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.

The involvement of hypoxia-inducible factor 1α (HIF1α)-stabilising factors in steroidogenic acute regulatory (STAR) protein-dependent steroidogenesis in murine KK1 granulosa cells in vitro

As a component of hypoxia-inducible factor1 (HIF1)-complexes, HIF1α regulates the expression of steroidogenic acute regulatory (STAR) protein in granulosa cells. However, severe hypoxia or exaggeratedly expressed HIF1α have detrimental effects. HIF1α is regulated by factor inhibiting HIF (FIH), prolyl hydroxylases (PHD1, 2, 3) and von Hippel-Lindau (VHL) suppressor protein. In this study, the expression of FIH, PHD1, 2, 3 and VHL was investigated in murine ovaries and immortalised KK1 granulosa cells. We found FIH, VHL and PHD2 transcripts predominantly in growing tertiary follicles. Functional aspects were assessed in KK1 cells exposed to decreasing O2 (20%, 10%, 1%), by determining HIF1α, FIH, VHL, PHD1–3 and STAR expression. The main findings indicated gradually increasing PHD2 under lowered O2. Functional blocking of PHDs revealed biphasic effects on STAR expression; concomitantly with increasing HIF1α, STAR expression, which was initially induced, decreased significantly when HIF1α was strongly stabilised. Finally, PHD2 in particular might act as a specific regulator of HIF1α and, thereby, of STAR availability in granulosa cells.

Protective effect of melatonin against methotrexate-induced testicular damage in the rat model: An experimental study

Background: Methotrexate (MTX) has been shown to affect the testes adversely, especially the seminiferous epithelium. As melatonin, an endocrine hormone, has been shown to normalize testicular function, its ability to prevent MTX-induced testicular damage should be considered. Objective: Based on the antioxidant, anti-inflammatory, and antiapoptotic activities of melatonin, this study aimed to investigate its protective effect against testicular damage induced by MTX. Materials and Methods: Forty adult male rats (200-230 g) were divided into five groups (n = 8/each). The rats in group I were injected with vehicle as a control. In group II, the rats were received intraperitoneal injections of melatonin (8 mg/kg) for 15 consecutive days. The rats in group III were intravenously injected with MTX (75 mg/kg) for 15 consecutive days. The remaining two groups received melatonin (8 mg/kgBW) for 15 (group IV) and 30 (group V) consecutive days, intraperitoneally, and then intravenously received MTX (75 mg/kgBW) on days 8 and 15 of the experimental period. Reproductive parameters, including epididymal sperm concentration, testicular tyrosine-phosphorylated protein expression, steroidogenic acute regulatory (StAR) protein expression, and caspase-3 and malondialdehyde levels, were examined. Results: The sperm concentrations (×106/ml) of groups IV (58.75 ± 1.28) and V (55.93 ± 2.57) were improved significantly (p = 0.032) compared with that of group II (32.92 ± 2.14). The seminiferous epithelium in groups IV and V also increased, while caspase- 3 expression decreased. In the melatonin-treated groups, the expression of tyrosinephosphorylated proteins at 32 kDa was decreased and that of proteins at 47 kDa was increased compared with the MTX group. StAR protein expression was not altered in any of the groups. Conclusion: Our results indicate that melatonin improves the epididymal sperm concentration by decreasing the expression of caspase-3 and increasing that of tyrosine-phosphorylated proteins in MTX-treated testes. Key words: Melatonin, Testis, Sperm, Methotrexate, Caspase-3, Tyrosine phosphorylation.

106 Randel Lecture: The history of the discovery of the Steroidogenic Acute Regulatory (StAR) Protein

This two-part presentation regarding acute regulation of steroid biosynthesis documents discovery of the StAR protein and resolves controversy regarding mitochondrial cholesterol transport. The acute regulation of steroid biosynthesis was known to require de novo synthesis of a regulator protein to mediate the transfer of cholesterol, the substrate for steroids, from the outer to the inner mitochondrial membrane where it was converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme. We discovered a novel protein that was tightly correlated with steroid biosynthesis and had the requisite characteristics for the putative acute regulator of cholesterol transfer for steroid synthesis. Further studies confirmed that StAR protein is an indispensable component in the process of mitochondrial uptake of the cholesterol substrate for steroidogenesis. The translocator protein (TSPO) is a mitochondrial outer membrane protein suggested to import cholesterol to the inner mitochondrial membrane. However, it was demonstrated in vivo in Leydig cell specific TSPO conditional knockout mice that TSPO was not required for testosterone production or fertility. Similarly, global TSPO knockout (TSPO/-) mice were viable and fertile with fecundity equivalent to TSPO floxed (TSPOfl/fl) controls. Adrenal and gonadal steroidogenesis did not differ between TSPOfl/ fl and TSPO-/- mice. In vitro use of different steroidogenic cell line models (MA-10, MLTC, Y-1, H295R and R2C) demonstrated that siRNA-knockdown of TSPO did not affect steroidogenesis. Also, CRISPR/ Cas9-mediated TSPO deletion did not affect MA-10 cell steroidogenesis. These results directly 1) refute the suggestion that TSPO is indispensable for viability and steroid hormone biosynthesis; and, 2) substantiate the primal role of the StAR protein as the rate limiting factor in steroid hormone biosynthesis.

Genomic Profiling of the Steroidogenic Acute Regulatory Protein in Breast Cancer: In Silico Assessments and a Mechanistic Perspective

Cancer is a multifactorial condition with aberrant growth of cells. A substantial number of cancers, breast in particular, are hormone sensitive and evolve due to malfunction in the steroidogenic machinery. Breast cancer, one of the most prevalent form of cancers in women, is primarily stimulated by estrogens. Steroid hormones are made from cholesterol, and regulation of steroid/estrogen biosynthesis is essentially influenced by the steroidogenic acute regulatory (StAR) protein. Although the impact of StAR in breast cancer remains a mystery, we recently reported that StAR protein is abundantly expressed in hormone sensitive breast cancer, but not in its non-cancerous counterpart. Herein, we analyzed genomic profiles, hormone receptor expression, mutation, and survival for StAR and steroidogenic enzyme genes in a variety of hormone sensitive cancers. These profiles were specifically assessed in breast cancer, exploiting The Cancer Genome Atlas (TCGA) datasets. Whereas StAR and key steroidogenic enzyme genes evaluated (CYP11A1, HSD3B, CYP17A1, CYP19A1, and HSD17B) were altered to varying levels in these hormone responsive cancers, amplification of the StAR gene was correlated with poor overall survival of patients afflicted with breast cancer. Amplification of the StAR gene and its correlation to survival was also verified in a number of breast cancer studies. Additionally, TCGA breast cancer tumors associated with aberrant high expression of StAR mRNA were found to be an unfavorable risk factor for survival of patients with breast cancer. Further analyses of tumors, nodal status, and metastases of breast cancer tumors expressing StAR mRNA displayed cancer deaths in stage specific manners. The majority of these tumors were found to express estrogen and progesterone receptors, signifying a link between StAR and luminal subtype breast cancer. Collectively, analyses of genomic and molecular profiles of key steroidogenic factors provide novel insights that StAR plays an important role in the biologic behavior and/or pathogenesis of hormone sensitive breast cancer.

Effect of follicle size and atresia grade on mitochondrial membrane potential and steroidogenic acute regulatory protein expression in bovine granulosa cells

SummaryDuring follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Oestrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens that arise from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GCs) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle walls and cumulus–oocyte complexes (COC) and assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped into small, medium or large follicles. After follicle opening, the COCs were morphologically classified for follicle atresia and the GCs were collected. Granulosa cells were fixed for immunofluorescence (IF) and TUNEL assay, frozen for western blotting (WB) or freshly maintained for MMP analyses. StAR protein expression was assessed using both IF and WB analyses. The follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. Granulosa cells collected from small non-atretic follicles showed a higher (P <0.01) MMP and WB-based StAR protein expression than small atretic follicles. For IF analysis, StAR protein expression in large atretic follicles was higher (P <0.05) than that in large non-atretic follicles. These results suggest a role played by mitochondria in GC steroidogenic activity, which declines in healthy follicles along with their growth. In large follicles, steroidogenic activity increases with atresia and is possibly associated with progesterone production.