StAR mRNA Expression Research Articles

The inhibition roles of RAB23 gene in granulosa cell proliferation and progesterone synthesis of hen ovarian prehierarchical follicles

ABSTRACT 1. The proliferation of granulosa cells is vital for the development and recruitment of hen ovarian prehierarchical follicles (PF). The RAB23 protein is a member of the Rab family, belonging to the GTPase family. This study studied the regulatory roles of the RAB23 gene in PF. 2. The expression of RAB23 was significantly increased in granulosa cells (GC) during PF growth and was highest in GC at 6–8 mm diameter (p < 0.05). The RAB23 protein was mainly expressed in the GC, oocytes (OC) as well as somatic cells (SC) of the PF. 3. The mRNA expression of FSHR, CCND1,CYP11A1, StAR and HSD3B1 was significantly increased in the siRNA RAB23 group (p < 0.05). Additionally, protein expression of FSHR, CCND1, CYP11A1, HSD3B1 was significantly increased (p < 0.05) after GC were transfected with RAB23-specific siRNA. Protein expression of StAR in the siRNA RAB23 group was numerically higher than that in the positive control (PC) and negative control (NC) groups. The GC proliferation rate and progesterone synthesis of the prehierarchical follicles in hen ovaries were markedly increased in vitro (p < 0.05). 4.This study revealed that RAB23 might play an inhibitory role in GC proliferation and progesterone synthesis during the prehierarchical follicles development in vitro.

Improvement in Testosterone Production by Acorus gramineus for the Alleviation of Andropause Symptoms.

Acorus gramineus has a number of beneficial effects, including protective effects against age-related disorders. In this study, the effects of A. gramineus on testosterone production and andropause symptoms were evaluated. We first treated TM3 mouse Leydig cells, responsible for testosterone production, with A. gramineus aqueous extract at different concentrations. In TM3 cells, the testosterone concentration increased in a concentration-dependent manner compared with those in the control. In addition, at 400 μg/mL extract, the mRNA expression level of the steroidogenic enzyme CYP11A1 was increased. Subsequently, 23-week-old Sprague-Dawley (SD) rats exhibiting an age-related reduction in serum testosterone (approximately 80% lower than that in 7-week-old SD rats) were administered A. gramineus aqueous extract for 8 weeks. Serum total testosterone and free testosterone levels were higher and serum estradiol, prostate-specific antigen levels, and total cholesterol levels were lower in the AG50 group (A. gramineus aqueous extract 50 mg/kg of body weight/day) than in the OLD (control group). The AG50 group also showed significant elevations in sperm count, grip strength, and mRNA expression of StAR, CYP11A1, 17β-HSD, and CYP17A1 compared with those in the OLD group. In conclusion, A. gramineus aqueous extract facilitated steroidogenesis in Leydig cells, elevated testosterone levels, lowered serum estradiol and total cholesterol levels, and increased muscle strength and sperm count, thus alleviating the symptoms of andropause. These findings suggest that A. gramineus aqueous extract is a potentially effective therapeutic agent against various symptoms associated with andropause.

Barley sprouts and D-Aspartic acid supplementation improves fertility, hatchability, and semen quality in aging male broiler breeders by up-regulating StAR and P450SCC gene expressions

At 50 wk of age, broiler breeder roosters exhibit a significant decline of fertility. Therefore, the aim of this study was to assess the impact of incorporating barley sprout (BS) powder, D-aspartic acid (DA), or their combination into the diet on fertility, hatchability, semen quality, and the relative expression of StAR and P450SCC genes in aging broiler roosters. Aging (50 wk) male broiler breeders (n=32) were randomly assigned to one of four dietary treatments (2 × 2 factorial) with 2 levels of BS (0 or 2% basal diet) and DA (0 or 200 mg/kg/BW) for 12 wk. Roosters were individually housed under a 14-h light and 10-h dark cycle, with 150 g/d feed allocation and free access to fresh water, then euthanized. Throughout the study, the body weight of the broiler breeders was measured, along with various parameters related to semen quality, on a weekly basis. Additionally, artificial insemination was performed during the last 2 wk to evaluate reproductive endpoints. The results revealed that both BS and DA decreased (P < 0.01) body weight. Interestingly, the inclusion of BS, either alone or in combination with DA, resulted in a significant increase in total and forward sperm motility. Furthermore, it was demonstrated that the seminal concentration of malondialdehyde, a marker of oxidative stress, was significantly decreased by more than 20% in all groups compared to the control. The combination of both BS and DA led to the highest levels of circulating testosterone, as well as the functionality and membrane integrity of sperms. Additionally, it resulted in increased sperm concentrations, production, and penetration, ultimately leading to improved fertility rate and hatchability percentage. Moreover, a positive association between total motility and fertility was observed (P < 0.01). Furthermore, the combined supplementation of BS and DA up-regulated the relative mRNA expression of P450scc and StAR (P < 0.01). To summarize, dietary inclusion of BS, DA, or their combination have a potential to improve various aspects of reproductive performance in aging roosters.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Effects of combined exposure to polystyrene microplastics and 17α-Methyltestosterone on the reproductive system of zebrafish

Polystyrene microplastics (PS-MPs) are important carriers of pollutants in water. 17α-Methyltestosterone (MT) is a synthetic environmental endocrine disrupting chemical (EDC) with androgenic effects. To study the effects of PS-MPs and MT on zebrafish reproductive systems, zebrafish were exposed to 0 or 50 ng L−1 MT, 0.5 mg∙L−1 PS-MPs, or 50 ng∙L−1 MT + 0.5 mg∙L−1 PS-MPs for 21 d. The results showed that the different exposure reagents caused varying degrees of damage to the reproductive systems in zebrafish, with the extent of damage increasing as the exposure duration increased. Histological analysis of the gonads revealed that the ratio of mature oocytes and mature spermatozoa in the gonad decreased gradually with increased exposure time, with the ratio being Control > PS-MPs > MT > MT + PS-MPs in decreasing order. The results of quantitative real-time PCR (qRT‒PCR) showed that in female fish treated for 7 d, the expression of cyp11a mRNA was significantly reduced in all three treatment groups(MT, PS-MPs, and MT + PS-MPs), while in the group treated for 14 d with MT + PS-MPs, the expression of cyp19a1a and StAR mRNA was significantly increased. In male fish exposed for 21 d, the expression of cyp11a, cyp17a1, cyp19a1a, StAR, 3β-HSD, and 17β-HSD3 mRNA was significantly decreased in MT + PS-MPs. ELISA results showed that after 14 d of exposure, the levels of E2, LH, and FSH in the ovaries of female fish were significantly reduced in all three treatment groups. Similarly, the levels of T, E2, LH, and FSH in the testis of male fish were significantly reduced after 14 d of exposure to PS-MPs and MT + PS-MPs. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. The cross-generational toxicity of PS-MPs themselves may be negligible, but it can exacerbate the toxicity of MT, making the cross-generational effects more pronounced in the offspring, causing offspring mortality and malformations. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. In addition, MT caused malformations such as pericardial edema, yolk cysts, and spinal deformities in zebrafish during the incubation period.

The relationship between CYP19A1 gene expression in luteinized granulosa cells and follicular estradiol output in women with endometriosis

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3βHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3βHSD gene expression. No difference in mRNA expression of CYP19A1, StAR and 3βHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522–1221 ng/ml)) and control patients (median=878, IQR=609–1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17β-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3βHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [β:− 1.214;95%CI: − 2.085 - (−0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [β:− 0.003; 95%CI:− 0.468–0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women.

Adipolin (C1QTNF12) is a new adipokine in female reproduction: expression and function in porcine granulosa cells.

Adipolin (C1QTNF12) has been described as a regulator of metabolism and is linked with the pathophysiology of PCOS. In this study, for the first time, we show the expression of C1QTNF12 in granulosa cells and its positive effect on porcine granulosa cell proliferation and steroid synthesis. Adipolin (C1QTNF12) is a recently discovered adipokine that plays an important role in glucose and insulin level regulation. Previous studies showed its reduced level in serum of women suffering from polycystic ovarian syndrome; however, whether C1QTNF12 regulates ovary function is still unknown. The aim of the study was first to determine the level of C1QTNF12 in the porcine ovarian follicles granulosa cells (Gc) and then its in vitro effect on proliferation and steroidogenesis as well as phosphorylation of several signalling pathways. Our results showed that the expression of C1QTNF12 was dependent on follicle size and was higher at the mRNA and protein level in Gc of small than large follicles from both prepubertal and mature animals. Similar pattern was observed for C1QTNF12 concentration in porcine follicular fluid. Additionally, we observed immunolocalisation of C1QTNF12 in Gc, theca cells and oocytes. We found that C1QTNF12 stimulated porcine Gc proliferation via the activation of protein kinase B (AKT). Moreover, C1QTNF12 enhanced progesterone, testosterone and oestradiol secretion by elevating STAR, CYP11A1, HSD3B and CYP19A1 mRNA expression and by activation of MAP3/1 pathway. Additionally, C1QTNF12 increased pMAP3/1-to-MAP3/1 protein expression ratio and enhanced IGF1-induced pTyr-IGF1Rβ-to-IGFR1β and pMAP3/1-to-MAP3/1 protein ratios. Taken together, C1QTNF12 could act directly on proliferation and steroid synthesis and serve as an important factor in in vivo ovarian follicle function, possibly regulating the course of folliculogenesis.

Sphingosine-1-phosphate mediates FSH-induced cell viability but not steroidogenesis in bovine granulosa cells

Follicle-stimulating hormone (FSH) stimulates the proliferation, survival, and estradiol synthesis of granulosa cells by binding to their G protein-coupled receptors. Although FSH activates sphingosine kinase-1 (SPHK1) to induce sphingosine-1-phosphate (S1P) synthesis, which is required to mediate the proliferative and survival effect of this gonadotrophin, the mechanisms, and the role of S1P in estradiol synthesis have not been reported. This study aimed to evaluate the importance of FSH-induced S1P synthesis as a mediator of the effects of this gonadotrophin on granulosa cell viability and steroidogenesis and to determine if FSH-induced S1P synthesis depends on estradiol, cAMP, PKA, or PKC. To achieve these objectives, we tested the effects of FSH, a sphingosine kinase-1 inhibitor (SKI-178), estradiol and inhibitors of aromatase, cAMP, PKA, and PKC (Formestane, MDL-12330A, H-89 dihydrochloride hydrate and Calphostin C respectively), on granulosa cell viability, S1P and estradiol production, and the mRNA expression of CYP19A1 and STAR in four in vitro culture experiments. The addition of FSH (1 ng/mL) increased (P < 0.05) granulosa cells number and S1P concentration in the culture media. Conversely, the addition of SKI-178 (10 μM) reduced (P < 0.05) S1P concentration negating the effect of FSH on cell viability. Inhibition of PKC and PKA, but not cAMP, reduced (P < 0.05) S1P secretion of FSH treated granulosa cells. It is important to note that the reduction in S1P secretion was strong (49 %) with the use of the PKC inhibitor. The use of formestane (10 μg) did not modify (P > 0.05) S1P secretion in FSH-treated cells; however, the addition of 5 or 10 ng/mL of estradiol increased (P < 0.05) S1P secretion. Finally, FSH increased (P < 0.05) estradiol concentration in the culture media, but this effect was not blocked by the inhibition of S1P synthesis. Similarly, FSH, SKI-178 or their combination did not modify the mRNA expression of CYP19A1 and STAR. In conclusion, S1P synthesis is stimulated FSH in granulosa cells and mediated mainly by PKC. S1P in turn promotes the granulosa cell viability, however, this does not influence estradiol synthesis. Additionally, estradiol synthesis induced by FSH is not essential for S1P synthesis, however high estradiol concentration may stimulate S1P production by granulosa cells.

Blumea balsamifera Leaf Extract Maintain Testosterone Levels in Hypercholesterolemic Rats Through Antioxidant Mechanism and Upregulation of StAR Gene Expression

High cholesterol levels can increase lipid peroxidation in tissues that are potentially toxic to reproductive organ cells, especially the Leydig cells that produce the testosterone hormone. Blumea balsamifera leaf extract (BBLE) has the main content in the form of flavonoid compounds with antihypercholesterolemic activity. This study aimed to determine the effect of BBLE administration on MDA levels, StAR mRNA expression, Leydig cell counts, and testosterone levels in hypercholesterolemic rats. A posttest-only control group design was utilized in this research. For 50 days, 36 male Wistar rats had been separated into two groups: 1) the control group (HCD + 1 ml/day sterile aquadest) and 2) the BBLE group (HCD + 4 mg/bb rats per day). After treatment, MDA testicular tissue levels, StAR mRNA expression, Leydig cell count, and testosterone levels were measured in both groups. The data collected were statistically examined using the Independent T-test and path analysis. The results indicated that the MDA level was lower in the BBLE group, though StAR gene expression, Leydig cell count, and testosterone levels were significantly greater (p0.05). StAR mRNA expression had a significant direct effect on testosterone levels. Administration of BBLE had been shown to improve testosterone hormone secretion in hypercholesterolemic rats by preventing oxidative stress in testicular tissue with the signs of lower MDA levels, up-regulation of the StAR mRNA, and Leydig cell regeneration.

Sodium arsenite exposure enhances H3K14 acetylation and impairs male spermatogenesis in rat testes

Histone modifications play important roles in the epigenetic regulation of spermatogenesis via mediating gene transcription. Steroidogenic regulatory enzymes control testosterone biosynthesis, which are essential for spermatogenesis. Arsenic exposure inhibits the expression of steroidogenic genes by significantly increasing tri-methylation of H3K9 (H3K9me3) level in rat testis, finally diminishes testosterone release and lowers the rat sperm quality. Acetylation of H3K14 (H3K14ac) is associated with testosterone production and spermatogenesis. Co-occurrence of H3K9me3/H3K14ac has been identified previously by mass spectrometry in histone H3 isolated from different human cell types. H3K9me3/H3K14ac dually marked regions are in a poised inactive state to inhibit the gene expression. Whereas, whether inorganic arsenic exposure affects spermatogenesis and steroidogenic regulatory enzymes via mediating H3K14ac level has not been studied. Thereupon, the male Sprague-Dawley (SD) rats were exposed to (NaAsO2) for 6 weeks, then the sperm density and motility, testosterone level in serum, arsenic in rat testis were detected. mRNA expression of steroidogenic regulatory enzymes Star, Cyp11a1, Hsd3b and Hsd17b were determined by RT-PCR. H3K14ac level and the expression of histone acetylases of H3K14 (KAT2A and EP300), histone deacetylases of H3K14 (HDAC6 and HDAC3), the reader of H3K14ac (BAZ2A) were determined. The results suggested arsenic enhances H3K14ac in rat testis, which was associated with repression of steroidogenic regulatory genes expression, further reduced testosterone production, and impaired the spermatogenesis.

Hypoxia activation attenuates progesterone synthesis in goat trophoblast cells via NR1D1 inhibition of StAR expression†.

Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH

To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8mm prehierarchal follicles were cultured with IGF1 (0, 10, 100ng/mL) in the presence or absence of FSH (0, 10ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Hypothalamus-pituitary-interrenal (HPI) axis signaling in Atlantic sturgeon (Acipenser oxyrinchus) and sterlet (Acipenser ruthenus)

In vertebrates, the hypothalamic–pituitaryadrenal/interrenal (HPA/HPI) axis is a highly conserved endocrine axis that regulates glucocorticoid production via signaling by corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Once activated by ACTH, Gs protein-coupled melanocortin 2 receptors (Mc2r) present in corticosteroidogenic cells stimulate expression of steroidogenic acute regulatory protein (Star), which initiates steroid biosynthesis. In the present study, we examined the tissue distribution of genes involved in HPI axis signaling and steroidogenesis in the Atlantic sturgeon (Acipenser oxyrinchus) and provided the first functional characterization of Mc2r in sturgeon. Mc2r of A. oxyrinchus and the sterlet sturgeon (Acipenser ruthenus) are co-dependent on interaction with the melanocortin receptor accessory protein 1 (Mrap1) and highly selective for human (h) ACTH over other melanocortin ligands. A. oxyrinchus expresses key genes involved in HPI axis signaling in a tissue-specific manner that is indicative of the presence of a complete HPI axis in sturgeon. Importantly, we co-localized mc2r, mrap1, and star mRNA expression to the head kidney, indicating that this is possibly a site of ACTH-mediated corticosteroidogenesis in sturgeon. Our results are discussed in the context of other studies on the HPI axis of basal bony vertebrates, which, when taken together, demonstrate a need to better resolve the evolution of HPI axis signaling in vertebrates.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

Selenium Nanoparticles Modulate Steroidogenesis-Related Genes and Improve Ovarian Functions via Regulating Androgen Receptors Expression in Polycystic Ovary Syndrome Rat Model.

Polycystic ovary syndrome (PCOS) occurs during the reproductive period in women and is characterized by reproductive, endocrine, and metabolic disorders. Androgen plays a decisive role in its pathogenesis due to the interaction between hyperandrogenism and insulin resistance, which might be improved by selenium nanoparticles (SeNPs). The present study aimed to clarify the effect of SeNPs on androgen synthesis and action in the PCOS model and the resulting effect on ovarian function. Fifty-five 7-week-old female albino rats (90-105g) were divided equally into five groups: control (C), fed a standard diet for 11weeks; high-fat diet (HFD) group, fed HFD for 11weeks; HFD and letrozole (L) (HFD + L), fed HFD for 11weeks and administrated orally with L, at a daily dose of 1mg/kg BW, for three weeks from the 7th to 9th week of the trial; HFD + L + 0.1SeNPs and HFD + L + 0.2SeNPs groups, treated the same as HFD + L group and orally gavaged SeNPs at daily doses of 0.1 and 0.2mg/kg BW, respectively, during the last 14day of the experiment. Daily determination of estrous cycle was performed, and at the end of the experimental period, BMI, serum glucose, insulin, HOMA-IR, lipid profile, sex hormones, TNF-α, IL6, oxidative stress biomarkers, ovarian mRNA expression of different proteins and enzymes involved in steroidogenesis, pathological examination, and immunohistochemical staining for androgen receptor (AR) were evaluated. Treatment of SeNPs restored estrous cyclicity, decreased BMI, and insulin resistance, improved dyslipidemia, reduced serum testosterone, and improved ovarian histopathology in PCOS rats. Furthermore, the anti-inflammatory and antioxidant impacts of SeNPs were remarkably noticed. Administration of SeNPs decreased androgen synthesis and expression of ovarian AR protein by decreasing the mRNA expression of STAR, Cyp11A1, Cyp17A1, and HSD17B3 and increasing the expression of Cyp19α1. Conclusively, SeNPs decreased androgen synthesis and blocked the vicious circle initiated by excessive androgen secretion via decreased AR expression. Thus, it may effectively treat PCOS cases by eliminating its reproductive, endocrine, and metabolic dysfunctions.

A multi-targeted investigation of Deepwater Horizon crude oil exposure impacts on the marine teleost stress axis

The toxicity of the polycyclic aromatic hydrocarbons (PAHs) in Deepwater Horizon (DWH) oil is well-established, but a knowledge gap exists regarding how this combination of PAHs affects the vertebrate stress axis. We hypothesized that (1) marine vertebrates exposed to DWH PAHs experience stress axis impairment, and co-exposure to an additional chronic stressor may exacerbate these effects, (2) serotonin (5-hydroxytryptamine; 5-HT) may act as a secondary cortisol secretagogue in DWH PAH-exposed fish to compensate for impairment, and (3) the mechanism of stress axis impairment may involve downregulation of cyclic adenosine monophosphate (cAMP; as proxy for melanocortin 2 receptor (MC2R) functionality), total cholesterol, and/or mRNA expression of CYP1A and steroidogenic proteins StAR, P450scc, and 11β-h at the level of the kidney. We found that in vivo plasma cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations in Gulf toadfish exposed to an environmentally relevant DWH PAH concentration (ΣPAH50= 4.6 ± 1.6 μg/L) for 7 days were not significantly different from controls, whether fish were chronically stressed or not. However, the rate of cortisol secretion by isolated kidneys after acute stimulation with ACTH was significantly lower in PAH-exposed toadfish compared to clean seawater (SW) controls. 5-HT does not appear to be acting as a secondary cortisol secretagogue, rather, PAH-exposed + stressed toadfish exhibited significantly lower plasma 5-HT concentrations than clean SW + stressed fish as well as a reduced sensitivity to 5-HT at the level of the kidney. There was a tendency for kidney cAMP concentrations to be lower in PAH-exposed fish (p = 0.069); however, mRNA expression of steroidogenic proteins between control and PAH-exposed toadfish were not significantly different and a significant elevation in total cholesterol concentration in PAH-exposed toadfish compared to controls was measured. Future work is needed to establish whether the slower cortisol secretion rate by isolated kidneys of PAH-exposed fish is detrimental, to determine the potential role of other secretagogues in compensating for the impaired kidney interrenal cell function, and to determine whether there is a reduction in MC2R mRNA expression or an impairment in the function of steroidogenic proteins.

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 17β (E2) assumes a central role in follicular development and selection by activating estrogen receptors β (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.

The Role of Reduced Oxygen Supply and Transcription Factors cJUN and CREB in Progesterone Production during the Corpus Luteum Rescue in Gilts.

Simple SummaryAppropriate function of the corpus luteum and progesterone secretion are fundamental for the establishment and maintenance of pregnancy. Still, there are gaps in the knowledge of mechanisms regulating the maintenance of steroidogenic machinery of porcine corpus luteum during early pregnancy. In the present study, we analyzed the in vitro effects of decreasing oxygen concentration on progesterone production, expression of hypoxia inducible factor-1 subunit alpha (HIF-1A) and expression of genes encoding key stimulators of steroidogenesis, STAR and VEGFA, in the luteal tissue collected from cyclic and early pregnant gilts. In vitro experiments indicate that: (1) progesterone production and genes expression are modulated by decreasing oxygen concentrations and (2) luteal tissue of cyclic and pregnant gilts is similarly sensitive to reduction of oxygen. We also determined the mRNA and protein expression of HIF-1α, STAR, and VEGFA as well as transcription factors-cJUN and CREB, which are known regulators of STAR expression, in the luteal tissue of cyclic and pregnant gilts ex vivo. The relatively high level of STAR, phosphorylated forms of cJUN and CREB and progesterone concentration in the luteal tissue of early pregnant gilts suggests the role of these transcription factors in extension of luteal function. This regulation may be important in supporting luteal rescue during early pregnancy to maintain the production of progesterone. A reduced concentration of oxygen could be involved in the process of corpora lutea regression in gilts.The corpus luteum plays a fundamental role in regulating reproduction via progesterone production. Still, there is little data on factors regulating the maintenance of luteal function during early pregnancy in gilts. Previous studies emphasize the role of hypoxia and HIF-1 in the regulation of steroidogenic and angiogenic genes expression and progesterone production by ovarian cells. Using the corpus luteum of cyclic and early pregnant gilts we analyzed: (1) the in vitro effects of reduced oxygen tension on progesterone production and mRNA expression of HIF1A and luteal function regulators, STAR and VEGFA; (2) the ex vivo profiles of mRNA and protein expression of HIF-1α, STAR, VEGFA and transcription factors-cJUN and CREB, regulating STAR expression, in the corpus luteum of cyclic and pregnant gilts. The synthesis of progesterone was gradually inhibited in cyclic or pregnant gilt luteal tissue (on day 13 of cycle or pregnancy) incubated in a decreasing concentration−20%, 10%, and 3% of oxygen (O2). Luteal tissues of pregnant gilts produced trace amounts of progesterone in 10% O2, which was similar to cyclic gilts in 3% O2. HIF1A expression increased after 24 h of incubation in tissues of cyclic gilts in 3% vs. 20% O2 (p < 0.01), whereas levels of STAR and VEGFA increased significantly in cyclic and pregnant gilt tissues incubated in 10% and 3% vs. 20% O2. The ex vivo mRNA expression of HIF1A and VEGFA was elevated (p < 0.001) on day 14 vs. day 12 of pregnancy. The protein expression of HIF-1 and VEGFA increased (p < 0.001), whereas the level of STAR (mRNA and protein) and progesterone dropped (p < 0.001) on day 14 of the estrous cycle vs. a parallel day of pregnancy and/or day 12 of the estrous cycle. The content of phosphorylated cJUN and CREB was elevated (p < 0.01) in the luteal tissue on day 12 or 14 of pregnancy vs. parallel days of the estrous cycle. These increases of phosphorylated cJUN and CREB may be involved in STAR expression in the luteal tissue during early pregnancy in gilts.

The protective effect of L-carnitine on testosterone synthesis pathway, and spermatogenesis in monosodium glutamate-induced rats

BackgroundMonosodium glutamate (MSG) is a food ingredient that is increasingly used commercially. MSG leads to oxidative stress, consequently suppressing steroid hormone production that causes defects in male reproductive system. This study aimed to evaluate the effect of L-carnitine as an antioxidant on testicular damage in MSG-induced male rats.MethodsSixty adult male Spargue-Dawley rats were randomly divided into six groups of ten as follows: control (water), sham (normal saline), L-carnitine (200 mg/kg b.w), MSG (3 g/kg b.w), MSG + L-carnitine 100 (3 g/kg b.w of MSG and 100 mg/kg b.w of L-carnitine), and MSG + L-carnitine 200 (3 g/kg b.w of MSG and 200 mg/kg b.w of L-carnitine). The treatment was administered by oral gavage for six months. Serum levels of Malondialdehyde (MDA), Total Anti-oxidant Capacity (TAC), LH, FSH, testosterone, and mRNA expressions of Star, Cyp11a1, and Hsd17b3 genes, and histological and stereological changes were assessed.ResultsL-carnitine led to a significant decrease in the level of MDA and a significant rise in the serum levels of TAC, LH, FSH, and mRNA expression of Star and Cyp11a1 compared to the MSG group (p < 0.05). Furthermore, stereological results indicated a significant increment in the number of sexual lineage cells, the total volume of the testis, length, diameter, and volume of seminiferous tubules, the height of the germinal epithelium, sperm count, and sperm motility (p < 0.05) in MSG + L-carnitine 200 compare to MSG group.ConclusionThe study’s findings demonstrated that L-carnitine due to its anti-oxidant properties, ameliorated the reproductive abnormalities in the male rats exposed to MSG.