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. 2011 Nov;142(5):723-31.
doi: 10.1530/REP-11-0093. Epub 2011 Sep 9.

Molecular Characterization of Bovine Placental and Ovarian 20α-hydroxysteroid Dehydrogenase

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Free PMC article

Molecular Characterization of Bovine Placental and Ovarian 20α-hydroxysteroid Dehydrogenase

Purevjargal Naidansuren et al. Reproduction. .
Free PMC article

Abstract

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2 kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37 kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

Figures

Figure 1
Figure 1
Comparison of the bovine 20α-HSD and related sequences. (A) The deduced bovine 20α-HSD amino acid sequence was aligned with the amino acid sequence of goat, deer, rabbit, human, mouse, and rat 20α-HSD. Amino acids are presented in the conventional single-letter code and are numbered on the right. The 1F and 3R PCR primers are boxed. Amino acid residues that are completely conserved across the species are indicated by dashed lines. (B) The phylogenetic tree of the bovine 20α-HSD amino acid sequence was compared with the 20α-HSD sequences from other vertebrate species. The other 20α-HSD amino acid sequences were obtained from GenBank, and the phylogenetic tree was constructed using the unrooted analysis with PHYLYP and Tree View.
Figure 2
Figure 2
Expression of the bovine 20α-HSD gene in the ovary throughout the estrous cycle, the pre-parturition placenta, and in cultured luteum cells. (A) We randomly collected ovaries at different times throughout the estrous cycle. The corpus luteum at the estrous cycle was divided according to CL stages. (B) Placentomes were obtained from cesarean delivery on day 283 of gestation from Korean native bovines. (C) Bovine 20α-HSD mRNA was detected in cultured corpus luteum cells. The corpus luteum cells of the CH2 stage were cultured until 120 h. (D) The relative expression of the 20α-HSD gene (mean±s.d.) present in three sets of samples evaluated by PCR. Total RNA was extracted and analyzed by RT-PCR. The PCR products were then separated by agarose gel electrophoresis. Representative PCR results are shown. The amplified products of 20α-HSD and GAPDH were separated by electrophoresis and stained with ethidium bromide. M, DNA size marker; CH, corpus hemarrhagicum; CL, corpus luteum.
Figure 3
Figure 3
Northern blot analysis of bovine 20α-HSD mRNA expression throughout the estrous cycle in the ovaries and pre-parturition placenta. (A) Northern blot analysis with 10 μg total RNA/lane. (B) The relative expression of 20α-HSD is presented. The blots shown are the results of a representative experiment. CH, corpus hemarrhagicum; CL, corpus luteum; Pl, placenta. 18S rRNA was used as a loading control.
Figure 4
Figure 4
Expression of bovine 20α-HSD protein in E. coli and CHO cell lines. (A) SDS–PAGE of purified recombinant 20α-HSD. E. coli transfected with pRSET+b20α-HSD was cultured, and the purified recombinant bovine 20α-HSD protein was separated by SDS–PAGE. (B) Bovine 20α-HSD recombinant protein produced in CHO-K1 cells was analyzed by immunoblot. The bands corresponding to the protein produced by pcDNA3+b20α-HSD and pcDNA4/HisMax+b20α-HSD vectors were detected. After gel electrophoresis, the proteins were transferred to a nitrocellulose membrane, and the protein was detected with bovine 20α-HSD antiserum followed by staining with goat anti-rabbit IgG-POD. Lane 1, pcDNA3+b20α-HSD; lane 2, pcDNA4/HisMax+b20α-HSD.
Figure 5
Figure 5
Western blot analysis of the bovine ovary during the estrous cycle and pre-parturition placenta. (A) Corpus luteum tissues throughout the estrous cycle (CH2, CH3, CL3, CL2, and CL1). (B) The placentome was collected on day 283 of pregnancy. Placentas were divided into the caruncle and cotyledon. β-Actin was used as the loading control; Caru, Caruncle; Coty, Cotyledon.
Figure 6
Figure 6
Catalytic activity of bacterially expressed purified 20α-HSD protein. The enzyme activity was measured by spectrophotometry. Each point represents the mean values of three separate experiments.
Figure 7
Figure 7
Localization of bovine 20α-HSD protein expression in the CL throughout the estrous cycle by immunohistochemistry. Representative immunohistochemical analyses using 20α-HSD antiserum (1:1000) and goat anti-rabbit secondary antibodies (1:500). Preimmune serum (1:1000) was used for primary antiserum as the negative control. Immunohistochemistry was performed by a Vectastain ABC kit. Ovarian sections are shown during the estrous cycle. Preimmune serum was used as the control for the CL stage 7 below the right panel. Black bar=100 μm. Red arrows indicate luteal cells.
Figure 8
Figure 8
Bovine 20α-HSD expression in cultured bovine and rat corpus luteum cells by immunofluorescence. Counter staining was performed with DAPI (blue). Bovine 20α-HSD expression was detected by Alexa 488 (green). The merged picture shows blue and green colors. (A) Bovine luteal cells. (B) Rat corpus luteal cells. White bars=100 μm.

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