Steroidogenic Enzyme Gene Expression Research Articles

Chrysin’s dose-dependent effects on steroidogenesis in female BALB/c mice: In vivo study of adrenal, ovarian, and uterine hormone regulation

Background: Chrysin is known for its pharmacological effects and structural resemblance to estrogen. The study explores the impact of chrysin on steroidogenesis focusing on adrenal and ovarian steroidogenic enzymes. Materials and methods: Thirty female BALB/c mice were divided into three groups: a control group and two chrysin-treated groups (50 mg and 100 mg). Gene expression of key steroidogenic enzymes was assessed in adrenal glands and ovaries using RT-qPCR. Uterine expression of estrogen receptor alpha (ERα) was also examined. Histological analysis of adrenal glands and ovaries was performed. Results: High-dose chrysin downregulated CYP17A1 expression in adrenal glands compared to control and low-dose groups. In contrast, 3β-HSD was significantly downregulated in the high-dose group. In ovaries, high dose chrysin reduced aromatase expression. Conclusion: Our findings revealed that chrysin’s impact on steroidogenesis is dose-dependent. By downregulating CYP17A1 in adrenal glands, potentially affecting androgen and estrogen synthesis, and enhancing aromatase expression in ovaries at lower doses.

Steroidogenic enzyme gene expression and testosterone production are developmentally modulated by bone morphogenetic protein receptor-1B in mouse testis.

Bone morphogenetic proteins (BMPs) and receptors (BMPR-1A, BMPR-1B, BMPR-2) have been shown to be vital for female reproduction, while their roles in males are poorly described. Our study was undertaken to specify the function of BMPR-1B in steroidogenic enzyme gene expression, testosterone production and reproductive development in male mice, given that Bmpr1b mRNA is expressed in mouse testis and Bmpr1b knockout results in compromised fertility. Male mice were passively immunized for 6 days with anti-BMPR-1B in the presence or absence of exogenous gonadotrophins. We then measured the effects of anti-BMPR-1B on testicular hydroxysteroid dehydrogenase isoforms (Hsd3b1, Hsd3b6, and Hsd17b3) and aromatase (Cyp19) mRNA expression, testicular and serum testosterone levels, and testis and seminal vesicle weight. In vitro testosterone production in response to anti-BMPR-1B was determined using testicular culture, and Leydig cell culture in the presence or absence of gonadotrophins. In Leydig cell culture the contribution of seminiferous tubules and Leydig cells were examined by preconditioning the media with these testicular constituents. In adult mice, anti-BMPR-1B increased testosterone and Hsd3b1 but decreased Hsd3b6 and Cyp19 mRNA. In adult testicular culture and seminiferous tubule conditioned Leydig cell culture, anti-BMPR-1B reduced testosterone, while in normal and Leydig cell conditioned Leydig cell culture it increased testosterone levels. In pubertal mice, anti-BMPR-1B reduced gonadotrophin stimulated seminal vesicle growth. In conclusion, BMPR-1B has specific developmental functions in the autocrine and paracrine regulation of testicular steroidogenic enzyme gene expression and testosterone production in adults and in the development of seminal vesicles during puberty.

Subacute exposure to Roundup® changes steroidogenesis and gene expression of the glutathione-glutaredoxin system in rat ovaries: Implications for ovarian toxicity of this glyphosate-based herbicide

Studies have indicated that glyphosate induces endocrine disruption and may adversely affect the male reproductive system. However, evidence of its effects on ovarian function is poorly understood so far, making further studies necessary on the mechanisms of the glyphosate toxicity in the female reproductive system. The aim of this work was to evaluate the effect of a subacute exposure (28 days) to the glyphosate-based formulation Roundup® (1.05, 10.5 and 105 μg/kg b.w. of glyphosate) on steroidogenesis, oxidative stress, systems involved in cell redox control and histopathological parameters in rat ovaries. Hence we quantify plasma estradiol and progesterone by chemiluminescence; non-protein thiol levels, TBARS, superoxide dismutase and catalase activity by spectrophotometry; gene expression of steroidogenic enzymes and redox systems by real-time PCR; and ovarian follicles by optical microscopy. Our results demonstrated that oral exposure increased progesterone levels and the mRNA expression of 3β-hydroxysteroid dehydrogenase. Histopathological analysis revealed a decrease in the number of primary follicles and an increase in the number of corpus luteum in rats exposed to Roundup®. An imbalance of the oxidative status was also evidenced by decreasing the catalase activity at all groups exposed to the herbicide. Increased lipid peroxidation and gene expression of glutarredoxin and decreased of glutathione reductase were also observed. Our results indicate that Roundup® causes endocrine disruption of hormones related to female fertility and reproduction and changes the oxidative status by altering antioxidant activity, inducing lipid peroxidation, as well as changing the gene expression of the glutathione-glutarredoxin system in rat ovaries.

Effect of anti-estrogen and anti-progesterone on spermatogenesis, testosterone production and expression of steroidogenic enzyme genes in adult male rats.

The present study was planned to investigate the anti-spermatogenic and anti-steroidogenic effects of Clomiphene Citrate (CC) an anti-estrogen and Mifepristone (MT) an anti-progesterone in the testis of male rats. Following the oral administration of 1.0mg and 5.0mg/kg b.w/day of each for the duration of 30 and 60 days, quantitation of spermatogenesis, RIA for serum and intra-testicular testosterone levels, western blotting and RT-PCR for expression of StAR, 3β-HSD and P450arom enzymes in the testis was done. Clomiphene Citrate at 5.0mg/kg b.w/day for 60 days significantly reduced testosterone (T) levels however the effect was not significant with the lower doses. Reproductive parameters in animals treated by Mifepristone remained mostly unaffected, however, a significant decline in testosterone levels and altered expression of selected genes was observed in 5.0mg for the 30d treatment group. Clomiphene Citrate at higher doses affected the weights of the testis and secondary sex organs. Seminiferous tubules revealed hypo-spermatogenesis with a significant decrease in the number of maturing germ cells and a reduction in tubular diameter. Attenuation in serum testosterone was associated with the downregulation of expression in StAR, 3β-HSD, and P450arom mRNA and protein levels in the testis even after 30 d of CC administration. The results indicate that the anti-estrogen (Clomiphene Citrate) but not anti-progesterone (Mifepristone) induces hypo-spermatogenesis in rats which are associated with a downregulation of expression of two of the steroidogenic enzymes, 3β-HSD and P450arom mRNA and StAR protein.

Effects of melatonin on a d-galactose-induced male reproductive aging mouse model

Understanding the aging mechanism of the male reproductive system and developing anti-aging interventions are essential for preventing age-related male infertility. The pineal hormone melatonin has been effectively used as an antioxidant and anti-apoptotic molecule in various cells and tissues. However, the effects of melatonin on d-galactose (D-gal)-induced aging have not been studied with regards to testicular function. Thus, we investigated whether melatonin suppresses the dysfunction of male reproductive function induced by D-gal treatment. The mice were divided into the following four groups receiving treatments for six weeks: phosphate-buffered saline (PBS) group, d-galactose (200 mg/kg) group, melatonin (20 mg/kg) group, and d-galactose (200 mg/kg)+ melatonin (20 mg/kg) group. At six weeks of treatments, sperm parameters, body and testes weight, gene and protein expression of germ cell and spermatozoa marker were analyzed. Our results showed that melatonin suppressed the decrease in body weight, sperm vitality, motility, and gene expression levels of spermatozoa markers such as Protamine 1, PGK2, Camk4, TP1, and Crem in the testis of D-gal-induced aging models. However, the gene expression levels of the pre-meiotic and meiotic markers in the testes did not change in the D-gal-injected model. The injection of D-gal impaired the decreased expression of steroidogenic enzyme genes, such as HSD3b1, Cyp17a1, and Cyp11a1, but melatonin inhibited the decrease in the expression of these genes. In addition, protein levels of spermatozoa and germ cell markers were evaluated by immunostaining and immunoblotting. Consistent with the qPCR results, PGK2 protein levels were decreased by d-galactose treatment. A decrease in PGK2 protein levels by D-gal was inhibited by melatonin treatment. In conclusion, melatonin administration improves testicular function with age.

Pathogen odors elicit distinct neurosteroid and behavioral responses in Rainbow trout (Oncorhynchus mykiss)

Rainbow trout can smell virus and bacteria cues that in turn activate an immune response in the brain in absence of a systemic response (i.e. the immune response is activated by the nervous system instead of the pathogen inside tissues or bloodstream). However, little is known if pathogen odors can elicit different physiological responses (i.e. behavioral and immune) and how are pathogen odors integrated into different responses in the brain. We hypothesize that pathogen odors not only activate immune responses but also induce avoidance behavior. Moreover, we hypothesized that pathogen olfactory responses are modulated by neurosteroids at different times in different parts of the brain. We used a two-choice maze test to determine whether inactivated Yersinia ruckeri elicits aversive behavior. Furthermore, to check if behavioral and immune responses are modulated by neurosteroids, we performed a second experiment where we nasally administered inactivated Y. ruckeri to rainbow trout. After that, the nose, olfactory bulb, cerebellum, and optic tectum were collected at four different time points (15 min, 4 hours, 24 hours, and 7 days) and samples were extracted to measure gene expression of several steroidogenic enzymes ( cyp19a1, 3βHSD, 11HSD, and 17HSD) and fish production of neurosteroids (e.g. cortisol, estradiol, and progesterone). We found that rainbow trout actively avoids pathogen odors. Moreover, we found a different expression of neuro steroidogenic enzymes at different brain areas and times post-exposure. Fish exposed to pathogen odor express a significant increase (ANOVA 2 factor, P<0.05) of cyp19a1 (involved in estradiol synthesis) as soon as 15 min in the nose and olfactory bulb, and 4 hours post-exposure in the cerebellum and optic tectum. This gene expression was parallel to a significant increase of estradiol in the nose, bulb, and rest of the brain between 15 min and 4 hours. Moreover, there was a significant increase of 11HSD and 17HSD (related to the production of testosterone, cortisol, and corticosterone) 4 hours after exposure in the nose and cerebellum but a significant decrease in the olfactory bulb and optic tectum. These gene expression patterns were parallel o an increase of cortisol in the nose and olfactory bulb. Finally, we found a significant increase of 3βHSD (involved in the first steps of steroidogenesis) after 24 hours in the nose and olfactory bulb, although we didn’t find an increase in steroid production in our preliminary assays. Our results showed that exposure to pathogen odors activates different neuro steroidogenic pathways at different times after exposure, and at different areas of the brain. These findings pinpoint the use of the sense of smell by fish to control behavioral and immune responses to pathogens, with neurosteroids playing a key role in modulating and integrating these olfactory responses. This material is based upon work supported by the National Science Foundation under Grant No. 1755348 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic effect of gossypetin against paraquat-induced testicular damage in male rats: a histological and biochemical study.

Paraquat (PQ) is an organic compound, which is commonly used as a herbicide in the agriculture sector, and it is also known to stimulate critical damages in the male reproductive system. Gossypetin (GPTN) is one of important members of the flavonoid family, which is an essential compound in flowers and calyx ofHibiscus sabdariffawithpotential pharmacological properties. The current investigation was aimed to examine the ameliorative potential of GPTN against PQ-instigated testicular damages. Adult male Sprague-Dawley rats (n = 48) were distributed into four groups: control, PQ (5mg/kg), PQ + GPTN (5mg/kg + 30mg/kg respectively), and GPTN (30mg/kg). After 56days of treatment, biochemical, spermatogenic indices, hormonal, steroidogenic, pro-or-anti-apoptotic, and histopathological parameters were estimated. PQ exposure disturbed the biochemical profile by reducing the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GSR), while it increased the concentration of reactive oxygen species (ROS) and malondialdehyde (MDA) level. Furthermore, PQ exposure decreased the sperm motility, viability, number of hypo-osmotic tail swelled spermatozoa, and epididymal sperm count; additionally, it increased sperm morphological (head mid-piece and tail) abnormalities. Moreover, PQ lessened the follicle-stimulating hormone (FSH), luteinizing hormone (LH), and plasma testosterone levels. Besides, PQ-intoxication downregulated the gene expression of steroidogenic enzymes (StAR, 3β-HSD, and 17β-HSD) and anti-apoptotic marker (Bcl-2), whereas upregulated the gene expression of apoptotic markers (Bax and Caspase-3). PQ exposure led to histopathological damages in testicular tissues as well. Nonetheless, GPTN inverted all the illustrated impairments in testes. Taken together, GPTN could potently ameliorate PQ-induced reproductive dysfunctions due to its antioxidant, androgenic, and anti-apoptotic potential.

Oxytocin modulates steroidogenesis-associated genes and estradiol levels in the placenta

Oxytocin (OXT) plays a significant role during pregnancy, especially toward the end of pregnancy. Some studies have reported that OXT is involved in the stimulation of steroidogenesis in several organs. However, the effects of OXT on placental steroidogenesis have not yet been established. In this study, we investigated the regulation of steroid hormones and steroidogenic enzymes by OXT-associated signaling in vitro and in vivo. OXT increased the gene expression of steroidogenic enzymes, which convert pregnenolone to progesterone and dehydroepiandrosterone (DHEA) in vitro. In OXT-administered pregnant rats, pregnenolone and DHEA levels were significantly enhanced in the plasma and the expression of the enzymes synthesizing DHEA, testosterone, and estradiol (E2) was increased in placental tissues. Furthermore, OXT was found to affect placental cell differentiation, which is closely related to steroid hormone synthesis. After treatment of the pregnant rats with atosiban, an antagonist of the OXT receptor, the concentration of E2 in the plasma and the expression of E2-synthesizing enzyme were reduced. This regulation may be due to OXT-mediated differentiation, because OXT increases the expression of corticotropin-releasing hormone, which is a biomarker of placental cell differentiation. Our findings suggest that OXT contributes to maintaining pregnancy by regulating the differentiation of placental cells and steroidogenesis during pregnancy.

The effects of irisin and leptin on steroidogenic enzyme gene expression in human granulosa cells: In vitro studies

Reproduction and energy metabolism are closely related, and fertility can be directly affected by either obesity or malnutrition. In this study, we investigated the in vitro effects of irisin and leptin, two hormones primarily involved in energy metabolism, on the expression of genes encoding key steroidogenic enzymes in primary cultures of human granulosa cells. Granulosa cells were purified from follicular fluid samples obtained during in vitro fertilization (IVF) procedure, cultured, and treated with irisin (125-2000 ng/ml) or leptin (25–400 ng/ml) for 1–3 days. mRNA expression levels of cytochrome P450 enzymes [CYP11A1, CYP19A1, CYP21A2], hydroxy-delta-5-steroid dehydrogenase, 3 beta and steroid delta-isomerase 1 (HSD3B1), and hydroxysteroid 17-beta dehydrogenase 3 (HSD17B3) were measured using qRT-PCR analysis. Irisin significantly upregulated CYP19A1 mRNA levels, while leptin upregulated CYP19A1 and HSD3B1 mRNA levels. These preliminary results show that irisin and leptin may directly affect the expression of the genes important for ovarian steroidogenesis and female reproduction.

Effect of Trans-Anethole on Gene Expression of Steroidogenic Enzymes in the Ovary of Polycystic Ovary Syndrome Model Rate:.

The process of steroidogenesis is crucial to the normal function of the ovaries. In individuals with polycystic ovary syndrome (PCOS), the activity of related enzymes in this process is disrupted. In the present study, the effect of trans-anethole was investigated on gene expression of steroidogenesis enzymes in PCOS model rats. In this experimental study, thirty female rats were divided into six groups (n=5 per group). Fifteen PCOS rats in three groups received intraperitoneal injections of distilled water, 50, and 80 mg/kg of trans-anethole, respectively. Also, 15 intact rats in three groups received intraperitoneal injections of distilled water, 50, and 80 mg/kg trans-anethole. The expression of steroidogenesis genes was determined using real-time reverse transcription polymerase chain reaction. The mRNA level of Cyp19 significantly increased in intact rats receiving 80 mg/kg trans-anethole compared to the control group. The Cyp19 level in PCOS groups was significantly reduced compared to the control group. The mRNA level of Cyp19 in PCOS groups that resived 50 or 80 mg/kg trans-anethole increased compared to PCOS rats, but this increase was not statistically significant. The mRNA level of Cyp17 did not significantly change in intact and PCOS rats that received trans-anethole compared to the control group. Trans-anethole may improve PCOS complications due to its involvement in regulating steroidogenesis.

Adiponectin affects uterine steroidogenesis during early pregnancy and the oestrous cycle: An in vitro study

Reproduction in females is an energetically demanding process. We assumed that adiponectin (ADPN), known for its role in energy balance maintenance, is also engaged in the regulation of uterine steroidogenesis in the pig. We determined the impact of ADPN alone or in combination with insulin (INS) on testosterone (T), estrone (E1) and estradiol (E2) secretion by porcine endometrium and myometrium, uterine expression of CYP17A1 and CYP19A3 genes, and endometrial abundance of P450C17 and P450AROM proteins during the peri-implantation period and the oestrous cycle, using radioimmunoassay, qPCR, and Western Blot, respectively. During pregnancy, in the endometrial explants from days 10–11, ADPN decreased CYP17A1 gene expression, P450C17 protein abundance and T secretion, whereas increased E1 secretion. On days 12–13 of pregnancy, ADPN decreased CYP17A1 and CYP19A3 expression, P450C17 and P450AROM protein abundance and E1 secretion, but stimulated T secretion. On days 15–16 of pregnancy, ADPN decreased P450C17 protein accumulation but enhanced CYP19A3 expression and E1 secretion. On days 27–28 of pregnancy, ADPN increased CYP17A1 and CYP19A3 mRNA content and T secretion in this tissue and decreased P450C17 content. ADPN effect on myometrial explants was dependent on stage of gestation or oestrous cycle. Moreover, INS treatment modulated basal and ADPN-affected steroidogenic enzymes gene and protein expression and steroids secretion. The results obtained indicate that ADPN may affect processes required for successful implantation such as steroidogenesis. ADPN and INS were also shown to modulate each other action, which indicates that the proper course of uterine steroidogenesis may be dependent on both hormones’ interaction.

Genome-Wide Identification and Characterization of the BRD Family in Nile Tilapia (Oreochromis niloticus).

Simple SummaryNile tilapia is a good model for genome-wide identification and examination of the expression and role of gene families. In this study, we identified 54 bromodomain genes (BRDs) divided into eight subfamilies in Nile tilapia. Phylogenetic analysis revealed a high conservation of the BRDs family in vertebrates, with BRDs expansion due to fish-specific duplications. Most of the BRDs displayed sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and 9 ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testes of treated fish.The bromodomain (BRD) proteins specifically recognize the N-acetyllysine motifs, which is a key event in the reading process of epigenetic marks. BRDs are evolutionarily highly conserved. Over recent years, BRDs attracted great interest because of their important roles in biological processes. However, the genome-wide identification of this family was not carried out in many animal groups, in particular, in teleosts. Moreover, the expression patterns were not reported for any of the members in this family, and the role of the BRD family was not extensively studied in fish reproduction. In this study, we identified 16 to 120 BRD genes in 24 representative species. BRDs expanded significantly in vertebrates. Phylogenetic analysis showed that the BRD family was divided into eight subfamilies (I–VIII). Transcriptome analysis showed that BRDs in Nile tilapia (Oreochromis niloticus) exhibited different expression patterns in different tissues, suggesting that these genes may play different roles in growth and development. Gonadal transcriptome analysis showed that most of the BRDs display sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and nine ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Consistent with transcriptomic data, the results of qRT-PCR and fluorescence in situ hybridization showed that brdt expression was higher in the testis than in the ovary, suggesting its critical role in the spermatogenesis of the tilapia. Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced, and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testis tissue of treated fish. Our data provide insights into the evolution and expression of BRD genes, which is helpful for understanding their critical roles in sex differentiation and gonadal development in teleosts.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRβ and RXRβ knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Association of DNA methylation with steroidogenic enzymes in Cushing's adenoma.

DNA methylation and demethylation regulate the transcription of genes. DNA methylation-associated gene expression of adrenal steroidogenic enzymes may regulate cortisol production in cortisol-producing adenoma (CPA). We aimed to determine the DNA methylation levels of all genes encoding steroidogenic enzymes involved in CPA. Additionally, the aims were to clarify the DNA methylation-associated gene expression and evaluate the difference of CPA genotype from others using DNA methylation data. Twenty-five adrenal CPA and six nonfunctioning adrenocortical adenoma (NFA) samples were analyzed. RNA sequencing and DNA methylation array were performed. The methylation levels at 118 methylation sites of the genes were investigated, and their methylation and mRNA levels were subsequently integrated. Among all the steroidogenic enzyme genes studied, CYP17A1 gene was mainly found to be hypomethylated in CPA compared to that in NFA, and the Benjamini-Hochberg procedure demonstrated that methylation levels at two sites in the CYP17A1 gene body were statistically significant. PRKACA mutant CPAs predominantly exhibited hypomethylation of CYP17A1 gene compared with the GNAS mutant CPAs. Inverse associations between CYP17A1 methylation in three regions of the gene body and its mRNA levels were observed in the NFAs and CPAs. In applying clustering analysis using CYP17A1 methylation and mRNA levels, CPAs with PRKACA mutation were differentiated from NFAs and CPAs with a GNAS mutation. We demonstrated that CPAs exhibited hypomethylation of the CYP17A1 gene body in CPA, especially in the PRKACA mutant CPAs. Methylation of CYP17A1 gene may influence its transcription levels.

Brown adipose tissue activation with ginsenoside compound K ameliorates polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disease affecting women of reproductive age. Due to its complex aetiology, there is no currently effective cure for PCOS. Brown adipose tissue (BAT) activity is significantly decreased in PCOS patients, and BAT activation has beneficial effects in animal models of PCOS. Here, we investigated the effect of ginsenoside compound K (CK) in an animal model of PCOS and its mechanism of BAT activation. Primary brown adipocytes, Db/Db mice and dehydroepiandrosterone (DHEA)-induced PCOS rats were used. The core body temperature, oxygen consumption, energy metabolism related gene and protein expression were assessed to identify the effect of CK on overall energy metabolism. Oestrous cycle, serum sex hormone, ovarian steroidogenic enzyme gene expression and ovarian morphology were also evaluated following CK treatment. Our results indicated that CK treatment could significantly protect against body weight gain in Db/Db mice via BAT activation. Furthermore, we found that CK treatment could normalize hyperandrogenism, oestrous cyclicity, normalize steroidogenic enzyme expression and decrease the number of cystic follicles in PCOS rats. Interestingly, as a potential endocrine intermediate, C-X-C motif chemokine ligand-14 protein (CXCL14) was significantly up-regulated following CK administration. In addition, exogenous CXC14 supplementation was found to reverse DHEA-induced PCOS in a phenotypically similar manner to CK treatment. In summary, CK treatment significantly activates BAT, increases CXCL14 expression and ameliorates PCOS. These findings suggest that CK might be a potential drug candidate for PCOS treatment.

P-567 Recombinant hCG (choriogonadotropin beta, CG beta) exerts a positive dose-dependent effect on human follicular steroidogenesis during ovarian stimulation using a constant rFSH administration

Abstract Study question How does increasing doses of CG beta augment follicular steroidogenesis and is it associated with gene expression in cumulus cells? Summary answer With the exception of progesterone, CG beta exerts dose-dependent effects on intrafollicular steroid hormone concentrations and CYP19a1 expression during constant rFSH administration. What is known already CG beta (FE 999302) is a newly developed rhCG, produced by a human cell line (PER.C6®). This variant contains the same amino acid sequence as the endogenous hCG but presents with a different glycosylation profile when compared to urinary hCG or rhCG derived from a CHO cell line. CG beta has also been shown to have a longer half-life and higher relative potency. It is well known that hCG like LH creates an intracellular response through the LHR expressed on theca cells and mature granulosa cells and hereby augment the production of androgens, progesterone, and oestrogen. Study design, size, duration This study is part of a randomised, double-blind, placebo-controlled trial to investigate the efficacy and safety of CG beta as an add-on treatment to rFSH in women undergoing ovarian stimulation during a long GnRH agonist protocol. The primary endpoint of this study was intrafollicular steroid levels in relation to CG beta administration. Secondary outcomes were gene expression of LHR, FSHR, CYP19a1, and androgen receptor (AR) and single nucleotide polymorphisms (SNPs) in LHR(312) and FSHR(307). Participants/materials, setting, methods 619 women with AMH levels 5-35 pmol/L were randomized to receive either placebo or 1, 2, 4, 8, or 12 µg CG beta from day one of stimulation combined with a constant individualized dose of rFSH. Follicular fluid (FF) (n = 558), granulosa (n = 498) and cumulus cells (n = 368) were collected at oocyte retrieval. Steroid FF hormones were measured using ELISAs, gene expression was analysed in cumulus cells by qRT-PCR, and SNP analysis was performed in granulosa cells. Main results and the role of chance This study found that CG beta has a pronounced positive dose-dependent effect on intrafollicular concentrations of 17-OH-progesterone, androstenedione, testosterone, and oestradiol during constant rFSH administration. As compared to the placebo group without CG beta administration the concentrations of steroids were significantly dose-dependently increased (p-value<0.0001) reaching up to 10 times higher values in the highest dose group without a clear plateauing. However, for progesterone, there was no statistically significant difference between the CG beta dose groups and placebo. The gene expression level of CYP19a1 in the cumulus cells collected at oocyte retrieval increased significantly (p-value=0.0325) for the highest dose of CG beta. However, the gene expression level of FSHR, LHR, and AR in cumulus cells were not affected by CG beta administration, as no dose-response trend with increasing dose of CG beta was observed. Additionally, this study revealed that there was no overall clear difference in the number of oocytes retrieved between the different CG beta dose groups based on either the FSHR(307) or LHR(312) genotypes. Limitations, reasons for caution The gene expression was assessed in cumulus cells, but it would also have been interesting to evaluate the mural granulosa cells. Analysis of the gene expression of 3βHSD and other important steroidogenic enzymes in the mural granulosa cells could reveal further explanation to the effect of CG beta administration. Wider implications of the findings To our knowledge, this is the first study to show a clear dose-dependent effect of rhCG on human steroidogenesis during constant rFSH administration in preovulatory follicles. This reflects the importance of the combined effect of FSH and hCG/LH on granulosa cell activity, follicle health, and potentially oocyte quality. Trial registration number 000289

Neurosteroid Role in the Integration of Olfactory Immune Responses in Rainbow Trout (Oncorhynchus Mykiss)

The objective of this study is to unravel the integration of olfactory signals involved in the nasal detection of pathogens and the activation of associated physiological responses in Rainbow trout.The infectious pathogen Yersinia ruckeri causes enteric redmouth disease in aquaculture farms worldwide. Recent research shows that olfactory organ of rainbow trout is highly susceptible external tissue like gills or skin due to direct contact with the microbiome‐rich environment. Recent findings have demonstrated that olfactory sensory cells play a key role in early pathogen detection and immune cell activation along with their primary role in detecting environmental stimuli.We hypothesize that olfactory immune responses elicited by Y ruckeri are modulated by neurosteroids. To test our hypothesis, we nasally exposed rainbow trout to Y. ruckeri vaccine or saline and took nose and brain samples at four different time periods (4 hours, 24 hours, 7 days, and 14 days) after exposure. We analyzed fish neurosteroids (cortisol, estradiol, and progesterone) and gene expression of steroidogenic enzymes (p450, 3β HSD, 11β HSD, and 17 β HSD) in nose, olfactory bulb, and rest of the brain. Exposure to Y. rukeri vaccine caused significant changes in tissue neurosteroid production and steroidogenic enzyme expression at different times after exposure.Within the olfactory epithelium (OE), we detected a significant decrease (p = 0.003) in estradiol after 15 min exposure with 26.1 pg/mg tissue and 391.0 pg/mg tissue in treated and control individual respectively. Progesterone (174.83 pg/mg tissue) was significantly (p = 0.004) higher after 4 hours compared to the control (26.5 pg/mg of tissue) and cortisol (10.95 pg/mg of tissue) was significantly lower (p = 0.003) after 14 days in Y. ruckeri‐treated fish compared to the control (78.76 pg/mg of tissue). Within the olfactory bulb (OB), lower concentrations of cortisol and progesterone (5.65 pg/mg tissue and 1.6 pg/mg tissue respectively) were observed after 15 minutes in the Y. ruckeri‐treated fish compared to the control (22.8 pg/mg of tissue and 1.9 pg/mg of tissue, respectively). There was also a higher concentration of estradiol after 15 minutes and 4 hours compared to the control. Lastly, within the rest of brain cortisol (9.1 pg/mg tissue) was significantly higher after 4 hours in vaccine treated fish compared to the control (4.9 pg/mg tissue) and estradiol was significantly lower after 15 minutes in the vaccine group (5.5 pg/mg of tissue), compared to the control (55.9 pg/mg of tissue). However, neurosteroid concentration in the rest of brain was relatively low compared to the OE and OB. The neurosteroids: cortisol, estradiol, and progesterone play many important roles in fish physiology including stress response, cognitive behavioral modulation, and immunological response. Our preliminary results also show a significant differential expression of steroidogenic enzymes after exposure to the vaccine. Altogether, our results demonstrate that neurosteroids play a critical role in the integration of immune olfactory responses in vertebrates.

Phospholipase D2 Loss Impairs Low Salt‐Induced Increases in Steroidogenic Gene Expression with No Effect on Serum Aldosterone Levels

Primary aldosteronism (PA), in which plasma aldosterone levels are normal or elevated relative to suppressed plasma renin levels, is the most frequent cause of secondary hypertension. PA accounts for 5‐10% of hypertension cases and up to 20% in those with resistant hypertension. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis; it is tightly regulated by the renin‐angiotensin‐aldosterone system. Our laboratory has previously shown that phospholipase D2 (PLD2) mediates angiotensin II‐induced aldosterone synthesis and secretion in vitro. Therefore, we hypothesized that PLD2 loss would result in decreased aldosterone production in vivo. To test this hypothesis, 11‐week‐old male and female wild‐type and PLD2‐/‐ mice were fed a high salt (1% NaCl) or a low salt (0.02% NaCl; to raise endogenous serum angiotensin II levels) diet for 24 hours. Adrenal steroidogenic gene expression and serum aldosterone, corticosterone, and electrolytes were measured by qRT‐PCR, radioimmunoassay, ELISA, and atomic absorption spectrometry, respectively. The PLD2‐/‐ male mice exhibited increased StAR, Dab2, and CYP11B1 expression; further, PLD2 loss impaired low salt‐induced increases in StAR, Dab2, and CYP11B1 expression; however, PLD2 loss had no effect on CYP11B2 expression or serum aldosterone, Na+, K+, and corticosterone levels in these male mice. These data suggest that increased steroidogenic enzyme expression may compensate for PLD2 loss in order to maintain aldosterone secretion in the males. On the other hand, the PLD2‐/‐ female mice exhibited decreased StAR and CYP11B1 expression and serum Na+ levels as well as impaired low salt‐induced increases in StAR, CYP11B2, Dab2, and CYP11B1 expression. PLD2 loss had no effect on serum aldosterone, K+, or corticosterone levels in these female mice. It is possible that other compensatory mechanisms (not involving steroidogenic enzymes) might be involved in maintaining aldosterone secretion in the females. Together, our findings suggest a likely importance of PLD2 in mediating aldosterone production and steroidogenic enzyme gene expression, with differential effects in male and female mice.

Chemoprotective effect of vitexin against cisplatin-induced biochemical, spermatological, steroidogenic, hormonal, apoptotic and histopathological damages in the testes of Sprague-Dawley rats

Cisplatin (CP) is one of the most widely used antineoplastic drugs, which possesses the potential to treat a variety of malignancies. However, it displays numerous side effects as well. Male reproductive dysfunction is one of the most adverse side effects of CP. Vitexin is a naturally occurring flavonoid, which exhibits remarkable antioxidant properties. Present study was designed to evaluate the protective effects of vitexin on CP-induced damages on testes. 48 Sprague-Dawley rats were equally distributed into 4 groups: control, cisplatin (CP), cisplatin + vitexin (CP + VIT) and vitexin (VIT). After 14 days of treatment, evaluation of biochemical, spermatogenic, steroidogenical, hormonal, apoptotic and histopathological parameters was carried out. CP damaged the biochemical profile by reducing activity of CAT, SOD, GPx and GSR, while level of MDA and ROS was increased. It also decreased sperm motility, viability, number of hypo-osmotic tail swelled spermatozoa and epididymal sperm count, besides increasing the sperm morphological anomalies. Moreover, levels of LH, FSH and plasma testosterone were reduced. CP reduced the gene expression of testicular anti-apoptotic marker (Bcl-2) and steroidogenic enzymes (3β-HSD, 17β-HSD and StAR), but upregulated the gene expressions of apoptotic markers (Bax and Caspase-3). Besides, CP led to histopathological damages in testicular tissues. However, vitexin reversed all aforementioned damages in testes. Therefore, it is concluded that vitexin could play an effective role as a therapeutic agent against CP-prompted testicular toxicity due to its antioxidant, anti-apoptotic and androgenic potential.

Perfluorooctanoic Acid Disrupts Ovarian Steroidogenesis and Folliculogenesis in Adult Mice.

Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 μg/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 μg/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.