P450 Aromatase mRNA Research Articles

Kisspeptin modulation of nonapeptide and cytochrome P450 aromatase mRNA expression in the brain and ovary of the catfish Heteropneustes fossilis: in vivo and in vitro studies.

In Heteropneustes fossilis, kisspeptins (Kiss) and nonapeptides (NPs; vasotocin, Vt; isotocin, Itb; Val8-isotocin, Ita) stimulate the hypothalamus-pituitary-gonadal (HPG) axis, and estrogen feedback modulates the expression of these systems. In this study, functional interactions among these regulatory systems were demonstrated in the brain and ovary at the mRNA expression level. Human KISS1 (hKISS1) and H. fossilis Kiss2 (HfKiss2) produced biphasic effects on brain and ovarian vt, itb and ita expression at 24h post injection: low and median doses produced inhibition, no change or mild stimulation, and the highest dose consistently stimulated the mRNA levels. The Kiss peptides produced an upregulation of NP mRNA expression at 24h incubation of brain and ovarian slices by increasing the concentration of hKISS1 and HfKiss2. The kiss peptides stimulated brain cyp19a1b and ovary cyp19a1a expression, both in vivo and in vitro. Peptide234, a Kiss1 receptor antagonist, inhibited basal mRNA expression of the NPs, cyp19a1b and cyp19a1a, which was prevented by the Kiss peptides, both in vivo and in vitro. In all the experiments, HfKiss2 was more effective than hKISS1 in modulating mRNA expression. The results suggest that the NP and E2 systems are functional targets of Kiss peptides and interact with each other.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes)

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

The mediation of pigeon egg production by regulating the steroid hormone biosynthesis of pigeon ovarian granulosa cells.

The aim of this study was to determine the molecular mechanism of miR-205b targeting 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) on the apoptosis and proliferation of granulosa cells (GC) of pigeons. Our previous studies suggested that HSD11B1 was the target gene of miR-205b and played a key role in steroid hormone biosynthesis and GC development. The adenovirus-miR-205b recombinant virus and adenovirus-cli-miR-205b-sh recombinant virus were generated, verified, and their characteristics determined. The recombinant viruses were used to infect the GC of pigeons, with real time quantitative PCR used to examine the expressions of HSD11B1 and related genes. The HSD11B1 antibody was obtained and verified, and Western blotting was used to detect the protein level of HSD11B1. The Cell Counting Kit–8 assay kit was used to detect cell viability, and the Annexin V-FITC/PI kit was used for the apoptosis assays. The expression of HSD11B1 was significantly lower in the overexpression (OE) than in OE negative control (OE-NC) treatments and significantly higher in short hairpin (SH) than in SH negative control (SH-NC) treatments. The expression levels of cytochrome P4503A5 was significantly higher in SH and lower in OE treatments, and the rhythms of cytochrome P450 aromatase mRNA levels were similar. The mRNA level of cytochrome P450scc in OE was lower than in OE-NC treatments and higher in SH than in SH-NC treatments. The protein expressions of HSD11B1 were decreased in the GC of OE, whereas increased in the SH group. The Cell Counting Kit–8 assay revealed that overexpression of miR-205b significantly suppressed proliferation of the GC of pigeons, whereas interference of miR-205b significantly induced the proliferation of the GC. The overexpression and the interference of miR-205b did not have a significant effect on cell cycle. The overexpression of miR-205b significantly increased the number of apoptotic cells, whereas the interference of miR-205b decreased the number of apoptotic cells. These findings indicated that miR-205b mediated pigeon egg production by regulating the steroid hormone biosynthesis of the pigeon ovarian GC by targeting HSD11B1, which may be useful in increasing pigeon egg production.

A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens.

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

Bone morphogenetic proteins and folliculogenesis: lessons from the Booroola mutation

The Booroola phenotype is associated with a point mutation in the kinase domain of the bone morphogenetic protein receptor 1 B (BMPR1 B), and is characterized by 'precocious' differentiation of ovarian follicles, leading to the production of large numbers of ovulatory follicles that are smaller in diameter than wild-type follicles. These smaller follicles attain differentiation markers, such as expression of mRNA for P450 aromatase and inhibin-betaA subunit, granulosa cell LH receptors and aromatase activity, earlier than follicles from wild-type ewes. However, the preovulatory follicles from mutant ewes collectively secrete similar quantities of oestradiol, androstenedione and inhibin A in exactly the same pattern as wild-type ewes, which result in similar concentrations of FSH. The available evidence strongly indicates that the Booroola mutation exerts its action at the ovary rather than by altering gonadotrophin secretion. The bone morphogenetic protein (BMP) receptors and putative ligands are ubiquitously expressed within the ovary and BMPs seem to be involved in the paracrine regulation of FSH action. Thus, if the mutation is causing a reduction in BMPR1 B signalling, it may act on an inhibitor of follicle differentiation. Further research in this area will concentrate on the elucidation of the natural ligands for BMPR1 B at different stages of follicle development and examine the effect of BMPR1 B mutation on the downstream signalling cascade.

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1–F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice.

Effect Of Microgravity On Aromatase Expression In Sertoli Cells

Cytochrome P450-aromatase catalyzes estrogen biosynthesis from C19 steroids. In the testis, Sertoli cells express P450-aromatase and represent the primary source of estrogen during prepuberal age. This study focused on the effect of simulated microgravity (SM) on aromatase expression in primary mouse Sertoli cells. When cultured in Rotary Cell Culture System (RCCS), Sertoli cells, formed multicellular three dimensional spheroids (3D). Biological properties were first analyzed in terms of viability, cell cycle, expression of cytoskeletal components and growth factors in comparison to Sertoli cells cultured in spheroids at unit gravity (G). SM did not affect cell viability and proliferation, nor expression of the main cytoskeleton proteins and of growth factors like Kit Ligand (KL) and glial derived neurotrophic factor (GDNF). On the other hand, SM caused a strong increase in P450 aromatase mRNA and protein expression. Interestingly, P450-aromatase was no more inducible by 8-Br-cAMP. The presence of a functional aromatase was confirmed by enrichment of 17β-estradiol released in the medium by androgen precursors. We concluded that SM causes a significant upregulation of aromatase gene expression in Sertoli cells, leading to a consequent increase in 17β-estradiol secretion. High level of 17β-estradiol in the testis could have potentially adverse effects on male fertility and testicular cancer.

Monocrotophos pesticide affects synthesis and conversion of sex steroids through multiple targets in male goldfish (Carassius auratus)

Monocrotophos (MCP) is an organophosphorus pesticide that is median-toxic to fish. MCP pesticide resulted in an increase of 17 beta estradiol following a decrease in testosterone in male goldfish (Carassius auratus). To fully understand the mechanism of MCP pesticide that causes the imbalance between male and female hormones, we determined the levels of plasma cholesterol, spermatic steroidogenic acute regulatory protein mRNA, steroidogenesis enzyme mRNA, plasma sex hormone synthesis intermediates, and effectual hormones in male goldfish exposed to MCP pesticide at nominal concentrations of 0.01, 0.10, and 1.00 mg/L for 21 days in a semi-static exposure system. The results indicated that MCP pesticide (a) led to decreased steroidogenic acute regulatory protein mRNA levels; (b) decreased mRNA levels of cholesterol side chain cleavage enzyme and cytochrome P450 17 alpha hydroxylase, which are steroidogenesis enzymes involved in androgen synthesis; and (c) increased cytochrome P450 aromatase mRNA levels, a steroidogenesis enzyme involved in the synthesis of effectual estrogen. The present study provides evidence that MCP pesticide affects synthesis and conversion of sex steroids through multiple targets in male goldfish.

Effects on Circulating Steroid Hormones and Gene Expression along the Hypothalamus-Pituitary-Gonadal Axis in Adult Japanese Quail Exposed to 17β-Trenbolone across Multiple Generations.

We investigated the effects of the androgenic growth promoter 17β-trenbolone (17βTB) on adult Japanese quail (Coturnix japonica) exposed across three generations. The F0 generation was exposed after sexual maturity to 0, 1, 5, 10, 20, and 40 ppm through feed. The F1 generation was exposed in ovo by maternal transfer and through feed at the same doses as their parents. The F2 generation was exposed in ovo only. Levels of plasma sex steroids, gonadal Cytochrome P450 aromatase (CYP19A1) mRNA and select brain neuroendocrine peptide mRNAs were measured. In males, testosterone levels did not differ in any generation from those in controls. Estradiol was significantly elevated in 17βTB treated F0 and F1 males. In F0 and F1 females, testosterone was suppressed by 17βTB, whereas estradiol was significantly higher at 40 ppm in F0 and at 10 ppm in F1 females. CYP19A1 expression in F1 males and females increased suggesting a compensatory response to the androgenic effects of 17βTB. Few significant effects were observed in the F2 birds indicating that in ovo exposure had limited effects on the monitored endpoints. Overall, our results confirmed endocrine disrupting effects of dietary 17βTB in Japanese quail but the response was dependent on sex, developmental stage at initiation of exposure, and dose.

An in vivo assay performed using multiple biomarkers related to testosterone synthesis and conversion for assessing the androgenic potency of refuse leachate

Refuse leachate is likely an important source of androgens. However, common in vitro bioassays underestimate the potential androgenic activity of leachate, owing to non-receptor-mediated mechanisms that modify the balance of sex hormones and promote the accumulation of endogenous androgens. This study aimed to develop an in vivo assay by using multiple biomarkers related to testosterone synthesis and conversion for assessing the potential androgenic activity of refuse leachate sampled from a municipal solid waste treatment plant in Qingdao, China. The results indicated that exposure to leachate increased the levels of testosterone and luteinizing hormone, but decreased those of 17β-estradiol in both male and female goldfish (Carassius auratus), suggesting a potential androgenic activity. Further, Leydig cell hyperplasia and decreased gonadal P450 aromatase mRNA levels were observed; these alterations might promote the biosynthesis of testosterone and hinder the conversion of testosterone to 17β-estradiol, which in turn enhance testosterone accumulation. Exposure to leachate also resulted in reproductive impairments, including decreased gonadosomatic index and plasma vitellogenin levels of female goldfish, as well as decreased testicular enzyme activities in male goldfish. The integrated use of biochemical, molecular, and histological markers not only improved our understanding of the androgenic effects of leachate but also verified the reliability and validity of the results. Therefore, the in vivo bioassay described in this study might allow the investigation of the androgenic effects of other complex contaminant mixtures in the future.

Ginkgo biloba extract EGb 761-mediated inhibition of aromatase for the treatment of hormone-dependent breast cancer

Ginkgo biloba has been used in herbal medicines for thousands of years. Although a standard G. biloba extract, EGb 761 has been used to improve cognition in breast cancer patients, its effects on breast cancer are unknown. Therefore, we investigated the antitumorigenic effects of EGb 761 using an in vitro cell model and an in vivo xenograft model. EGb 761 significantly inhibited aromatase activity in aromatase over-expressing MCF-7 cells (MCF-7 AROM). In addition, EGb 761 exposure reduced cytochrome p450 aromatase (CYP19) mRNA and protein expression; CYP19 promoter I.3 and PII expression particularly decreased. These inhibitory effects on aromatase were accompanied by reduced 17β-estradiol levels in MCF-7 AROM cells. For elucidating antitumorigenic effects, MCF-7 AROM cells were implanted in BALB/c nude mice prior to oral EGb 761 treatment for 3 weeks. EGb 761 reduced the tumor size and significantly reduced tumor CYP19 mRNA expression. Taken together, our results indicated that EGb 761 inhibited aromatase and exerted antitumor effects on breast cancer cells both in vitro and in vivo. These findings suggest that EGb761 may be a useful aromatase inhibitor for the treatment for estrogen-sensitive breast cancer.

Estrogen synthesis and secretion during postnatal testicular development in male goats: In situ aromatase mRNA expression

The role of testosterone in males has been extensively studied. However, studies in several species suggest that estrogens are also important for the development and function of male brain, bone, gonads as well as for spermatogenesis and sexual behavior. Data related to testicular P450-aromatase expression and estrogen receptors α and β in several species had been published. However, to our knowledge there is no information on testicular mRNA aromatase expression, estrogen synthesis, or estradiol-17β concentrations in the circulation of the male goat. Thus, the objective of this work was to conduct in vivo and in vitro studies related to estrogen synthesis and serum concentrations during the postnatal testicular development in goat bucks. To do this, circulating concentrations of estradiol-17β and testosterone were evaluated in male goat during the first year of life, starting at 15 days of age. Besides, in order to assess the estrogen-producing ability of testicles from prepubertal male goats (four months), testicular cells were incubated with tritiated androgen precursors (3H-androstenedione and 3H-testosterone) in the presence or absence of the P450-aromatase inhibitor formestane, using TLC to identify sex steroids. Finally, the expression and tissue distribution of P450-aromatase mRNA in the testis of 3–5 month old males was evaluated by in situ RT-PCR. Results indicate that estradiol-17β is present in the serum of male goats at 0.5 months of age, and that there are significant increases at 3 and 10 months of age. The in vitro synthesis of tritiated estradiol-17β was inhibited by the P450-aromatase inhibitor formestane. Messenger RNA for P450-aromatase was abundantly expressed both in Sertoli cells in the testis of goats of ages three, four and five months. The expression of P450 mRNA was also localized in Leydig cells. It is concluded that the prepubertal goat has measurable quantities of serum estradiol-17β and that their testis has the capacity to synthesize estrogens during this period.

Expression levels of mRNA for neurosteroidogenic enzymes 17β-HSD, 5α-reductase, 3α-HSD and cytochrome P450 aromatase in the fetal wild type and SF-1 knockout mouse brain

The presence of steroidogenic enzymes in the brain suggests de novo synthesis of steroid hormones in the brain. The current study was designed to determine the developmental profiles of cytochrome p450 aromatase (cyp19), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-reductase type I and 3α-hydroxysteroid dehydrogenase (3α-HSD) mRNA expression levels in the fetal mouse brain and potential influence of peripheral steroids, and the steroidogenic factor 1 (SF-1) gene on their expression. Brains were collected from WT and SF-1 knockout male and female fetuses at embryonic (E) days E12, E14, E16, and E18. Quantitative PCR analyses revealed age related increases in the expression levels of 17β-HSD and 5α-reductase. Differences between genotypes in the expression levels of 17β-HSD and 5α-reductase were detected on E14, with reduced levels of expression in SF-1 KO males and females for 17β-HSD and only between females for 5α-reductase. Expression of 3α-HSD mRNA did not differ significantly between sexes, age groups or genotypes with the exception of SF-1 KO males, which had an unexplained increase in mRNA for this enzyme on day E18. Expression of cyp19 was at the limit of detection and could not be analyzed effectively. There were no sex differences and, with the exception of small difference on E14 for 17β-HSD and 5α-reductase, no differences between genotypes. The results suggest that gonadal steroids do not influence the production of neurosteroids in the fetal brain, nor does SF-1 play a major role in the regulation of steroidogenic enzyme expression in the brain.

Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells.

Exerting a broad range of biological effects in various tissues, activins are homo- or heterodimers of activin/inhibin β-subunits (βA, βB, βC, and βE in humans). Although activins A (βAβA), B (βBβB), AB (βAβB), and AC (βAβC) have been demonstrated in the female reproductive system, little is known about their individual functions in the ovary. To investigate the biological roles and activities of activins in regulating steroidogenesis in human granulosa cells. Human granulosa-lutein cells obtained from 32 patients undergoing in vitro fertilization were used to investigate the effects of activin A, B, AB, and AC on the expression of steroidogenic enzymes and steroid production. An academic research center. mRNA and protein levels were examined by reverse transcription quantitative real-time PCR and Western blot analysis, respectively. The production of estradiol and progesterone was measured by enzyme immunoassay. P450 aromatase, FSH receptor, and estradiol levels were increased, whereas steroidogenic acute regulatory protein (StAR), LH receptor, and progesterone levels were decreased after treatment with activin A, B, and AB, but not activin AC. FSH or LH induced the production of aromatase/estradiol and StAR/progesterone; however, pretreatment with activin A, B, or AB enhanced the effects of gonadotropins on aromatase/estradiol, but suppressed their effects on StAR/progesterone. Treatment with activin A, B, or AB induced the phosphorylation of SMA- and MAD-related proteins (SMAD2 and 3), whereas activin AC had no such effects. Furthermore, co-culture of activin AC (1-100 ng/mL) with activin A (25 ng/mL) did not alter the effects of activin A on P450 aromatase or StAR mRNA levels. Activin A, B, and AB have similar effects on steroidogenesis in human granulosa cells. In contrast, activin AC is not biologically active and does not act as a competitive antagonist.

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles.

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100ng/ml), TGF-β (10ng/ml), or TGF-β + FSH for 18d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.

Endocrine disruption and reproduction impairment in zebrafish after long‐term exposure to DE‐71

The objective of the present study was to investigate the impact of polybrominated diphenyl ethers (PBDEs) on fish reproduction over 2 generations. Zebrafish (Danio rerio) embryos (F0) were exposed to low concentrations (3 µg/L, 10 µg/L, and 30 µg/L) of the PBDE mixture DE-71 until they were sexually mature, and steroid hormone production, expression of genes involved in steroidogenesis, gonadal development, and gamete characteristics were examined. Exposure of female zebrafish to DE-71 resulted in lower estradiol production and downregulation of cytochrome P450 aromatase mRNA. In males, exposure to DE-71 resulted in greater testosterone production and greater cytochrome P450 c17 α-hydroxylase,17,20-lase mRNA expression. Moreover, hepatic vitellogenin mRNA and estrogenic receptor β gene transcription were downregulated in females and males. Expression of the follicle-stimulating hormone β gene in the pituitary was upregulated, and the expression of luteinizing hormone β was downregulated in both sexes. Histological examination showed inhibition of oocyte maturation in females and retarded spermiation in males. The average number of eggs (F1) produced was also reduced. Additionally, exposure of F0 embryos to DE-71 did not result in developmental toxicity, whereas delayed hatching, reduced survival, and decreased growth were observed in the F1 embryos derived from parent fish exposed to DE-71. Therefore, long-term exposure to low concentrations of PBDEs in zebrafish could cause reproductive impairment, suggesting that PBDEs might have significant adverse effects on fish population in the highly PBDEs-contaminated aquatic environment.

PPARγ Activation Inhibits Growth and Survival of Human Endometriotic Cells by Suppressing Estrogen Biosynthesis and PGE2 Signaling.

Endometriosis is a chronic inflammatory disease of reproductive age women leading to chronic pelvic pain and infertility. Current antiestrogen therapies are temporizing measures, and endometriosis often recurs. Potential nonestrogenic or nonsteroidal targets are needed for treating endometriosis. Peroxisome proliferator-activated receptor (PPAR)γ, a nuclear receptor, is activated by thiazolidinediones (TZDs). In experimental endometriosis, TZDs inhibit growth of endometriosis. Clinical data suggest potential use of TZDs for treating pain and fertility concurrently in endometriosis patients. Study objectives were to 1) determine the effects of PPARγ action on growth and survival of human endometriotic epithelial and stromal cells and 2) identify the underlying molecular links between PPARγ activation and cell cycle regulation, apoptosis, estrogen biosynthesis, and prostaglandin E2 biosynthesis and signaling in human endometriotic epithelial and stromal cells. Results indicate that activation of PPARγ by TZD ciglitazone 1) inhibits growth of endometriotic epithelial cells 12Z up to 35% and growth of endometriotic stromal cells 22B up to 70% through altered cell cycle regulation and intrinsic apoptosis, 2) decreases expression of PGE2 receptors (EP)2 and EP4 mRNAs in 12Z and 22B cells, and 3) inhibits expression and function of P450 aromatase mRNA and protein and estrone production in 12Z and 22B cells through EP2 and EP4 in a stromal-epithelial cell-specific manner. Collectively, these results indicate that PGE2 receptors EP2 and EP4 mediate actions of PPARγ by incorporating multiple cell signaling pathways. Activation of PPARγ combined with inhibition of EP2 and EP4 may emerge as novel nonsteroidal therapeutic targets for endometriosis-associated pain and infertility, if clinically proven safe and efficacious.

Effect of nutrition on plasma lipid profile and mRNA levels of ovarian genes involved in steroid hormone synthesis in Hu sheep during luteal phase1

Ovarian steroid hormones regulate follicular growth and atresia. This study aims to determine whether key ovarian sterol-regulatory genes are differentially expressed in Hu sheep under different short-term nutritional regimens. Estrus was synchronized using intravaginal progestagen sponges. The ewes were assigned randomly to 3 groups. On d 6 to 12 of their estrous cycle, the control (CON) group received a maintenance diet (1.0×M), the supplemented (SUP) group received 1.5×M, and the restricted (R) group received 0.5×M. On d 7 to 12, blood samples were taken. The sheep were slaughtered at the end of the treatment, and their organs and ovaries were collected. The plasma concentrations of urea (P<0.01), total cholesterol (P<0.01), low-density lipoprotein cholesterol (P<0.01), NEFA (P<0.01), FSH (P<0.05), and estradiol (P<0.05) increased with decreasing dietary intake, whereas plasma triglyceride (P<0.01) and triiodothyronine (T3) concentrations decreased (P<0.05). The ewes in the R group had higher spleen weight and percentage of spleen to BW and lower liver and small intestine weights and percentage of liver/stomach to BW than the SUP group ewes (P<0.05). Nutritional restriction decreased the cytochrome p450 (CYP17A1) and estrogen receptor 1 (ESR1) mRNA expression (P<0.05) and increased the cytochrome p450 aromatase (CYP19A1) mRNA expression (P<0.05) in follicles>2.5 mm. Follicle size affected the mRNA expression of very low density lipoprotein receptor (VLDLR), estrogen receptor 2 (ESR2), FSH receptor (FSHR), CYP17A1, and CYP19A1 (P<0.05). In conclusion, we suggest that a potential mechanism by which short-term negative energy balance inhibits follicular growth may involve responses to disrupted reproductive hormone concentrations and influenced the intrafollicular expression of CYP17A1, CYP19A1, and ESR1. This result may be due to increased plasma urea and lipid concentrations.

Activin-A promotes the development of goat isolated secondary follicles in vitro

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 μm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

Activin stimulates CYP19A gene expression in human ovarian granulosa cell-like KGN cells via the Smad2 signaling pathway

Activin, a transforming growth factor β family member, has a wide range of physiological roles during embryonic development and organogenesis. In the ovary, activin, secreted from ovarian granulosa cells, not only acts on the pituitary gland to regulate the gonadotropin secretion from the pituitary gland in an endocrine manner but also acts on granulosa cells in a paracrine/autocrine manner to regulate folliculogenesis. Previously, we showed that activin signals through activin type IB receptor (ActRIB) and up-regulates follicle-stimulating hormone receptor expression and P450 aromatase activity in human ovarian granulose cell-like KGN cells. In the current study, we demonstrate the direct involvement of Smad2 as a downstream signal mediator of ActRIB in the transcriptional regulation of the P450 aromatase gene (CYP19A) in KGN cells. Upon activin stimulation, Smad2 activation and an increase in P450 aromatase messenger RNA (mRNA) were observed in KGN cells. Interestingly, Smad2 phosphorylation correlated well with the increase in P450 aromatase mRNA. Reciprocally, knockdown of Smad2 mRNA in KGN cells led to a decrease in the P450 aromatase mRNA expression, suggesting that Smad2 regulates CYP19A gene expression. Further analysis of CYP19A promoter activity revealed that the 5′ upstream region between −2069 and −1271bp is required for the activation by Smad2. Finally, we provide compelling evidence that Smad2 shows follicular stage-specific expression, which is high in granulosa cells of preantral or early antral follicles in mice. Our results suggest that activin signaling through the ActRIB-Smad2 pathway plays a pivotal role in CYP19A expression and thus in follicular development.