Interaction between orexin A and bone morphogenetic protein system on progesterone biosynthesis by rat granulosa cells

Abstract

The involvement of orexins in reproductive function has been gradually uncovered. However, the functional role of orexins in ovarian steroidogenesis remains unclear. In the present study, we investigated the effects of orexin A on ovarian steroidogenesis by using rat primary granulosa cells that express both OX1 and OX2 receptors for orexins. Treatment with orexin A enhanced progesterone, but not estradiol, biosynthesis induced by FSH, whereas it did not affect basal levels of progesterone or estradiol. In accordance with the effects on steroidogenesis, orexin A increased the mRNA levels of progesterogenic enzymes, including StAR, P450scc and 3βHSD, but not P450arom, and cellular cAMP synthesis induced by FSH. Under the condition of blockage of endogenous BMP actions by noggin or BMP-signaling inhibitors, orexin A failed to increase levels of progesterone synthesis induced by FSH treatment, suggesting that endogenous BMP activity in granulosa cells might be involved in the enhancement of progesterone synthesis by orexin A. Treatment with orexin A impaired Smad1/5/9 activation as well as Id-1 mRNA expression stimulated by BMP-6 and BMP-7, the latter of which was reversed by treatment with an OX1 antagonist. It was also found that orexin A suppressed the mRNA expression of both type-I and -II receptors for BMPs and increased that of inhibitory Smad6 and Smad7 in granulosa cells. On the other hand, treatments with BMP-6 and -7 suppressed the expression of OX1 and OX2. Collectively, the results indicated that orexin A enhances FSH-induced progesterone production, at least in part, by downregulating BMP signaling in granulosa cells. Thus, a new role of orexin A in facilitating progesterone synthesis and functional interaction between the orexin and BMP systems in granulosa cells were revealed.

Interaction of ovarian steroidogenesis and clock gene expression modulated by bone morphogenetic protein-7 in human granulosa cells

“…In the present experiments, it was found that BMP-7 affected the levels of 3βHSD expression rather than StAR, though this discrepancy could be due to the characteristic differences between rat primary granulosa cells and human KGN cells. As other humoral modulators related to ovarian steroidogenesis, androgens [15], incretins [16], orexins [17] and melatonin [35], which can affect endogenous BMP-Smad signaling activity in granulosa cells [36,37], may also be involved in the modulation of Clock gene expression in the ovary. Further studies are necessary to determine the functional interaction between the activities of Clock-related molecules and ovarian steroidogenesis.…”

“…KGN cells (1 × 10 5 cells/mL) were treated with forskolin (1 μM) or BMPs (100 ng/mL) in 12-well plates containing serum-free DMEM/F12 for the indicated periods. Concentrations of forskolin and BMP ligands used in the current experiments were selected based on our earlier data obtained from the same in vitro experiments [14][15][16][17]. Total cellular RNA was extracted using TRI Reagent ® (Cosmo Bio Co., Ltd., Tokyo, Japan) and the concentration of extracted RNA were determined by NanoDrop TM One spectrophotometer (Thermo Fisher Scientific, Waltham, MA).…”

“…Total cellular RNA was extracted using TRI Reagent ® (Cosmo Bio Co., Ltd., Tokyo, Japan) and the concentration of extracted RNA were determined by NanoDrop TM One spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Primer pairs for detecting the following genes: steroidogenic acute regulatory protein (StAR), steroid side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), aromatase (P450arom), and a housekeeping gene, ribosomal protein L19 (RPL19), were utilized as we earlier reported [13,[15][16][17][18][19]. Other primers were also selected from different exons to eliminate PCR products originated from chromosomal DNA as follows: 966-985 and 1,099-1,118 for Bmal1 (from GenBank accession #AB000812); 2,072-2,091 and 2,262-2,281 for Clock (NM_001267843); 1,614-1,633 and 1,870-1,889 for Per2 (NM_0022817); and 2,203-2,222 and 2,461-2,480 for Cry1 (NM_ 004075).…”

“…Other primers were also selected from different exons to eliminate PCR products originated from chromosomal DNA as follows: 966-985 and 1,099-1,118 for Bmal1 (from GenBank accession #AB000812); 2,072-2,091 and 2,262-2,281 for Clock (NM_001267843); 1,614-1,633 and 1,870-1,889 for Per2 (NM_0022817); and 2,203-2,222 and 2,461-2,480 for Cry1 (NM_ 004075). Reverse transcription using ReverTra Ace ® (TOYOBO CO., LTD., Osaka, Japan) was performed and proceeded to real-time PCR using the LightCycler ® Nano real-time PCR system and LightCycler ® 96 System (Roche Diagnostic Co., Tokyo, Japan) after optimizing annealing conditions and amplification efficiency [17]. Determination of target gene mRNA level was performed by the method using Δ threshold cycle (Ct), in which subtraction of the Ct value of RPL19 from that of the target genes was utilized to calculate the ΔCt value.…”

Effect of the interaction of metformin and bone morphogenetic proteins on ovarian steroidogenesis by human granulosa cells

“…The extracted RNA (0.5e1 mg) was subjected to reverse transcription (RT) with ReverTra Ace ® (TOYOBO CO., LTD., Osaka, Japan). Quantitative real-time PCR was performed using the LightCycler ® Nano real-time PCR system (Roche Diagnostic Co., Tokyo, Japan) under the optimized annealing conditions and amplification efficiency [14]. The mRNA levels of target genes were analyzed by the D threshold cycle (Ct) method, in which DCt values were obtained by subtracting the Ct value of RPL19 from that of the target.…”