Expression Of 3β-HSD Research Articles

Reconsidering reproductive patterns in a model dissociated species, the red-sided garter snake: Sex-specific and seasonal changes in gonadal steroidogenic gene expression.

Sex steroid hormones are powerful regulators of reproductive behavior and physiology in vertebrates, and steroidogenesis has distinct sex- and season-specific patterns ultimately dictated by the expression of key enzymes. Most comparative endocrinology studies, however, focus only on circulating levels of sex steroids to determine their temporal association with life-history events in what are termed associated reproductive patterns. The red-sided garter snake (Thamnophis sirtalis parietalis) is a notable exception; this species exhibits maximal sex behavior decoupled from maximal sex steroid production and gametogenesis in what is termed a dissociated reproductive pattern. And while this is true for male red-sided garter snakes and their production of testosterone, females have maximal estradiol production during peak breeding (spring) but only immediately after mating. Here, we demonstrate that expression of ovarian aromatase (conversion of androgens to estrogens) matches the established seasonal hormone pattern in females. Additionally, steroidogenic gene expression in the ovary is broadly reduced if not suppressed compared to the testis throughout the active year. Bizarrely, male red-sided garter snakes demonstrate an unexplained pattern of steroidogenic gene expression in the testis. StAR (import of cholesterol to steroidogenesis) is maximally expressed in spring, yet Hsd17b3 expression (conversion of androstenedione to testosterone) is highest in summer, with the latter matching the established summer peak in male testosterone. The function of elevated StAR in spring is unknown, but our results suggest a decoupling between maximal StAR expression and testosterone biosynthesis (Hsd17b3 expression). We also purport that the reproductive pattern binary should be reassessed given its lack of fit for many vertebrate species that demonstrate seasonal, mixed patterns of (a)synchrony between circulating sex hormones and reproductive behavior.

Effects of four hormones on the mitigation of ovarian damage in tilapia (Oreochromis niloticus) after copper and cadmium exposure

Female tilapia of the Genetic Improvement of Farmed Tilapia (GIFT) strain were selected as an animal model to study the effects of four hormonal drugs in mitigating ovarian damage following exposure to copper and cadmium. After combined exposure to copper and cadmium in aqueous phase for 30 d, tilapia were randomly injected with oestradiol (E2), human chorionic gonadotropin (HCG), luteinizing hormone releasing hormone (LHRH), or coumestrol and raised in clear water for 7 d Ovarian samples were collected after combined exposure to heavy metals for 30 d and after recovery for 7 d Gonadosomatic index (GSI), copper and cadmium levels in the ovary, reproductive hormone levels in serum, and mRNA expression of key reproductive regulatory factors were determined. After 30 d of exposure to the combined copper and cadmium in aqueous phase, the Cd2+ content in tilapia ovarian tissue increased by 1,242.46% (p < 0.05), whereas the Cu2+ content, body weight, and GSI decreased by 68.48%, 34.46%, and 60.00% (p < 0.05), respectively. Additionally, E2 hormone levels in tilapia serum decreased by 17.55% (p < 0.05). After drug injection and recovery for 7 d, compared to the negative control group, the HCG group exhibited an increase of 39.57% (p < 0.05) in serum vitellogenin levels. Increases of 49.31%, 42.39%, and 45.91% (p < 0.05) in serum E2 levels were observed, and mRNA expression of 3β-HSD increased by 100.64%, 113.16%, and 81.53% (p < 0.05) in the HCG, LHRH, and E2 groups, respectively. The mRNA expression of CYP11A1 in tilapia ovaries increased by 282.26% and 255.08% (p < 0.05) and mRNA expression of 17β-HSD increased by 109.35% and 111.63% in the HCG and LHRH groups, respectively (p < 0.05). All four hormonal drugs, particularly HCG and LHRH, promoted the restoration of tilapia ovarian function to varying degrees after injury induced by combined exposure to copper and cadmium. This study presents the first hormonal treatment protocol for the mitigation of ovarian damage in fish exposed to combined aqueous phases of copper and cadmium as a strategy to prevent and treat fish ovarian damage induced by heavy metals.

Down-Regulation of the Mineralocorticoid Receptor (MR) and Up-Regulation of Hydroxysteroid 11-Beta Dehydrogenase Type 2 (HSD11B2) Isoenzyme in Critically Ill Patients.

Objective: The mineralocorticoid receptor (MR) has two ligands, aldosterone and cortisol. Hydroxysteroid 11-beta dehydrogenase (HSD11B) isoenzymes regulate which ligand will bind to MR. In this study we aimed to evaluate the expression of the MR and the HSD11B isozymes in peripheral polymorphonuclear cells (PMNs) in critical illness for a 13-day period.Design: Prospective studySetting: One multi-disciplinary intensive care unit (ICU)Participants: Forty-two critically ill patientsMethods: Messenger RNA (mRNA) expression of MR, HSD11B1, and HSD11B2, aldosterone levels, and plasma renin activity (PRA) were measured in 42 patients on ICU admission and on days 4, 8, and 13. Twenty-five age and sex-matched healthy subjects were used as controls.Results: Compared to healthy controls, MR expression in critically ill patients was lower during the entire study period. HSD11B1 expression was also lower, while HSD11B2 expression was higher. In patients, PRA, aldosterone, the aldosterone:renin ratio, and cortisol remained unaltered during the study period.Conclusion: Our results suggest that, in our cohort of critically ill patients, local endogenous cortisol availability is diminished, pointing towards glucocorticoid resistance. Aldosterone probably occupies the MR, raising the possibility that PMNs might be useful to study to gain insights into MR functionality during pathological states.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

Protective effects of MNQ against Lipopolysaccharide-induced inflammatory damage in bovine ovarian follicular granulosa cells in Vitro

Inflammation of the reproductive tract in dairy cows lead to functional disorders of follicular granulosa cells (GCs) in mammalian ovaries resulting in infertility and serious losses to the livestock industry. Lipopolysaccharide (LPS) can induce an inflammatory response in follicular granulosa cells in vitro. The aim of this study was to investigate the cellular regulatory mechanism of MNQ (2-methoxy-1,4-naphthoquinone) on eliminating the inflammatory response and restoring normal functions for bovine ovarian follicular GCs cultured in vitro exposed to LPS. The cytotoxicity of MNQ and LPS on GCs were detected by MTT method to determine the safe concentration. The relative expression of inflammatory factors and steroid synthesis-related genes were detected by qRT-PCR. The concentration of steroid hormones in the culture broth were detected by ELISA. Differential gene expressions were analyzed by RNA-seq. There were no toxic effects on GCs at MNQ and LPS concentrations of less than 3 µM and 10 µg/mL, respectively and treated in 12 h. The relative expressions of IL-6, IL-1β and TNF-α were significantly higher in the LPS group compared with the CK group when GCs cultured in vitro were treated with the above concentrations and times (P < 0.05), but significantly lower in the MNQ+LPS group compared with the LPS group (P < 0.05). The levels of E2 and P4 in the culture solution were significantly reduced in the LPS group compared to the CK group (P < 0.05), and restored in the MNQ+LPS group. The relative expressions of CYP19A1, CYP11A1, 3β-HSD, and STAR were significantly decreased in the LPS group compared with the CK group (P < 0.05), while the MNQ+LPS group also recovered to some extent. There were 407 differential genes shared by LPS vs CK and MNQ+LPS vs LPS by RNA-seq analysis, which were mainly enriched in steroid biosynthesis and TNF signaling pathway. We screened 10 genes for analysis and found consistent results for RNA-seq and qRT-PCR. In this study, we confirmed the protective effect of MNQ, an extract from Impatiens balsamina L, on LPS-induced inflammatory responses in bovine follicular granulosa cells in vitro as well as functional damage, and acted through steroid biosynthesis and TNF signaling pathways.

Comparison of Placental HSD17B1 Expression and Its Regulation in Various Mammalian Species.

During mammalian gestation, large amounts of progesterone are produced by the placenta and circulate for the maintenance of pregnancy. In contrast, primary plasma estrogens are different between species. To account for this difference, we compared the expression of ovarian and placental steroidogenic genes in various mammalian species (mouse, guinea pig, porcine, ovine, bovine, and human). Consistent with the ability to synthesize progesterone, CYP11A1/Cyp11a1, and bi-functional HSD3B/Hsd3b genes were expressed in all species. CYP17A1/Cyp17a1 was expressed in the placenta of all species, excluding humans. CYP19A/Cyp19a1 was expressed in all placental estrogen-producing species, whereas estradiol-producing HSD17B1 was only strongly expressed in the human placenta. The promoter region of HSD17B1 in various species possesses a well-conserved SP1 site that was activated in human placental cell line JEG-3 cells. However, DNA methylation analyses in the ovine placenta showed that the SP1-site in the promoter region of HSD17B1 was completely methylated. These results indicate that epigenetic regulation of HSD17B1 expression is important for species-specific placental sex steroid production. Because human HSD17B1 showed strong activity for the conversion of androstenedione into testosterone, similar to HSD17B1/Hsd17b1 in other species, we also discuss the biological significance of human placental HSD17B1 based on the symptoms of aromatase-deficient patients.

Utero-placental expression and functional implications of HSD11B1 and HSD11B2 in canine pregnancy†.

Glucocorticoids modulate the feto-maternal interface during the induction of parturition. In the dog, the prepartum rise of cortisol in the maternal circulation appears to be erratic, and information about its contribution to the prepartum luteolytic cascade is scarce. However, the local placental upregulation of glucocorticoid receptor (GR/NR3C1) at term led to the hypothesis that species-specific regulatory mechanisms might apply to the involvement of cortisol in canine parturition. Therefore, here, we assessed the canine uterine/utero-placental spatio-temporal expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; reduces cortisone to cortisol), and -2 (HSD11B2; oxidizes cortisol to the inactive cortisone). Both enzymes were detectable throughout pregnancy. Their transcriptional levels were elevated following implantation, with a strong increase in HSD11B2 post-implantation (days 18-25 of pregnancy), and in HSD11B1 at mid-gestation (days 35-40) (P < 0.05). Interestingly, when compared pairwise, HSD11B2 transcripts were higher during post-implantation, whereas HSD11B1 dominated during mid-gestation and luteolysis (P < 0.05). A custom-made species-specific antibody generated against HSD11B2 confirmed its decreased expression at prepartum luteolysis. Moreover, in mid-pregnant dogs treated with aglepristone, HSD11B1 was significantly higher than -2 (P < 0.05). HSD11B2 (protein and transcript) was localized mostly in the syncytiotrophoblast, whereas HSD11B1 mRNA was mainly localized in cytotrophoblast cells. Finally, in a functional approach using placental microsomes, a reduced conversion capacity to deactivate cortisol into cortisone was observed during prepartum luteolysis, fitting well with the diminished HSD11B2 levels. In particular, the latter findings support the presence of local increased cortisol availability at term in the dog, contrasting with an enhanced inactivation of cortisol during early pregnancy.

Exposure to dipentyl phthalate in utero disrupts the adrenal cortex function of adult male rats by inhibiting SIRT1/PGC-1α and inducing AMPK phosphorylation.

Di-n-pentyl phthalate (DPeP) is an endocrine-disrupting phthalate plasticizer. The objective of this study was to investigate the effect of DPeP on adrenocortical function in adult male rats following in utero exposure. DPeP (0, 10, 50, 100, and 500 mg/kg/day) was administered by gavage to pregnant Sprague-Dawley rats from gestational day 14 to 21. The morphology and function of the adrenal cortex in 56-day-old male offspring were studied. DPeP at 100 and 500 mg/kg/day significantly reduced serum aldosterone levels and at 500 mg/kg/day markedly reduced corticosterone and adrenocorticotropic hormone levels. DPeP at 10-500 mg/kg markedly reduced the thickness of zona glomerulosa without affecting the thickness of zona fasciculata. DPeP significantly downregulated the expression of Agtr1a, Mc2r, Scarb1, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a2, and Bcl2 genes as well as their proteins. DPeP at 500 mg/kg/day significantly increased phosphorylated AMPK, while DPeP at 100 mg/kg/day and higher doses reduced phosphorylated AKT1 and total SIRT1 level. DPeP at 100 and 500 μM markedly induced reactive oxygen species and apoptosis in H295R cells after 24 h of culture. In conclusion, in utero exposure to DPeP disrupts adrenocortical function of the adult male offspring by (1) increasing AMPK phosphorylation and decreasing AKT1 phosphorylation and SIRT1 levels, (2) reducing adrenocorticotropic hormone levels, and (3) possibly inducing oxidative stress and apoptosis.

Citrinin inhibits the function of Leydig cells in male rats in prepuberty

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21–28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1–5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10–100 μM. Citrinin at 50 μM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role.

Oxytocin enhances progesterone production with upregulation of BMP-15 activity by granulosa cells

To elucidate the reproductive role of oxytocin (OXT) in ovarian steroidogenesis and its functional interaction with bone morphogenetic proteins (BMPs), the effects of OXT on ovarian steroidogenesis were investigated by utilizing primary culture of rat granulosa cells and human granulosa KGN cells. Here we revealed that the OXT receptor was expressed in both rat and human granulosa cells and that OXT treatment significantly increased follicle-stimulating hormone (FSH)- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat and human granulosa cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA expression of CYP11A1 and HSD3B2 induced by FSK in human granulosa cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the transcription of ID1 induced by BMP-15, but not those induced by BMP-6, in human granulosa cells. It was also revealed that OXT treatment upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by human granulosa cells. Collectively, it was shown that OXT accelerates ovarian progesterone synthesis with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis (186 words).

Persistence of foetal testicular features in patients with defective androgen signalling.

Congenital defects of androgen synthesis or action in 46, XY individuals can result in impaired virilisation, despite the apparent testicular development. In a recent case report of a young adult with complete androgen insensitivity syndrome (CAIS), tumorous gonadal tissue was shown to express HSD17B3 in Sertoli cells (SCs) and not in Leydig cells (LCs). This expression pattern differs from the typical adult human testis and resembles foetal mouse testis, suggesting an underlying testicular development and function defect. Here, we investigate the effect of altered androgen signalling in gonads from five 46, XY individuals with defects in androgen synthesis or action. Gonadal tissue sections from four patients with CAIS, one with CYP17A1 deficiency, and one control were immunostained for LC developmental and steroidogenic markers. The expression of some of these markers during development was investigated by reanalysing previously published single-cell RNA sequencing (scRNA-seq) data from normal human testicular tissues. All gonadal tissues from the patients show exclusive expression of HSD17B3 in SCs and expression of the foetal/immature LC marker DLK1 in a subset of LCs, suggesting an androgen-dependent differentiation defect of adult SCs and LCs. Furthermore, reanalysis of scRNA-seq data reveals an expression of HSD17B3 in foetal and neonatal SCs that is downregulated in adult SCs. Androgen signalling may affect the differentiation of adult but possibly not foetal SCs or LCs and may induce a shift of testosterone production from the tubular compartment in the foetal phase to the interstitial compartment in the adult phase.

DNA methylation-mediated inhibition of MGARP is involved in impaired progeny testosterone synthesis in mice exposed to DBP in utero.

The dibutyl phthalate (DBP) has been detected in fetuses and infants and can cause damage to the reproductive system in adulthood, but the exact mechanism remains unclear. Here, we aim to investigate the effects of intrauterine DBP exposure on offspring reproductive function and explore possible mechanisms. SPF C57BL/6 pregnant mice were given DBP (0.5, 5, 75 mg/kg/d) or corn oil from day 5 to day 19 by gavage. After weaning, the pups were fed a standard diet for 5 weeks. In addition, TM3 Leydig cell cultures were used to study the relevant mechanisms in vitro. The results showed that intrauterine DBP exposure could reduce sperm density and sperm motility, cause testicular tissue damage, down-regulate serum T and LH levels, and up-regulate serum FSH levels at 75 mg/kg/d. Western blot and methylation detection revealed intrauterine exposure to DBP down-regulated testosterone synthesis-related proteins StAR, P450scc, 3β-HSD, PKA, and PKC expression, while up-regulated the levels of methyltransferase proteins expression and DNA 5-methylcytosine (5mC) in testicular tissue of mouse offspring at 75 mg/kg/d. Further detection found in utero 75 mg/kg/d DBP exposure down-regulated MGARP protein expression, and induced incomplete methylation of the MGARP gene. An in vitro analysis showed that MGARP inhibition is involved in an impaired testosterone synthesis in TM3 cells. Cell culture results suggest that MGARP down-regulation may be involved in impaired testosterone production in monobutyl phthalate-treated cells. The present study revealed that 75 mg/kg/d DBP exposure in utero resulted in testosterone synthesis disorders and reproductive function impairment in mouse offspring, and the mechanism may be related to DNA methylation-mediated down-regulation of MGARP in the testis.

Dual role of NR4A1 in porcine ovarian granulosa cell differentiation and granulosa-lutein cell regression in vitro

This study aimed to investigate the role of NR4A1 in forskolin (FSK)-induced granulosa cell (GC) differentiation and PGF2α-induced granulosa-lutein cell (GLC) regression. For experiment 1, primary porcine GCs were pre-cultured for 6 d before induced-differentiation by FSK with or without siNR4A1, and changes in GC proliferation, lipid droplets (LDs), and P4 level were detected. For experiment 2, the GLC model was established by FSK as in experiment 1, and then PGF2α was utilized to induce GLC regression with or without siNR4A1, changes in P4 secretion, apoptosis proteins, and associated signaling pathway members were detected. Results showed that in experiment 1, FSK up-regulated NR4A1 expression during GC differentiation and decreased GC proliferation activity, which was reversed by siNR4A1. siNR4A1 inhibited the FSK-induced decreases in Cyclin B1/D1 and CDK1/2 mRNA abundances, and increases in P21/P27 mRNA abundances, and FSK-induced LD accumulation. FSK up-regulated P4 secretion and StAR, CYP11A1 and HSD3B expression, decreased CYP19A1 expression, which were reversed by siNR4A1 except for StAR expression. In experiment 2, PGF2α induced NR4A1 expression and reduced GLC viability, which were reversed by siNR4A1. Compared with PGF2α group, the levels of P4 secretion and StAR expression were higher in PGF2α+siNR4A1 group, while CYP11A1 and HSD3B expressions held at low levels. siNR4A1 inhibited PGF2α-induced expression of apoptosis proteins (caspase3, Bax, Fas, TNFa), ATF3, and phosphorylated MAPKs (ERK1/2, P38, JNK). In summary, NR4A1 is involved in regulating porcine GC differentiation and GLC regression as well as the changes in cell proliferation, apoptosis, steroidogenesis, and MAPK pathways, which provide a theoretical basis for further understanding of the mechanism of porcine luteal formation and regression.

Progesterone modulates HSD11B1-mediated cortisol production in luteinized bovine granulosa cells.

Progesterone (P4) and cortisol production increase in luteinized granulosa cells (LGCs) during the periovulatory period, but their interaction is not well established. Therefore, we investigated their interaction in cultured bovine LGCs. Granulosa cells were collected from follicles of 2-5 mm in diameter and cultured in DMEM/F-12 supplemented with 10% fetal calf serum for up to 14 days. P4 production and the expression of steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) rapidly increased until day 10 and remained high thereafter. No de novo of cortisol from P4 was detected during the culture period. The expression of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts cortisone to cortisol, increased dramatically on day two, decreased until day 8, and remained relatively constant. To investigate how P4 and cortisol influence each other's production, LGCs were treated with trilostane (a P4 synthesis inhibitor), nomegestrol acetate (NA, a synthetic progestogen), P4, and/or cortisol for 24 h on days 6 and 12 of culture. Trilostane suppressed P4 and STAR expression while elevating HSD11B1 and HSD3B1 expression and cortisol production. Concomitant treatment with NA or P4 dose-dependently decreased cortisol production and HSD11B1 and HSD3B1 expression but elevated STAR expression in both days 6 and 12. Conversely, cortisol treatment increased HSD11B1 and HSD3B1 expression and decreased STAR expression without influencing P4 production. These results indicate that progestogens suppress cortisol production by modulating HSD11B1 expression and that progestogens and cortisol differentially regulate STAR, HSD3B1, and HSD11B1 expression in bovine LGCs.

Glucagon-like peptide-1 promotes Leydig cell regeneration from stem cells in rats.

Glucagon-like peptide-1 stimulates stem Leydig cell development. Glucagon-like peptide-1 stimulates stem Leydig cell differentiation without affecting its proliferation. The regulators of stem Leydig cell (SLC) development remain largely unknown. The effect of glucagon-like peptide-1 (GLP-1) on rat SLC proliferation and differentiation was investigated using a 3D tissue culture system and an ethane dimethane sulfonate (EDS)-treated in vivo LC regeneration model. RNA-seq analysis was performed to analyze pathways in which GLP-1 may be involved. GLP-1 (3 and 30 nmol/L) significantly increased medium testosterone abundances and upregulated the expression of Scarb1, Cyp11a1, and Hsd11b1. GLP-1 in vitro did not affect SLC proliferation by 5-Ethynyl-2'- deoxyuridine (EdU) incorporation assay. Intratesticular injection of GLP-1 (10 and 100 ng/testis) into the LC-depleted testis from day 14 to day 28 post-EDS significantly increased serum testosterone abundances and upregulated the expression of Cyp11a1, Hsd3b1, and Hsd11b1. It did not affect the number of HSD11B1+ and CYP11A1+ LCs. RNA-seq analysis revealed that GLP-1 upregulated several pathways, including cAMP-PKA-EPAC1 and MEK/ERK1/2. GLP-1 stimulates SLC differentiation without affecting its proliferation, showing its novel action and mechanism on rat SLC development.

Health risk assessment, water quality indices and reproductive hormone profile among Abakpa citizens in Enugu metropolis

Numerous substances has been attributed to elicit endocrine-disruption and may potentially affect healthy reproductive functions. This study was carried out to determine the health risk assessment, water quality indices and reproductive hormone profile among citizens of Abakpa in Enugu metropolis. Ten (10) water sampling points comprising of wells and streams were analyzed for chemical properties such BOD, COD, DO, pH, TSS, TDS, Total Hardness, and Alkalinity and Heavy metal composition such as Zinc (Zn), Lead (Pb), Chromium (Cr), Cadmium (Cd) and Mercury (Hg) using the American Public Health Association’s (APHA) guidelines and Atomic absorption spectrophotometer (AAS) respectively. Five (5) blood samples comprising males and females was collected across the sampling areas and assayed for reproductive hormone profile such as Testosterone, Follicle Stimulating Hormone, Progesterone, Prolactin, Estradiol and the enzyme Aromatase was using the ELISA kit method. The result of chemical properties such as TSS, TDS, COD, Total hardness and Alkalinity were all within World Health Organization (WHO) stipulated limits, whereas DO, BOD and pH were higher than WHO limits across all sampling points from the sampling area. Some parameters showed significance difference while some sample did not at different sampling locations at 95% confidence level. The result of heavy metals showed very high concentrations of analyzed metals with Hg present in five water samples from Abakpa at concentrations ranging from 0.01 to 1.96 ppm. Pb concentration ranged between 0.06-0.35 ppm; Zn 24.93- 33.65 ppm; Cr 0.00 – 0.23 ppm; Cd. 0.11 – 0.17 ppm across all sampling points. All the result of reproductive hormone showed lower concentration of testosterone in men (1.99 to 2.19 ng/ml) and high concentration in females (2.03 to 2.06 ng/ml) across all sampling points. The concentration of FSH was in the range of 0.67 to 0.77 ng/ml in males, while the level was between 0.71 to 0.83 ng/ml in female. Prolactin results was 3.49 to 3.86 ng/ml for males and 3.44 to 3.59 ng/ml in females. Estradiol levels were 8.48 to 9.54 ng/ml and 9.61 to 9.84 ng/ml for females and males respectively. However, progesterone level were between 34.08 to 37.45 ng/ml for females and 36.48 to 39.56 ng/ml. The aromatase enzyme was averagely below 4.0 Pmol/mg in both male and female. The risk assessment showed low risk exposure but mercury had high risk exposure tendencies of 16.333 when compared to other heavy metals from all samples. Consumption of water in sampled area’s may pose a serious health challenge; high concentration of heavy metals could be attributed to the hormonal imbalance recorded in the research as most heavy metals is seen to reduce the expression and activity of 3β-HSD and 17β-HSD, which could be facilitated by high acidic nature of the water. Thus, interfering with steroidogenesis. Therefore, continuous monitoring of water quality in Abakpa in Enugu metropolis is highly recommended in order to ensure the health safety of humans living in such environment.

Male Reproductive Dysfunction and Pharmacological Action of Curcumin: A Concise Review

Background: Male reproductive dysfunctions are the results of the malfunctioning of one or more of these cellular components due to several factors among which are exposure to heavy metals, reactive oxygen species, smoking and alcohol, genital infection, lifestyle, hormonal disorders, and unhealthy diet. However, it has been widely reported that curcumin possesses pharmacological potential which could ameliorate the effects of these factors on male reproductive dysfunctions. Introduction: This review discusses the pharmacological activities of curcumin about its ameliorating effects on male reproductive dysfunction especially on induced sperm disorders, steroidogenic toxicity, immune-regulatory activity, apoptotic toxicity, and oxidative toxicity, along with their possible mechanisms on male reproductive dysfunction. Method: The terms such as "Curcumin" together with "testis", "sperm", " male reproductive function", and "testosterone" were searched on Google Scholar, Pubmed, and Scopus. Relevant literature was also assessed. Result: Curcumin mitigated the adverse effects on hormone levels, cellular apoptotic activity, oxidative stress, and histopathological damages. Curcumin also inhibited the activation of the mitogen-activated protein kinase (MAPK) pathway, increased semen quality, sperm motility, and concentration, and restored the IL-1β, TNF-α, and MCP-1 level in the reproductive tissues. Furthermore, curcumin has been shown to increase mRNA, cytoplasmic Bcl-2, 3β-HSD, and 17β-HSD and Nrf2 expression, ɣ-GCS, and GSH-Px levels, the number of TUNEL-positive cells, and to reduce levels of LDH-x, and LDH activities. Conclusion: Curcumin exerts pharmacological activities which improved the male reproductive functions as it pinpoints how curcumin affects various testicular processes. This will be imperative as a measure to enhance male fertility.

Effects of melatonin on development and hormone secretion of sheep theca cells in vitro

Theca cells (TCs) play a unique role in the structure and function of the ovary. They are not only the structural basis of the follicle but also the androgen-secreting cells in female mammals, which can affect the normal development and atresia of the follicle. The results showed that melatonin receptor (MTR) MT1 and MT2 were expressed on sheep TCs. In the present study, the effects of different concentrations of MT at 0, 10−10, 10−8, 10−6 and 10−4 M/L on sheep TCs with regards to the antioxidant levels, proliferation, apoptosis and steroid hormone secretion were investigated. The results showed that in sheep TCs, all concentrations of MT significantly decreased reactive oxygen species (ROS) concentration and BAX expression; increased Cat, Sod1, and BCL-2 expression. The proliferation viability of TCs was significantly inhibited in all groups except for 10−10 M/L MT, and the expression of cyclin D1 and CDK4 was significantly reduced. MT significantly increased StAR expression and progesterone secretion in TCs, but there was no significant effect on androgen secretion and CYP11A1, CYP17A1 and 3β-HSD expression in all groups. MT-induced progesterone secretion was completely inhibited by Luzindole (a nonspecific MT1 and MT2 inhibitor) and partially inhibited by 4p-PDOT (specific MT2 inhibitor). MT-induced progesterone secretion can be inhibited by LY294002 (PI3K/AKT pathway inhibitor). This study indicated that MT inhibits apoptosis and proliferation of in vitro cultured sheep TCs, which has implications for slowing ovarian atresia and aging. MT activates the PI3K/Akt pathway to mediate the synthesis and secretion of progesterone by TCs. This study provides a basis for further exploration of the role of TCs on follicle development and ovarian steroid hormone secretion.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4–6 mm; <0.5 ng/mL) (ii) F2 (7–9 mm; 0.5–5 ng/mL) (iii) F3 (10–13 mm; 5–40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

HSD3B1 Expression Is Upregulated by Interleukin 4 in HT-29 Colon Cancer Cells via Multiple Signaling Pathways.

3β-Hydroxysteroid dehydrogenase/isomerase is essential for the synthesis of active steroid hormones. Interleukin 4 (IL4) induces the expression of HSD3B1 in various human cancer cell lines. Here, we demonstrated that administration of IL4 to an HT-29 colon cancer cell line induced high expression of HSD3B1 at the mRNA and protein levels. In the HT-29 cells, IL4 stimulated the activity of signal transducer and activator of transcription 6 (STAT6) and promoted its binding to the STAT6-binding site in the HSD3B1 promoter. The STAT6 inhibitor significantly suppressed HSD3B1 induction by IL4 in a dose-dependent manner. Moreover, inhibition of the PI3-kinase/AKT pathway strongly suppressed the IL4-induced HSD3B1 expression. Glycogen synthase kinase 3 (GSK3), a downstream target of AKT, had a stimulatory effect on the IL4-induced HSD3B1 expression. However, IL4 stimulated the phosphorylation of AKT, which inhibited the GSK3 activity at the early stage. Hence, GSK3 potentiated the HSD3B1 levels at the late stage of the IL4 stimulation. Additionally, inhibitors of mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, but not of JNK, partly reduced the HSD3B1 expression following the IL4 stimulation. We further demonstrated that IL4 potently promoted steroid synthesis. Our results indicate that IL4 induces HSD3B1 expression via multiple signaling pathways in HT-29 cells and may play a role in the regulation of steroid synthesis.