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.

Equine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone. eCG, over 40%, is a heavily glycosylated glycoprotein than other glycoprotein hormones. eCG is composed of non-covalently linked α and β subunit. The α subunit is common to all glycoprotein hormones, whereas the β subunit was known distinct sequences and specific receptor binding. Unusually, eCG shows both FSH and LH activities in other species. eCG α subunit has two N-glycosylation sites (Asn56, Asn82) and β subunit has one N-glycosylation site (Asn13) and about 13 O-glycosylation sites in the C-terminal region. We constructed 3 type mutants (βα△56: α-subunit Asn56→ Gln56; β-Da: β-subunit C-terminal deletion; β-Dα△56: β C-terminal deletion & α Asn56→Gln56) in the tethered eCGβα (wild type) and all mutants included myc-tag between first and second amino acid of β subunit. The plasmid DNAs cloned into pcDNA3 mammalian expressing vector were transiently transfected into CHO-Suspension cells. We also constructed rat LH/CG receptor and rat FSH receptor into pcDNA3 expression vector. These receptors were transiently transfected into CHO-K1 cell. Each receptor cells were used for further assays at 3 days after transfection. cAMP and IP-one were evaluated by CISBIO cAMP HiRange and IP-one kits using the rec-eCGβα mutants. According to cAMP assay results, IC50 values of 4 type ligand treatment in the rat FSH receptor cells were: eCGβα: IC50_16.8841; eCGβα56: IC50_95.6099; eCGβ-Dα: IC50_395.0087; eCGβ-Dα56: IC50_1439.8702. In the rat LH/CG receptor cells of 4 types ligand treatments, cAMP results were: eCGβα: IC50_0.9760; eCGβα56: IC50_8.3884; eCGβ-Dα: IC50_9.2550; eCG β-Dα56: IC50_45.9439. As seen in these data, β C-terminal region and α Asn56 play an important role in rat FSHR and rat LH/CGR, respectively. And rat LHCG receptor cells was remarkably stronger than rat FSH receptor cells. According to IP-one assay, IC50 values in rat FSH receptor cells, the results were: eCGβα: IC50_561.4490; eCGβα56: IC50_361.3005; eCGβ-Dα: IC50_911.8577; eCGβ-Dα56: IC50_139.1193. And in rat LH/CG receptor cells, IP-one results were: eCGβα: IC50_422.7315; eCGβα56: IC50_406.4915; eCGβ-Dα: IC50_537.8300; eCGβ-Dα56: IC50_254.2004. As shown in these data, IP-one result was a little different to cAMP result. The β eCGβ-Dα56 of IC50 value was shown generally high signal. Now we are trying to analyse role of C-terminal region of eLH/CGR with cAMP, IP-one and ERK signal transduction assays.