Expression Of 3β-HSD Research Articles

Phosphorylation of GATA4 serine 105 but not serine 261 is required for testosterone production in the male mouse.

GATA4 is a transcription factor essential for male sex determination, testicular differentiation during fetal development, and male fertility in the adult. GATA4 exerts part of its function by regulating multiple genes in the steroidogenic enzyme pathway. In spite of these crucial roles, how the activity of this factor is regulated remains unclear. Studies in gonadal cell lines have shown that GATA4 is phosphorylated on at least two serine residues-serine 105 (S105) and serine 261 (S261)-and that this phosphorylation is important for GATA4 activity. The objective of the present study is to characterize the endogenous role of GATA4 S105 and S261 phosphorylation in the mouse testis. We examined both previously described GATA4 S105A mice and a novel GATA4 S261A knock-in mouse that we generated by CRISPR/Cas9 gene editing. The male phenotype of the mutants was characterized by assessing androgen-dependent organ weights, hormonal profiles, and expression of multiple testicular target genes using standard biochemical and molecular biology techniques. The fecundity of crosses between GATA4 S105A mice was reduced but without a change in sex ratio. The weight of androgen-dependent organs was smaller when compared to wild-type controls. Plasma testosterone levels showed a 70% decrease in adult GATA4 S105A males. This decrease was associated with a reduction in Cyp11a1, Cyp17a1, and Hsd17b3 expression. GATA4 S261A mice were viable and testis morphology appeared normal. Testosterone production and steroidogenic enzyme expression were not altered in GATA4 S261A males. Our analysis showed that blocking GATA4 S105 phosphorylation is associated with decreased androgen production in males. In contrast, S261 phosphorylation by itself is dispensable for GATA4 function. These results confirm that endogenous GATA4 action is essential for normal steroid production in males and that this activity requires phosphorylation on at least one serine residue.

Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression.

Steroidogenic enzymes are essential for prostate cancer development. Enzymes inactivating potent androgens were not investigated thoroughly, which leads to limited interference strategies for prostate cancer therapy. Here we characterized the clinical relevance, significance, and regulation mechanism of enzyme HSD17B2 in prostate cancer development. HSD17B2 expression was detected with patient specimens and prostate cancer cell lines. Function of HSD17B2 in steroidogenesis, androgen receptor (AR) signaling, and tumor growth was investigated with prostate cancer cell lines and a xenograft model. DNA methylation and mRNA alternative splicing were investigated to unveil the mechanisms of HSD17B2 regulation. HSD17B2 expression was reduced as prostate cancer progressed. 17βHSD2 decreased potent androgen production by converting testosterone (T) or dihydrotestosterone (DHT) to each of their upstream precursors. HSD17B2 overexpression suppressed androgen-induced cell proliferation and xenograft growth. Multiple mechanisms were involved in HSD17B2 functional silencing including DNA methylation and mRNA alternative splicing. DNA methylation decreased the HSD17B2 mRNA level. Two new catalytic-deficient isoforms, generated by alternative splicing, bound to wild-type 17βHSD2 and promoted its degradation. Splicing factors SRSF1 and SRSF5 participated in the generation of new isoforms. Our findings provide evidence of the clinical relevance, significance, and regulation of HSD17B2 in prostate cancer progression, which might provide new strategies for clinical management by targeting the functional silencing mechanisms of HSD17B2.See related commentary by Mostaghel, p. 1139.

Cellular localization and seasonal variation in BMP15 expression in ovary of the catfish Clarias batrachus and its role in ovarian steroidogenesis

Despite important actions of BMP15 (a TGFβ superfamily member) in follicular development in vertebrates, studies are mostly limited to mammals. The folliculogenic processes in lower vertebrates, particularly, fishes are quite different from mammals. It was, therefore, decided to detect the presence of BMP15 in ovarian follicles and what are its role in folliculogenesis and steroidogenesis in catfish? BMP15 protein was detected in different cellular compartments of ovarian follicles of the catfish collected during different reproductive phases, using immunohistochemical and Western blotting methods, with concurrent measurement of ovarian steroids through ELISA. In vitro effects of rhBMP15 on ovarian steroids, expression and activities of steroidogenic enzymes and StAR were also analyzed using established immunoblotting and spectrophotometric methods. BMP15 was localized distinctly in the nest of oogonia, perinucleolar oocytes and in oocytes as well as follicular cells of the primary ovarian follicles, which started diminishing gradually with the progression of folliculogenesis in oocytes-II and finally it was greatly reduced in the oocytes-III (fully grown follicles). BMP15 expression in follicles showed negative correlation with ovarian steroid levels. Further BMP15 also inhibited steroids production by suppressing the expression and activities of ovarian 3β-HSD, 17β-HSD and aromatase, however, it did not influence the expression of StAR. The findings of study suggests that BMP15 help in maintaining the early-stage oocytes in catfish and inhibits follicular growth by reducing ovarian steroidogenesis through suppression of the expression and activities of steroidogenic enzymes.

Extraction of RNA from formalin-fixed, paraffin-embedded equine placenta.

Archived formalin-fixed, paraffin-embedded (FFPE) samples represent a valuable resource for the determination of gene expression for physio/pathological conditions. In the present study, we validated a protocol for the extraction of RNA from FFPE samples collected from healthy and diseased equine placenta. The quality and quantity of the extracted RNA from the FFPE and matching RNAlater™-preserved samples and expression levels of common housekeeping genes and reference microRNAs were evaluated. Precision of the expression data was evaluated by comparing relative expression of CYP19A1 and HSD3B1 in FFPE and RNAlater™ samples. The median RNA concentration recovered from FFPE samples was 316.8ng/mm3 of tissue (ranging between 61.6 and 917.4ng/mm3 ), average RNA integrity number was 2.3±0.9 (mean±standard deviation), and 84% of samples had RNA fragments longer than 200 nucleotides (DV200 ). RNA concentrations and CT values for GAPDH, ACTB, miR-8908a and miR-369 in FFPE samples were significantly correlated (r=-0.8, -0.7, -0.4 and -0.4, respectively; p<0.001). Expression pattern of normalized CYP19A1 and HSD3B1 in paired FFPE and RNAlater™ samples was significantly correlated (r=0.97 for CYP19A1 and HSD3B1; p<0.001). This study demonstrates that RNA can be extracted from FFPE equine placental tissue and used for downstream transcriptomic analysis. Similar RNA expression patterns were obtained using RNAlater™ and FFPE tissue samples.

The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells†.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3β-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3β-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3β-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

MicroRNA-150 promote apoptosis of ovine ovarian granulosa cells by targeting STAR gene

To better understand the function of oar-miR-150 in the ovine granulosa cells (GCs) during the estrus cycle, the five turpan sheep were selected for detection of the expression of oar-miR-150 in follicular and luteal ovaries, respectively. Then the granulosa cells treated with oar-miR-150 mimics or negative control (NC) were analyzed by qPCR to assess the expression of genes involved in steroidogenic and apoptosis. Expression of oar-miR-150 was increased in follicular phase compared with that in luteal ovaries. STAR was a target gene of miR-150 with oar-miR-150 mimic or inhibitor and luciferase reporter assay. Overexpression of oar-miR-150 promoted expression of bax, bcl2 and Casp3 expression and declined the expression of STAR, Cyp11a1 and HSD3B1 in vitro. Taken together, these results demonstrate that the oar-miR-150 promote GCs apoptosis through modifying the expression of genes involved in progesterone synthesis.

Characterization of 17β-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1.

In bacteria, the enzyme catalyzing the transformation of 17β-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17β-hydroxysteroid dehydrogenase (17β-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17β-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17β-estradiol and catalyzed the oxidization reaction at the C17 site of 17β-estradiol efficiently. Its Km value was 0.068mM, and its Vmax value was 56.26μmol/min/mg; over 98% of 17β-estradiol was oxidized into estrone in 5min, indicating higher efficiency than other reported bacterial 17β-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17β-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17β-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17β-hsd gene directly, and binding of OxyR could be released by 17β-estradiol. OxyR repressed the expression of 17β-hsd by its specific binding to the conserved motif of GATA-N9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N11-A. Therefore, this 17β-HSD transformed 17β-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.

Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS.

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assays. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique, and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, Baicalin decreased the testosterone concentrations in a dose-and time-dependent manner in NCI-H295R cells. GEP revealed that 3β-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity, and decreased the expression of HSD3B2 in ovarian. In summary , our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows.

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Pre-pubertal exposure to high temperature impairs ovarian and adrenal gland function in female rats.

The influence of different levels of heat exposure on the functions of ovarian and adrenal gland were investigated in pre-puberty female rats. Three-week old female rats were treated with control (26°C) or three higher temperatures (38, 40 and 42°C) for 2hr/day. After 9 days of treatment, blood samples were collected for measurement of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol-17β, corticosterone, cholesterol and triglyceride. Adrenal glands, ovaries and liver were collected for analyzing gene expressions. Body and liver weight were significantly low in the 42°C heating group. Circulating LH and triglyceride in the 42°C heating group were significantly lower, and estradiol-17β, corticosterone and cholesterol were significantly higher than those of the control group. The gene expression of 3β-HSD and P450c21 in the adrenal gland; 3β-HSD, receptors of LH, FSH and estrogen in the ovary were significantly low in heated rats. The liver gene expressions of caspase 3 and NK-κB were significantly high in 42°C heated rats, suggesting that the ability of liver metabolic function reduced in the 42°C heated rats. These results demonstrated that the high temperature is responsible for suppression of ovarian function by decreasing the expression of steroidogenic enzymes, estrogen and gonadotropin receptors in the ovary. Increase in circulating estradiol-17β in the heated rats may be due to accumulate this hormone in circulation by potential changes in liver metabolism during the heat stress.

The Non-Aromatic Δ5-Androstenediol Derivative of Dehydroepiandrosterone Acts as an Estrogen Agonist in Neonatal Rat Osteoblasts through an Estrogen Receptor α-related Mechanism

ABSTRACTPurpose: It has been proposed that DHEA influences bone formation through, bioconversion to 17β-estradiol; however, DHEA is converted to Δ5-androstenediol (Δ5-Adiol), a metabolite with estrogenic potential involved in diverse biological process. To gain new insight into the role of Δ5-Adiol in bone cells, we examined DHEA and Δ5-Adiol effects in neonatal rat and human hFOB1.19 osteoblasts.Methods: Osteoblast activity was assessed by analyzing proliferation, alkaline phosphatase activity, and expression of OSX and ALPL. We also examined binding affinities for osteoblast-ER and transcriptional activation of human (h)ERα, hERβ or hAR in U2-OS cells.Results: The most striking finding was that Δ5-Adiol had greater stimulatory effect than DHEA on rat osteoblast proliferation and differentiation, as well as ALPL expression in human osteoblasts. Interestingly, the Δ5-Adiol or DHEA-induced effects were not precluded with letrozole or trilostane, consistent with bioconversion of DHEA to Δ5-Adiol due to elevated expression of Hsd17b1 in neonatal rat osteoblasts, suggesting a high level of 17β-hydroxysteroid dehydrogenase type 1 activity. Conversely, Δ5-Adiol and DHEA-induced proliferative effects were inhibited with ICI 182780 alone or combined with trilostane, which correlates with the higher binding affinity of Δ5-Adiol for ER compared to DHEA. Furthermore, Δ5-Adiol showed a greater relative agonist activity for hERα than for hERβ or hAR.Conclusion: This study is the first to show that a bioactive DHEA derivative stimulates E2-dependent osteoblast activities, including proliferation and differentiation in rat and human osteoblasts, through ERα-related mechanisms.

Venlafaxine and carvedilol ameliorate testicular impairment and disrupted spermatogenesis in rheumatoid arthritis by targeting AMPK/ERK and PI3K/AKT/mTOR pathways

Testicular impairment has been commonly described in long-standing rheumatoid arthritis (RA) patients. Since depression and cardiovascular disorders are the most disturbing co-morbidities of RA, investigating the efficacy of the anti-depressant venlafaxine or the beta-blocker carvedilol in RA-associated testicular dysfunction may add to their clinical utility for RA patients. Previously, both agents have demonstrated significant in vivo anti-oxidant and anti-inflammatory actions. In the current study, venlafaxine (50 mg/kg/day) and carvedilol (10 mg/kg/day) were orally administered to adjuvant arthritic rats for 20 days. Interestingly, venlafaxine and carvedilol effectively suppressed paw edema and mitigated the testicular histopathological aberrations and the disrupted spermatogenesis. Both drugs enhanced testicular steroidogenesis through upregulation of 3β-HSD, 17β-HSD and StAR gene expression with concomitant augmentation of serum testosterone. They also blunted the inflammatory burden via attenuation of myeloperoxidase, TNF-α and the protein expression of NF-κBp65 along with elevation of IL-10. They attenuated testicular oxidative perturbations via lowering lipid peroxides and nitric oxide and boosting glutathione levels. With regard to apoptosis, the two agents lowered the protein expression of caspase-3, cleaved caspase-3, cleaved PARP, Bax and p53, promoting germ cell survival. They also modulated the AMPK/ERK signaling via lowering of p-AMPK and upregulation of p-ERK1/2 along with PI3K/AKT/mTOR transduction by enhancing the PI3Kp110α, p-AKT and p-mTOR protein expression. Together, the present work demonstrates the beneficial effects of venlafaxine and carvedilol in RA testicular dysfunction and impaired spermatogenesis via modulation of AMPK/ERK and PI3K/AKT/mTOR signaling and intervention with the testicular oxidative stress, inflammation and apoptosis.

Evidence for the involvement of FXR signaling in ovarian granulosa cell function.

Farnesoid X receptor (FXR) is mainly present in enterohepatic tissues and regulates cholesterol, lipid, and glucose homeostasis in coordination with target genes such as SHP and FABP6. Although FXR has been revealed to be expressed in reproductive tissues, FXR function and expression levels in the ovary remain unknown. In this study, we investigated FXR expression in mouse ovaries and its target genes in ovarian granulosa cells. In situ hybridization and immunohistochemical staining showed that FXR was mainly distributed in secondary and tertiary follicles. The agonist-induced activation of FXR in cultured granulosa cells induced the expression of SHP and FABP6, while siRNA targeting of FXR decreased CYP19a1 and HSD17b1 expression. Upon examination of the roles of SHP and FABP6 in granulosa cells, we found that SHP overexpression significantly decreased StAR, CYP11a1, and HSD3b gene expression. In addition, siRNA targeting of FABP6 decreased CYP19a1 and HSD17b1 expression, while FABP6 overexpression increased CYP19a1 expression. In conclusion, the present study demonstrates the presence of FXR signaling in the ovary and reveals that FXR signaling may have a role in function of granulosa cells.

Regulatory role of microRNA-320a in the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells by targeting estrogen-related receptor γ.

Recently, microRNAs (miRNAs) have been demonstrated to participate in many physiological and biological processes, especially by acting as circulating biomarkers or modulators in cell differentiation. Therefore, the aim of the current study was to clarify whether microRNA-320a (miR-320a) regulates the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells. In this study, miR-320a mimics and inhibitors were transfected into HTR.8/SVneo cells and human umbilical vein endothelial cells (HUVECs) using liposomes. Subsequently, the expression of miR-320a and estrogen-related receptor γ (ERRγ) mRNA was detected by a reverse transcription quantitative polymerase chain reaction, whereas the protein expression of ERRγ, vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1), human 3beta-hydroxysteroid dehydrogenase type 1 (HSD3B1), and human chorionic gonadotropin (HCG) was detected by western blot analysis. Furthermore, the proliferation, invasion/migration, and apoptosis of cells were analyzed by the cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The results showed that overexpression of miR-320a decreased the optical density (OD) values and the proliferation rate of HTR.8/SVneo cells and HUVECs, while inhibiting the expression of VEGF, Ang-1, HSD3B1, and HCG in these cells. Furthermore, miR-320a reduced the ability of cell invasion and migration, while increasing the rate of cell apoptosis. After cotransfecting the cells with miR-320a and ERRγ small (or short) interfering RNA (siRNA), the decreased ERRγ expression led to inhibited proliferation, migration, and invasion, but increased apoptosis of HTR.8/SVneo cells and HUVECs. Our results further revealed that miR-320a induced the apoptosis of trophoblasts and endothelial cells while inhibiting their proliferation, migration, and invasion by decreasing the expression of ERRγ and by indirectly suppressing the expression of VEGF, Ang-1, HSD3B1, and HCG.

Effects of glyphosate on the ovarian function of pregnant mice, the secretion of hormones and the sex ratio of their fetuses

Glyphosate is the active ingredient of the commercial formulation Roundup®, which is used worldwide. This study aimed to investigate the toxic effects of pure glyphosate or Roundup® on pregnant mice and their fetuses during pregnancy. From gestation days (GDs) 1–19, ICR mice were orally administered distilled water, 0.5% glyphosate solution or 0.5%-glyphosate Roundup® solution. The ovaries and serum were collected at GD19. The results showed decreases in body weight gain and, ovary and liver weight in glyphosate-treated mice. Additionally, histopathological alterations in the ovary including increased atretic follicles, interstitial fibrosis and decreased mature follicles were observed in the groups treated with glyphosate. The serum concentrations of both progesterone and estrogen were markedly altered after glyphosate exposure, and there were also changes in the expression of GnRH, LHR, FSHR, 3β-HSD and Cyp19a1 genes at the hypothalamic-pituitary-ovarian axis. Furthermore, oxidative stress was observed in the treated mice, increasing the activity of T-AOC, CAT and GSH-Px, as well as the MDA content in both the serum and ovary. With regard to litters, the sex ratio was significantly altered by pure glyphosate. These results show that glyphosate is able to cause several effects on pregnant mice, such as ovarian failure, interference with hormone secretion by affecting the steroidogenesis-related gene expression, and oxidative stress. The sex ratio of litters was also influenced by prenatal exposure to pure glyphosate.

Molecular identification of steroidogenesis-related genes in scallops and their potential roles in gametogenesis

Sex steroids are crucial for controlling gametogenesis and germ cell maturation in vertebrates. It has been proposed that Yesso scallop (Mizuhopecten yessoensis) has the same sex steroids as those animals, but the scallop biosynthetic pathway is unclear. In this study, we characterized several steroidogenesis-related genes in M. yessoensis and proposed a putative biosynthetic pathway for sex steroids that is similar to that of vertebrates. Specifically, we identified several steroidogenesis-related gene sequences that encode steroid metabolizing enzymes: StAR-related lipid transfer (START) protein, 17α-hydroxylase, 17,20-lyase (cyp17a), 17β-hydroxysteroid dehydrogenase (hsd17b), and 3β-hydroxysteroid dehydrogenase (hsd3b). We sampled adult scallops throughout their reproductive phase to compare their degree of maturation with their intensity of mRNA expression. Semi-quantitative RT-PCR analysis revealed a ubiquitous expression of transcripts for steroid metabolizing enzymes (i.e., star, cyp17a, hsd17b, and hsd3b) in peripheral and gonadal tissues. Real-time PCR analysis revealed a high level of expression of star3 and cyp17a genes in gonadal tissues at the early stage of cell differentiation in scallops. Interestingly, mRNA expression of hsd3b and hsd17b genes showed a synchronous pattern related to degree of gonad maturity. These results indicate that both hsd3b and hsd17b genes are likely involved in steroidogenesis in scallops. We therefore believe that these steroid-metabolizing enzymes allow scallops to endogenously produce sex steroids to regulate reproductive events.

Age-related changes in the bovine corpus luteum function and progesterone secretion.

Decreased fertility associated with maternal ageing is a well-known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age-associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age-dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8months), middle (80.1months) and aged (188.9months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6months) and aged (183.1months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β-HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation- and senescence-related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age-dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.

Molecular mechanism of mercuric chloride inhibiting progesterone secretion in ovarian granulosa cells of laying hens.

This study investigated the effect of mercury (Hg) on progesterone secretion in ovarian granulosa cells of laying hens. The gene expressions of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and intracellular calcium ion (Ca2+ ) were further investigated to uncover the molecular mechanism. Results revealed that the cell viability was gradually decreased after Hg exposure from 0 to 24hr. Besides, progesterone secretion was significantly decreased (p<0.05) as the concentration of Hg increased from 0 to 4μM followed by a plateau in 6μM Hg group at 12-hr time point. Compared with 0μM Hg group, 4 and 6μM Hg for 48hr had significantly decreased progesterone secretion (p<0.05), while Hg exposure for 6 and 24hr had no apparent effect on progesterone secretion. In addition, positive correlations occurred among intracellular progesterone, cAMP, PKA, mRNA expressions of StAR, P450scc and 3β-HSD at 12-h and 24-h time points. On the contrary, intracellular Ca2+ level was negatively related to cAMP level at 6 time point and was negatively correlated with progesterone and PKA level at 48 time point. It could be concluded that Hg dose- and time-dependently inhibited progesterone secretion by means of attenuating cAMP-PKA signal pathway, gene expressions of StAR, P450scc and 3β-HSD and enhancing intracellular Ca2+ in ovarian granulosa cells of laying hens.

4-Bromodiphenyl ether delays pubertal Leydig cell development in rats

Polybrominated diphenyl ethers are a class of brominated flame retardants that are potential endocrine disruptors. 4-Bromodiphenyl ether (BDE-3) is the most abundant photodegradation product of higher polybrominated diphenyl ethers. However, whether BDE-3 affects Leydig cell development during puberty is still unknown. The objective of this study was to explore effects of BDE-3 on the pubertal development of rat Leydig cells. Male Sprague Dawley rats (35 days of age) were gavaged daily with BDE-3 (0, 50, 100, and 200 mg/kg body weight/day) for 21 days. BDE-3 decreased serum testosterone levels (1.099 ± 0.412 ng/ml at a dose of 200 mg/kg BDE-3 when compared to the control level (2.402 ± 0.184 ng/ml, mean ± S.E.). BDE-3 decreased Leydig cell size and cytoplasmic size at a dose of 200 mg/kg, decreased Lhcgr, Star, Dhh, and Sox9 mRNA levels at ≥ 100 mg/kg and Scarb1, Cyp11a1, Hsd17b3, and Fshr at 200 mg/kg. BED-3 also decreased the phosphorylation of AKT1, AKT2, ERK1/2, and AMPK at 100 or 200 mg/kg. BDE-3 in vitro induced ROS generation, inhibited androgen production, down-regulated Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Srd5a1, and Akr1c14 expression in immature Leydig cells after 24-h treatment. In conclusion, the current study indicates that BDE-3 disrupts Leydig cell development via suppressing AKT, ERK1/2, and AMPK phosphorylation and inducing ROS generation.

Myostatin is expressed in bovine ovarian follicles and modulates granulosal and thecal steroidogenesis

Myostatin plays a negative role in skeletal muscle growth regulation but its potential role in the ovary has received little attention. Here, we first examined relative expression of myostatin (MSTN), myostatin receptors (ACVR1B, ACVR2B and TGFBR1) and binding protein, follistatin (FST), in granulosa (GC) and theca (TC) cells of developing bovine follicles. Secondly, using primary GC and TC cultures, we investigated whether myostatin affects steroidogenesis and cell number. Thirdly, effects of gonadotropins and other intrafollicular factors on MSTN expression in GC and TC were examined. MSTN, ACVR1B, TGFBR1, ACVR2B and FST mRNA was detected in both GC and TC at all follicle stages. Immunohistochemistry confirmed follicular expression of myostatin protein. Interestingly, MSTN mRNA expression was lowest in GC of large oestrogen-active follicles whilst GC FST expression was maximal at this stage. In GC, myostatin increased basal CYP19A1 expression and oestradiol secretion whilst decreasing basal and FSH-induced HSD3B1 expression and progesterone secretion and increasing cell number. Myostatin also reduced IGF-induced progesterone secretion. FSH and dihydrotestosterone had no effect on granulosal MSTN expression whilst insulin-like growth factor and tumour necrosis factor-alpha suppressed MSTN level. In TC, myostatin suppressed basal and LH-stimulated androgen secretion in a follistatin-reversible manner and increased cell number, without affecting progesterone secretion. LH reduced thecal MSTN expression whilst BMP6 had no effect. Collectively, results indicate that, in addition to being potentially responsive to muscle-derived myostatin from the circulation, myostatin may have an intraovarian autocrine/paracrine role to modulate thecal and granulosal steroidogenesis and cell proliferation/survival.