The effect of orexin B on steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase gene expression, and progesterone and androstenedione secretion by the porcine uterus during early pregnancy and the estrous cycle

Abstract

The aim of this study was to investigate the effect of orexin B (OXB) on progesterone (P4) and androstenedione (A4) secretion by porcine endometrial and myometrial tissue explants and on the expression of key steroidogenic proteins and enzymes involved in steroid production. The hormones secretion and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase (HSD3B1) were analyzed on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy and during the luteal phase of the estrous cycle (days 10 to 11). Endometrial and myometrial explants were cultured in vitro in the presence of OXB (1, 10, or 100 nM) and OXB (1, 10, or 100 nM) with 1 µM of JNJ (OX2R antagonist). Gene expression was examined by real-time PCR, and steroid secretion was determined by radioimmunoassay. Orexin B modulated StAR, CYP11A1, HSD3B1 mRNA content depending on the type of uterine tissue, the applied OXB dose, and the stage of pregnancy or the estrous cycle (P < 0.05). Orexin B increased P4 secretion in all stages of early gestation (P < 0.05). Orexin B enhanced the release of A4 on days 12 to 13, 15 to 16, and 27 to 28 of gestation, whereas on days 10 to 11 of early pregnancy, A4 secretion decreased in the endometrium and increased in the myometrium (P < 0.05). These results indicate that OXB affects the expression of key steroidogenic regulators and the secretion of steroid hormones in the porcine uterus during early pregnancy.

Figure 1.

The influence of OXB (1, 10, and 100 nM) in the presence or absence of the selective antagonist of orexin receptor type 2 (JNJ—JNJ10397049) on the relative expression of StAR gene in endometrial (A) and myometrial (B) tissue explants harvested from pigs on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy, and on days 10 to 11 of the estrous cycle preincubated for 2 h and subsequently incubated for 24 h. The expression levels were normalized with the geometric mean expression of reference genes: cyclophilin and β-actin. Data are expressed as the mean ± standard error of the mean (n = 5). Bars with different letters are significantly different at P < 0.05.

Figure 2.

The influence of OXB (1, 10, and 100 nM) in the presence or absence of the selective antagonist of orexin receptor type 2 (JNJ—JNJ10397049) on the relative expression of CYP11A1 gene in endometrial (A) and myometrial (B) tissue explants harvested from pigs on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy, and on days 10 to 11 of the estrous cycle preincubated for 2 h and subsequently incubated for 24 h. The expression levels were normalized with the geometric mean expression of reference genes: cyclophilin and β-actin. Data are expressed as the mean ± standard error of the mean (n = 5). Bars with different letters are significantly different at P < 0.05.

Figure 3.

The influence of OXB (1, 10, and 100 nM) in the presence or absence of the selective antagonist of orexin receptor type 2 (JNJ—JNJ10397049) on the relative expression of HSD3B1 gene in endometrial (A) and myometrial (B) tissue explants harvested from pigs on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy, and on days 10 to 11 of the estrous cycle preincubated for 2 h and subsequently incubated for 24 h. The expression levels were normalized with the geometric mean expression of reference genes: cyclophilin and β-actin. Data are expressed as the mean ± standard error of the mean (n = 5). Bars with different letters are significantly different at P < 0.05.

Figure 4.

The influence of OXB (1, 10, and 100 nM) in the presence or absence of the selective antagonist of orexin receptor type 2 (JNJ—JNJ10397049) on P4 secretion by endometrial (A) and myometrial (B) tissue explants harvested from pigs on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy, and on days 10 to 11 of the estrous cycle preincubated for 2 h and subsequently incubated for 24 h. Data are expressed as the mean ± standard error of the mean (n = 5). Bars with different letters are significantly different at P < 0.05.

Figure 5.

The influence of OXB (1, 10, and 100 nM) in the presence or absence of the selective antagonist of orexin receptor type 2 (JNJ—JNJ10397049) on A₄ secretion by endometrial (A) and myometrial (B) tissue explants harvested from pigs on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy, and on days 10 to 11 of the estrous cycle preincubated for 2 h and subsequently incubated for 24 h. Data are expressed as the mean ± standard error of the mean (n = 5). Bars with different letters are significantly different at P < 0.05.