In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α-OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase known to be inhibited by prolactin. In this study, we focused on the analysis of transgenic mice expressing EGFP under control of monkey 20α-HSD promotor in mice testis. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis, respectively. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blotting analysis and EGFP was found at 27-kDa in the testis of TG mice. Also EGFP and 20a-HSD protein expression on 1, 2, 4, 6 and 8 weeks after birth were assessed. Both of them were increased the expression level time-dependently. 20α-HSD were strongly expressed in seminiferous tubule from 1 week after birth as seen in Immunohistochemical analysis. However, EGFP was strongly expressed in the seminiferous epithelial cells. Then, we determined the expression of EGFP mRNA in mice testis. Using primers specific for mouse EGFP, mRNA expression levels were analyzed by RT-PCR. The EGFP molecular weights is 400bp, qRT-PCR results using EGFP primer, The Cq value of the ratio decreased as the age increased. On this basis, mRNA were increased the expression level time-dependently. In conclusion, these observations suggest that the 20α-HSD in testis could be play a pivotal role in the spermatogenesis.

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. Aldo-keto reductases (AKRs) belong to a superfamily of NADPH-dependent reductases that act on a wide range of substrates, including simple carbohydrates, steroid hormones, and endogenous prostaglandins. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α -OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase is known to be inhibited by prolactin. We have been reporting on the molecular characterizations of placental and ovarian 20α-HSD in the bovine, pig, deer and monkey. In this study, we focused on the 20α-HSD expression in testis(6, 9, 12, 18 and 21 days after birth) of miniature pig. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blot analysis. Also the RNA expression were detected by Reverse Transcription-PCR and quantification PCR. Additionally, We are going to analysis the function and role of 20α-HSD in the pig testis.

20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) enzyme converts progesterone into biological inactive steroid, thus playing a key role in the termination of pregnancy or estrus cycle and allowing parturition and ovulation to occur in most mammalian animals. However, function and regulation of this enzyme has not known well in primate reproductive physiology. We previously demonstrated the expression level and localization of the 20α-HSD in the reproductive tissues of macaque monkeys of pre-ovulation and pre-parturition period. Also, we amplified about 2005 bp 5'-flanking region from placenta genomic DNA and examined methylation pattern and promoter activity. In present study, we focus on the analysis of molecular characterization of the promoter region by using reporter assay systems. We constructed of deleted mutants (— 890 bp; HSF-2), (— 513 bp; XFD), (— 276 bp; Ap-1) and (— 72 bp; Sp-1) and each mutants were cloned into pGL3-basic vector. These deletion mutants were transfected into CHO cells and co-transfected with Sp-1 or Ap-1 transcription factor plasmids. Compared to — 890 bp and 513 bp promoter fragments alone, transcription activity increased when these constructs were co-transfected with Sp-1 and Ap-1 factor. However, for the absence Ap-1 factor binding site in 276 bp fragment activity dramatically decreased in both transfections. Next, we constructed of 306 bp fragment which is including of Ap-1 binding site and nucleotides converted mutants of the Ap-1 factor binding site. In this result, 306 bp fragment's transcription activity was high as wild type. However, the mutant activity which converted Ap-1 site’s all nucleotide was significantly decreased. These findings are confirmed by gel-shift assay examining Ap-1 binding site on the 20 α-HSD gene upstream region and expression of Ap-1 factor was determined by RT-PCR and Western blot in pre-parturition period placenta and CHO-K1 cell line. Our results indicate that Ap-1 site (— 281 → — 274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20 α- HSD gene transcription.