Effect of Curcuma longa extract on reproduction function in mice and testosterone production in Leydig cells.

Curcuma longa, best known for its culinary application as the main constituent of curry powder, has shown potential impact on the reproductive system. This study aimed to investigate the efficacy of Curcuma longa extract (CLE) on Kidney-Yang deficiency mice induced by hydrocortisone and the possible roles in testosterone secretion in Leydig cells. We evaluated male sexual behaviour, reproductive organ weight, testosterone levels, and histological tissue changes in hydrocortisone-induced mice. CLE effectively reversed hydrocortisone-induced Kidney-Yang deficiency syndrome by improving sexual behaviour, testis and epididymis weight, testosterone levels and reducing pathological damage. Our in vitro study further indicated that CLE stimulated testosterone production via upregulating the mRNA and protein expression of steroidogenic enzymes in Leydig cells. It significantly improved H89-inhibited protein expression of StAR and cAMP-response element-binding (CREB), as well as melatonin-suppressed StAR protein expression. The data obtained from this study suggest that CLE could alleviate Kidney-Yang deficiency symptoms and stimulate testosterone production by upregulating the steroidogenic pathway. This research identifies CLE as a potential nutraceutical option for addressing testosterone deficiency diseases.

Butorphanol inhibits androgen biosynthesis and metabolism in rat immature Leydig cells in vitro.

Butorphanol is a synthetic opioid analgesic medication that is primarily used for the management of pain. Butorphanol may have an inhibitory effect on androgen biosynthesis and metabolism in rat immature Leydig cells. The objective of this study was to investigate the influence of butorphanol on androgen secretion by rat Leydig cells isolated from the 35-day-old male rats. Rat Leydig cells were cultured with 0.5-50 µM butorphanol for 3 h in vitro. Butorphanol at 5 and 50 µM significantly inhibited androgen secretion in immature Leydig cells. At 50 µM, butorphanol also blocked the effects of luteinizing hormone (LH) and 8bromo-cAMP-stimulated androgen secretion and 22R-hydroxycholesterol- and pregnenolone-mediated androgen production. Further analysis of the results showed that butorphanol downregulated the expression of genes involved in androgen production, including Lhcgr (LH receptor), Cyp11a1 (cholesterol side-chain cleavage enzyme), Srd5a1 (5α-reductase 1), and Akr1c14 (3α-hydroxysteroid dehydrogenase). Additionally, butorphanol directly inhibited HSD3B1 (3ß-hydroxysteroid dehydrogenase 1) and SRD5A1 activity. In conclusion, butorphanol may have side effects of inhibiting androgen biosynthesis and metabolism in Leydig cells.

Sudachitin Alleviates Paraquat Instigated Testicular Toxicity in Albino Rats via Regulating Nrf-2/Keap-1, Inflammatory, Steroidogenic, and Histological Profile.

Paraquat (PQ) is a noxious herbicide which adversely affects the vital organs including male reproductive system. Sudachitin (SCN) is a naturally occurring flavonoid that demonstrates a wide range of biological potentials. The current study was designed to investigate the alleviative potential of SCN to avert PQ-induced testicular toxicity in rats. Forty-eight male rats (Rattus norvegicus) were apportioned into four groups including control, PQ (5 mg/kg), PQ + SCN (5 mg/kg + 30 mg/kg), and SCN (30 mg/kg) only treated group. Our findings elucidated that PQ treatment reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its antioxidant genes as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSR) and glutathione peroxidase (GPx), while elevating the levels of reactive oxygen species (ROS), and malondialdehyde (MDA). Furthermore, PQ intoxication upregulated the expressions of Keap-1 while downregulating the expression of 3-beta hydroxysteroid dehydrogenase (3ß-HSD), 17-beta hydroxysteroid dehydrogenase (17ß-HSD), and steroidogenic acute regulatory protein (StAR). Moreover, sperm anomalies were increased following the exposure to PQ. Besides, PQ exposure decreased the levels of plasma testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) while increasing the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), interleukin-1beta (IL-1ß), and cyclooxygenase-2 (COX-2). Additionally, PQ treatment escalated the expressions of cysteinyl aspartate-specific proteases-3 (Caspase-3) and Bcl-2-associated X-protein (Bax) while downregulating the expressions of B-cell lymphoma-2 (Bcl-2). Furthermore, PQ exposure disrupted the normal architecture of testicular tissues. However, SCN treatment remarkably protected the testicular tissues via regulating the aforementioned disruptions owing to its antioxidant, anti-inflammatory, and androgenic potential.

Diagnostic Value of Autoantibodies against Steroidogenic Enzymes and Hormones in Infertile Women with Premature Ovarian Insufficiency.

The objective of the study was to evaluate the profile and diagnostic significance of serum autoantibodies in infertile patients with premature ovarian insufficiency (POI). The pilot study included 26 patients of reproductive age with POI and diminished ovarian reserve who received complex treatment using new surgical technologies (Group 1) and 18 patients without POI (Group 2). The profile of serum autoantibodies, including anti-ovarian antibodies, antibodies against thyroid peroxidase (TPO), steroidogenic enzymes, and steroid and gonadotropic hormones, was studied using modified ELISAs and human recombinant steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2). Patients in Group 1 had higher levels of IgG autoantibodies against steroidogenic enzymes, estradiol, progesterone, and TPO than those in Group 2. Tests for IgG antibodies against CYP11A1, CYP19A1, and CYP21A2 exhibited high sensitivity (65.4-76.9%), specificity (83.3-89.9%), and AUC values (0.842-0.910) for POI, the highest in the first test. Three-antibodies panel screening showed higher diagnostic accuracy (84.1% versus 75-79.6%). The levels of these antibodies correlated with menstrual irregularities and a decrease in the antral follicle count. Thus, antibodies against CYP11A1, CYP19A1, and CYP21A2 have a high diagnostic value for POI. Three-antibody panel screening may improve the accuracy of POI diagnosis and be useful for identifying high-risk groups, early stages of the disease, and predicting POI progression.

Time- and glucose-dependent differentiation of 3T3-L1 adipocytes mimics dysfunctional adiposity.

The 3T3-L1 murine adipocyte cell line remains one of the most widely used models to study the mechanisms of obesity and related pathologies. Most studies investigate such mechanisms using mature adipocytes that have been chemically induced to differentiate for 7 days in media containing 25 mM glucose. However, the dysfunctional characteristics commonly observed in obesity including adipocyte hypertrophy, increased expression of inflammatory markers, enhanced production of reactive oxygen species (ROS), increased steroidogenic enzyme expression/activity and production of steroid hormones, are not necessarily mimicked in these cells. The aim of this study was to provide an inexpensive model which represents the well-known characteristics of obesity by manipulating the time of adipocyte differentiation and increasing the concentration of glucose in the cell media. Our results showed a glucose- and time-dependent increase in adipocyte hypertrophy, ROS production and gene expression of the pro-inflammatory cytokine interleukin-6 (IL-6), as well as a time-dependent increase in lipolysis and in the gene expression of the chemokine monocyte chemoattractant protein 1 (MCP1). We also showed that gene expression of the steroidogenic enzymes 11-beta-hydroxysteroid dehydrogenase type 1 (11ßHSD1), 17ßHSD type 7 and 12, as well as CYP19A1 (aromatase), were significantly higher in the hypertrophic model relative to the control adipocytes differentiated using the conventional method. The increase in 11ßHSD1 and 17ßHSD12 expression was consistent with the enhanced conversion of cortisone and androstenedione to cortisol and testosterone, respectively. As these characteristics reflect those commonly observed in obesity, hypertrophic 3T3-L1 adipocytes are an appropriate in vitro model to study mechanisms of adipocyte dysfunction in an era where the rise in obesity incidence is a global health concern, and where access to adipose tissue from obese patients are limited.

Sex and seasonal differences in neural steroid sensitivity predict territorial aggression in Siberian hamsters.

Many animals display marked changes in physiology and behavior on a seasonal timescale, including non-reproductive social behaviors (e.g., aggression). Previous studies from our lab suggest that the pineal hormone melatonin acts via steroid hormones to regulate seasonal aggression in Siberian hamsters (Phodopus sungorus), a species in which both males and females display increased non-breeding aggression. The neural actions of melatonin on steroids and aggressive behavior, however, are relatively unexplored. Here, we housed male and female hamsters in long-day photoperiods (LDs, characteristic of breeding season) or short-day photoperiods (SDs, characteristic of non-breeding season) and administered timed melatonin (M) or control injections. Following 10 weeks of treatment, we quantified aggressive behavior and neural steroid sensitivity by measuring the relative mRNA expression of two steroidogenic enzymes (aromatase and 5α-reductase 3) and estrogen receptor 1 in brain regions associated with aggression or reproduction [medial preoptic area (MPOA), anterior hypothalamus (AH), arcuate nucleus (ARC), and periaqueductal gray (PAG)] via quantitative PCR. Although LD-M and SD males and females displayed increased aggression and similar changes in gene expression in the ARC, there were sex-specific effects of treatment with melatonin and SDs on gene expression in the MPOA, AH, and PAG. Furthermore, males and females exhibited different relationships between neural gene expression and aggression in response to melatonin and SDs. Collectively, these findings support a role for melatonin in regulating seasonal variation in neural steroid sensitivity and aggression and reveal how distinct neuroendocrine responses may modulate a similar behavioral phenotype in male and female hamsters.

Pyridoxamine protects human granulosa cells against advanced glycation end-products-induced steroidogenesis disturbances.

BACKGROUND: Ovarian advanced glycation end-products (AGEs) accumulation is associated with ovarian granulosa cells (GCs) dysfunction. Vitamin B6 derivatives positively affected reproduction. The current study was conducted to elucidate the AGEs effects on human luteinized mural GCs steroidogenesis in the presence or absence of pyridoxamine (PM). METHODS AND RESULTS: Isolated GCs of 50 healthy women were divided into four parts and treated with media alone (Control), PM alone, or human glycated albumin (HGA) with/without PM. Main steroidogenic enzymes and hormones were assessed by qRT-PCR and ELISA. The AGE receptor (RAGE) protein was also determined using Western blotting. The non-toxic concentration of HGA increased the expression of RAGE, StAR, 3ß-HSD, and 17ß-HSD (P < 0.0001 for all) but decreased the expression of CYP19A1 at mRNA levels. The increased RAGE protein expression was also confirmed by western blot analysis. These effects resulted in declined estradiol (E2), slightly, and a sharp rise in progesterone (P4) and testosterone (T) levels, respectively. PM, on its own, ameliorated the HGA-altered enzyme expression and, thereby, corrected the aberrant levels of E2, P4, and T. These effects are likely mediated by regulating the RAGE gene and protein expression. CONCLUSION: This study indicates that hormonal dysfunctions induced by the AGEs-RAGE axis in luteinized GCs are likely rectified by PM treatment. This effect is likely acquired by reduced expression of RAGE. A better understanding of how AGEs and PM interact in ovarian physiology and pathology may lead to more targeted therapy for treating ovarian dysfunction.

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17ß (E2) assumes a central role in follicular development and selection by activating estrogen receptors ß (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

Icariin promotes mouse Leydig cell testosterone synthesis via the Esr1/Src/Akt/Creb/Sf-1 pathway.

Icariin (ICA), extracted from Epimedium, is a flavonoid used in traditional Chinese medicine. Di(2-ethylhexyl) phthalate (DEHP) is a phthalate used in commercial products as a plasticizer that can influence the human endocrine and reproduction system. We previously found that ICA reversed DEHP-induced damage through the prevention of reactive oxygen species accumulation and promotion of testosterone secretion. Here we investigated the mechanisms of ICA in promoting testosterone secretion from murine Leydig cells. We used ICA, DEHP, the Akt agonist SC-79, the Akt inhibitor MK2206, and the Creb inhibitor KG501 to determine the effect of these treatments on the expression levels of the steroidogenic enzymes, Cyp11a1 and Hsd3b, which play critical roles in androgen production, in Leydig cells. Bioinformatic analysis was used to search for ICA-targeted proteins and their associated pathways. We found that icariin interacted with estrogen receptor on the cell membrane, leading to increased phosphorylation levels of Akt and Creb proteins and enhanced transcription of genes encoding steroidogenic enzymes and testosterone synthesis. We further investigated ICA activity in vivo using male mice pretreated with 100 mg/kg ICA and then treated with 750 mg/kg DEHP. ICA pretreatment reversed the reduced protein expression levels of Cyp11a1 and Hsd3b induced by DEHP in Leydig cells in vivo. Furthermore, while the phosphorylation levels of Akt and Creb were decreased in testes of mice exposed to DEHP alone, these effects were reversed by ICA pretreatment. These findings indicate that ICA promotes testosterone synthesis via the Esr1/Src/Akt/Creb/Sf-1 signaling pathway.

Impaired negative feedback and death following acute stress in glucocorticoid receptor knockout Xenopus tropicalis tadpoles.

Blood glucocorticoid levels are regulated by the hypothalamo-pituitary-adrenal/interrenal axis (HPA axis in mammals, HPI axis in amphibians), and negative feedback by glucocorticoid signaling is a key player in that regulation. Glucocorticoid and mineralocorticoid receptors (GR and MR) mediate negative feedback in mammals, but little is known about nuclear receptor-mediated feedback in amphibians. Because amphibians have only one corticosteroidogenic cell type responsible for glucocorticoid and mineralocorticoid production, we hypothesized that GR knockout (GRKO) tadpoles have elevated levels of glucocorticoids and mineralocorticoids as well as axis components regulating their production. We also examined the response to stress and potential for increased aldosterone signaling in GRKO tadpoles. We found that GRKO tadpoles have severe hyperactivity of the HPI axis, namely high mRNA expression levels of pomc, cyp17a1, cyp21a2, cyp11b2, and star, and high tissue content of corticosterone, aldosterone, 17-hydroxyprogesterone, 21-deoxycortisol, and progesterone. Such aberrant HPI activity was accompanied by reduced survival after acute temperature shock and shaking stress. Like mammalian models of HPA hyperactivity, GRKO tadpoles have high MR mRNA expression levels in brain, kidney, heart, and skin and high levels of the inflammatory cytokine tnf-α and the profibrotic factor tgf-ß in kidneys. This study showed GR is critical for negative feedback to the amphibian HPI axis and for survival from acute stressors. This study also showed GRKO tadpoles exhibit altered expression/overproduction of regulators of salt-water homeostasis and associated biomarkers of kidney disease.

Investigation of the effect of hesperidin on some reproductive parameters in testicular toxicity induced by Bisphenol A.

Bisphenol A (BPA) is one of the chemicals that cause dysfunction and infertility in testicles. Therefore, it is crucial to develop effective treatments against this damage. In this study, the effects of Hesperidin (HESP), a flavonoid in testicular toxicity induced by BPA in rats, on oxidative stress, inflammation, apoptosis, histological damage, spermatogenesis, steroidogenic enzymes and reproductive hormones were investigated. Our study used 52 Sprague Dawley male rats weighing 250-300 g, and four experimental groups were formed. From the experimental groups, 1 ml of olive oil was administered to the control group, HESP at a dose of 50 mg/kg to the HESP group, BPA at a dose of 100 mg/kg to the BPA group, HESP at a dose of 50 mg/kg to the BPA + HESP group and 100 mg/kg BPA was administered intragastrically (ig) for 14 days. We determined that BPA administration causes apoptosis, histological damage, inflammation, oxidative stress and toxic effects on spermatogenesis and steroidogenic enzymes in testicles. We observed that the administration of HESP with BPA attenuated oxidative stress, inflammation and apoptosis resulting in therapeutic effects on both steroidogenic enzymes and spermatogenesis and reproductive hormones (FSH, LH and testosterone). Our findings from this study clearly showed that while HESP treatment alleviates oxidative damage, inflammation and apoptosis in testicles of rats treated with BPA, it has regulatory effects on steroidogenic enzymes, spermatogenesis and serum reproductive hormones.

Leydig Cells in Immunocastrated Polish Landrace Pig Testis: Differentiation Status and Steroid Enzyme Expression Status.

Porker immunocastration against gonadoliberin (GnRH) secretion has been utilized since 2009; however, consumers are still skeptical of it. This is due to not having full information available on the problem of a boar taint, as well as a lack of research on morphological and molecular changes that may occur in the animal reproductive system and other body systems. The present study aimed to explore the functional status of steroidogenic Leydig cells of the testicular interstitial tissue in immunocastrated Polish Landrace pigs. Analyses were performed using Western blot, immunohistochemistry for relaxin (RLN), insulin-like 3 protein (INSL3), pelleted growth factor receptor α (PDGFRα), cytochrome P450scc, 3ß- and 17ß-hydroxysteroid dehydrogenases (3ß-HSD, 17ß-HSD), cytochrome P450arom, and 5α-reductase (5α-RED). Immunoassay ELISA was used to measure the androstenone, testosterone, and estradiol levels in the testis and serum of immunocastrates. We revealed disturbances in the distribution and expression of (i) RLN, indicating an inflammatory reaction in the interstitial tissue; (ii) INSL3 and PDGFRα, indicating alterations in the differentiation and function of fetal, perinatal, or adult Leydig cell populations; (iii) P450scc, 3ß-HSD, 17ß-HSD, P450arom, and 5α-RED, indicating disturbances in the sex steroid hormone production and disturbed functional status of Leydig cells; as well as (iv) decreased levels of androstenone, testosterone, and estradiol in testicular tissue and serum, indicating the dedicated action of Improvac to reduce boar taint at both the hypothalamic-hypophysis-gonadal axis and local level (Leydig cells). In summary, our study provides a significant portion of knowledge on the function of Leydig cells after immunocastration, which is also important for the diagnosis and therapy of testis dysfunction due to GnRH action failure and/or Leydig cell differentiational-functional alterations.

Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos.

Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERß, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERß, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERß, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERß, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.

Role of sex steroids in fish sex determination and differentiation as revealed by gene editing.

The involvement of sex steroids in sex determination and differentiation is relatively conserved among non-mammalian vertebrates, especially in fish. Thanks to the advances in genome sequencing and genome editing, significant progresses have been made in the understanding of steroidogenic pathway and hormonal regulation of sex determination and differentiation in fish. It seems that loss of function study of single gene challenges the traditional views that estrogen is required for ovarian differentiation and androgen is needed for testicular development, but it is not so in essence. Steroidogenic enzymes can be classified into two categories based on expression and enzyme activities in fish. One type, encoded by star2, cyp17a1 and cyp19a1a, is involved in estrogen production and exclusively expressed in the gonads. Mutation of these genes results in the up-regulation of male pathway genes and sex reversal from genetic female to male. The other type, encoded by the duplicated paralogs of the above genes, including star1, cyp11a1, cyp17a2 and cyp19a1b, as well as cyp11c1 gene, is dominantly expressed both in gonads and extra-gonadal tissues. Mutation of these genes alters the steroids (androgen, DHP and cortisol) production and spermatogenesis, fertility, secondary sexual characteristics and sexual behavior, but usually does not affect the sex differentiation. For the estrogen receptors (esr1, esr2a and esr2b), single mutation failed to, but double and triple mutation leads to sex reversal from female to male, indicating that at least Esr2a and Esr2b are required to mediate the role of estrogen in sex determination proved by gene editing experiments. Taken together, results from gene editing enrich our understanding of steroid synthesis pathways and further confirm the critical role of estrogen in female sex determination by antagonizing the male pathway in fish.

Rutin inhibits androgen synthesis and metabolism in rat immature Leydig cells in vitro.

In the early stage of androgen-sensitive prostate cancer, cancer cells require androgens to grow. Hormone therapy that lowers androgen output or blocks androgen receptor can suppress the growth of this type of prostate cancer. Rutin, a flavonoid derivative of many plants, has numerous pharmacological effects. The objective of this study was to investigate the effect of rutin on androgen biosynthesis in Leydig cells isolated from the testes of pubertal rats. Immature Leydig cells isolated from 35 days-old male Sprague-Dawley rats were cultured in vitro with 0.5-50 µM rutin for 3 hr. Rutin significantly inhibited androgen secretion at 0.5, 5 and 50 µM under basal condition (medium only). At 50 µM, rutin also markedly compromised androgen secretion stimulated by 10 ng/ml luteinising hormone and 10 mM 8-bromoadenosine 3', 5'-cyclic monophosphate. Further analysis demonstrated that rutin compromised the transcript levels of Scarb1, Cyp11a1 and Hsd3b1 and their proteins expression. Rutin directly inhibited rat testicular CYP17A1, HSD17B3 and AKR1C14 activities at 50 µM. Rutin did not alter mitochondrial membrane potential at up to 50 µM. In conclusion, rutin suppresses androgen biosynthesis in Leydig cells through multiple mechanisms, thereby having benefits for the treatment of androgen-sensitive prostate cancer.

Follicular hormone dynamics during the midcycle surge of gonadotropins in women undergoing fertility treatment.

Changes in concentrations of intra-follicular hormones during ovulation are important for final oocyte maturation and endometrial priming to ensure reproductive success. As no human studies have investigated these changes in detail, our objective was to describe the dynamics of major follicular fluid (FF) hormones and transcription of steroidogenic enzymes and steroid receptors in human granulosa cells (GCs) during ovulation. We conducted a prospective cohort study at a public fertility clinic in 2016-2018. Fifty women undergoing ovarian stimulation for fertility treatment were included. From each woman, FF and GCs were collected by transvaginal ultrasound-guided follicle puncture of one follicle at two specific time points during ovulation, and the study covered a total of five time points: before ovulation induction (OI), 12, 17, 32 and 36 h after OI. Follicular fluid concentrations of oestradiol, progesterone, androstenedione, testosterone, 17-hydroxyprogesterone, anti-Mullerian hormone, inhibin A and inhibin B were measured using ELISA assays, and a statistical mixed model was used to analyse differences in hormone levels between time points. Gene expression of 33 steroidogenic enzymes and six hormone receptors in GCs across ovulation were assessed by microarray analysis, and selected genes were validated by quantitative reverse transcription PCR. We found that concentrations of oestradiol, testosterone, progesterone, AMH, inhibin A and inhibin B (P < 0.001) and gene expression of 12 steroidogenic enzymes and five receptors (false discovery rate < 0.0001) changed significantly during ovulation. Furthermore, we found parallel changes in plasma hormones. The substantial changes in follicular hormone production during ovulation highlight their importance for reproductive success.

Seasonal expression and cellular distribution of star and steroidogenic enzymes in quail testis.

The quail Coturnix coturnix is a seasonal breeder with a physiological switch on/off of gonadic activity. Photoperiod and temperature are the major environmental factors regulating the spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we have investigated the localization of StAR protein and steroidogenic enzymes (3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red) as well as androgen and estrogen levels, in the testis of reproductive and nonreproductive quails. We demonstrated that StAR, 3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red were always present in the somatic (Leydig and Sertoli cells) and germ cells (spermatogonia, spermatocytes I and II, spermatids, and spermatozoa). In addition, by western blot analysis, we demonstrated that 17ß-HSD, P450 aromatase, and 5α-Red showed the highest expression levels during the reproductive testis compared with nonreproductive one. Accordingly, we also found that during the reproductive phase the highest titres of testosterone, 17ß-estradiol, and 5α-dihydrotestosterone are recorded. In conclusion, our findings demonstrated that in C. coturnix: (a) both somatic and germ cells are involved in the local synthesis of sex hormones; (b) 17ß-HSD, P450 aromatase, and 5α-Red expressions, as well as testicular androgens and estrogens, increased in reproductive quail testis. This study strongly indicates that the steroidogenic process in quail testis exhibits seasonal changes with the promotion of both androgenic and estrogenic pathways in the reproductive period, suggesting their synergic mechanism in the spermatogenesis regulation.

The effect of seminal plasma ß-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells.

BACKGROUND: Nerve growth factor (ß-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified ß-Nerve Growth Factor (ß-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. METHODS: Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 µg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama ß-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or ß-NGF treatment. RESULTS: Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified ß-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified ß-NGF with respect to the control group. All treatments except ß-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or ß-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified ß-NGF (10 and 20 h). Experiment II: Only ß-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with ß-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or ß-NGF treatment. CONCLUSIONS: Systemic administration of purified ß-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or ß-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes.

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 -9 and 10 -7 M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.

Effect of mice Taenia crassiceps WFU cysticerci infection on the ovarian folliculogenesis, enzyme expression, and serum estradiol.

The murine infection with Taenia crassiceps WFU (T. crassiceps WFU) cysticerci has been widely used as an experimental model to better understand human cysticercosis. Several reports have established that the host hormonal environment determines the susceptibility and severity of many parasite infections. Female mice are more susceptible to infection with T. crassiceps cysticerci suggesting that a rich estrogen environment facilitates their reproduction. Ovarian androgens and estrogens are synthesized by key enzymes as P450-aromatase and 17α-hydroxilase/17, 20 lyase (P450C17). The aim of this study was to determine the effect of chronic intraperitoneal infection of T. crassiceps WFU cysticerci on mice ovarian follicular development, ovulation, the expression of ovarian P450-aromatase and P450C17, and serum 17ß-estradiol, key enzymes of the ovarian steroidogenic pathway. To perform this study ovaries and serum were obtained at two, four and six months from T. crassiceps WFU cysticerci infected mice, and compared to those of healthy animals. The ovaries were fixed and processed for histology or lysed in RIPA buffer for Western blot using specific antibodies for P450C17 and P450-aromatase. 17ß-estradiol serum concentration was measured by ELISA. The results showed that the infection with T. crassiceps WFU cysticerci significantly reduced the number of primordial and primary follicles after two months of infection. Through the course of the study, the corpus luteum number began to decrease, whereas atretic follicles increased. The expression of ovarian P450C17 and P450-aromatase as well as serum E2 concentration were significantly increased in the infected group compared to control. These findings show that chronic infection with Taenia crassiceps WFU may alter the reproductive functions of the female mice host.