Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 20;9:139.
doi: 10.1186/1477-7827-9-139.

Expression of Aldo-Keto Reductase Family 1 Member C1 (AKR1C1) Gene in Porcine Ovary and Uterine Endometrium During the Estrous Cycle and Pregnancy

Affiliations
Free PMC article

Expression of Aldo-Keto Reductase Family 1 Member C1 (AKR1C1) Gene in Porcine Ovary and Uterine Endometrium During the Estrous Cycle and Pregnancy

Kyeong-Seok Seo et al. Reprod Biol Endocrinol. .
Free PMC article

Abstract

Background: The 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 20 alpha-hydroxysteroid dehydrogenase (20 alpha-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.

Methods: Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-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 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary.

Results: The porcine 20 alpha-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 20 alpha-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.

Conclusions: Our 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.

Figures

Figure 1
Figure 1
Amino acid alignment of pig AKR1C1 and other animal 20α-HSD proteins. A C-terminal amino acid region of pig AKR1C1 sequence was compared that of bovine, goat, deer, human, rabbit, mouse, and rat 20α-HSD. Dashed lines denote the amino acids identities. The numbers denote the amino acids before the stop codon. Porcine AKR1C1 denotes the results reported previously [7]. Bold and underlined AKR1C1 was shown sequence cloned in this paper.
Figure 2
Figure 2
AKR1C1 mRNA expression in the ovary during the estrous cycle. A. AKR1C1 mRNA was detected by RT-PCR. B. Real-time PCR results. Ovarian tissues were obtained by laparotomy under general anesthesia on days 0, 5, 10, and 15 of the estrous cycle. Total RNA was extracted and then subjected to RT-PCR and real-time PCR. The amplified products of the AKR1C1 and GAPDH genes were separated on agarose gel and stained with ethidium bromide. Representative results are shown; graphs show the average ± SEM of 3 independent experiments.
Figure 3
Figure 3
Northern blot analysis of pig AKR1C1 mRNA expression in the ovary during the estrous cycle and pregnancy. A. Ovarian tissues were obtained by laparotomy under general anesthesia on days 5, 10, and 15 of the estrous cycle. B. Ovaries were collected by the same method on days 0, 30, and 60 of pregnancy, and before parturition. Blots shown are the results of a representative experiment; graphs show the average ± SEM of 3 independent experiments. P-part: before parturition.
Figure 4
Figure 4
Northern blot analysis of pig AKR1C1 mRNA expression in the ovary and uterus during the estrous cycle and pregnancy. A. Total RNA was isolated from ovaries on day 12 of the estrous cycle and pregnancy. B. Uteri were collected on days 12 and 15 of the estrous cycle, and on days 12, 15, and 30 of pregnancy. Blots shown are the results of a representative experiment; graphs show the average ± SEM of 3 independent experiments. C: estrous cycle; P: pregnancy.
Figure 5
Figure 5
Western blot analysis of pig AKR1C1 protein in the ovary during the estrous cycle and pregnancy. A. Pig ovaries on days 0, 5, 10, and 15 of the estrous cycle were obtained by laparotomy under general anesthesia. B. Uteri were collected on days 12, and 30 of pregnancy. The proteins were transferred onto a PVDF membrane. Proteins on the blot were detected with rabbit anti-bovine 20α-HSD antibody, followed by staining with secondary antibody linked to anti-rabbit IgG-peroxide. M, marker; p-part, before parturition.
Figure 6
Figure 6
Immunoblot analysis of AKR1C1 recombinant protein in CHO-K1 cells. The bands corresponding to the protein produced by pcDNA3 + pAKR1C1 and pcDNA4/HisMax + pAKR1C1 vectors were detected. After gel electrophoresis, the proteins were transferred to a nitrocellulose membrane. The protein on the blot was detected with rabbit anti-bovine 20α-HSD, followed by staining with anti-rabbit IgG-POD. Lane 1, pcDNA3 + pAKR1C1; lane 2, pcDNA4/HisMax + pAKR1C1.
Figure 7
Figure 7
Immunohistochemical localization of pig AKR1C1 protein in the ovary on days 2, 5, 10, and 15 of the estrous cycle and before parturition. A. Ovary on day 2 of the estrous cycle (negative control). B. Ovary on day 2 of the estrous cycle. C. Ovary on day 5 of the estrous cycle. D. Ovary on day 10 of the estrous cycle. E. Ovary on day 15 of the estrous cycle. F. Ovary before parturition. Representative immunohistochemical analyses are shown for anti-bovine 20α-HSD (1:1,000). Anti-rabbit IgG (1:500) was used as a secondary antibody. For the negative control, normal rabbit serum (1:100) was used instead of the secondary antibody. Scale bar: 200 um (A, B, C, D, E, and F); 100 um (A', B', C', D', E', and F'), and 50 um (A'', B'', C'', D'', E'', and F''). Green arrow indicated the granulose layer and black arrow indicate the large luteal cells. Red arrow indicated the luteal cells.

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Penning TM. Molecular endocrinology of hydroxysteroid dehydrogenase. Endocr Rev. 1997;18(3):281–305. doi: 10.1210/er.18.3.281. - DOI - PubMed
    1. Jez JM, Bennett MJ, Schlegel BP, Lewis M, Penning TM. Comparative anatomy of the aldoketo reductase superfamily. Biochem J. 1997;326(Pt3):625–636. - PMC - PubMed
    1. Penning TM, Jin Y, Steckelbroeck S, Lanisnik Lizner T, Lewis M. Structure-function of human 3 alpha-hydroxysteroid dehydrogenases: genes and proteins. Mol Cell Endocrinol. 2004;215(1-2):63–72. doi: 10.1016/j.mce.2003.11.006. - DOI - PubMed
    1. Zhang Y, Dufort I, Rheault P, Luu-The V. Characterization of a human 20α-HSD. J Mol Endocrinol. 2000;25(2):221–228. doi: 10.1677/jme.0.0250221. - DOI - PubMed
    1. Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA, Penning TM, Febbo PG, Balk SP. Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res. 2006;66(5):2815–2825. doi: 10.1158/0008-5472.CAN-05-4000. - DOI - PubMed

MeSH terms

LinkOut - more resources

Feedback