Molecular Cloning of Goat 20.ALPHA.-Hydroxysteroid Dehydrogenase cDNA

Abstract

Abstract. 20α-Hydroxysteroid dehydrogenase (20α-HSD), which catalyzes the conversion of progesterone to its inactive form 20α-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20α-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20α-HSD cDNA were performed. The full nucleotide sequence of 20α-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20α-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20α-HSD cDNA had 20α-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20α-HSD protein showed 20α-HSD enzyme activity. A tissue distribution study demonstrated that 20α-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20α-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy. Key words: Goat, 20α-Hydroxysteroid dehydrogenase (20α-HSD), Placenta, Ovary (J. Reprod. Dev. 50: [323][324][325][326][327][328][329][330][331] 2004) 0α-Hydroxysteroid dehydrogenase (20α-HSD) is a steroid metabolizing enzyme, which catalyzes the reaction that converts progesterone, a potent progestin, to its metabolically inactive form 20α-dihydroprogesterone (20α-OHP). This enzyme belongs to the aldo-keto reductase (AKR) superfamily according to the protein phylogeny [1]. Members of this family prefer NADP/NADPH as a cofactor.Progesterone is essential for the initiation and maintenance of pregnancy in mammals. In goats, placenta does not produce sufficient amount of progesterone, and therefore goats are fully dependent on luteal progesterone for maintenance of pregnancy. As in most animals, plasma progesterone concentration in goats is high during the gestation period and decreases abruptly before parturition [2][3][4][5][6]. In addition, increased levels of 20α-OHP have also been observed in the general circulation during pregnancy in goats [2]. In rats, it has been shown that 20α-OHP concentration decreases with the increase in the progesterone concentration during early pregnancy while it increases with the reduction of the progesterone levels around the parturition [7].

Expression and Possible Role of 20.ALPHA.-Hydroxysteroid Dehydrogenase in the Placenta of the Goat

“…This is consistent, at least partially, with previous reports showing the presence of mRNA [9,10,12,21,22], protein [8] and the activity [8,11] of 20α-HSD in the placenta and/or uterus of several mammalian species. The expression of 20α-HSD in the intrauterine tissue in goats was especially prominent in the latter half of pregnancy, when the maternal serum progesterone levels were high.…”

“…We have also shown that bacterially expressed protein using cloned goat 20α-HSD cDNA indeed possesses 20α-HSD activity [10]. In addition, we detected expression of 20α-HSD mRNA in the goat placenta, but not in the corpus luteum, liver, spleen or adrenal gland, at day 90 of pregnancy by means of RT-PCR [10].…”

“…We have also shown that bacterially expressed protein using cloned goat 20α-HSD cDNA indeed possesses 20α-HSD activity [10]. In addition, we detected expression of 20α-HSD mRNA in the goat placenta, but not in the corpus luteum, liver, spleen or adrenal gland, at day 90 of pregnancy by means of RT-PCR [10]. To determine the possible physiological roles of 20α-HSD in the maintenance of pregnancy in the goat, 20α-HSD gene expression in the placenta together with changes in progestin concentrations in the maternal and fetal sera and amniotic fluid during pregnancy of the goat were investigated in the present study.…”

“…DNA fragments of about 1 kilobase, containing the entire coding region of the goat 20α-HSD [10], were removed from the pGEM R -T vector (Promega, Madison, WI) by Pst I and Sph I digestion and purified with a QIA quick gel extraction kit (QIAGEN Inc, Valencia, CA). An aliquot of the above sample was used as a template to prepare the probe.…”

“…The cDNAs for both rat [17] and mouse 20α-HSD [9] and the genomic DNA for mouse 20α-HSD [18] have been cloned in our laboratory, and we also recently cloned goat 20α-HSD cDNA, which has 74% and 76% homology to rat and mouse 20α-HSD cDNAs, respectively [10]. We have also shown that bacterially expressed protein using cloned goat 20α-HSD cDNA indeed possesses 20α-HSD activity [10].…”