Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Leptin siRNA promotes ovarian granulosa cell apoptosis and affects steroidogenesis by increasing NPY2 receptor expression.

Leptin has been found to be involved in the ovarian granulosa cell apoptosis and steroidogenesis. Loss of neuropeptide Y (NPY) can correct the obesity syndrome of mutant mice lacking of leptin (ob/ob). However, the association of NPY and leptin in ovarian granulosa cells and ovarian steroidogenesis has not been investigated. Here, C57BL/6J ob/ob mice and C57BL/6J (control) mice were intraperitoneally injected with PBS, leptin (0.4µg/g bodyweight) or BIIE0246 (NPY2 receptor [NPY2R] antagonist, 30µg/kg bodyweight) every day for 15days. We found that NPY2R mRNA expression in mouse ovary was suppressed by leptin treatment, but increased by leptin deficiency. Leptin or BIIE0246 treatment significantly increased E2, but notably decreased progesterone in both mice. A lower level of E2 and a higher level of progesterone was observed in ob/ob mice than in control mice. Further, we then knocked down leptin expression in human ovarian granulosa cells by siRNA transfection and treated the cells with DMSO or BIIE0246. In vitro experiments confirmed the findings in mice. siLeptin treatment decreased the secretion of E2, anti-Mullerian hormone (AMH), insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß, and the cell proliferation, but increased the secretion of progesterone and cell apoptosis. Western blotting analysis of PCNA, Bcl-2 and Bax confirmed the results of cell proliferation and apoptosis. Activation of JAK2 and STAT3 was also suppressed by knocking down leptin. All the effects of siLeptin on ovarian granulosa cells were partially reversed by BIIE0246. In conclusion, knockdown of leptin significantly affected ovarian steroidogenesis and ovarian function through NPY. siLeptin transfection impaired the activation of JAK2/STAT3 and contributed to ovarian granulosa cell apoptosis partially through up-regulating NPY2R expression.

Association of TLR2 S450S and ICAM1 K469E polymorphisms with polycystic ovary syndrome (PCOS) and obesity.

Toll-like receptors (TLRs) are activated by inflammatory stimuli and influence endothelial functions, contributing to the pathogenesis of atherosclerosis. We investigate the influence of polymorphisms in the genes encoding toll-like receptor 2 (TLR2) and 4 (TLR4) and endothelial adhesion molecules on polycystic ovary syndrome (PCOS) and its interaction with obesity. Ten single nucleotide polymorphisms were genotyped in 305 women with PCOS and 166 non-hyperandrogenic control women. In obese women, TLR2 S450S and ICAM1 K469E polymorphisms differently influenced metabolic variables and PCOS, respectively. Irrespective of PCOS, variant alleles of TLR2 S450S increased triglycerides, fasting insulin levels, and insulin resistance in obese women. TLR2 S450S interacted with obesity and PCOS on androstenedione levels, mutant alleles were associated with increased androstenedione concentrations in all women, with the exception of obese patients with PCOS (P=0.034). Regarding ICAM1 K469E, homozygosis for K469 alleles was more frequent in PCOS, but only in obese women (P=0.014). K469 alleles were also related to increased body mass index (P=0.017) and diastolic blood pressure (P=0.034). Moreover, ICAM1 K469E interacted with obesity and PCOS on serum triglyceride levels (P=0.019) and with PCOS on serum sex hormone-binding globulin concentrations (P=0.006). In conclusion, TLR2 S450S and ICAM1 K469E polymorphisms may be associated with PCOS and metabolic comorbidities in obese women.

Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment.

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) restricts the application of the microbial transformation of phytosterols in the industry. A novel atmospheric and room temperature plasma (ARTP) treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30 °C for 7 d, (6.28±0.11) g/L of AD and (0.82±0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83±0.13) g/L of AD and (2.34±0.06) g/L of ADD by the original strain, M. neoaurum ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.

The endometrial cancer cell lines Ishikawa and HEC-1A, and the control cell line HIEEC, differ in expression of estrogen biosynthetic and metabolic genes, and in androstenedione and estrone-sulfate metabolism.

Estrogens have important roles in the pathogenesis of endometrial cancer. They can have carcinogenic effects through stimulation of cell proliferation or formation of DNA-damaging species. To characterize model cell lines of endometrial cancer, we determined the expression profiles of the estrogen receptors (ERs) ESR1, ESR2 and GPER, and 23 estrogen biosynthetic and metabolic genes, and investigated estrogen biosynthesis in the control HIEEC cell line and the Ishikawa and HEC-1A EC cell lines. HIEEC and Ishikawa expressed all ERs to different extents, while HEC-1A cells lacked expression of ESR1. Considering the estrogen biosynthetic and metabolic enzymes, these cells showed statistically significant different gene expression profiles for SULT2B1, HSD3B2, CYP19A1, AKR1C3, HSD17B1, HSD17B7, HSD17B12, CYP1B1, CYP3A5, COMT, SULT1A1, GSTP1 and NQO2. In these cells, E2 was formed from E1S and E1, while androstenedione was not converted to estrogens. HIEEC and Ishikawa had similar profiles of androstenedione and E1 metabolism, but hydrolysis of E1S to E1 was weaker in Ishikawa cells. HEC-1A cells were less efficient for activation of E1 into the potent E2, but metabolized androstenedione to other androgenic metabolites better than HIEEC and Ishikawa cells. This study reveals that HIEEC, Ishikawa, and HEC-1A cells can all form estrogens only via the sulfatase pathway. HIEEC, Ishikawa, and HEC-1A cells expressed all the major genes in the production of hydroxyestrogens and estrogen quinones, and in their conjugation. Significantly higher CYP1B1 mRNA levels in Ishikawa cells compared to HEC-1A cells, together with lack of UGT2B7 expression, indicate that Ishikawa cells can accumulate more toxic estrogen-3,4-quinones than HEC-1A cells, as also for HIEEC cells. This study provides further characterization of HIEEC, Ishikawa, and HEC-1A cells, and shows that they differ greatly in expression of the genes investigated and in their capacity for E2 formation, and thus they represent different in vitro models.

Gonadectomy-related adrenocortical tumors in ferrets demonstrate increased expression of androgen and estrogen synthesizing enzymes together with high inhibin expression.

The 2 objectives of this study were to (1) measure by quantitative polymerase chain reaction the expression of genes involved in steroid and inhibin synthesis in adrenocortical tumors of gonadectomized ferrets and (2) localize by immunohistochemistry several proteins that are key to adrenal steroidogenesis. Relative to the control adrenals, expression of the messenger RNAs encoding StAR (steroidogenic acute regulatory protein; P = 0.039), CYP11A (P = 0.019), CYP21 (P = 0.01), and 3ß-HSD (P = 0.004), all involved in the synthesis of mineralocorticoids and glucocorticoids, were decreased in the adrenocortical tumors. In contrast, expression of cytochrome B5 (CytB5; P = 0.0001) and aromatase (P = 0.003), involved in androgen and estrogen synthesis, and both inhibin α-subunit (P = 0.002) and ßB-subunit (P = 0.001) were upregulated. In tumors, immunostaining of CYP21 was low, whereas staining of Cyp17 and CytB5, necessary for androgen synthesis, was present. It is concluded that ferret adrenocortical tumors express genes for androgen production. In addition, the expression of aromatase and inhibin suggests an even more gonadal differentiation, which is reminiscent to the fact that both gonads and adrenals are derived from a common urogenital primordial cell.

Inner mitochondrial translocase Tim50 interacts with 3ß-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis.

In the adrenals, testes, and ovaries, 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3ßHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3ßHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3ßHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3ßHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3ßHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3ßHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3ßHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3ßHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3ßHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3ßHSD2 expression and activity, representing a new role for translocases in steroidogenesis.

Molecular mechanism for repression of 17alpha-hydroxylase expression and androstenedione production in granulosa cells.

CONTEXT: According to the traditional two-cell two-gonadotropin model of follicular steroidogenesis, androgen production arises exclusively from theca cells. The granulosa cells, in turn, utilize androstenedione and testosterone, which are aromatized into estrone and estradiol, respectively. Differential expression of the activator protein-1 (AP-1) transcription factor, c-fos, has been postulated to result in distinct patterns of steroidogenesis in the theca and granulosa cell compartments. We hypothesize that c-fos functions to inhibit the production of 17alpha-hydroxylase 17,20 lyase (CYP17) in granulosa cells, thereby suppressing androgen synthesis. OBJECTIVE: Our objective was to define the role of c-fos in the regulation of CYP17 production in granulosa cells. DESIGN AND METHODS: Human luteinized granulosa (HGL5) cells were utilized for all experiments. The following techniques were used: mRNA extraction, steroid quantification, small interfering RNA silencing, microarray analysis, and immunohistochemistry. RESULTS: Immunohistochemistry studies demonstrated significant staining of c-fos in the granulosa cell layer, but absent staining for CYP17. Conversely, the theca cell layer did not stain for c-fos, but staining was evident for CYP17. Treatment of HGL5 cells with the MAPK kinase inhibitor PD98059 resulted in an 11-fold increase in CYP17 mRNA levels. In c-fos gene silenced cells, CYP17 mRNA levels increased 8-fold. Androstenedione production was increased 13-fold after treatment with PD98059. CONCLUSIONS: These results suggest that the AP-1 transcription factor, c-fos, may be one of the factors responsible for CYP17 repression and hence suppression of androstenedione production in granulosa cells. This may provide an explanation for the lack of CYP17 in granulosa cells.

Effect of days after calving on insulin-like growth factor-I, insulin-like growth factor binding proteins, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant follicles of postpartum beef cows.

The effect of days after calving on IGF-I, IGFBP, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant ovarian follicles (DF) was evaluated in Angus x Hereford cows. Growth of DF (>9 mm) was monitored daily by ultrasonography and fluid from DF was collected in vivo at either 30+/-2 d or 47+/-2 d postpartum. Follicular fluid (FF) was also aspirated from DF of contemporary ovulatory cows at proestrus. Estrous behavior was monitored continuously using the HeatWatch system, and progesterone in plasma collected twice weekly was used to assess luteal activity. Anovulatory DF aspirated 30 and 47 d postpartum had similar concentrations of IGF-I, IGFBP, progesterone, estradiol and androstenedione in FF and IGF-I and IGFBP in plasma. The intervals from aspiration to estrus were similar for cows aspirated 30 and 47 d postpartum. Proestrous follicles had greater (P<0.01) estradiol (435+/-79 ng/mL) than DF at 30 d (107+/-63 ng/mL) or 47 d (68+/-53 ng/mL) after calving. Concentrations of androstenedione in FF were also greater (P<0.01) in proestrous follicles than in DF aspirated at 30 or 47 d after calving. Concentrations of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding proteins (IGFBP) in FF and plasma, and aromatase mRNA in granulosa cells were similar for anovulatory and proestrous cows. In conclusion, estradiol production by DF of postpartum anovulatory cows may be limited by inadequate production of androstenedione during the postpartum anovulatory interval and this may influence follicular maturation. Concentrations of IGF-I and IGFBP were similar in anovulatory and proestrous cows, an indication that alterations in the IGF-I system in the DF at 30-47 d after calving are not associated with delayed follicular development in postpartum beef cows.