Sirtuin proteins are evolutionary conserved Sir2-related NAD+-dependent deacetylases and regulate many of cellular processes such as metabolism, inflammation, transcription, and aging. Sirtuin contains activity of either ADP-ribosyl-transferase or deacetyltranfease and their activity is dependent on the localization in cells. However, the expression pattern of Sirtuins has not been well studied. To examine the expression levels of Sirtuins, RT-PCR was performed using total RNAs from various tissues including liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Sirtuins were highly expressed in most of tissues including the testis. Immunostaining assay showed that Sirt1 and Sirt6 were mainly located in the nucleus of germ cells, spermatocytes, and spermatids in the seminiferous tubules, whereas Sirt2 and Sirt5 were exclusively present in the cytoplasm of germ cells and sperma-tocytes. Our results indicate that Sirtuins may function as regulators of spermatogenesis and their activities might be dependent on their location in the seminiferous tubules.

The spermatogenesis and oogenesis-specific helix-loop-helix transcription factor 2 (Sohlh2) is exclusively expressed in germ cells of male and female gonad. Sohlh2 acts as a transcriptional factor via its specific DNA binding site, E-box to regulate target genes such as Lhx8, Zp genes, Ngn3. Sohlh2 localize in the female oocyte and in the male spermatogonia. However, the regulatory mechanism of Sohlh2 was poorly understood. In this study, we examine the patterns interacting with Sohlh2. First, we performed immunoprecipitation with the antibody against Sohlh2 protein extracts from the testis. Two-dimensional SDS-gel showed sexual distinguishable protein including Fkbp12, 13, 3, 59. Among them, Fkbp3 is a member of the immunophilin protein family, which play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. Protein is a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin. It has a higher affinity for rapamycin than for FK506 and thus may be an important target molecule for immunosuppression by rapamycin. In the expression analysis of Fkbp3 is detected in the multiple tissues; intestine, stomach, kidney, spleen, liver, heart, brain, lung, uterus, ovary, testis. Here, we identified that Fkbp3 mRNA is detected in the ovary and testis, kidney, liver, heart, brain, lung. Immunostaning assay shows that Fkbp3 is localized at the spermatogonia in testis. In further studies, in order to confirm the interaction between Fkbp3 and Sohlh2, we will perform immunoprecipitation.

Tdrd family members contain Tudor domain repeat which is found in polar granules in Drophila. Tdrd12 is one of Tdrd family members. Tdrd12 contains a DEAD-box and a Tudor domain. However, the molecular mechanism and physiological function of Tdrd12 has not been described. To examine the expression pattern of Tdrd12, RT-PCR and Northern blot analysis were performed using total RNAs extracted from tissues; liver, intestine, heart, brain, kidney, lung, brain, uterus, ovary, and testis. The full-length of Tdrd12 was amplified from total RNA from mouse testis and cloning into the cloning vector. Cloned PCR products were purified,sequenced and analyzed using the ABI Prism Sequencer 3130XL. To look into Tdrd12 protein location, rabbit antibodies against mouse Tdrd12 were made using two epitopes: 1st epitope: (318~334)- SQRPNEKPLRLTEKKDC and 2nd epitope: (737~750)- LEAKEDKKARRPLC, and its specificity was tested using tissue extracts including the gonad. Here, we identified that Tdrd12 mRNA is detected in the ovary and testis, but not in other tissues. The size of its transcript is about 4.5kb on the northern blot. Antibody against Tdrd12 detects about 150 kDa protein on the western blot analysis. Immunostaining assay shows that Tdrd12 is localized at the spermatid in the seminiferous tubules. The current study is the first to investigate Tdrd12 expression is limited in the gonad. Thissuggest that Tdrd12 plays a role in the gonad like other known Tdrd family members, Tdrd1, Tdrd6, Tdrd7, and Tdrd9.

The spermatogenesis and oogenesis-specific helix-loop-helix transcription factor 2 (Sohlh2) is exclusively expressed in germ cells of male and female gonad. Sohlh2 acts as a transcriptional factor via its specific DNA binding site, E-box to regulate target genes such as Lhx8, Zp genes, Ngn3. Sohlh2 localize in the female oocyte and in the male spermatogonia. In recent studies, Sohlh2 knockout (KO) mice occurs abnormal spermagoenesis resulting in sperm defect. Sohlh2 KO male mice were infertility due to disruption of numerous gene expression. However, the gene profiles of Sohlh2 KO testes were not characterized and the regulatory mechanism of Sohlh2 was poorly understood. In this study, we analyzed the gene profiles and examined the possible mechanism of Sohlh2 in the spermatogenesis. First, we performed histological analysis such as Hematoxylin and eosin stain, Tunel assay, and Immunohistochemistry to show the onset of disruption of Sohlh2 KO testes. These results showed that Sohlh2 KO testes have atrophic seminiferous tubule due to increased apoptosis at 2 weeks old. And then we analyzed the whole gene profiles in the Sohlh2 KO testes at 2 weeks old. We found that 91 genes were regulated at least 5-fold in knockout testes. Among these, several genes are involved in meiotic process. Quantitative-PCR results are shown that several meiotic factors are significantly down-regulated in 2-weeks-old Sohlh2 KO testes compared with that of wild type mice. Through chromosome spreading assay, we observed that the formation of synaptonemal complex of homologous chromosome during the meiosis in Sohlh2 KO testes was not completed. These suggest that Sohlh2 is critical for regulation of numerous factors including meiotic factors either directly or indirectly. Therefore, mis-regulation of meiotic factors at prophase I of meiosis during spermatogenesis leads to disruption of spermatogenesis in Sohlh2 KO testes. Further studies are needed to look at the mechanism of Sohlh2 for regulation of target genes in detail.