Steroidogenic Gene Expression Research Articles

New insights into the mechanisms underlying 5-hydroxymethylfurfural-induced suppression of testosterone biosynthesis in vivo and in vitro

5-hydroxymethylfurfural (HMF), produced by the Maillard reaction, can indicate thermal processes in food. Previous research has examined the cytotoxic, genotoxic, mutagenic, and carcinogenic characteristics of HMF and its derivatives in different organs. Nevertheless, there is currently no available evidence about the impact of HMF on male reproductive toxicity. In this study, the effects of HMF on testosterone biosynthesis in both mouse testis and TM3 Leydig cells were investigated. HMF was administered to mice at doses of 30 and 300 mg/kg/day for 21 days and to Leydig cells at concentrations of 0.1, 1, and 10 mM for 24 h. The mechanism of action of HMF on testosterone biosynthesis in both mouse testis and Leydig cells was revealed by measuring the amount of testosterone, 3′,5′-cyclic adenosine monophosphate (cAMP) levels, and the expression level of some important genes in the steroidogenic pathway. In addition, its effects on general testis were examined through histopathological evaluations. Upon examination of the results, it was observed that HMF had a significant impact on reducing testosterone and cAMP levels. Furthermore, HMF inhibited the expression of steroidogenic genes, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxy dehydrogenase, and 17β-hydroxy dehydrogenase, as well as transcription factors, such as steroidogenic factor-1, GATA binding protein-4, and nerve growth factor IB. HMF-administrated groups had germinal epithelium degradation, vacuolization, and disorders in the interstitial area. Consequently, it has been proven for the first time that HMF can damage the male reproductive system by detrimentally impacting the production of testosterone.

Myo-Inositol and D-Chiro-Inositol Reduce DHT-Stimulated Changes in the Steroidogenic Activity of Adult Granulosa Cell Tumors.

Considering the properties of myo-inositol (MI) and D-chiro-inositol (DCI), which are well known in polycystic ovary syndrome therapy, and the limitations of adult granulosa cell tumor (AGCT) treatment, especially for androgen-secreting tumors, we studied the role of MI and DCI in the androgen-rich environment of AGCTs. For this purpose, we analyzed the mRNA expression of steroidogenic genes and the secretion of progesterone (P4) and 17β-estradiol (E2) in an unstimulated and/or dihydrotestosterone (DHT)-stimulated environment under MI and DCI influence. Thus, we used the HGrC1 and KGN cell lines as in vitro models of healthy and cancerous granulosa cells. We found that DHT, the most potent androgen, increased E2 secretion and steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage gene (CYP11A1) mRNA expression without affecting 450 aromatase (CYP19A1) in AGCTs. However, after the MI and DCI treatment of KGN cells, both compounds strongly reduced StAR and CYP11A1 expression. Interestingly, in DHT-stimulated KGN cells, only DCI alone and its cotreatment with MI reduced both CYP11A1 mRNA and E2 secretion. These findings suggest that CYP11A1 is responsible for the antiestrogenic effect of DCI in the androgen-rich environment of AGCTs. Therefore, MI and DCI could be used as effective agents in the adjuvant treatment of AGCT, but further detailed studies are needed.

The senolytic drug ABT-263 accelerates ovarian aging in older female mice

Previous studies have reported that senolytic drugs can reverse obesity-mediated accumulation of senescent cells in the ovary and protect against cisplatin-induced ovarian injury by removing senescent cells. Early intervention with ABT-263 has been shown to mitigate ovarian aging. However, it remains unknown whether treatment with ABT-263 could rejuvenate the aged ovary in reproductively old females. Therefore, the current study was aimed to investigate whether advanced age intervention with ABT-263 could ameliorate age-related decline in ovarian function. Fourteen 16-month-old mice with a C57/BL6 background were treated with ABT-263 (N = 7) or vehicle (N = 7) for two weeks. Mice were initially treated with ABT-263 (60 mg/kg/d) or vehicle for 7 consecutive days. After a 7-day break, the treatment was repeated for another 7 consecutive days. Six 2-month-old mice with C57BL/6 were used as a young control. The hormonal levels, estrus cycles, ovarian reserve, ovarian cell proliferation and apoptosis, ovarian fibrosis, and steroidogenic gene expression of ovarian stromal cells were evaluated. ABT-263 treatment did not rescue abnormal estrus cycles and sex hormonal levels, or inhibit the formation of multinucleated giant cells and ovarian stromal cell apoptosis in aged ovaries. However, it reduced ovarian fibrosis and preserved the steroidogenic gene expression of ovarian stromal cells in aged ovaries. Importantly, ABT-263 treatment further depleted ovarian follicles in aged mice. In conclusion, ABT-263 treatment accelerated the depletion of ovarian follicles in aged mice, suggesting that senolytic drugs for reproductively old female may adversely affect female fertility.

Selenium Improves Arsenic-Induced Male Reproductive Dysfunction by Regulating H3K14ac Level.

Arsenic exposure has been known to be associated with male reproduction injury. Exploring the antidote of arsenic and ascertaining proper dose of antidote are important for detoxifying the male reproductive toxicity of arsenic. Selenium, which is essential for the male reproduction and spermatogenesis, can alleviate the toxicity of many environmental toxins, such as metals, and fluoride (F). Selenium relieves arsenic-induced reductions in spermatogenesis index and testicular function marker enzymes via promoting the antioxidative ability of rats. Our previous study has found that arsenic can induce male reproductive toxicity by affecting the level of H3K14ac in the testis, so we further investigate whether selenium can antagonize arsenic-induced male reproductive toxicity through the H3K14ac pathway and ascertain the appropriate dose of selenium. The results show that selenium intervention reduces the accumulation of arsenic in rat testis probably attributing to promote the excretion of arsenic from rat, then improves the testis injury induced by arsenic. Selenium intervention enhances sperm quality, testosterone level, and expression of steroidogenic genes by regulating H3K14ac level and expression of its associated enzymes (KAT2A, BAZ2A, and HDAC6), and thus alleviates the male reproductive toxicity of arsenic, and the proper dose of Se for mitigating arsenic male reproductive toxicity is 1mg/kg.

Shatavarin-IV rescues the Di (2-ethylhexyl) phthalate (DEHP) induced oxidative stress in rat granulosa cells in vitro

Studies provide notable evidence that oxidative stress (OS) mediated reactive oxygen species (ROS) disturb reproductive health. We have shown in our previous publication that exposure of Di-(2-ethylhexyl) phthalate (DEHP), induces OS mediated ROS generation which inhibits steroid synthesis. In the present study, we demonstrated the ameliorative/protective effects of one of the steroidal saponins, i.e., Shatavarin-IV, isolated from the roots of Asparagus racemosus against DEHP induced OS in rat granulosa cells. Granulosa cells were exposed with DEHP alone (400μM), Shatavarin-IV alone (8μg/ml), and a combination of DEHP + Shatavarin-IV (400μM + 8μg/ml) in vitro for 24 hrs. Intracellular ROS, OS/hypoxia, mitochondrial membrane potential, steroid-responsive genes expression were analyzed. The results revealed that the effective dose of DEHP (400µg) significantly increased OS compared to the control by increasing ROS levels, mitochondrial membrane potential, and β-galactosidase activity with a higher level of apoptotic genes (Bax, Caspase-3) expression at mRNA level. Further, DEHP significantly (p<0.05) reduced mRNA expression of steroidogenic responsive genes (StAR, CYP17A1 and CYP19A1) in granulosa cells treated with above combination compared to control. Interestingly, co-treatment of DEHP + Shatavarin-IV significantly suppressed the DEHP induced OS, ROS, β-galactosidase levels and enhanced steroidogeneic and apoptotic gene expression activities, which suggests that Shatavarin-IV rescued DEHP-induced changes that may useful for the prevention of DEHP- induced reproductive toxicity.

Azoospermia and multi-organ damage in juvenile rats exposed to α-Terpineol from weaning to sexual maturity

This study aimed to evaluate the repeated oral administration of α-terpineol in juvenile Wistar rats over a 70-day period. The objective was to assess the potential systemic and reproductive toxicity of α-terpineol when administered by gavage at doses of 75, 150, and 300 mg/kg/day to juvenile Wistar rats for 70 days from postnatal day 24. The control group received corn oil for 70 days. During the study, various parameters were evaluated, including clinical signs, body weight, food intake, neurobehavioral observations, haematology, serum biochemistry, organ weights, steroidogenic gene expression, and histopathological examination. No toxicity-related changes were observed in body weight, food intake, neurobehavioral observations, or steroidogenic gene expression. However, sperm evaluation revealed a complete absence of sperm and delayed sexual maturation. Total cholesterol was significantly elevated in both sexes, and serum testosterone was reduced at the 150 and 300 mg/kg doses. Microscopic examination showed severe pathological changes in the testes, epididymis, liver, and kidneys of both males and females. After the 14-day recovery period, total cholesterol levels returned to the normal range, but no recovery was observed in the other organs. The no-observed-adverse-effect level was 75 mg/kg/day for male rats based on the histopathological findings in the testes, liver, and kidneys, and for female rats based on the kidney and liver histopathology.

Impact of the novel chlorinated polyfluorinated ether sulfonate, F-53B, on gill structure and reproductive toxicity in zebrafish

6:2 Chlorinated polyfluorinated ether sulfonate, commonly known as F-53B, is widely used as a mist suppressant in various industries and is frequently detected in the environment. Despite its prevalent presence, the adverse effects of F-53B are not well understood and require future investigation. This study utilized zebrafish embryos and adults to examine the toxic effects of F-53B. Our findings revealed that F-53B impaired gill structure and increased erythrocyte numbers in adult zebrafish. Notably, F-53B demonstrated a higher sensitivity for inducing mortality (LC50 at 96 h) in adult zebrafish compared to embryos. Additionally, F-53B disrupted the expression of critical steroidogenic genes and hindered sex hormone production, which negatively affecting egg production. In conclusion, this study underscores the detrimental impact of F-53B on gill structure and reproductive toxicity in zebrafish, providing valuable insights into its overall toxicity.

A STAR for the ages: a 30-year historical perspective of the role of transcription factors in the regulation of steroidogenic acute regulatory gene expression.

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.

Characterization of urethra closure in female neonatal mice at histological and molecular levels.

Female hypospadias is a little-known and poorly studied birth defect. This research establishes an anatomical and molecular foundation for future research to investigate the origins of this defect. Hypospadias is a congenital anomaly of the external genitalia where the urethra does not properly close. In humans, hypospadias is mostly reported in male newborns, whereas in females hypospadias is rare, although it is generally considered to be under-reported. Improper urethra closure in the female genitalia can cause recurrent genitourinary tract infections and infertility. In mice, female hypospadias was induced by exposure to exogenous estrogenic compounds. Aside from the link between estrogen exposure and female hypospadias, the process of female urethra closure is largely unstudied, with the precise timing of urethra closure and associated molecular mechanisms remaining poorly understood. To address this gap, we determined when urethra closure occurs and identified gene expression patterns during the process of urethra closure in female neonatal mice from postnatal day (PND) 5 to 10. Using whole mount imaging and histology, we discovered that the initiation of urethra closure begins at PND7, and urethra closure is fully completed by PND10. To identify the genes associated with urethra closure, we conducted bulk RNA sequencing on female external genitalia prior to and after urethra closure. Gene ontology analyses revealed an increase in steroidogenic gene expression (Star, Hsd3b6, and Cyp17a1) during urethra closure, suggesting that the female genitalia locally produce steroids which could facilitate steroid signaling within the genitalia. With this study, we establish an anatomical timeline of female urethra closure and hypothesize a paracrine steroid signaling mechanism of urethra closure. These observations provide entry points to aid in further understanding external genital abnormalities, like hypospadias, in females.

A common phthalate replacement disrupts ovarian function in young adult mice.

Di-2-ethylhexyl terephthalate (DEHTP) is a replacement for its structural isomer di-2-ethylhexyl phthalate (DEHP), a known endocrine disrupting chemical and ovarian toxicant. DEHTP is used as a plasticizer in polyvinyl chloride products and its metabolites are increasingly found in biomonitoring studies at levels similar to phthalates. However, little is known about the effects of DEHTP on the ovary. In this research, we tested the hypothesis that DEHTP is an ovarian toxicant and likely endocrine disrupting chemical like its isomer DEHP. The impact of environmentally relevant exposure to DEHTP and/or its metabolite mono-2-ethylhexyl terephthalate (MEHTP) on the mouse ovary was investigated in vivo and in vitro. For the in vivo studies, young adult CD-1 mice were orally dosed with vehicle, 10 µg/kg, 100 µg/kg, or 100 mg/kg of DEHTP for 10 days. For the in vitro studies, isolated untreated ovarian follicles were exposed to vehicle, 0.1, 1, 10, or 100 µg/mL of DEHTP or MEHTP. Follicle counts, hormone levels, and gene expression of steroidogenic enzymes, cell cycle regulators, and apoptosis factors were analyzed. In vivo, DEHTP exposure increased primordial follicle counts at 100 µg/kg and 100 mg/kg and decreased primary follicle counts at 100 mg/kg compared to control. DEHTP exposure also decreased expression of cell cycle regulators and apoptotic factors compared to control. In vitro, follicle growth was reduced by 1 µg/mL DEHTP and 1, 10, and 100 µg/mL MEHTP compared to controls, and expression of the cell cycle regulator Cdkn2b was increased. Steroid hormone levels and steroidogenic enzyme gene expression trended toward decreases in vivo, whereas progesterone was significantly increased by exposure to 100 µg/mL MEHTP in vitro. Overall, these results suggest that DEHTP and MEHTP may be ovarian toxicants at low doses and should be subjected to further scrutiny for reproductive toxicity due to their similar structures to phthalates.

The Effects of Human Chorionic Gonadotropin and Gonadotropin-Releasing Hormone Receptor Antagonist on Testicular Steroidogenesis in Normal and Obese Rats.

We studied the effect of a high-fat, high-carbohydrate diet (HFHCD) on basal testosterone levels in the blood and testosterone, its precursors, and expression of steroidogenic genes in the testes of rats treated with human chorionic gonadotropin (hCG, 10 IU/rat, subcutaneously, once), gonadotropin-releasing hormone receptor antagonist cetrorelix (75 μg/kg, subcutaneously, 3 days), and their combination. In HFHCD rats, no obvious signs of androgen deficiency were observed and the response of the testes to hCG stimulation was preserved. Unlike control rats (normal diet), the expression of the luteinizing hormone receptor gene in these rats did not change in response to hCG stimulation and cetrorelix administration; they also showed a paradoxical, more pronounced response to hCG administration under conditions of suppression of the gonadotropin secretion by cetrorelix. This suggests that the etiology and pathogenesis of obesity may have different effects on the hormonal status of the male reproductive system.

Impacts of Acrylamide on testis and spermatozoa.

Acrylamide (ACR) is an industrial chemical used to produce polyacrylamide, a synthetic polymer with a wide range of applications. Depending on the dosage, its presence in occupational and environmental sources poses potential health risks to humans and animals. ACR can be formed in starchy foods cooked at high temperatures. Its effects on human sperm are not well understood. Animal studies indicate that ACR induces toxicity in the male reproductive system through oxidative stress mechanisms. Exposure to ACR alters the normal structure of testicular tubules, leading to congestion, interstitial edema, degeneration of spermatogenic cells, formation of abnormal spermatid giant cells, and necrosis and apoptosis. It also disrupts the balance of important biomarkers such as malondialdehyde, nitric oxide, superoxide dismutase, catalase, and glutathione. ACR has a negative impact on mitochondrial function, antioxidant enzymes, ATP production, and sperm membrane integrity, resulting in decreased sperm quality. Furthermore, it interferes with the expression of steroidogenic genes associated with testosterone biosynthesis. This review explores the detrimental effects of ACR on sperm and testicular function and discusses the potential role of antioxidants in mitigating the adverse effects of ACR on male reproduction.

Star1 gene mutation reveals the essentiality of 11-ketotestosterone and glucocorticoids for male fertility in Nile Tilapia (Oreochromis niloticus)

Steroidogenic acute regulatory protein (Star) plays an essential role in the biosynthesis of corticosteroids and sex steroids by mediating the transport of cholesterol from the outer to the inner membrane of mitochondria. Two duplicated Star genes, namely star1 and star2, have been identified in non-mammalian vertebrates. To investigate the roles of star genes in fish steriodogenesis, we generated two mutation lines of star1−/− and star1−/−/star2−/− in Nile tilapia (Oreochromis niloticus). Previous studies revealed that deficiency of star2 gene caused delayed spermatogenesis, sperm apoptosis and sterility in male tilapia. Our present data revealed that mutation of star genes impaired male fertility. Disordered seminiferous lobules and spermatic duct obstruction were found in the testis of both types of mutants. Moreover, significant decline in semen volume, sperm abnormality and impaired fertility were also detected in star1−/− and star1−/−/star2−/− males. In star1−/− male fish, lipid accumulation, up-regulation of steroidogenic enzymes, and significant decline of androgens were found. Additionally, hyperplasic interrenal cells, elevated steroidogenic gene expression level and decline of serum glucocorticoids were detected in star1 mutants. Intriguingly, either 11-KT or cortisol supplementation successfully rescued the impaired fertility of the star1−/− mutants. Taken together, these results further indicate that Star1 might play critical roles in the production of both 11-KT and glucocorticoids, which are indispensable for the maintenance of male fertility in fish.

DUSP8-attenuated ERK1/2 signaling mediates lipogenesis and steroidogenesis in chicken granulosa cells

The lipogenesis and steroidogenesis of granulosa cells are crucial during follicular development, yet it remains unclear whether dual-specificity phosphatase 8 (DUSP8) is involved. In this study, the specific role of DUSP8 in lipogenesis and steroidogenesis was investigated through culturing chicken granulosa cells in vitro. The results revealed that the expression levels of adipogenic genes were elevated after DUSP8 overexpression and reduced after knockdown. The same was observed for lipid deposition in granulosa cells. Meanwhile, the steroidogenic gene expression and progesterone synthesis were promoted after DUSP8 overexpression and inhibited after knockdown. In addition, we also found that DUSP8 blocked the phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2). Based on the previous results that activated ERK1/2 signaling inhibited lipid deposition and progesterone synthesis in chicken granulosa cells, we demonstrated that DUSP8 promoted lipid deposition and progesterone synthesis through mediating the ERK1/2 signaling pathway. The results will improve our understanding of the molecular regulatory mechanisms regarding lipid metabolism and progesterone synthesis in chicken granulosa cells.

Dynamics of gonadotropin and thienopyrimidine derivative TP03 effects on ovulation and ovarian steroidogenesis in Follimag-stimulated immature female rats

BACKGROUND: Gonadotropin preparations, particularly human chorionic gonadotropin (hCG), are commonly used to induce ovulation and treat reproductive disorders in women, albeit with associated side effects. Low-molecular-weight allosteric agonists of the luteinizing hormone receptor (LHR), such as thieno[2,3-d]pyrimidine derivatives, offer a potential alternative. AIM: This study aims to compare the effects of thieno[2,3-d]pyrimidine TP03 and hCG on ovarian weight, corpus luteum formation, and plasma levels of estradiol, progesterone, and luteinizing hormone in immature female rats pre-treated with Follimag®. It also examines their impact on ovarian gene expression related to LHR and steroidogenesis. MATERIALS AND METHODS: TP03 and hCG were administered 48 h after the Follimag® injection at a dose of 20 mg/kg (i.p.) and 15 IU/rat (s.c.), respectively. Parameters were assessed at 1, 2, 4, 8, 16, and 24 h after TP03 and hCG administration. Plasma hormone levels were measured via ELISA, and ovarian gene expression was analyzed using real-time PCR. RESULTS: TP03 increased ovarian weight, progesterone levels in the blood, and expression of steroidogenic genes encoding the cholesterol-transporting protein StAR and the cytochromes CYP11A1 and CYP17A1. TP03 also stimulated corpus luteum formation (16–24 h after treatment). The temporal dynamics of its stimulating effects were similar to those of hCG, although their magnitude was slightly inferior to those of gonadotropin. TP03-induced decrease in blood estradiol levels and aromatase gene expression in the ovaries was also more moderate. Unlike hCG, which suppressed LHR gene expression 8 h after treatment, TP03 maintained a high LHR gene expression, preserving ovarian sensitivity to endogenous luteinizing hormone. CONCLUSIONS: TP03 exhibits potential as an ovulation inducer with milder stimulating effects on ovarian steroidogenesis than hCG, which reduces the risks of developing ovarian hyperstimulation syndrome and resistance to gonadotropins.

Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression.

Epidemiologic studies suggested the association between prenatal di-(2-ethylhexyl) phthalate (DEHP) exposure and disorders of sex development (DSD), adult male disorders, and reproductive aging. Inhibiting testosterone synthesis by interfering with steroidogenic gene expression induces testicular toxicity, however, whether prenatal DEHP exposure induces testicular toxicity through this mechanism remains uncertain. C57BL/6JGpt male mice underwent different doses (0, 100, 500, 1,000 mg/kg) of prenatal DEHP exposure during gestational day 10 to delivery day, the testicular toxicity (genital development, testosterone, semen quality, and morphology of testis tissue) in the neonatal, post-puberal and middle-aged stages was observed, and the steroidogenic gene (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd3b2) expression was analyzed by quantitative polymerase chain reaction (qPCR) and Western blot (WB). The interference of steroidogenic gene expression in TM3 cells after mono-(2-ethylhexyl) phthalate (MEHP) exposure was also explored for verification. Prenatal DEHP exposure induced immediate testicular injury in the neonatal stage [reduced anogenital distance (AGD) and intratesticular testosterone], DSD in the post-puberal stage (poor genital development), and reproductive aging in the middle-aged stage (obesity, reduced testosterone and semen quality, and atrophic seminiferous tubules), especially in the high dose. Prenatal DEHP exposure continuously interfered with steroidogenic gene expression (Hsd3b2, Hsd17b3) in RNA and protein levels. MEHP inhibited testosterone synthesis of TM3 cells by interfering with steroidogenic gene expression (Hsd3b2, Hsd17b3) in RNA and protein levels. Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression, thus indicating the association between prenatal exposure and DSD, adult male disorders, and reproductive aging.

In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis.

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3β1, HSD3β6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 106 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant (P < 0.01) increase in testosterone production and up-regulation (P < 0.05) of CYP11A1, CYP17A1, HSD3β1 and HSD3β6 gene expression. In summary, the present study underscored the effects of UcOCN on testosterone biosynthesis, expression of crucial steroidogenic genes and interaction with GPRC6A receptors in buffalo Leydig cells, emphasizing its potential implications in andrology.

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/β-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3β-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2mU/L). Cells were cultured at 37°C in DMEM, supplemented with 5% FBS. The cells were treated with 1nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.