miR-10a-5p inhibits steroid hormone synthesis in porcine granulosa cells by targeting CREB1 and inhibiting cholesterol metabolism.

During growth, proliferation, differentiation, atresia, ovulation, and luteinization, the morphology and function of granulosa cells (GCs) change. Estrogen and progesterone are steroid hormones secreted by GCs that regulate the ovulation cycle of sows and help maintain pregnancy. miR-10a-5p is highly expressed in GCs and can inhibit GC proliferation. However, the role of miR-10a-5p in the steroid hormone synthesis of porcine GCs is unclear. In this study, miR-10a-5p agomir or antagomir was transfected into GCs. Overexpression of miR-10a-5p in GCs inhibited steroid hormone secretion and significantly downregulated steroid hormone synthesis via 3ß-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1. Interference with miR-10a-5p had the opposite effect. Bodipy and Oil Red O staining showed that overexpression of miR-10a-5p significantly reduced the formation of lipid droplets. Overexpression significantly inhibited the content of total cholesterol esters in GCs. The mRNA and protein levels of 3-hydroxy-3-methylglutaryl-CoA reductase and scavenger receptor class B member 1 decreased significantly, and the opposite effects were seen by interference with miR-10a-5p. Bioinformatic analysis of potential targets identified cAMP-responsive element binding protein 1 as a potential target and dual-luciferase reporter system analysis confirmed that miR-10a-5p directly targets the 3' untranslated region. These findings suggest that miR-10a-5p inhibits the expression of 3ß-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1 to inhibit the synthesis of steroid hormones in GCs. In addition, miR-10a-5p inhibits the cholesterol metabolism pathway of GCs to modulate steroid hormone synthesis.

Fetal Estrogens are not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits.

AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

Molecular characterization, expression profile and transcriptional regulation of the CYP19 gene in goose ovarian follicles.

Cytochrome P450 Family 19 (CYP19) is a crucial enzyme to catalyze the conversion of androgens to estrogens. However, the regulatory mechanism of goose CYP19 gene remains poorly understood. The present study attempted to obtain the full-length coding sequence (CDS) and 5'-flanking sequence of CYP19 gene, to investigate its expression and distribution profiles in different sized follicles, and to analyze the transcriptional regulatory mechanism of CYP19 gene in goose. Results showed that its CDS consisted of 1512 nucleotides and the encoded amino acid sequence contained a classical P450 structural domain. Homology analysis showed that there were high homologies of nucleotide and amino acid sequences between goose and other avian species. Its promoter sequence spanned from -1925 bp to the transcription start site (ATG) and several transcriptional factors were predicted in this region. Further analysis from luciferase assay showed that the luciferase activity was the highest spanning from -118 to -1 bp by constructing deletion promoter reporter vector. In addition, result from quantitative real-time polymerase chain reaction indicated that the mRNA level of CYP19 gene were highly expressed in theca layer of the fifth largest follicle, and the cellular location was in the theca externa cells by immunohistochemistry. Taken together, it could be concluded that the transcription activity of CYP19 gene was activated by transcriptional factors in its proximal region of promoter to promote the synthesis of estrogens, regulating the selection of pre-hierarchical into hierarchical follicle in goose.

The UV filter benzophenone 3, alters early follicular assembly in rat whole ovary cultures.

Our goal was to study the effect of BP3 (benzophenone 3) in the follicular assembly and the potential involvement of Foxl2 pathway using whole ovary cultures. Ovaries were collected from Wistar rats at birth, treated in vitro with vehicle (0.01% DMSO), BP3 (5.8 nM, 276 nM, 576 nM and 876 nM) or ESR2 inhibitor (0.1 nM), and cultured for 7 days. Nest breakdown, follicular assembly and the expression of several regulators of these processes (p27, Foxl2, Sox9, Bmp2, Cyp19 and Fst) were evaluated. In vitro exposure to BP3 (5.8 nM) decreased the population of total oocytes, the number of nests per ovary and early primary follicles population. In addition, BP3 (5.8 nM) induced overexpression of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. We also observed that the number of p27-positive oocytes was decreased after BP3 (5.8 nM). On the other hand, exposure to BP3-276 increased total oocytes, the number of nests per ovary and decreased primary follicles. In addition, BP3-276 induced no changes of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. In conclusion, our study clearly shows that exposure to BP3 is to perturb the early events of germ cell development as showed here in whole ovary cultures.

Short-term rapamycin treatment increases ovarian lifespan in young and middle-aged female mice.

Although age-related ovarian failure in female mammals cannot be reversed, recent strategies have focused on improving reproductive capacity with age, and rapamycin is one such intervention that has shown a potential for preserving the ovarian follicle pool and preventing premature ovarian failure. However, the application is limited because of its detrimental effects on follicular development and ovulation during long-term treatment. Herein, we shortened the rapamycin administration to 2 weeks and applied the protocol to both young (8 weeks) and middle-aged (8 months) mouse models. Results showed disturbances in ovarian function during and shortly after treatment; however, all the treated animals returned to normal fertility 2 months later. Following natural mating, we observed prolongation of ovarian lifespan in both mouse models, with the most prominent effect occurring in mice older than 12 months. The effects of transient rapamycin treatment on ovarian lifespan were reflected in the preservation of primordial follicles, increases in oocyte quality, and improvement in the ovarian microenvironment. These data indicate that short-term rapamycin treatment exhibits persistent effects on prolonging ovarian lifespan no matter the age at initiation of treatment. In order not to disturb fertility in young adults, investigators should in the future consider applying the protocol later in life so as to delay menopause in women, and at the same time increase ovarian lifespan.

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation.

Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.

Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.

STUDY QUESTION: What are the direct effects and physiological role of anti-Müllerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis? SUMMARY ANSWER: AMH actions in the primate ovary may be stage-dependent, directly promoting pre-antral follicle growth while inhibiting antral follicle maturation and dominant follicle selection. WHAT IS KNOWN ALREADY: AMH is expressed in the adult ovary, particularly in developing follicles. Studies in mice suggest that AMH suppresses pre-antral follicle growth in vitro, and inhibits primordial follicle recruitment and FSH-stimulated antral follicle steroidogenesis. STUDY DESIGN, SIZE, DURATION: For in vitro study, secondary follicles were isolated from ovaries of 12 rhesus macaques and cultured for 5 weeks. For in vivo study, intraovarian infusion was conducted on five monkeys for the entire follicular phase during two spontaneous menstrual cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: For in vitro study, individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to treatments of recombinant human AMH protein or neutralizing anti-human AMH antibody (AMH-Ab). Follicle survival, growth, steroid production, steroidogenic enzyme expression, and oocyte maturation were assessed. For in vivo study, ovaries were infused with control vehicle or AMH-Ab during the follicular phase of the menstrual cycle. Cycle length, serum steroid levels, and antral follicle growth were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: AMH exposure during culture weeks 0-3 (pre-antral stage) promoted, while AMH-Ab delayed, antrum formation of growing follicles compared with controls. AMH treatment during culture weeks 3-5 (antral stage) decreased (P < 0.05) estradiol (E2) production, as well as the mRNA expression of cytochrome P450 family 19 subfamily A polypeptide 1, by antral follicles relative to controls, whereas AMH-Ab increased (P < 0.05) follicular mRNA levels of the enzyme. Intraovarian infusion of AMH-Ab during the follicular phase of the menstrual cycle increased (P < 0.05) the average levels of serum E2 compared with those of the control cycles. Three of the five AMH-Ab-treated ovaries displayed multiple (n = 2-9) medium-to-large (2-8 mm) antral follicles at the mid-cycle E2 peak, whereas only one large (4-7 mm) antral follicle was observed in all monkeys during their control cycles. The average levels of serum progesterone were higher (P < 0.05) during the luteal phase of cycles following the AMH-Ab infusion relative to the vehicle infusion. LIMITATIONS, REASONS FOR CAUTION: The in vitro study of AMH actions on cultured individual macaque follicles was limited to the interval from the secondary to small antral stage. A sequential study design was used for in vivo experiments, which may limit the power of the study. WIDER IMPLICATIONS OF THE FINDINGS: The current study provides novel information on direct actions and role of AMH during primate follicular development, and selection of a dominant follicle by the late follicular phase of the menstrual cycle. We hypothesize that AMH acts positively on follicular growth during the pre-antral stage in primates, but negatively impacts antral follicle maturation, which is different from what is reported in the mouse model. STUDY FUNDING/COMPETING INTERESTS: NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Regulation of cancer-related genes - Cyp1a1, Cyp1b1, Cyp19, Nqo1 and Comt - expression in ß-naphthoflavone-treated mice by miroestrol.

OBJECTIVE: The effects of miroestrol (MR), an active phytoestrogen from Pueraria candollei var. mirifica, on expression of cancer-related genes were determined. METHODS: Seven-week-old female ICR mice (n = 5 each) were subcutaneously administered estradiol (E2, 0.5 mg/kg/day) or MR (0.5 or 5 mg/kg/day) daily for 7 days. Some were given ER or MR in combination with ß-naphthoflavone (BNF, 30 mg/kg/day) for the last 3 days. The expression of cancer-related genes including cytochrome P450 1A (Cyp1a), cytochrome P450 1B1 (Cyp1b1), aromatase P450 (Cyp19), NAD(P)H: quinone oxidoreductase 1 (Nqo1) and catechol-O-methyltransferase (Comt) were evaluated. KEY FINDINGS: In the presence of BNF, MR suppressed hepatic CYP1A1 activity and CYP1A2 activity, expression of CYP1B1 mRNA and expression of CYP1A1/2 and CYP1B1 protein. E2, by contrast, did not. MR restored expression levels of hepatic NQO1 and uterine COMT in BNF-treated mice. Furthermore, MR increased expression of uterine CYP19 to the same extent as E2. CONCLUSION: MR may be superior to E2 as it downregulates expression of CYP1. Moreover, MR normalized expression of both NQO1 and COMT, the protective enzymes, in murine liver and uteri. These results support the use of MR as an alternative supplement for menopausal women, MR having the extra benefit of reducing cancer risk.