonemal complex (SC) spreading immunofluorescence was used and the progression of meiosis I prophase was examined. Results showed that some of the DNA double-strand branks (DSBs) on autosome in FADD(S191D) pachytene spermatocyte had not been repaired, and that the numbers of MLH1 focis which marks recombination sites on autosome per pachytene spermatocyte decreased significantly compared to control. We speculated that the phosphorylation of FADD may affect recombination repair during meiosis, leading to apoptosis of spermatocytes, then subsequently impaired spermatogenesis in testis.Then how does the permanent phosphorylation of FADD cause the abnormal DNA repair? Previous studies indicated that FADD could interact with MLH1 and methyl-CpG binding domain protein 4 (MBD4), both could regulate genome surveillance/DNA repair. So we speculate that FADD and its interaction with the nuclear localization of MLH1 and MBD4 may regulate DNA-damaging response. In order to verify the existence of this interaction in germ cells, cytoplasm and nuclear protein from FADD (S191D) and FADD^+/- mice testis were extracted respectively, and Western Blot was performed to examine the expression of the three proteins in testis cytoplasm and nuclear. Results showed that FADD was expressed mainly in the nucleus of mouse testis. The expression levels of MBD4 and MLH1 increased in cytoplasm while decreased in nucleus, which indicated that the phosphorylation of FADD might affect the transport of MBD4 and MLH1 from testis cytoplasm to nucleus, so as to decrease the nuclear expression of them and weaken the role of DNA repair function they played.Taken together, through analysis of the phenotype of FADD (S191D) mice on male reproductive system, we found that the permanent phosphorylation of FADD may impair spermatogenesis and finally lead to infertility. Further preliminary study for relevant mechanisms showed that phosphorylation of FADD may affect the nuclear expression of MBD4 and MLH1, then affect DNA repair in spermatocytes, which finally lead to cell apoptosis and damaged spermatogenesis. All these researches about FADD(S191D)mouse may help us better understand the function of FADD and its Ser191 phosphorylation on male reproduction.