Steroidogenic Acute Regulatory Protein Expression Research Articles

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

The involvement of let-7 in hCG-induced progesterone synthesis via regulating p27Kip1 and p21Cip1 expression

Progesterone is essential in females to maintain a regular menstrual cycle and pregnancy. The luteinizing hormone (LH) surge induces the luteinization of granulosa cells and thecal cells to form the corpus luteum, which is responsible for progesterone synthesis. However, the specific mechanism of how hCG, the analog of LH, regulates progesterone synthesis has yet to be fully discovered. In this study, we found that progesterone level was increased in adult wild-type pregnant mice 2 and 7 days post-coitum, along with a decrease in let-7 expression compared with the estrus stage. Besides, the let-7 expression was negatively correlated with progesterone level in post-delivery day 23 wild-type female mice after being injected with PMSG and hCG. Then, using let-7 transgenic mice and a human granulosa cell line, we found that overexpression of let-7 antagonized progesterone level via targeting p27Kip1 and p21Cip1 and steroidogenic acute regulatory protein (StAR) expression, which is a rate-limiting enzyme in progesterone synthesis. Furthermore, hCG suppressed let-7 expression by stimulating the MAPK pathway. This study elucidated the role of microRNA let-7 in regulating hCG-induced progesterone production and provided new insights into its role in clinical application.

Polystyrene nanoplastics inhibit StAR expression by activating HIF-1α via ERK1/2 MAPK and AKT pathways in TM3 Leydig cells and testicular tissues of mice.

Microplastics (MPs) and nanoplastics (NPs) are widely found in water, food and air, and have been found in human blood, lung and feces. Several studies in vivo have shown that MPs and NPs decrease testosterone level. However, the molecular mechanism of MPs and NPs leading to testosterone reduction remains unclear. In the present study, mice were treated with 50 μg/kg·day polystyrene (PS)-NPs by tail vein injection once daily for two consecutive days, the mRNA and protein levels of steroidogenic acute regulatory protein (StAR) decreased significantly in testis. TM3 Leydig cells were treated with non-toxic doses of PS-NPs, hypoxia-inducible factor-1α (HIF-1α) mRNA translation was induced by PS-NPs through mTOR/4E-BP1 pathway, which was activated by the ERK1/2 MAPK and AKT pathways. Simultaneously, increased HIF-1α protein inhibited StAR transcription. Additionally, reactive oxygen species production induced by PS-NPs played a central role in the activation of ERK1/2 MAPK/mTOR and AKT/mTOR signaling pathways. These results suggest that PS-NPs down-regulate StAR expression by increasing HIF-1α, which is induced by activation of mTOR/4E-BP1 through the ERK1/2 MAPK and AKT signaling pathways. Our findings provide new insight into the potential molecular mechanism by which PS-NPs impair testosterone synthesis and male reproductive function.

Rhamnetin alleviates polystyrene microplastics-induced testicular damage by restoring biochemical, steroidogenic, hormonal, apoptotic, inflammatory, spermatogenic and histological profile in male albino rats.

The current research was performed to evaluate the ameliorative effects of Rhamnetin (RHM) on polystyrene microplastics (PS-MPs)-instigated testicular dysfunction in male albino rats. 48 albino rats were distributed in four groups, i.e., control, PS-MPs treated, PS-MPs + RHM co-treated and RHM only supplemented group. PS-MPs exposure considerably reduced anti-oxidant enzymes i.e., catalase (CAT), glutathione peroxidase (GSR), superoxide dismutase (SOD) and glutathione reductase (GPx) activities. Whereas, reactive oxygen species (ROS) level along with malondialdehyde (MDA) was considerably escalated in PS-MPs treated rats as well as a potential decline was observed in sperm progressive motility. Additionally, a substantial upsurge was noticed in the count of dead sperms, deformity in the tail, mid-piece and head of sperms in PS-MPs treated rats. PS-MPs exposure also decreased steroidogenic enzymes, 17β-hydroxysteroid dehydrogenase (17β-HSD), steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) expressions. Moreover, the levels of inflammatory indices i.e., Interleukin-6 (IL-6), Nuclear factor kappa-B (NF-κB), Interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) activity were also increased in PS-MPs administrated group. Besides it increased the expression of apoptotic markers (Bax and caspase-3) expression. Whereas, anti-apoptotic marker i.e., Bcl-2 expression was reduced. Moreover, luteinizing hormone (LH), follicle-stimulating hormone (FSH) as well as plasma testosterone levels were also decreased. PS-MPs exposure also led to a substantial histopathological damage in testicular tissues. However, RHM supplementation potentially reduced the damaging effects of PS-MPs in the reproductive tissues of male albino rats. Thus, the current study revealed, RHM possesses potential to prevent PS-MPs-induced testicular damage due to its anti-oxidant anti-apoptotic, anti-inflammatory as well as androgenic properties.

Effect of maternal periconceptional undernutrition in sheep on cortisol regulation in offspring from mid-late gestation, through to adulthood.

Maternal periconceptional undernutrition (PCUN) alters fetal hypothalamic-pituitary-adrenal axis (HPAA) function and placental glucocorticoid metabolism in sheep. The effects of PCUN on HPAA function in adult life are not known. We investigated the effects of PCUN on fetal adrenal development across gestation and on cortisol regulation in adult offspring. Ewes were undernourished from 61 days before to 30 days after conception ('PCUN') or fed ad libitum ('N'). mRNA expression in the fetal adrenal gland of ACTH receptor (ACTHR), steroidogenic acute regulatory protein (STAR), cytochrome P450 17A1 (CYP17A1), 11beta-hydroxysteroid-dehydrogenase type 2 (11βHSD2), insulin-like growth factor-2 (IGF2), and in the fetal hippocampus of 11βHSD1, 11βHSD2, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) was determined at 50 (adrenal only), 85, 120 and 131 days of gestation (term=148 days). In adult offspring (≥ 3 years, N; 10 female, 5 male, PCUN; 10 female, 10 male) a combined arginine vasopressin (AVP, 0.1 μg/kg) and corticotropin-releasing hormone (CRH, 0.5 μg/kg) challenge and a metyrapone (40 mg/kg) challenge were undertaken. mRNA expression of ACTHR, STAR and CYP17A1 were determined in adult adrenals. Fetal adrenal STAR, CYP17A1 and IGF2 mRNA expression were not different between groups in early gestation but were higher in PCUN than N at 131 days' gestation (all p<0.01). PCUN reduced fetal hippocampal MR and GR mRNA expression by 50% at 85 day, but not in later gestation. Adult offspring plasma cortisol responses to AVP+CRH or metyrapone were not different between groups. Plasma ACTH response to AVP+CRH was lower in PCUN males but ACTH response to metyrapone was not different between groups. Adult adrenal ACTHR, STAR, and CYP17A1 mRNA expression were not affected by PCUN. We conclude that the effects of PCUN on fetal HPAA function that became apparent in late gestation, are not reflected in adrenal cortisol secretion in mid-adulthood.

Progesterone modulates HSD11B1-mediated cortisol production in luteinized bovine granulosa cells.

Progesterone (P4) and cortisol production increase in luteinized granulosa cells (LGCs) during the periovulatory period, but their interaction is not well established. Therefore, we investigated their interaction in cultured bovine LGCs. Granulosa cells were collected from follicles of 2-5 mm in diameter and cultured in DMEM/F-12 supplemented with 10% fetal calf serum for up to 14 days. P4 production and the expression of steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) rapidly increased until day 10 and remained high thereafter. No de novo of cortisol from P4 was detected during the culture period. The expression of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts cortisone to cortisol, increased dramatically on day two, decreased until day 8, and remained relatively constant. To investigate how P4 and cortisol influence each other's production, LGCs were treated with trilostane (a P4 synthesis inhibitor), nomegestrol acetate (NA, a synthetic progestogen), P4, and/or cortisol for 24 h on days 6 and 12 of culture. Trilostane suppressed P4 and STAR expression while elevating HSD11B1 and HSD3B1 expression and cortisol production. Concomitant treatment with NA or P4 dose-dependently decreased cortisol production and HSD11B1 and HSD3B1 expression but elevated STAR expression in both days 6 and 12. Conversely, cortisol treatment increased HSD11B1 and HSD3B1 expression and decreased STAR expression without influencing P4 production. These results indicate that progestogens suppress cortisol production by modulating HSD11B1 expression and that progestogens and cortisol differentially regulate STAR, HSD3B1, and HSD11B1 expression in bovine LGCs.

The ERK Signaling Cascade Inhibits Gonadotropin-Stimulated Steroidogenesis.

The response of granulosa cells to Luteinizing Hormone (LH) and Follicle- Stimulating Hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the Steroidogenic Acute Regulatory Protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells

BackgroundHeparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments.ResultsWestern blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway.ConclusionThis study increases the understanding of the physiological role of HB-EGF in human luteal functions.7G2SU9-j2App9A69cYCW26Video

Astragalus polysaccharide protects sepsis model rats after cecum ligation and puncture.

To investigate the protective effect and mechanism of Astragalus polysaccharide (APS) on septic rats, the present project applied APS at concentrations of 400, 600, and 800mg/kg/d to rats for prophylactic administration for 7d, and a rat sepsis model was constructed by the cecum ligation and puncture (CLP) method. Forty-eight rats were divided into six groups of eight each. Each experiment was repeated at least three times. Rat serum levels of VD3, 25(OH)D3, 1,25(OH)2D3, IL-6, TNF-α, CRP, sICAM-1, corticosterone (CORT), and short-chain fatty acids (SCFAs) in each group were detected, and renal damage was observed by H&E. We also determined the protein expression of CYP27B1, CYP24A1, vitamin D receptor (VDR), steroidogenic acute regulatory protein (STAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP21A2, CYP17A1, and CYP11B1. An operational taxonomic unit (OTU) was used to determine the gut microbiota diversity of septic rats after prophylactic administration and before modeling. Results revealed that APS markedly increased the contents of 25(OH)D3 and 1,25(OH)2D3 but greatly decreased those of TNF-α, IL-6, CRP, sICAM-1, and CORT. APS alleviated renal tubular dilation and vascular congestion in rat kidneys and substantially reduced renal cell apoptosis. Moreover, the expression of CYP24A1, VDR, CYP11B1, CYP21A2, CYP17A1, STAR, and 3β-HSD in the kidneys of the H-APS group was substantially decreased compared to that of the model group, whereas CYP27B1 was markedly increased. GC-MS detection indicated a substantial increase in SCFAs and acetic acid content in the H-APS group versus model group. Through 16S sequencing, the abundance of genus and gut microbiota species increased in the APS groups compared to that of the control group. Taken together, APS increased the activity of the vitamin D axis, inhibited the production of inflammatory factors in the body, altered the structure of rat intestinal flora, and increased the amount of acetic acid and SCFAs in rats, thereby effectively hindering inflammation and organ damage in septic rats.

Prevention of Testicular Damage by Indole Derivative MMINA via Upregulated StAR and CatSper Channels with Coincident Suppression of Oxidative Stress and Inflammation: In Silico and In Vivo Validation.

Cis-diamminedichloroplatinum (II) (CDDP) is a widely used antineoplastic agent with numerous associated side effects. We investigated the mechanisms of action of the indole derivative N’-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide (MMINA) to protect against CDDP-induced testicular damage. Five groups of rats (n = 7) were treated with saline, DMSO, CDDP, CDDP + MMINA, or MMINA. Reproductive hormones, antioxidant enzyme activity, histopathology, daily sperm production, and oxidative stress markers were examined. Western blot analysis was performed to access the expression of steroidogenic acute regulatory protein (StAR) and inflammatory biomarker expression in testis, while expression of calcium-dependent cation channel of sperm (CatSper) in epididymis was examined. The structural and dynamic molecular docking behavior of MMINA was analyzed using bioinformatics tools. The construction of molecular interactions was performed through KEGG, DAVID, and STRING databases. MMINA treatment reversed CDDP-induced nitric oxide (NO) and malondialdehyde (MDA) augmentation, while boosting the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymis and testicular tissues. CDDP treatment significantly lowered sperm count, sperm motility, and epididymis sperm count. Furthermore, CDDP reduced epithelial height and tubular diameter and increased luminal diameter with impaired spermatogenesis. MMINA rescued testicular damage caused by CDDP. MMINA rescued CDDP-induced reproductive dysfunctions by upregulating the expression of the CatSper protein, which plays an essential role in sperm motility, MMINA increased testosterone secretion and StAR protein expression. MMINA downregulated the expression of NF-κB, STAT-3, COX-2, and TNF-α. Hydrogen bonding and hydrophobic interactions were predicted between MMINA and 3β-HSD, CatSper, NF-κβ, and TNFα. Molecular interactome outcomes depicted the formation of one hydrogen bond and one hydrophobic interaction between 3β-HSD that contributed to its strong binding with MMINA. CatSper also made one hydrophobic interaction and one hydrogen bond with MMINA but with a lower binding affinity of -7.7 relative to 3β-HSD, whereas MMINA made one hydrogen bond with NF-κβ residue Lys37 and TNF-α reside His91 and two hydrogen bonds with Lys244 and Thr456 of STAT3. Our experimental and in silico results revealed that MMINA boosted the antioxidant defense mechanism, restored the levels of fertility hormones, and suppressed histomorphological alterations.

Amylin regulates testosterone levels via steroidogenesis-related enzymes in the central nervous system of male mice

Amylin is a peripheral satiation signal polypeptide co-secreted with insulin by pancreatic β-cells in response to nutrient ingestion. Amylin participates in the eating-inhibitory effect and regulates energy metabolism by acting on the central nervous system (CNS). However, the role of amylin in regulating the biosynthesis of steroid hormones, such as testosterone, through the hypothalamic-pituitary-gonadal axis (HPG) remains unexplored. However, only limited evidence is available on the involvement of amylin in steroid synthesis, we hypothesize that amylin regulates testosterone levels via steroidogenesis-related enzymes in the CNS. In this study, we elucidated the effect of intraperitoneal injection of amylin on the protein expression of steroidogenesis-related enzymes, including 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17A1 (CYP17A1), and steroidogenic acute regulatory protein (StAR), and phospho-extracellular signal-regulated kinase (pERK). Additionally, the effect of amylin on testosterone levels in male mice was examined. Our results suggested that 3β-HSD and CYP17A1 neurons were widely expressed in the CNS of male mice, whereas StAR neurons were mainly expressed in the zona incerta (ZI) and locus coeruleus (LC) regions. Intraperitoneal injection of amylin significantly reduced (p < 0.01) the expression of 3β-HSD, CYP17A1, and StAR in ZI and other areas near the third ventricle (3 V) but increased (p < 0.01) pERK expression, brain testosterone levels, serum FSH, serum LH, and decreased (p < 0.01) serum testosterone levels in mice. In conclusion, amylin regulates testosterone levels via steroidogenesis-related enzymes in the central nervous system of male mice.

The aphrodisiac potential of β-cyclodextrin–curcumin via stimulating cAMP-PKA pathway in testicular Leydig cells

The water-soluble β-cyclodextrin–curcumin (CDC) is used in pharmaceutical applications and as a natural food colorant. The previous study revealed that curcumin potentially impacted the reproductive system. The present study investigated the possible roles of the CDC in testosterone secretion in Leydig cells and mice. Primary Leydig cells were treated with the CDC to determine their effect on cell proliferation, testosterone levels, the protein and mRNA expression of the transcription factor, and steroidogenic enzymes. Our data showed that CDC stimulated testosterone production via upregulating transcription factor steroidogenic factor-1 (NR5A1), cAMP-response element-binding protein (CREB), and steroidogenic enzymes steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1), 17-alpha-hydroxylase/17,20-lyase (CYP17A1), 3β-/17β-hydroxysteroid dehydrogenase type 1 (3β/17β-HSD, HSD3b1/HSD17b1). CDC could significantly stimulate H89-suppressed StAR and CREB expression but not reverse melatonin-suppressed StAR expression. We further detected the hormonal activity with transgenic yeast, and CDC showed potential androgenic antagonistic activity. Meanwhile, we investigated its aphrodisiac effect on hydrocortisone-induced mice. Exposure to hydrocortisone decreased the mating ability, reproductive organs, and testosterone level and disrupted testicular histology. However, all of these effects were significantly improved by CDC treatment. In conclusion, these results indicated that mechanisms of CDC in stimulating testosterone production involve upregulating the cAMP-PKA pathway.

Interplay between polycystic ovary syndrome and hypothyroidism on serum testosterone, oxidative stress and StAR gene expression in female rats.

IntroductionEndocrine disorders such as polycystic ovary syndrome (PCOS) and hypothyroidism can cause infertility. There are evidence that they happen jointly in some circumstances. It still remains unknown, how these two illnesses interact and influence the body.MethodsAccordingly, a five‐group was designed, first is the control group, followed by the PCOS group. Estradiol valerate (EV) induced PCOS, the second group had only PCOS and the third, fourth and fifth groups were given varied dosages of propylthiouracil (PTU) to cause hypothyroidism after induction of PCOS. Steroidogenic acute regulatory protein (StAR) expression was measured in the ovaries, and serum was obtained to determine testosterone levels, as well as superoxide dismutase (SOD) as an antioxidant and malondialdehyde (MDA) as an oxidant.ResultsBased on radioimmunoassay data, testosterone levels were significantly higher in the PCOS group than the control group, and significantly lower (p ˂ .05) in PTU groups comparing with the PCOS group. According to the quantitative real‐time polymerase chain reaction (qRT‐PCR) data, the same results were obtained for the StAR gene as well. The data also indicated a positive correlation between these two. Although both oxidant and antioxidant level increased in PCOS group compared than control group, after hypothyroidism, oxidant level increased significantly (p ˂ .05), meanwhile antioxidant level decreased significantly (p ˂ .05).ConclusionsThe results of this study illustrate that the presence of both PCOS and hypothyroidism alters the situation more than just PCOS. They also indicate that this situation is associated with imbalanced oxidative/antioxidative status.

Immunocastration in adult boars as a model for late-onset hypogonadism.

BackgroundWhile immunocastration has been studied in male pre‐pubertal pigs, data on older, sexually mature animals are limited. To understand the physiological effects of androgen deprivation in the late sexual development phase, we compared mature immunocastrated boars (n = 19; average age = 480 days) to young male immunocastrated pigs (n = 6; average age = 183 days) and young entire males (n = 6; average age = 186 days) as positive and negative controls, respectively.ObjectivesWe hypothesized that the timing of gonadotropin‐releasing hormone suppression (early or late sexual development phases) influences the extent of reproductive function inhibition, histological structure of testicular tissue, and expression levels of selected genes related to steroid metabolism.Materials and methodsAntibody titer, hormonal status, and histomorphometric analysis of testicular tissue were subjected to principal component analysis followed by hierarchical clustering to evaluate the immunocastration effectiveness in mature boars.ResultsHierarchical clustering differentiated mature immunocastrated boars clustered with young immunocastrated pigs from those clustered with entire males. Although all mature immunocastrated boars responded to vaccination, as evidenced by the increased gonadotropin‐releasing hormone antibody titers (p < 0.001), decreased serum luteinizing hormone concentrations (p = 0.002), and changes in testicular tissue vascularization (lighter and less red testicular parenchyma; p ≤ 0.001), the responses were variable. Sharp decreases in testes index (p < 0.001), Leydig cell volume density (p < 0.001), Leydig cell nucleus‐to‐cytoplasm ratio (p < 0.001), and testosterone concentration (p < 0.001) were observed in mature immunocastrated boars clustered with young immunocastrated pigs compared with those that clustered with entire males. Additionally, mature immunocastrated boars clustered with young immunocastrated pigs showed lower hydroxysteroid 17‐beta dehydrogenase 7 expression than entire males (p < 0.05). The young immunocastrated pigs group showed higher follicle‐stimulating hormone receptors than the entire males and mature immunocastrated boars, lower steroidogenic acute regulatory protein expression levels compared with entire males, and mature immunocastrated boars clustered with entire males (p < 0.01).ConclusionThe two‐dose vaccination regime resulted in progressive but variable regression of testicular function in adult (post‐pubertal) pigs; however, it was insufficient to induce a complete immunocastration response in all animals.

Low-dose rapamycin prevents Ang-II-induced toxicity in Leydig cells and testicular dysfunction in hypertensive SHR model.

Hypertension is a common chronic cardiovascular disease reported among both men and women. Hypertension in males affects the testis and reproduction function; however, the pathogenesis is poorly understood. Rapamycin has been reported to have a variety of beneficial pharmacological effects; however, high-doses rapamycin does have side effects such as immunosuppression. The present study investigates whether low-dose rapamycin can reduce the damage caused by hypertension to the testis of spontaneously hypertensive rats (SHRs) and further examines molecular mechanism of low-dose rapamycin in preventing testicular toxicity induced by angiotensin II (Ang II). Low rapamycin dose restores the testicle size, histological alterations, 3β-hydroxysteroid dehydrogenase (3β-HSD) expression, and prevents apoptosis in SHR rats. Ang II downregulates angiotensin-converting enzyme-2 (ACE2) expression through AT1R, p-ERK, and MAS receptor in LC-540 Leydig cells in a dose-dependent manner. Low doses of rapamycin effectively upregulate steroidogenic enzymes, steroidogenic acute regulatory protein and 3β-HSD expression in Leydig cells. Rapamycin upregulates ACE2 expression through p-PKAc and p-PI3k in Ang II-treated cells. Further, rapamycin curbs mitochondrial superoxide generation and depleted mitochondrial membrane potential induced by Ang II through activation of Nrf2-mediated Gpx4 and superoxide dismutase 2 expression. Our results revealed the involvement of ACE2, AT1R, AT2R, PKAc, and oxidative stress in Ang-II-induced testicular toxicity, suggesting low-dose rapamycin could be a potential therapeutic candidate to attenuate testicular toxicity.

1457-P: Cholesterol Accumulation in Islets Increases Steroidogenic Acute Regulatory (StAR) Protein Expression and Decreases Islet Cell Viability and ß-Cell Function

Diabetes is associated with elevated plasma cholesterol (Chol) , with its intracellular accumulation resulting in β-cell dysfunction. In steroidogenic tissues, StAR facilitates Chol transport from the outer to inner mitochondrial membrane for subsequent metabolism. We have shown that StAR is expressed in β cells and its upregulation increases mitochondrial Chol content and decreases mitochondrial function. To determine whether excess Chol per se increases StAR expression and reduces islet viability, we cultured mouse islets for 24 hours in Chol and quantified mRNA levels of Star and genes involved in Chol synthesis (Hmgcr) , uptake (Ldlr) and efflux (Abca1) , cell viability and glucose stimulated insulin secretion (GSIS) . Islet Chol content increased with increasing Chol concentrations (27.1±1.7, 55.3±0.5 and 72.7±5.2 µg/mg protein for 0, 0.25 and 0.5 mM respectively; n=3, p&amp;lt;0.0by ANOVA) , with mitochondrial Chol increasing 2.4-fold in 0.5 mM (9.1±0.2 to 22.4±1.9 µg/mg protein; n=3, p&amp;lt;0.005) . This was associated with increased Star mRNA expression (1.0±0.1, 3.0±0.9 and 7.0±1.5; n=3, p=0.014) . Further, mRNA expression of Hmgcr tended to decrease (1.0±0.2, 0.8±0.3 and 0.3±0.0; n=3, p=0.088) , whereas expression of Ldlr and Abca1 did not change (Ldlr: 1.7±0.8, 1.1±0.7 and 0.5±0.3; n=3, p=0.508; Abca1: 1.1±0.3, 1.9±0.7 and 1.1±0.1; n=3, p=0.413) . These changes were associated with reductions in cell viability (100±15.7%, 68.0±12.6% and 57.3±9.6%; n=2, p=0.025) and GSIS in response to 20 mM glucose (fold increase over 2.8 mM: 0 mM Chol 28.5±0.6, 0.25 mM Chol 8.9±0.5, 0.5 mM Chol 10.8±0.9; n=2, p&amp;lt;0.0001) . In summary, islet cholesterol accumulation is associated with increased StAR expression, decreased cell viability and reduced β-cell function. These data suggest that elevated cholesterol in diabetes may contribute to β-cell dysfunction by increasing StAR expression and mitochondrial cholesterol accumulation. Disclosure R.Akter: None. M.F.Hogan: Employee; NanoString. N.Esser: None. J.J.Castillo: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. S.Zraika: None. S.E.Kahn: Advisory Panel; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Inc., Merck &amp; Co., Inc., Novo Nordisk, Pfizer Inc. Funding VA (I01BX001060) ,VA (IBX004063) ,NIDDK (P30 DK017047) ,NIDDK (T32 DK007247)

MiR-466b-3p/HDAC7 meditates transgenerational inheritance of testicular testosterone synthesis inhibition induced by prenatal dexamethasone exposure

The adverse environment during pregnancy could change epigenetic modifications in germ cells, leading to transgenerational effects. This study aims to explore transgenerational-inheritance of inhibited testicular testosterone synthesis induced by prenatal dexamethasone exposure (PDE) and its mechanism. Wistar pregnant rats were subcutaneously injected with 0.2 mg/kg.d dexamethasone at gestational-day (GD) 9–20. Blood and testes of F1/F3 were obtained by maternal inheritance before and after birth. Meanwhile, Leydig cells were treated with dexamethasone and related interventions to confirm its direct effect and mechanism. The results showed that, in PDE F1 fetus, the serum testosterone level decreased, steroidogenic acute regulatory protein (StAR) decreased, and imprinted miR-466b-3p reduced, histone deacetylase 7 (HDAC7) upregulated, histone 3 lysine 9 acetylation (H3K9ac) level of StAR promoter decreased in testis. Meanwhile, consistent changes were observed in F1/F3 testes. In addition, miR-466b-3p expression in PDE F1/F2 oocytes also decreased. Further, series cell interventions confirmed that dexamethasone could reduce imprinted miR-466b-3p expression, increase HDAC7 expression, and reduce StAR acetylation level and expression by glucocorticoid receptor, finally causing testosterone synthesis inhibition. This study provided an experimental basis for confirming the developmental toxicity in offspring testis induced by PDE and its maternal transgenerational inheritance, offering a potential target for early warning and intervention therapy.

Activation of Autophagy by Low-Dose Silica Nanoparticles Enhances Testosterone Secretion in Leydig Cells.

Silica nanoparticles (SNPs) can cause abnormal spermatogenesis in male reproductive toxicity. However, the toxicity and toxicological mechanisms of SNPs in testosterone synthesis and secretion in Leydig cells are not well known. Therefore, this study aimed to determine the effect and molecular mechanism of low doses of SNPs in testosterone production in Leydig cells. For this, mouse primary Leydig cells (PLCs) were exposed to 100 nm Stöber nonporous spherical SNPs. We observed significant accumulation of SNPs in the cytoplasm of PLCs via transmission electron microscopy (TEM). CCK-8 and flow cytometry assays confirmed that low doses (50 and 100 μg/mL) of SNPs had no significant effect on cell viability and apoptosis, whereas high doses (more than 200 μg/mL) decreased cell viability and increased cell apoptosis in PLCs. Monodansylcadaverine (MDC) staining showed that SNPs caused the significant accumulation of autophagosomes in the cytoplasm of PLCs. SNPs activated autophagy by upregulating microtubule-associated protein light chain 3 (LC3-II) and BCL-2-interacting protein (BECLIN-1) levels, in addition to downregulating sequestosome 1 (SQSTM1/P62) level at low doses. In addition, low doses of SNPs enhanced testosterone secretion and increased steroidogenic acute regulatory protein (StAR) expression. SNPs combined with rapamycin (RAP), an autophagy activator, enhanced testosterone production and increased StAR expression, whereas SNPs combined with 3-methyladenine (3-MA) and chloroquine (CQ), autophagy inhibitors, had an opposite effect. Furthermore, BECLIN-1 depletion inhibited testosterone production and StAR expression. Altogether, our results demonstrate that low doses of SNPs enhanced testosterone secretion via the activation of autophagy in PLCs.

Low H3K27 acetylation of SF1 in PBMC: a biomarker for prenatal dexamethasone exposure-caused adrenal insufficiency of steroid synthesis in male offspring.

Dexamethasone is widely used to treat pregnancy disorders related to premature delivery. However, lots of researches have confirmed that prenatal dexamethasone exposure (PDE) could increase the risk of offspring multiple diseases. This study was designed to elucidate the epigenetic mechanism of adrenal developmental programming and explore its early warning marker in peripheral blood mononuclear cells (PBMC). We found the adrenal morphological and functional changes of PDE male offspring rats before and after birth, which were mainly performed as the decreased serum corticosterone concentration, steroidogenic acute regulatory (StAR) protein expression, and histone 3 lysine 27 acetylation (H3K27ac) level of steroidogenic factor 1 (SF1) promoter region and its expression. Simultaneously, the expressions of glucocorticoid receptor (GR) and histone acetylation enzyme 5 (HDAC5) in the PDE male fetal rats were increased. In vitro, dexamethasone reduced the expression of SF1, StAR, and cortisol production and still increased the expression of GR and HDAC5, the binding between GR and SF1 promoter region, and protein interaction between GR and HDAC5. GR siRNA or HDAC5 siRNA was able to reverse the above roles of dexamethasone. Furthermore, in vivo, we confirmed that H3K27ac levels of SF1 promoter region and its expression in PBMC of the PDE group were decreased before and after birth, showing a positive correlation with the same indexes in adrenal. Meanwhile, in clinical trials, we confirmed that prenatal dexamethasone application decreased H3K27ac of SF1 promoter region and its expression in neonatal PBMC. In conclusion, PDE-caused adrenal insufficiency of male offspring rats was related to adrenal GR activated by dexamethasone in uterus. The activated GR, on the one hand, increased its direct binding to SF1 promoter region to inhibit its expression, on the other hand, upregulated and recruited HDAC5 to decrease H3K27ac level of SF1 promoter region, and strengthened the inhibition of SF1 and subsequent StAR expression.