Abstract
BackgroundThe aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy.MethodsRapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary.ResultsThe porcine 20alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition.ConclusionsOur study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.
Highlights
The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPHdependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins
Determination of AKR1C1 C-terminal region nucleotide sequence The complete cDNA sequence (957 bp) for porcine AKR1C1 was obtained by assembly of the sequences from the PCR and Rapid amplification of cDNA ends (RACE) fragments
We sequenced 20 clones from the 3’-RACE experiment to confirm the sequence accuracy. This result was different from the 337 amino acids sequence of pig AKR1C1 that was previously reported, and there was a deletion of 4 amino acids compared with other animal 20a-hydroxysteroid dehydrogenases (HSDs) sequences reported to date (Figure 1)
Summary
The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPHdependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The aldo-keto reductase (AKR) superfamily are monomeric oxidoreductases that catalyze the NADP(H)-dependent reduction of a wide variety of substrates, including steroids, prostaglandins, bile acids, carbohydrates, and xenobiotics [1]. A group of AKRs known as hydroxysteroid dehydrogenases (HSDs) play a pivotal role in the modulation and regulation of steroid hormones [2], such as androgens, estrogens, and progestins, and are considered important targets for drug design [3]. AKR1C1, a member of the AKR1C subfamily (that shows both 20aand 3a-HSD activities), plays a major role in progesterone metabolism and maintenance of pregnancy through the formation of progestin. The process of AKR-dependent whole-cell biotransformation has been established, to be used for the production of human AKR metabolites on a large scale [8]
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