Comparison of Placental HSD17B1 Expression and Its Regulation in Various Mammalian Species

Abstract

During mammalian gestation, large amounts of progesterone are produced by the placenta and circulate for the maintenance of pregnancy. In contrast, primary plasma estrogens are different between species. To account for this difference, we compared the expression of ovarian and placental steroidogenic genes in various mammalian species (mouse, guinea pig, porcine, ovine, bovine, and human). Consistent with the ability to synthesize progesterone, CYP11A1/Cyp11a1, and bi-functional HSD3B/Hsd3b genes were expressed in all species. CYP17A1/Cyp17a1 was expressed in the placenta of all species, excluding humans. CYP19A/Cyp19a1 was expressed in all placental estrogen-producing species, whereas estradiol-producing HSD17B1 was only strongly expressed in the human placenta. The promoter region of HSD17B1 in various species possesses a well-conserved SP1 site that was activated in human placental cell line JEG-3 cells. However, DNA methylation analyses in the ovine placenta showed that the SP1-site in the promoter region of HSD17B1 was completely methylated. These results indicate that epigenetic regulation of HSD17B1 expression is important for species-specific placental sex steroid production. Because human HSD17B1 showed strong activity for the conversion of androstenedione into testosterone, similar to HSD17B1/Hsd17b1 in other species, we also discuss the biological significance of human placental HSD17B1 based on the symptoms of aromatase-deficient patients.

Keywords: HSD17B1; androstenedione; human; ovine; placenta; testosterone.

Figure 1

Pathways for producing androgens and estrogens in mammalian ovary and placenta. Human, bovine, ovine, and porcine steroidogenic enzymes are indicated totally by capital letters, whereas rodent counterparts are indicated only initially by a capital letter.

Figure 2

Expression of steroidogenic genes in ovary and placenta of various mammalian species. RT-PCR analyses of each gene in the ovary and placenta of a mouse (A), guinea pig (B), porcine (C), ovine (D), bovine (E), and human (F).

Figure 3

Analyses of HSD17B1 promoter region (A) Alignment for the nucleotide sequence of HSD17B1 gene and its 5′-flanking region in human, mouse, porcine, bovine, and ovine. The asterisks (*) are the conserved nucleotides among all species. The conserved SP1 site is shown by a redline box. Putative transcription start and translation start sites are shown by the yellow and blue boxes, respectively. Cytosine bases in CpG dinucleotides of ovine HSD17B1 promoter region are shown by green boxes. (B) Activation of human and ovine HSD17B1 promoter in human placenta-derived JEG-3 cells. Each vector was transfected by lipofection into JEG-3 cells. At 48 h after transfection, luciferase assays were performed using the cell lysates. Values of the pGL4.10 Basic vector were defined as 1. Data are the mean  ±  SEM values of four independent experiments * p < 0.05 vs. pGL4.10. (C) Methylation analysis of the promoter region (−79 to +41) of the HSD17B1 gene in the ovine placenta. The methylation pattern of individual bisulfite-sequenced clones is indicated. Each circle denotes cytosine bases in CpG dinucleotides, and filled circles represent methylated and unmethylated cytosines, respectively.

Figure 4

Comparison of enzymatic activities of HSD17B1/Hsd17b1 in various species. (A) Western blot analyses were performed with the antibodies against FLAG-tag and GAPDH using lysates of GFP- and FLAG-tagged HSD17B1/Hsd17b1 (mouse, porcine, ovine, bovine, and human)-introduced HEK293 cells. (B) Activation of AR-mediated transcription by culture media from GFP or each HSD17B1/Hsd17b1-introduced HEK293 cells collected at 2 h after A4 addition. CV-1 cells were transfected with ARE-Luc and AR expression vectors. At 24 h post-transfection, cells were incubated with vehicle (lane C), A4 (1 nM), T (1 nM), or culture medium from GFP- and each species HSD17B1/Hsd17b1-expressing HEK293 cells for 24 h. Values of the vehicle were defined as 1. Data represent the mean ± SEM of four independent experiments. Values marked by the different letters are significantly different from each other (p < 0.05).