Steroidogenic Function Research Articles

Discovery of the First-in-Class Dual TSPO/Carbonic Anhydrase Modulators with Promising Neurotrophic Activity.

In searching for putative new therapeutic strategies to treat neurodegenerative diseases, the mitochondrial 18 kDa translocator protein (TSPO) and cerebral isoforms of carbonic anhydrase (CA) were exploited as potential targets. Based on the structures of a class of highly affine and selective TSPO ligands and a class of CA activators, both developed by us in recent years, a small library of 2-phenylindole-based dual TSPO/CA modulators was developed, able to bind TSPO and activate CA VII in the low micromolar/submicromolar range. The interaction with the two targets was corroborated by computational studies. Biological investigation on human microglia C20 cells identified derivative 3 as a promising lead compound worthy of future optimization due to its (i) lack of cytotoxicity, (ii) ability to stimulate TSPO steroidogenic function and activate CA VII, and (iii) ability to effectively upregulate gene expression of the brain-derived neurotrophic factor.

The Effects of Bisphenol A and its Analogs on Steroidogenesis in MA-10 Leydig Cells and KGN Granulosa Cells†.

Bisphenols are a family of chemicals used in the manufacture of consumer products containing polycarbonate plastics and epoxy resins. Studies have shown that exposure to bisphenol A (BPA) may disrupt steroidogenesis and induce adverse effects on male and female reproduction, but little is known about BPA replacements. We determined the effects of six bisphenols on the steroidogenic function of MA-10 Leydig cells and KGN granulosa cells by measuring the levels of progesterone and estradiol produced by these cells as well as the expression of transcripts involved in steroid and cholesterol biosynthesis. MA-10 and KGN cells were exposed for 48hours to one of six bisphenols (0.01-50μM): BPA, bisphenol F, bisphenol S, bisphenol AF, bisphenol M, or bisphenol TMC under both basal and dibutyryl cAMP (Bu2cAMP)-stimulated conditions. In MA-10 cells, most bisphenols increased the Bu2cAMP-stimulated production of progesterone. In KGN cells, there was a general decrease in progesterone production, while estradiol levels were increased following exposure to many bisphenols. qRT-PCR analyses revealed that all six bisphenols (≥1μM) upregulated the expression of STAR, a cholesterol transporter, in both cell lines after stimulation. Key transcripts directly involved in steroid and cholesterol biosynthesis were significantly altered in a cell line, chemical, and concentration-dependent manner. The expression of upstream genes was also differentially affected. Thus, BPA and five of its analogs can disrupt steroid production in two steroidogenic cell lines and alter the levels of transcripts involved in this process. Importantly, BPA replacements do not appear to have fewer effects than BPA.

Polystyrene microplastics cause reproductive toxicity in male mice

Microplastics are a common environmental pollutant that disrupts the reproductive system of living organisms. We investigated the reproductive toxicity of 2μm polystyrene microplastics (PS-MPs) in mice and treated them with PS-MPs for 6 weeks. We demonstrated that PS-MPs decreased the gonadosomatic index and the serum concentration of pituitary-testicular axis hormones (Follicle-stimulating hormone, Luteinizing hormone, and testosterone). The PS-MPs treatment also reduced viable epididymal sperm number and sperm motility. Our results also demonstrated a marked decrease in tubular differentiation index, spermatogenesis index, repopulation index, and steroidogenic foci. The PS-MPs treated mice demonstrated marketed tissue damage in the testis. We also found that reproductive abnormality in PS-MPs treated mice accompanied by reduced antioxidant capacity elevated oxidative stress, and, elevated apoptotic signaling. It was observed that Endoplasmic reticulum (ER) stress markers, including GRP78 and Chop, were upregulated. Based on these findings, oxidative stress and endoplasmic reticulum stress may contribute to the decline in the steroidogenic function of Leydig cells with PS-MPs treated.

Exposure of antral follicles to medroxyprogesterone acetate during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed cycles.

Does medroxyprogesterone acetate (MPA) exposure in progestin-primed ovarian stimulation (PPOS) cycles cause molecular perturbations in the steroidogenic function and gonadotropin responsiveness of the granulosa cells? PPOS cycles are identical to traditional GnRH antagonist cycles not only for clinical IVF characteristics but also for gonadotropin receptor expression, response to gonadotropins, and steroidogenic function at the molecular level. PPOS is increasingly used as an alternative to GnRH antagonists due to the inhibitory effect of progesterone on LH release by reducing GnRH pulsatility at the hypothalamic level. Although a growing body of evidence from clinical studies did not indicate significant differences between PPOS and antagonist protocols for IVF cycle characteristics and obstetrical outcomes, it is still unknown whether exposure of the antral follicle cohort to progesterone or its synthetic derivatives during ovarian stimulation causes any subtle molecular aberrations in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed comparative molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from normal responding IVF patients undergoing PPOS and antagonist cycles. A clinical translational research study was conducted with IVF patients. This study included 55 normal responding IVF patients who underwent ovarian stimulation with either PPOS using MPA (5 mg twice daily) or GnRH antagonist cetrorelix acetate. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation triggering, respectively. Luteinized mural GCs obtained during the oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging, and hormone assays were used. Demographic and IVF cycle characteristics of the patients undergoing ovarian stimulation with PPOS and GnRH antagonist were similar, including ovarian response, mature oocyte yield, and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, SCC, 3β-HSD, 17β-HSD, aromatase) and the uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase, and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to those of the antagonist cycles. The expression of the receptors for gonadotropins, estrogen, and progesterone hormones was also similar. Basal and hCG-induced increases in 3β-HSD expression and progesterone production and basal and FSH-induced increases in aromatase expression and E2 output of the GCs from PPOS patients did not exhibit any meaningful differences when compared with GCs from antagonist cycles. Furthermore, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other important genes regulating cumulus expansion, ovulation, and luteal function [Relaxin, ADAMTS-1, and epidermal growth factor (EGF)-like growth factor amphiregulin] in the GCs of the PPOS and antagonist cycles were similar. N/A. Caution should be exercised when interpreting our data which was derived from normally responding patients whose ovulation was triggered with hCG. It is unclear whether the molecular parameters assessed vary according to infertility etiologies, magnitude of ovarian response, mode of trigger, and any other underlying ovarian pathologies or systemic diseases. MPA was the progestin used for PPOS and whether these findings can be generalized to other progestins is unknown. This study provides reassuring molecular evidence that exposure of antral follicle cohorts to MPA during the follicular growth phase does not have any detrimental effects on steroidogenic, ovulatory, and luteal functions when compared with GnRH antagonist cycles. This study was funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koç University Research Center for Translational Medicine (KUTTAM), and equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest. N/A.

O-299 Exposure of antral follicles to progestins during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed IVF cycles

Abstract Study question Does progestin exposure cause any molecular perturbations in the steroidogenic features and gonadotropin responsiveness of the granulosa cells in progestin-primed ovarian stimulation (PPOS) cycles? Summary answer No. PPOS cycles are identical to antagonist cycles for gonadotropin receptor expression, response to gonadotropins, steroidogenic function at molecular level in normal responding IVF patients. What is known already PPOS is commonly used in IVF cycles as an alternative to antagonist cycles. Although accumulating evidence from clinical studies did not indicate any particular differences between these two types of stimulation protocol for IVF cycle characteristics, it is still unknown if exposure of antral follicle cohorts to synthetic progesterone derivatives during ovarian stimulation causes any subtle molecular aberrations in PPOS cycles in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from the normal responding patients undergoing PPOS vs. antagonist IVF cycles. Study design, size, duration A translational research study in IVF patients. Participants/materials, setting, methods 55 normal responding IVF patients underwent ovarian stimulation either with PPOS using medroxy progesterone acetate (5mg twice daily) or, with GnRH antagonist cetrorelix acetate were included in the study. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation trigger, respectively. Luteinized mural granulosa cells obtained during oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging and hormone assays were used. Main results and the role of chance Demographic and IVF characteristics of the patients undergoing ovarian stimulation with PPOS vs. GnRH antagonist were similar including ovarian-response, mature oocyte yield and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, aromatase, 3B-HSD, 17B-HSD) and uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to the antagonist IVF cycles. Both cycles were also similar for the expression of FSH and LH receptors at transcriptional and translational level. Basal and hCG-induced increases in 3B-HSD expression and P4 production, and basal and FSH-induced increases in aromatase expression and E2 output of the GCs of the PPOS patients did not show any meaningful differences from the antagonist cycles. Similarly, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake rate did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other genes regulating cumulus expansion, ovulation and luteal function (Relaxin, ADAMTS-1 and EGF-like growth factor amphiregulin (AREG)) in the GCs of the PPOS cycles were comparable to the antagonist cycles. Limitations, reasons for caution Caution should be exercised when interpreting our data, which was produced in normal responders. It is unclear if the molecular parameters assessed here vary according to infertility etiologies, ovarian response type, mode of trigger and any other underlying ovarian pathology or systemic diseases that are concomitantly present. Wider implications of the findings This study provides reassuring molecular evidence that exposure of antral cohorts to progestins follicular growth phase does not appear to have any detrimental effect on their steroidogenic, ovulatory and luteal functions, suggesting that PPOS IVF cycles are not different from traditional antagonist cycles. Trial registration number not applicable

P-661 Comparative and descriptive molecular analysis of cumulus and luteinized mural granulosa cells for steroidogenic function in IVF patients

Abstract Study question Do cumulus granulosa cells (CCs) have steroidogenic function and gonadotropin response like luteinized mural granulosa cells (LMGCs)? Summary answer Yes. Both types of granulosa cells have similar expression levels for the steroidogenic enzymes and hCG/LH receptors. What is known already CCs and LMGCs have distinct roles in follicular development, oocyte growth and maturation, cumulus expansion, ovulation, and luteinization. CCs support oocytes, especially during meiotic resumption, ovulation, and fertilization. They exhibit specialized functions in response to hormones like LH and participate in bidirectional communication with the oocyte. Although LMGCs are primarily responsible for steroidogenic function and progesterone (P4) production, very limited data is available as to whether CC themselves express the steroidogenic enzymes and gonadotropin receptors and produce sex steroids at basal or after gonadotropin stimulation in humans. We aimed to address this issue in the current study. Study design, size, duration An in-vitro basic science study was designed to compare CCs and LGMCs in the context of vitality, steroidogenic capabilities (both in baseline and response to rec-hCG (10 IU/ml), and EGF pathway responses. The assessment was planned to compare hormonal assay in spent culture and transcriptional levels. The study’s duration encompassed the period of ovarian stimulation, oocyte retrieval, and subsequent cellular analysis. Participants/materials, setting, methods 24 IVF patients undergoing ovarian stimulation with progestin primed (PPOS) and GnRH antagonist protocols for different infertility etiologies were included. Ovarian stimulation and ovulation trigger were done using rec-FSH and rec-hCG, respectively. CCs were collected from the denudation plate. LMGCs were obtained from follicular aspirates during the OPU and cultured individually for each patient without pooling in culture plates. After overnight incubation, the cells were treated with rec-hCG overnight. RNA isolation was performed. Main results and the role of chance The expression of the steroidogenic enzymes (StAR, SCC, 17B-HSD, 3B-HSD, Aromatase), LH receptor and EGF-like amphiregulin (AREG) in CCs were comparable to LMGCs on quantitative RT-PCR analysis. In line with this finding, we found they produced similar amounts of estradiol (E2) and progesterone (P4) in culture. Furthermore, similar to LMGCs, hCG treatment of the CCs resulted in a significant increase in P4 output, indicative of the presence of functional LH receptors. When the culture period was extended to six days, CCs retained their vitality and steroidogenic function like LMGCs and produced detectable E2 and P4. Limitations, reasons for caution Caution should be exercised when interpreting our data as it is unclear if the parameters analyzed here vary according to the infertility etiology, stimulation protocol, mode of trigger and any underlying disease that concomitantly present. Wider implications of the findings Prominent steroidogenic activity of CCs might have other important implications for a successful pregnancy. Moreover, similar expression profiles of steroidogenic pathways and hCG/LH receptors between CCs and LMGCs indicate that CCs can also be used to understand better the luteal function and intricate crosstalk between in oocyte microenvironment. Trial registration number N/A

O-310 PARP inhibitors have no toxic effect on follicle reserve, mitotic and steroidogenic function of granulosa cells in human ovary

Abstract Study question Do the PARP inhibitors have gonadotoxic effects on human ovary? Summary answer No. The PARP inhibitor drugs Olaparib and Niraparib do not appear to have any toxic effects on the human ovary. What is known already The Poly (ADP-ribose) polymerase (PARP) family has many essential functions in cellular processes, including the regulation of transcription, apoptosis, and the DNA damage response. PARP has Poly (ADP-ribose) activity, which facilitates the recruitment of other repair proteins to promote the repair of DNA single-strand breaks when DNA damage occurs. PARP inhibitors are approved for the treatment of BRCA1/2 mutated breast and ovarian cancers. No data is available regarding the gonadotoxic potentials, if any, of these drugs on the human ovary. We aimed to address this issue in the current study. Study design, size, duration A clinical translational research study that utilizes ex-vivo and in-vitro experimental models on human ovarian cortical samples, primary luteinized non-mitotic granulosa cells, and immortalized human granulosa cells. Participants/materials, setting, methods Ovarian cortical samples were obtained from young women undergoing laparoscopic surgery for benign ovarian cysts (n = 5). Human non-mitotic luteinized granulosa cells (HLGCs) obtained from 10 IVF patients during the oocyte retrieval procedure and immortalized mitotic non-luteinized granulosa cell line (HGrC1), and immortalized granulosa cell tumor cell line (COV434) were used for the experiments. Cell/tissue culture, confocal imaging, flow cytometry, immunoblotting, cell viability/proliferation and hormone assays were applied. Main results and the role of chance Treatment of ovarian tissue samples and granulosa cells with PARP inhibitors at five different therapeutic concentrations (1, 5, 10, 20, and 40 µM/mL) up to 96 hours in culture resulted in resulted in the cleavage of PARP, validating in vitro activity of the drugs. The primordial follicle reserve and steroidogenic activity of the tissue samples remained stable at the end of culture period. Similar results were obtained in the granulosa cells, which retained their viability and steroidogenic functions after exposure to the PARP inhibitors. Real-time impedance-based cell proliferation assay with xCelligence system revealed that granulosa cells continued to proliferate normally and reached log phase in the presence of PARP inhibitors without any discernible cytotoxic effect. In immunoblot analysis, the expression of steroidogenic enzymes (StAR, aromatase, 3B-HSD and 17B-HSD) was comparable to control cells and cleaved caspase-3 expression was absent in the granulosa cells exposed to PARP inhibitors. Limitations, reasons for caution Caution should be exercised before extrapolating our data because these findings were obtained in ex-vivo and in-vitro models. They need to be confirmed using in-vivo experimental models such as human ovarian tissue xenografting in nude mice. Wider implications of the findings This study provides the first reassuring molecular evidence that PARP inhibitor drugs do not appear to have a significant gonadotoxic effect on human ovary. Trial registration number not applicable

Increased adrenal steroidogenesis and suppressed corticosteroid responsiveness in critical COVID-19

BackgroundThe effect of coronavirus disease 2019 (COVID-19) on adrenal endocrine metabolism in critically ill patients remains unclear. This study aimed to investigate the alterations in adrenal steroidogenic activity, elucidate underlying mechanisms, provide in situ histopathological evidence, and examine the clinical implications. MethodsThe comparative analyses of the adrenal cortices from 24 patients with fatal COVID-19 and 20 matched controls were performed, excluding patients previously treated with glucocorticoids. SARS-CoV-2 and its receptors were identified and pathological alterations were examined. Furthermore, histological examinations, immunohistochemical staining and ultrastructural analyses were performed to assess corticosteroid biosynthesis. The zona glomerulosa (ZG) and zona fasciculata (ZF) were then dissected for proteomic analyses. The biological processes that affected steroidogenesis were analyzed by integrating histological, proteomic, and clinical data. Finally, the immunoreactivity and responsive genes of mineralocorticoid and glucocorticoid receptors in essential tissues were quantitatively measured to evaluate corticosteroid responsiveness. FindingsThe demographic characteristics of COVID-19 patients were comparable with those of controls. SARS-CoV-2-like particles were identified in the adrenocortical cells of three patients; however, these particles did not affect cellular morphology or steroid synthesis compared with SARS-CoV-2-negative specimens. Although the adrenals exhibited focal necrosis, vacuolization, microthrombi, and inflammation, widespread degeneration was not evident. Notably, corticosteroid biosynthesis was significantly enhanced in both the ZG and ZF of COVID-19 patients. The increase in the inflammatory response and cellular differentiation in the adrenal cortices of patients with critical COVID-19 was positively correlated with heightened steroidogenic activity. Additionally, the appearance of more dual-ZG/ZF identity cells in COVID-19 adrenals was in accordance with the increased steroidogenic function. However, activated mineralocorticoid and glucocorticoid receptors and their responsive genes in vital tissues were markedly reduced in patients with critical COVID-19. InterpretationCritical COVID-19 was characterized by potentiated adrenal steroidogenesis, associated with increased inflammation, enhanced differentiation and elevated dual-ZG/ZF identity cells, alongside suppressed corticosteroid responsiveness. These alterations implied the reduced effectiveness of conventional corticosteroid therapy and underscored the need for evaluation of the adrenal axis and corticosteroid sensitivity.

Effect of melatonin on reproductive function in propylthiouracil induced hypothyroidism in adult male rats.

Thyroid hormones are essential for regulating metabolism, reproduction, and growth. Hypothyroidism is connected with lower sperm count and motility, leading to male infertility. Oxidative stress is likely to be linked to this interaction. Melatonin, being known as an oxidative scavenger, may offer a feasible treatment method for reproductive dysfunction accompanying hypothyroidism in adult male rats. The purpose of this investigation was to determine the mechanism by which melatonin treatment affected spermatogenic and steroidogenic function in an experimental model-induced hypothyroidism in adult male rats. Twenty-one male albino adult rats weighing between 150 and 210 g were used in this experiment. Rats were split into three groups and studied for 11 weeks. The control euthyroid group, in which rats received 0.9% Sodium Chloride (NaCl) solution by intraperitoneal injection [solvent for 6-propyl 2-thouracil (PTU)], 6 days/week for 8 weeks; the PTU-induced hypothyroid group, in which chemical thyroidectomy was induced by intraperitoneal injection of PTU at a dose of 10 mg/kg body weight, 6 days/week for 8 weeks; and the melatonin-treated hypothyroid group, which received 3 mg/kg melatonin intraperitoneally daily for 21 days plasma free Triiodothyronine (T3), free Thyroxin (T4), thyroid stimulating hormone (TSH), free testosterone, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and prolactin were measured. Also, semen analysis, testicular tissue malondialdehyde (MDA), and tumor necrosis factor-α (TNF-α) were accessed. The results indicated that melatonin significantly increased sperm viability and motility compared to the untreated PTU-induced hypothyroid group (p<0.001). Testicular MDA and TNF-α showed a significant decrease in the melatonin-treated hypothyroid group compared with the PTU-induced hypothyroid group (p<0.05). In addition, plasma testosterone levels were significantly increased, accompanied by a significant reduction of plasma prolactin levels compared to the untreated hypothyroid group (p<0.05 for both). Based on the study findings, melatonin could mitigate gonadal dysfunction induced by hypothyroidism by improving several components of semen analysis, such as sperm motility and sperm viability, as well as by enhancing testosterone production focusing on oxidative and inflammatory stress as the underlying mechanisms.

The effects of prenatal azithromycin exposure on offspring ovarian development at different stages, doses, and courses

Azithromycin, a commonly used macrolide antibiotic for treating chlamydial infections during pregnancy, has sparked investigations into its potential effects on offspring development. Despite these inquiries, there remains uncertainty about the specific impact of prenatal azithromycin exposure (PAzE) on offspring ovarian development and the precise "effect window". Pregnant mice, following clinical guidelines for azithromycin dosing, were orally administered azithromycin at different gestational stages [(gestational day, GD) 10–12 or GD 15–17], doses (50, 100, or 200 mg/kg·d), and courses (single or multiple). On GD 18, we collected offspring blood and ovaries to examine changes in fetal serum estradiol (E2) levels, fetal ovarian morphology, pre-granulosa cell function, and oocyte development. Multiple courses of PAzE resulted in abnormal fetal ovarian morphological development, disorganized germ cell nests, enhanced ovarian cell proliferation, and reduced apoptosis. Simultaneously, multiple courses of PAzE significantly increased fetal serum E2 levels, elevated ovarian steroidogenic function (indicated by Star, 3β-hsd, and Cyp19 expression), disrupted oocyte development (indicated by Figlα and Nobox expression), and led to alterations in the MAPK signal pathway in fetal ovaries, particularly in the high-dose treatment group. In contrast, a single course of PAzE reduced fetal ovarian cell proliferation, decreased steroidogenic function, and inhibited oocyte development, particularly through the downregulation of Mek2 expression in the MAPK signal pathway. These findings suggest that PAzE can influence various aspects of fetal mouse ovarian cell development. Multiple courses enhance pre-granulosa cell estrogen synthesis function and advance germ cell development, while a single terminal gestation dose inhibits germ cell development. These differential effects may be associated with changes in the MAPK signal pathway.

Use of in vitro methodology to investigate phthalate effects on the differentiation of seminiferous tubule-associated stem cells to form Leydig cells and on the Leydig cells derived from the stem cells.

Introduction: Leydig cells isolated from the testis are able to sustain high levels of testosterone production in vitro, but only for up to 3days. Such cells are valuable for addressing the acute effects of chemicals on steroidogenic function, but not for repeated or chronic effects. Methodology is now available by which adult Leydig cells can be derived in vitro from seminiferous tubule-associated stem cells. In contrast to isolated Leydig cells, the Leydig cells derived in this way can synthesize and secrete high levels of testosterone for months. Herein, we asked whether this system might be used to address the effect of mono-(2-ethylhexyl) phthalate (MEHP) exposure on the formation of Leydig cells from tubule-associated stem cells, and on the Leydig cells after their formation. Methods: Adult Brown Norway rats received an intraperitoneal injection of ethane dimethanesulfonate (EDS) to eliminate the existing Leydig cells. Seminiferous tubules then were isolated and cultured in medium containing Insulin-Transferrin- Selenium (ITS), Smoothened Agonist (SAG), and luteinizing hormone (LH). Results: Culture of the tubules for 8weeks resulted in the formation of cells on the surfaces of the tubules that stained for CYP11A1 and STAR and produced high levels of testosterone. When the tubules were cultured in medium containing increasing concentrations of MEHP, concentration-dependent effects on Leydig cell formation occurred. To determine the effect of MEHP on newly produced Leydig cells, tubules were cultured for 8weeks in the absence of MEHP, resulting in the formation of adult Leydig cells, and then in medium containing increasing concentrations of MEHP. Concentration-dependent decreases in testosterone production by the adult Leydig cells were seen, and these decreases proved to be reversible. Discussion: The use of this new system should make it possible to determine the mechanisms by which acute, repeated, or chronic exposures to increasing concentrations of MEHP and/or exposure to other chemicals affect the formation of Leydig cells from stem cells, as well as the steroidogenic function of adult Leydig cells.

Abundance of selected genes implicated in testicular functions in Camelus dromedarius with high and low epididymal semen quality.

Studying testicular genes' expression may give key insights into precise regulation of its functions that influence epididymal sperm quality. The current study aimed to investigate the abundance of candidate genes involved in the regulation of testicular functions specially those regulate sperm function (PLA2G4D, SPP1, and CLUAP1), testicular steroidogenic function (ESR1 and AR), materials transport (AQP12B and LCN15), and defense mechanisms (DEFB110, GPX5, SOCS3, and IL6). Therefore, blood samples and testes with epididymis were collected from mature middle-aged (5-10years) dromedary camels (n= 45) directly prior and after their slaughtering, respectively, during breeding season. Sera were evaluated for testosterone level and testicular biometry was measured with caliper. The epididymal tail semen was evaluated manually. Samples were distinguished based on testosterone level, testicular biometry, as well as epididymal semen features into high and low fertile groups. Total RNA was isolated from testicular tissues and gene expression was done using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Results revealed that testosterone levels were significantly (P <0.005) higher in camels with good semen quality than those of low quality. There was a significant (P <0.0001) increase in testicular weight, length, width, thickness, and volume in high fertile than low fertile camels. PLA2G4D, SPP1, CLUAP1, ESR1, AR, AQP12B, LCN15, DEFB110, GPX5, and SOCS3 genes were upregulated (P <0.001), and IL6 gene was downregulated (P <0.01) in the testes of high fertile camels compared to the low fertile one. Thus, it could be concluded that examined genes might be valuable monitors of testicular functional status and fertility in dromedary camels.

Sex steroid hormones and steroid secretory cells in the gonads of cyclostomes and fish

. The purpose of this work is to generalize data on the content of sex steroid hormones in cyclostomes and fish in connection with the different state of the gonads, as well as on the localization and ultrastructural organization of steroid secretory cells (SCs). Method used: generalization of literature data. Novelty: for the first time, data on the development of SC in the gonads of juvenile fish are presented on the scale of a literature review; changes in their localization and functional activity are shown. Results: Most of the data on the steroidogenic function of the gonads in fish were obtained in connection with the study of sex differentiation (inversion) and sexual maturation (spawning). The issues of the synthesis of sex steroid hormones and their molecular structure, blood levels in various states of the gonads, as well as the localization and ultrastructural organization of SCs are considered. It has been noted that SCs appear in the gonads long before sex differentiation and can be present among stromal, granulosa, and theca cells. In cyclostomes and fish — juvenile protogynous hermaphrodites in the gonads of genetic males, SCs do not appear in the membranes around the oocytes of the previtellogenesis period, which determines their deficiency of estrogen hormones and, as a result, sex inversion. With induced sex reversal, the development of the steroidogenic function of the gonads proceeds in the opposite direction to its natural development: in males, oocytes appear and steroid activity shifts from the stroma of the gonads into granulosa and theca; in females, on the contrary, secretory activity in the follicular membranes decreases and increases in the stroma of the gonads. Practical significance: the results of the analysis will give insight into the nature of sex steroid hormones, their dynamics in the blood, and their role in the implementation of various processes of gonadogenesis and gametogenesis before their practical use in fish farms.

OR19-05 Human Induced Pluripotent Stem Cell-derived Granulosa-like Cells From Ligand-based Differentiation Express FOXL2 After Inhibition Of DKK1

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Premature ovarian insufficiency (POI) is characterized by the loss of ovarian function prior to an individual reaching the age of 40. POI can have serious ramifications on health, reducing average life expectancy, and can develop from iatrogenic treatments or genetic variants. POI patients can receive hormone replacement therapies (HRTs), but current methods are not dynamic, responsive, or comprehensive. Contrastingly, cell-based therapies have the potential to produce hormones other than estrogen or progesterone and respond dynamically to each patient. Using human induced pluripotent stem cells (hiPSCs) to generate such cell-based therapies may mitigate rejection upon transplantation and allow for personalized therapies for POI patients. Therefore, we propose to differentiate hiPSCs into granulosa like cells (GLCs) that are functionally the same as granulosa cells (GCs), which are responsible for aromatizing androgens into estrogens. One factor critical to steroidogenesis in GCs is FOXL2, which in mice is suppressed by DKK1, a WNT inhibitor. Therefore, we hypothesized that FOXL2 expression and subsequent steroidogenic functions in our GLCs will be influenced by DKK1. To generate GLCs, we treated the cells with WNT agonist CHIR99021 for two days which has been shown to induce the intermediate mesoderm, and then BMP4 and FST for three days to induce the ovarian somatic lineage. We analyzed our cultures for self-renewal (POU5F1), intermediate mesoderm (PAX2, LHX1, WT1), coelomic epithelium (EMX2), genital ridge (GATA4, NR5A1), and GC (FOXL2) markers using qPCR to assess correct lineage commitment. At day 2, we observed approximately 4000-fold induction of PAX2, 20-fold induction of LHX1, 35-fold increase of WT1, 3000-fold induction of GATA4, and 3.5-fold induction in FOXL2 transcript expression. At day 5, we observed 10-fold induction of NR5A1. We also observed a 7000-fold increase of DKK1 at day 2. Addition of Gallocyanine, a DKK1 inhibitor, from days 2 to 5 resulted in induction of FOXL2 protein expression at day 5, captured by immunofluorescence imaging. Analysis of the images showed that Gallocyanine-treated cells were 42.4% positive for FOXL2, while cells untreated were 0.325% positive for FOXL2. This data suggests that DKK1 does suppress FOXL2 expression in these hiPSC-derived GLCs and demonstrates the potential of these cells as a model to investigate mechanisms important to GC development and maintenance. Future steps include testing the steroidogenic abilities of the GLCs and identification of culture conditions needed to maximize steroidogenesis. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), Warren and Eloise Batts Endowment (MML), a Debicki Foundation Grant (MML), the Burroughs Wellcome Fund Career Award at the Scientific Interface (MML), Gesualdo Family Research Scholar (MML), and NIH/NCI training grant T32 CA009560 (HK). Presentation Date: Saturday, June 17, 2023

Steroidogenic Factor-1 form and function: From phospholipids to physiology

Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer.

Predictive role of 17α-hydroxy-progesterone serum levels of response to follicle-stimulating hormone in patients with abnormal sperm parameters

To study the possible role of serum 17α-hydroxy-progesterone (17αOH-P) levels in predicting favorable responses to follicle-stimulating hormone (FSH) administration in patients with normal serum FSH levels and idiopathic abnormal sperm parameters. Prospective cohort study. University-affiliated fertility center. Fifty patients with oligozoospermia, asthenozoospermia, and/or teratozoospermia and normal serum levels of gonadotropins and total testosterone (TT). Treatment with exogenous FSH is administered subcutaneously at a dose of 150 IU 3 times a week for 3 consecutive months. Luteinizing hormone levels, FSH levels, TT levels, 17αOH-P levels, testicular volume, conventional sperm parameters, and seminal spermatid concentration were evaluated before and after therapy. To evaluate the predictive role of pretreatment serum 17αOH-P levels on FSH responsiveness, the doubling of sperm concentration at the end of the FSH administration was considered a positive outcome. After therapy, patients showed a significant increase in sperm concentration, total sperm count (TSC), progressive motility, percentage of normal forms, FSH levels, TT levels, and testicular volume. There was a negative correlation between pretreatment 17αOH-P levels and the posttreatment increase in sperm concentration, TSC, progressive motility, and normal morphology, and a positive correlation with the posttreatment increase in spermatids. Predictive analysis showed that 17αOH-P levels (<1.18 ng/mL) foretold a doubling of sperm concentration with a sensitivity of 90.0% and a specificity of 73.3%, and of TSC with a sensitivity of 91.3% and a specificity of 81.48%. The results of this study suggest that pretreatment serum levels of 17αOH-P, a marker of steroidogenic function, appear to be able to predict the success of subcutaneous administration of exogenous FSH in terms of spermatogenesis improvement. Receiver operating characteristic curves indicated that 17αOH-P levels (<1.18 ng/mL) predict a doubling of sperm concentration and TSC after exogenous FSH administration to patients with idiopathic abnormal sperm parameters and normal gonadotropin levels.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Steroidogenic potential of the gonad during sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Prior studies demonstrated that gonadal differentiation in the rice field frog, Hoplobatrachus rugulosus, was of an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the steroidogenic potential of the gonad is still unknown. In this study, H. rugulosus were obtained by stimulating fertilization in the laboratory under natural light and temperature conditions. The gonads were collected and their steroidogenic potential was evaluated by determining the expression level of messenger RNA (mRNA) encoding for cytochrome P450 17-hydroxylase/C17-20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) using quantitative real-time RT-PCR and the localization of CYP17 mRNA in tissues by in situ hybridization. The CYP17 mRNA levels in males at 4-11 weeks postmetamorphosis were higher than in female and intersex gonads. This corresponded to their localization in the gonadal tissues, where CYP17 signals were specifically detected in the Leydig cells of the testis at 5-16 weeks postmetamorphosis but was undetectable in all ovary samples. The CYP19 mRNA levels in females at 4-11 weeks postmetamorphosis was higher than in male and intersex gonads, which corresponded with gonadal development, indicating the potential steroidogenic function of the ovary. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after gonadal sex differentiation and the steroidogenic potential of the gonads exhibited a sexual dimorphic pattern. These results provide a crucial basis for further research on the developmental biology in anuran species.

Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-β1 via the Hippo signaling pathway

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-β1 (TGF-β1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-β1 (Ad-sh-TGF-β1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-β1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-β1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD. Similar to Ad-sh-TGF-β1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-β1, Ad-ADM improved LPS-induced TGF-β1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-β1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-β1 through Hippo signaling.

Modulating Effect of the Leptin Fragment 116–122 on Testicular Steroidogenesis in Male Rats

Adipokine leptin plays an important role in the regulation of the reproductive system. It stimulates the activity of the hypothalamic-pituitary-gonadal axis by indirectly acting on GnRH-secreting neurons and modulates testicular steroidogenic function by binding to leptin receptors in Leydig cells. A leptin fragment 116–122 (LF) including the main receptor-binding determinants of this adipokine, normalizes metabolic parameters in animals with diet-induced obesity. However, its ability to influence the steroidogenic function of the testes, including through interaction with GnRH neurons of the hypothalamus, has not been studied. The aim of this work was to study the effects of a single and three-day intranasal administration of LF (200 μg/rat) on the blood testosterone level and the expression of steroidogenic genes in the testes in mature male Wistar rats. To evaluate the effect of LF on testicular steroidogenesis upon stimulation with human chorionic gonadotropin (hCG, 15 IU/rat, s/c), a stimulator of testosterone synthesis and an antagonist of the GnRH receptor cetrorelix (75 μg/kg, s.c.) an inhibitor of testicular steroidogenesis. It has been shown that LF increases the level of testosterone in the blood after a single injection, and after a three-day administration it enhances the steroidogenic effect of hCG. LF and hCG increased the expression of the Star gene encoding the key regulatory protein of steroidogenesis StAR. Administration of cetrorelix reduced testosterone levels and Star expression, and compensatory increased expression of the luteinizing hormone receptor gene. The potentiating effect of LF on hCG-induced stimulation of testosterone levels and Star expression was not detected under conditions of GnRH antagonist treatment. Thus, LF is capable of stimulating steroidogenesis in rat testis by itself and potentiating the steroidogenic effects of hCG. Since its effects are suppressed in the presence of a GnRH antagonist, there is reason to assume that the effect of LF is realized through stimulation of hypothalamic GnRH neurons.