Gigantol Improves Cholesterol Metabolism and Progesterone Biosynthesis in MA-10 Leydig Cells

In aging males, androgen production by testicular Leydig cells decreases at a rate of approximately 1% per year. Phenolic compounds may enhance testosterone biosynthesis and delay the onset of male hypogonadism. Gigantol is a bibenzyl compound isolated from several types of orchids of the genus Dendrobium. This compound has various biological activities, including antioxidant activity. However, its capacity to regulate gene expression and steroid production in testicular Leydig cells has never been evaluated. We investigated the effect of gigantol on MA-10 Leydig cells’ gene expression using an RNA-Seq approach. To further investigate the structure-function relationship of the hydroxy-methoxyphenyl moiety of gigantol, experiments were also performed with ferulic acid and isoferulic acid. According to transcriptomic analysis, all genes coding for cholesterol biosynthesis-related enzymes are increased in response to gigantol treatment, resulting in increased lipid droplets accumulation. Moreover, treatments with 10 μM gigantol increased StAR protein levels and progesterone production from MA-10 Leydig cells. However, neither ferulic acid nor isoferulic acid influenced StAR protein synthesis and progesterone production in MA-10 Leydig cells. Thus, our findings indicate that gigantol improves cholesterol and steroid biosynthesis within testicular Leydig cells.

miR-199-3p Suppresses Cellular Migration and Viability and Promotes Progesterone Production in Goose Ovarian Follicles Before Selection by Targeting ITGB8 and Regulating Other ECM-related Genes

ABSTRACTThe extracellular matrix (ECM) constitutes the follicular basal lamina and is also present between follicular cells. Remodeling of the ECM is believed to be a key event in follicular development, especially follicular selection, and plays important roles in cell migration, survival, and steroidogenesis. miR-199-3p is differentially expressed in the goose follicular granulosa layer during follicular selection and is reported to play a primary role in inhibiting cell migration and invasion. Nevertheless, the effect of miR-199-3p on ovarian follicles and its role in follicular cellular migration are not understood. In this study, we demonstrated by qRT-PCR that miR-199-3p was differentially expressed in the granulosa layer from goose ovarian follicles before and after follicular selection. Additionally, we found that miR-199-3p overexpression could significantly suppress cell viability and migration, as well as elevate both the concentration of progesterone and the expression of key progesterone production genes in cultured granulosa cells (GCs) from goose pre-hierarchical follicles. Furthermore, using dual-fluorescence reporter experiments on 293T cells, we confirmed that miR-199-3p downregulated the expression of the ECM gene ITGB8 by directly targeting its mRNA three prime untranslated region (3′ UTR). Finally, we found that miR-199-3p overexpression in the GCs of goose pre-hierarchical follicles inhibited the expression of two ECM-related genes (MMP9 and MMP15) yet promoted the expression of another two ECM-related genes (COL4A1 and LAMA1). Taken together, these findings suggest that miR-199-3p participates in granulosa cell migration, viability, and steroidogenesis in goose ovarian follicles before selection by targeting ITGB8 and modulating other ECM-related genes. These data highlight the key roles of miR-199-3p in follicular cell migration, viability, and steroidogenesis by regulating ECM-related genes and thus contribute to a better understanding of the mechanisms underlying follicle selection in birds.

Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β (TGFβ) family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GC and its interaction with gremlin or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GC but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GC. In small-follicle GC, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but gremlin inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GC. In large-follicle GC, BMP4 (10-30 ng/mL) increased (P < 0.05) GC numbers and gremlin (100 ng/mL) blocked this effect. In large-follicle GC, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, gremlin, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large versus small follicles.

Synergistic Regulatory Effect of Inhibin and Anti-Müllerian Hormone on Fertility of Mice

Inhibin (INH) and anti-Müllerian hormone (AMH) are essential in ovarian folliculogenesis and play an inhibitory role in mammalian fertility. However, the interactive effect of INH and AMH on the animal reproduction remains unknown. This study aimed to determine the possible interaction and synergy between INH and AMH in steroidogenesis by primary granulosa cells, and investigate their synergistic effect on fertility in mice. In in vitro granulosa cell culture system, we found that the treatment of either INHA or AMH had no significant effect on basal estradiol and progesterone production, whereas both significantly attenuated FSH-induced steroid hormone secretion. Importantly, combined treatment with INHA and AMH showed additive inhibitory effect on FSH-induced estradiol and progesterone production, accompanying a significant downregulation in the expression of FSH-stimulated CYP19A1, HSD3B, CYP11A1, StAR transcripts. The interrelationship of INH and AMH combinations was further investigated through active immune neutralization strategy. Female mice were immunized against INH and AMH eukaryotic expression plasmids, and the litter size was recorded after successfully mating. We observed that both INH and AMH plasmids were able to induce either anti-AMH or anti-INH antibodies in the immunized mice. In comparison with the control group, co-immunization with INH and AMH plasmids induced higher levels of estradiol, resulting in more litter size. Moreover, there was no significant difference on the offspring's weight between each group. Collectively, the results of the present study suggest that INH and AMH have synergistic effect in regulating steroidogenesis and the litter size in mice.

Cloprostenol, a synthetic analogue of prostaglandin F2α induces functional regression in cultured luteal cells of felids†

In the present study, we investigated the effect of the synthetic analogue of prostaglandin F2α (PGF2α)—cloprostenol—on cultured steroidogenic luteal cells of selected felid species over a two day culture period. The changes induced by cloprostenol were measured based on progesterone concentration and mRNA expression analysis of selected genes. Cloprostenol significantly reduced concentration of progesterone in cell culture medium of small luteal cells isolated from domestic cat corpora lutea (CL) at the development/maintenance stage (p < 0.05), but did not influence progesterone production in cultured cells from the regression stage. A decrease or complete silencing of progesterone production was also measured in cultured luteal cells of African lion (formation stage) and Javan leopard (development/maintenance stage). Gene expression analysis by real-time PCR revealed that treatment with cloprostenol did not have an influence on expression of selected genes coding for enzymes of steroidogenesis (StAR, HSD3B, CYP11A1) or prostaglandin synthesis (PTGS2, PGES), nor did it effect hormone receptors (AR, ESR1, PGR, PTGER2), an anti-oxidative enzyme (SOD1) or factors of cell apoptosis (FAS, CASP3, TNFRSF1B, BCL2) over the studied period. Significant changes were measured only for expressions of luteinizing hormone (P < 0.05), prolactin (P < 0.05) and PGF2α receptors (P < 0.005) (LHCGR, PRLR and PTGFR). The obtained results confirm that PGF2α/cloprostenol is a luteolytic agent in CL of felids and its impact on progesterone production depends on the developmental stage of the CL. Cloprostenol short term treatment on luteal cells was associated only with functional but not structural changes related to luteal regression.

FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells

Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured in vitro, FSH treatment increased the level of the autophagic marker, LC3-II, as well as increased the number of autophagic vacuoles in GCs. The underlying molecular mechanism and biological functions were then investigated in porcine GCs. Our results demonstrated that FSH could upregulate Beclin1 levels in porcine GCs; however, this effect was blocked by LY294002 (a PI3K/AKT inhibitor) and SP600125 (SAPK/JNK inhibitor). Further research confirmed that the transcriptional factor, c-Jun, was phosphorylated by FSH, then translocated into the nucleus from the cytoplasm and bound to the BECLIN1 promoter region, and that LY294002, SP600125, or c-Jun knockdown prevented the increase in Beclin1 levels induced by FSH. Interestingly, inhibition of autophagy using chloroquine or SP600125 decreased progesterone production in porcine GCs treated with FSH, although the expression of StAR and P450scc was not disturbed. Moreover, FSH treatment reduced the average number and size of LDs in porcine GCs, but these effects were eliminated by knocking down the key autophagy genes, ATG5 and BECLIN1; in addition, the effect of FSH on promoting progesterone secretion by the cells was also reduced significantly. Based on the above results, we concluded that FSH promoted progesterone production by enhancing autophagy through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs, independent of the classical steroidogenic pathway.