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Transfer RNA-derived small RNAs (tsRNAs) are novel regulatory small non-coding RNAs that have been found to modulate many life activities in recent years. However, the exact functions of tsRNAs in follicle development remain unclear. Follicle development is a remarkably complex process that follows a strict hierarchy and is strongly associated with reproductive performance in ducks. The process of converting small yellow follicles into hierarchal follicles is known as follicle selection, which directly determines the number of mature follicles. We performed small RNA sequencing during follicle selection in ducks and identified tsRNA-00764 as the target of interest based on tsRNA expression profiles in this study. Bioinformatics analyses and luciferase reporter assays further revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) was the target gene of tsRNA-00764. Moreover, tsRNA-00764 knockdown promoted estrogen and progesterone synthesis and lipid deposition in duck granulosa cells, while a PPAR-γ inhibitor reversed the above phenomenon. Taken together, these results demonstrate that tsRNA-00764, differentially expressed in pre-hierarchal and hierarchy follicles, modulates estrogen and progesterone synthesis and lipid deposition by targeting PPAR-γ in duck granulosa cells, serving as a potential novel mechanism of follicle selection. Overall, our findings provide a theoretical foundation for further exploration of the molecular mechanisms underlying follicle development and production performance in ducks. [ABSTRACT FROM AUTHOR]

Cumulus cells (CCs) synthesize estrogens that are essential for follicular development. However, the effects of androgen on estrogen production in buffalo CCs remain unknown. In the present study, the impacts of testosterone on estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes were investigated. The results showed that testosterone supplementation improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 17β-HSD) and the secretion levels of estradiol in buffalo CCs surrounding in vitro-matured oocytes. Furthermore, testosterone treatment enhanced the sensitivity of buffalo CCs surrounding in vitro-matured oocytes to follicle-stimulating hormone (FSH). This study indicated that testosterone supplementation promoted the estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes mainly through strengthening the responsiveness of CCs to FSH. The present study serves as a foundation of acquiring high-quality recipient oocytes for buffalo somatic cell nuclear transfer. [ABSTRACT FROM AUTHOR]

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of androgen on estrogen production in buffalo GCs remain unclear. In this study, the impacts of testosterone on estrogen synthesis in buffalo GCs were examined. The results showed that testosterone that was added to cell medium at a concentration of 10−7 mol/L and applied to GCs for 48 or 72 h enhanced the estrogen synthesis of buffalo GCs. This study provides a theoretical basis for further exploration of ovarian endocrine mechanism for steroidogenesis. [ABSTRACT FROM AUTHOR]

MicroRNA-7a2 (miR-7a2) plays fundamental roles in the female reproductive axis, and estrogen is indispensable for maintaining ovary function. However, the interaction between miR-7a2 and ovarian function is unclear. The present study aimed to determine whether and how miR-7a2 functions in estrogen synthesis. Firstly, the results verified that miR-7a was highly expressed in ovarian granulosa cells. The knockout (KO) of miR-7a2 caused infertility and abnormal ovarian function in mice. Concomitantly, the Cyp19a1 expression and estrogen synthesis were significantly inhibited, which was validated in primary granulosa cells. The mice transplanted with miR-7a2 KO ovaries showed similar results; however, estrogen supplementation reversed infertility. In the in vitro experiment, follicle-stimulating hormone (FSH) significantly improved the expression of miR-7a and Cyp19a1 and the synthesis of estrogen. However, the miR-7a2 KO markedly reversed the function of FSH. Also, FSH upregulated miR-7a by activating the (c-Jun N-terminal kinase) JNK signaling pathway. In addition, Golgi apparatus protein 1 (Glg1) was shown to be the target gene of miR-7a2. These findings indicated that miR-7a2 is essential for ovarian functions with respect to estrogen synthesis through the targeted inhibition of the expression of Glg1 and then promoting Cyp19a1 expression; the physiological process was positively regulated by FSH via the JNK signaling pathway in granulosa cells. [ABSTRACT FROM AUTHOR]

Endogenous microRNAs (miRNAs), often distributed in clusters, can enable the subtle and precise regulation in gene expression via a variety of ways and different regions. In this study, multiple regulatory patterns for the estrogenic cyp19a1a and hsd17b12a genes by miR-17-92 cluster (miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a) were investigated in ovaries of Japanese flounder (Paralichthys olivaceus), the important marine cultured fish in North Asia with sexual dimorphism in growth and sex reversal scenario. Through dual-luciferase reporter assay, the regulation of P. olivaceus estrogenic genes by miR-17-92 cluster could be the combined multi-effect between 3′-UTR and 5′-ends targeting. For cyp19a1a , miR-17/20a induced its expression by binding to the promoter but simultaneously inhibited cyp19a1a through 3′-UTR targeting, while miR-92a activated cyp19a1a by binding to the promoter, which made the overall up-regulation of cyp19a1a more significant. For hsd17b12a , the binding of miR-17/20a and miR-92a to the promoter and the binding of miR-18a to 3′-UTR all inhibited its expression, which ultimately reduced the hsd17b12a level. Both in vitro and in vivo experiments in P. olivaceus ovaries confirmed these canonical and non-canonical regulatory patterns of miR-17-92 cluster, which coordinately reduced the estradiol (E2) level. Moreover, E2 could act as a repressor of miR-17-92 cluster, which may form a regulatory feedback loop whereby E2 regulates estrogenic process through miR-17-92 cluster. These results contributed to a deeper understanding of the miR-17-92 cluster function in teleosts and provided more insights into the molecular mechanism of fish reproductive regulation. • MiR-17-92 cluster regulates estrogenic genes by targeting both 3′-UTR and 5′-ends. • The multiple effects of miR-17-92 members coordinately reduced E2 level. • E2 could act as a repressor of miR-17-92 cluster. • E2 regulates estrogenic process through miR-17-92 cluster as a feedback loop. [ABSTRACT FROM AUTHOR]

The circadian system performs an important role in mammalian reproduction with significant effects on hormone secretion. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor in the circadian system and affects granulosa cells (GCs), but how it regulates estrogen synthesis has not been clarified. We investigated the effect of NR1D1 on estrogen synthesis and found that NR1D1 was highly expressed in GCs, mainly in cell nuclei. Additionally, the expression of NR1D1 and estrogen synthesis key genes CYP19A1 , CYP11A1 and StAR showed rhythmic changes in porcine ovarian GCs. Activation of NR1D1 enhances its ability to inhibit the transcriptional activity of CYP19A1 by binding to the RORE on the CYP19A1 promoter, resulting in a decrease in estradiol content. Interference with NR1D1 can eliminate the transcriptional inhibition of CYP19A1 and promote the synthesis of estradiol. The results suggest that the hormone secretion of the ovary itself is also regulated by the biological clock, and any factors that affect the circadian rhythm can affect the endocrine and reproductive performance of sows, so the natural rhythm of sows should be maintained in production. • NR1D1 was highly expressed in granulosa cells, mainly in cell nuclei. • NR1D1 inhibits the synthesis of estradiol in granulosa cells. • NR1D1 regulates estrogen synthesis through affecting the transcription of CYP19A1. [ABSTRACT FROM AUTHOR]

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca 2+ ) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfolded protein response (UPR er ), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca 2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1 pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca 2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research., (© 2024 Wiley Periodicals LLC.)

MicroRNA-7a2 (miR-7a2) plays fundamental roles in the female reproductive axis, and estrogen is indispensable for maintaining ovary function. However, the interaction between miR-7a2 and ovarian function is unclear. The present study aimed to determine whether and how miR-7a2 functions in estrogen synthesis. Firstly, the results verified that miR-7a was highly expressed in ovarian granulosa cells. The knockout (KO) of miR-7a2 caused infertility and abnormal ovarian function in mice. Concomitantly, the Cyp19a1 expression and estrogen synthesis were significantly inhibited, which was validated in primary granulosa cells. The mice transplanted with miR-7a2 KO ovaries showed similar results; however, estrogen supplementation reversed infertility. In the in vitro experiment, follicle-stimulating hormone (FSH) significantly improved the expression of miR-7a and Cyp19a1 and the synthesis of estrogen. However, the miR-7a2 KO markedly reversed the function of FSH. Also, FSH upregulated miR-7a by activating the (c-Jun N-terminal kinase) JNK signaling pathway. In addition, Golgi apparatus protein 1 (Glg1) was shown to be the target gene of miR-7a2. These findings indicated that miR-7a2 is essential for ovarian functions with respect to estrogen synthesis through the targeted inhibition of the expression of Glg1 and then promoting Cyp19a1 expression; the physiological process was positively regulated by FSH via the JNK signaling pathway in granulosa cells.

In Eriocheir sinensis aquaculture, crabs feeding on an artificial diet without steroid hormones available in ice-fresh fish or snails show delayed ovarian maturation and late listing. Icariin (ICA), a safe plant-derived monomer compound, can effectively promote estradiol (E2) synthesis and ovarian development in mammals, but its effect in crustaceans has not been reported. We investigated the effects of ICA on estrogen synthesis, vitellogenesis, and oocyte development in Chinese mitten crabs fed with one of four diets containing 0, 50, 100, and 200 mg/kg ICA, respectively, for 8 weeks. Results showed that vitellogenin (VTG) contents, gonadosomatic indexes (GSIs), and oocyte volumes in ICA50 and ICA 100 crabs were significantly higher than those in the control. The highest upregulated expression of VTG in hepatopancreases and ovaries was detected in ICA100 crabs. Hemolymph E2 contents were also significantly increased in ICA100 crabs. Enzyme-linked immunosorbent assay (ELISA) and western blot analysis indicated that the increase in E2 concentrations was attributable to an elevation in aromatase protein levels in ovaries through the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element binding (CREB) protein pathway. These findings suggest that ICA could serve as a natural additive to resolve the problem of delayed ovarian maturation of crabs raised in ponds.

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.

Estrogens are known to act rapidly, probably via membrane estrogen receptors, to induce fast effects on physiological and behavioral processes. Engaging in some of these behaviors, such as sexual behavior, results in an acute modulation of the production of estrogens in the brain by regulating the efficiency of the estrogen synthase enzyme, aromatase. We recently demonstrated that aromatase activity (AA) in the male quail brain is rapidly inhibited in discrete brain regions including the medial preoptic nucleus (POM) following exposure to a female. Evidence from in vitro studies point to glutamate release as one of the mechanisms controlling these rapid regulations of the aromatase enzyme. Here, we show that (a) the acute injection of the glutamatergic agonist kainate into the POM of anesthetized male quail inhibits AA and (b) glutamate is released in the POM during copulation. These results provide the first set of in vivo data demonstrating a role for glutamate release in the rapid control of AA in the context of sexual behavior. [ABSTRACT FROM AUTHOR]

It is well known that a connection between xenobiotics inhalation, especially tobacco combustion and Lung Cancer development is strongly significant and indisputable. However, recent studies provide evidence indicating that another factors such as, estrogens are also involved in lung carcinoma biology and metabolism. Although the status of estrogen receptors (ER), in both cancerous and healthy lung tissue has been well documented, there is still inconclusive data with respect of which isoform of the receptor is present in the lungs. However according to several studies, ERβ appears to be predominant form. Apart from ERs, estrogens can work through a recently discovered G-coupled estrogen receptor. Binding with both types of the receptors causes a signal, which leads to i.e. enhanced cell proliferation. There are many published reports which suggest that estrogen can be synthesized in situ in lung cancer. Some disturbances in the activity and expression levels of enzymes involved in estrogen synthesis were proved. This suggests that increased amounts of sex-steroid hormones can affect cells biology and be the reason of the accelerated development and pathogenesis of lung cancer. There also exist phenomena which associate estrogenic metabolism and tobacco combustion and its carcinogenic influence on the lungs. Compounds present in cigarette smoke induce the activity of CYP1B1, the enzyme responsible for estrogenic metabolism and synthesis of their cateholic derivatives. These structures during their redox cycle are able to release reactive oxygen species or form DNA adduct, which generally leads to destruction of genetic material. This process may explain the synergistic effect of smoking and estrogens on estrogen-dependent lung cancer development. [ABSTRACT FROM AUTHOR]

Ovarian cancer is still the deadliest of all gynecologic malignancies in women worldwide. This is attributed to two main features of these tumors, namely, i) a diagnosis at an advanced tumor stage, and, ii) the rapid onset of resistance to standard chemotherapy after an initial successful therapy with platin- and taxol-derivatives. Therefore, novel targets for an early diagnosis and better treatment options for these tumors are urgently needed. Epidemiological data show that induction and biology of ovarian cancer is related to life-time estrogen exposure. Also experimental data reveal that ovarian cancer cells share a number of estrogen regulated pathways with other hormone-dependent cancers, e.g. breast and endometrial cancer. However, ovarian cancer is a heterogeneous disease and the subtypes are quite different with respect to mutations, origins, behaviours, markers and prognosis and respond differently to standard chemotherapy. Therefore, a characterization of ovarian cancer subtypes may lead to better treatment options for the various subtypes and in particular for the most frequently observed high-grade serous ovarian carcinoma. For this intention, further studies on estrogen-related pathways and estrogen formation in ovarian cancer cells are warranted. The review gives an overview on ovarian cancer subtypes and explains the role of estrogen in ovarian cancer. Furthermore, enzymes active to synthesize and metabolize estrogens are described and strategies to target these pathways are discussed.

This study aims to examine the effect of superfine powder and aqueous extract of Polygonati Rhizomaon on natural perimenopausal syndrome in rats and explore the underlying mechanism. To be specific, a total of 60 female SD rats(14-15 months old) with estrous cycle disorder were screened by the vaginal smear and randomized into model control group, β-estradiol 3-benzoate group(0.1 mg·kg~(-1)), superfine powder of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)) and aqueous extract of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)), and another 10 female SD rats(14-15 months old) were selected as the youth control group. The administration lasted 6 weeks. Then the perimenopausal syndrome-related indexes such as body temperature, microcirculatory blood flow of face and ear, vertigo period, salivary secretion, grip force, and bone strength were determined and open field test was conducted. The immune system-related indexes such as the wet weight and index of thymus and spleen, percentage of T lymphocytes and subgroups in peripheral blood, and hematological indexes were measured. In addition, the ovary-related indexes such as estrous cycle, the wet weight and index of uterus and ovary, ovarian tissue morphology, and cell apoptosis were determined. Moreover, hypothalamus-pituitary-ovary axis(HPO)-related indexes such as serum sex hormone levels, cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and cytochrome P450 family 17 subfamily A member 1(P450 17A1) in ovarian tissue were measured. The results showed that the superfine powder and aqueous extract of Polygonati Rhizoma significantly decreased body temperature(anal, facial and dorsal temperature), microcirculatory blood flow in the ear, and vertigo period, increased salivary secretion, grip force, bone strength, total distance and total speed in the open field test, wet weight and index of thymus and spleen, lymphocyte ratio, CD3~+ level, and CD4~+/CD8~+ ratio, reduced neutrophil number and ratio, estrous cycle disorder ratio, and number of ovarian apoptotic cells, raised wet weight and index of uterus, wet weight of ovary, levels of inhibin B(INHB), estradiol(E_2), anti-müllerian hormone(AMH), and ovarian CYP11A1 and CYP19A1, decreased follicle-stimulating hormone(FSH) and luteinizing hormone(LH) content, and improved ovarian tissue morphology. It is suggested that the superfine powder and aqueous extract of Polygonati Rhizoma can improve the symptoms associated with natural perimenopausal syndrome in rats and enhance ovarian function and immune function. The mechanism is that they regulate HPO axis function by increasing estrogen synthesis.

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 ( 3 H-androstenedione and 3 H-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. [ABSTRACT FROM AUTHOR]

The women during the menopause period have an increased tendency for the obesity, which represents the more fat production than during the premenopausal period. Although this is not beneficial overall, it could provide a compensatory source for the estrogen production for the menopausal women. So it would be meaningful to find an agent that could inhibit the fat production while does not disturb the total estrogen production by fat tissues. In the present study, the effect of oleanolic acid (OA) on the fat production and the total estrogen production of the differentiating mouse preadipocyte 3T3-L1 as well as the mechanisms behind those effects were preliminarily investigated. The cell line 3T3-L1 was chosen as the model cell because it is usually used for the research about the obesity. During the induced differentiation of 3T3-L1 cells, cells were intervened continuously with OA. The fat production was determined with the oil red staining assay and the total estrogen production was measured with the ELISA assay. Finally, the expression patterns for important genes of the fat production and the estrogen production were studied, respectively with the real-time fluorescence quantitative PCR (qPCR). The results showed that for the differentiating 3T3-L1 cells, OA could significantly inhibit the fat production and did not disturb the total estrogen production significantly. In the mechanism studies, OA was found to significantly down-regulate ACC, the key gene for fat synthesis, which could explain the inhibitory effect of OA on the fat production; OA was also found to significantly up-regulate CYP11A1, CYP17, CYP19, the key genes for the estrogen synthesis and significantly down-regulate CYP1A1, the key gene for the estrogen decomposition, which preliminarily explained the lack of the effect of OA on the total estrogen production. In conclusion, OA was found able to inhibit the fat production while maintaining the total estrogen level and the mechanisms for the above findings were preliminarily clarified, which suggests that OA may be useful to treat the menopausal obesity. [ABSTRACT FROM AUTHOR]

It has been observed previously that compared with women of European ancestry (EA), those of African ancestry (AA) are more likely to develop estrogen receptor (ER)-negative breast cancer, although the mechanisms have not been elucidated. We tested the associations between breast cancer risk and a targeted set of 20 genes known to be involved in estrogen synthesis, metabolism, and response and potential gene-environment interactions using data and samples from 1307 EA (658 cases) and 1365 AA (621 cases) participants from the Women's Circle of Health Study (WCHS). Multivariable logistic regression found evidence of associations with single-nucleotide polymorphisms (SNPs) in the ESR1 gene in EA women (rs1801132, odds ratio (OR)=1.47, 95% CI=1.20-1.80, P=0.0002; rs2046210, OR=1.24, 95% CI=1.04-1.47, P=0.02; and rs3020314, OR=1.43, 95% CI=1.19-1.70, P=0.00009), but not in AA women. The only other gene associated with breast cancer risk was CYP1A2 in AA women (rs2470893, OR=1.42, 95% CI=1.00-2.02, P=0.05), but not in EA women. When stratified by ER status, ESR1 rs1801132, rs2046210, and rs3020314 showed stronger associations in ER-positive than in ER-negative breast cancer in only EA women. Associations with the ESR1 SNPs in EA women also appeared to be stronger with longer endogenous estrogen exposure or hormonal replacement therapy use. Our results indicate that there may be differential genetic influences on breast cancer risk in EA compared with AA women and that these differences may be modified by tumor subtype and estrogen exposures. Future studies with a larger sample size may determine the full contribution of estrogen-related genes to racial/ethnic differences in breast cancer. [ABSTRACT FROM AUTHOR]

Ovarian cancer is still the deadliest of all gynecologic malignancies in women worldwide. This is attributed to two main features of these tumors, namely, (i) a diagnosis at an advanced tumor stage, and, (ii) the rapid onset of resistance to standard chemotherapy after an initial successful therapy with platin- and taxol-derivatives.Therefore, novel targets for an early diagnosis and better treatment options for these tumors are urgently needed. Epidemiological data showthat induction and biology of ovarian cancer is related to life-time estrogen exposure. Also experimental data reveal that ovarian cancer cells share a number of estrogen regulated pathways with other hormone-dependent cancers, e.g., breast and endometrial cancer. However, ovarian cancer is a heterogeneous disease and the subtypes are quite different with respect to mutations, origins, behaviors, markers, and prognosis and respond differently to standard chemotherapy. Therefore, a characterization of ovarian cancer subtypes may lead to better treatment options for the various subtypes and in particular for the most frequently observed high-grade serous ovarian carcinoma. For this intention, further studies on estrogen-related pathways and estrogen formation in ovarian cancer cells are warranted. The review gives an overview on ovarian cancer subtypes and explains the role of estrogen in ovarian cancer. Furthermore, enzymes active to synthesize and metabolize estrogens are described and strategies to target these pathways are discussed. [ABSTRACT FROM AUTHOR]

Experimental evidence reveals that phytoestrogens have many targets through which they inhibit growth of breast cancer cells. These targets include cyclins, inhibitors of cyclin-dependent kinases (p21/p27), the tumour-suppressor gene p53, and pro-apoptotic and anti-apoptotic genes including Bax and Blc-2. This review covers the experimental evidence for mechanisms through which isoflavones and resveratrol may inhibit the growth of breast cancer cells, including the action of phytoestrogens on estrogen receptors and the epigenome. Although there is much evidence revealing that phytoestrogens can inhibit or stimulate these targets involved in inhibiting the growth of breast cancer cells, there is a large discrepancy between the high-μmol L doses required to observe such anti-tumorigenic effects, and the blood concentrations of unbound 'free' phytoestrogens that would be achieved by dietary intake. It is suggested that more experiments investigating low-dose mixtures of phytoestrogens and chronic exposure to phytoestrogens-a closer model of dietary intake-are required to substantiate claims that phytoestrogens can protect against breast cancer. [ABSTRACT FROM AUTHOR]

Estrogen synthesis in adipose tissue is associated with the development of breast cancer. Tumors are preferentially found in breast quadrants with strongest expression of the cytochrome P450 aromatase (encoded by the gene CYP19A1). Several promoters regulated by various hormonal factors drive aromatase expression in human breast adipose fibroblasts (BAFs). As adipose tissue is a major source of retinoids, in this study, we investigated their role in the regulation of aromatase expression. The retinoids all-trans-retinoic acid (at-RA) and 9-cis-RA induce aromatase activity in human BAFs. In BAFs, at-RA induces aromatase gene expression via promoter I.4. In 3T3-L1 cells, both retinoids specifically drive luciferase reporter gene expression under the control of aromatase promoter I.4, whereas other promoters active in human adipose tissue are insensitive. Activation by retinoids depends on a 467 bp fragment (K256/C211) of promoter I.4 containing four putative retinoic acid response elements (RAREs). Site-directed mutagenesis revealed that only RARE2 (C91/C105) mediates the retinoid-dependent induction of reporter gene activity. In 3T3-L1 preadipocytes and human BAFs, RA receptor a (RARa (RARA)) expression is predominant, whereas RARb (RARB) or RARg (RARG) expression is low. Electrophoretic mobility shift assays with nuclear extracts obtained from human BAFs and 3T3-L1 cells identified a specific RARE2-binding complex. Retinoids enhanced complex formation, whereas pre-incubation with anti-RARa antibodies prohibited the binding of RARa to RARE2. Chromatin immunoprecipitation showed RA-dependent binding of RARa to the RARE2-containing promoter region in vivo. Furthermore, we provide evidence that RARE2 is also necessary for the basal activation of promoter I.4 in these cells. Taken together, these findings indicate a novel retinoid-dependent mechanism of aromatase activity induction in adipose tissue. [ABSTRACT FROM AUTHOR]

Summary: It is increasingly recognized that estrogens produce rapid and transient effects at many neural sites ultimately impacting physiological and behavioral endpoints. The ability of estrogens to acutely regulate cellular processes implies that their concentration should also be rapidly fine-tuned. Accordingly, rapid changes in the catalytic activity of aromatase, the limiting enzyme for estrogen synthesis, have been identified that could serve as a regulatory mechanism of local estrogen concentrations. However, the precise anatomical localization, time-course, triggering stimuli and functional significance of these enzymatic changes in vivo are not well understood. To address these issues as to where, when and why aromatase activity (AA) rapidly changes after sexual interactions, AA was assayed in six populations of aromatase-expressing cells microdissected from the brain of male quail that experienced varying durations of visual exposure to or copulation with a female. Sexual interactions resulted in a rapid AA inhibition. This inhibition occurred in specific brain regions (including the medial preoptic nucleus), in a context-dependent fashion and time-scale suggestive of post-translational modifications of the enzyme. Interestingly, the enzymatic fluctuations occurring in the preoptic area followed rather than preceded copulation and were tied specifically to the female''s presence. This pattern of enzymatic changes suggests that rapid estrogen effects are important during the motivational phase of the behavior to trigger physiological events essential to activate mate search and copulation. [Copyright &y& Elsevier]

Estrogens are the primary female sex hormones and play important roles in both reproductive and non-reproductive systems. Estrogens can be synthesized in non-reproductive tissues such as liver, heart, muscle, bone and brain, and tissue-specific estrogen synthesis is consistent with a diversity of estrogen actions. In this article we review tissue and cell-specific estrogen synthesis and estrogen receptor signaling in three parts: (i) synthesis and metabolism, (ii) the distribution of estrogen receptors and signaling, and (iii) estrogen functions and related disorders, including cardiovascular diseases, osteoporosis, Alzheimer''s disease (AD), and Parkinson disease (PD). This comprehensive review provides new insights into estrogens by giving a better understanding of the tissue-specific estrogen effects and their roles in various diseases. [Copyright &y& Elsevier]

Inspired by the localization, on 15q21.2 of the CYP19A1 gene in the linkage region of speech and language disorders, and a rare translocation in a dyslexic individual that was brought to our attention, we conducted a series of studies on the properties of CYP19A1 as a candidate gene for dyslexia and related conditions. The aromatase enzyme is a member of the cytochrome P450 super family, and it serves several key functions: it catalyzes the conversion of androgens into estrogens; during early mammalian development it controls the differentiation of specific brain areas (e.g. local estrogen synthesis in the hippocampus regulates synaptic plasticity and axonal growth); it is involved in sexual differentiation of the brain; and in songbirds and teleost fishes, it regulates vocalization. Our results suggest that variations in CYP19A1 are associated with dyslexia as a categorical trait and with quantitative measures of language and speech, such as reading, vocabulary, phonological processing and oral motor skills. Variations near the vicinity of its brain promoter region altered transcription factor binding, suggesting a regulatory role in CYP19A1 expression. CYP19A1 expression in human brain correlated with the expression of dyslexia susceptibility genes such as DYX1C1 and ROBO1. Aromatase-deficient mice displayed increased cortical neuronal density and occasional cortical heterotopias, also observed in Robo1−/− mice and human dyslexic brains, respectively. An aromatase inhibitor reduced dendritic growth in cultured rat neurons. From this broad set of evidence, we propose CYP19A1 as a candidate gene for human cognitive functions implicated in reading, speech and language. [ABSTRACT FROM AUTHOR]

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity. [ABSTRACT FROM AUTHOR]

Abstract: Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16α-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E1S) uptake by these transporters stably expressed in FlpIn™-HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E1S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E1S by OAT4 was partly Na+-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na+-independent. Kinetic analysis of the initial uptake rates of E1S by OAT4 and OATP2B1 gave very similar values for K m (about 20μM) and V max (about 600pmol/(min×mg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K m >200μM) then for OAT4 (K m =29μM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation. [Copyright &y& Elsevier]

Abstract: Estrogens exert a wide variety of actions on reproductive and non-reproductive functions. These effects are mediated by slow and long lasting genomic as well as rapid and transient non-genomic mechanisms. Besides the host of studies demonstrating the role of genomic actions at the physiological and behavioral level, mounting evidence highlights the functional significance of non-genomic effects. However, the source of the rapid changes in estrogen availability that are necessary to sustain their fast actions is rarely questioned. For example, the rise of plasma estrogens at pro-estrus that represents one of the fastest documented changes in plasma estrogen concentration appears too slow to explain these actions. Alternatively, estrogen can be synthesized in the brain by the enzyme aromatase providing a source of locally high concentrations of the steroid. Furthermore, recent studies demonstrate that brain aromatase can be rapidly modulated by afferent inputs, including glutamatergic afferents. A role for rapid changes in estrogen production in the central nervous system is supported by experiments showing that acute aromatase inhibition affects nociception as well as male sexual behavior and that preoptic aromatase activity is rapidly (within min) modulated following mating. Such mechanisms thus fulfill the gap existing between the fast actions of estrogen and their mode of production and open new avenues for the understanding of estrogenic effects on the brain. [Copyright &y& Elsevier]

Abstract: Due to limitations of the currently used enzymatic assays, it is difficult to determine aromatase activity in testicular tissue of amphibians. Quantitative reverse transcription polymerase chain reaction (Q-RT PCR) is a sensitive and reliable technique to detect low amounts of mRNA for specific genes. This study was designed to develop and optimize a SYBR Green I-based Q-RT PCR method to quantify CYP19 mRNA in testicular tissue from male Xenopus laevis. Four quantification methods for measuring CYP19 mRNA expression were compared. The established test system proved to be highly sensitive (detectable mRNA copies <10), reproducible (interassay CV<5.4%, intraassay CV<0.9%), precise and specific for the CYP19 gene. To confirm the validity of the applied test system, an ex vivo testicular and ovarian explant study with a known inducer of aromatase, forskolin, was conducted. Forskolin induced CYP19 gene expression in both ovarian (3.7-fold) and testicular (2.6-fold) explants. Of the four quantification methods, the absolute standard curve and the comparative CT method appear to be optimal as indicated by their highly significant correlation (r2 =0.998, p<0.001). In conclusion, we recommend the comparative CT method over the standard curve method because it is more economical in terms of both cost and labor. Although both aromatase activity and CYP19 mRNA were clearly detectable in testes of X. laevis, both aromatase enzyme activity and CYP19 gene expression were very low. Also, no significant relationships were found between aromatase enzyme activity and gene expression. This is likely due the fact that the aromatase enzyme may have been dormant at the developmental stage the frogs were in during the experiment. [Copyright &y& Elsevier]

Abstract: Neurosteroids are still found in the brain after steroidogenic glands were removed, indicating that they are synthesized either de novo or from endogenous precursors by enzymes present in the CNS. In fact, steroidogenic acute regulatory protein, and aromatase, two molecules essential for estrogen synthesis, are expressed in the hippocampus. We recently showed, for the first time, that estrogens are synthesized de novo in hippocampal neurons and that these hippocampus-derived estrogens are essential for synaptic plasticity. Both estrogen receptor isoforms, estrogen receptor α and estrogen receptor β, are expressed in the hippocampus, and estradiol treatment of the cultures leads to an upregulation of estrogen receptor α. This finding confirmed the presence of functional estrogen receptors in hippocampal neurons and showed the responsiveness of the cultured hippocampal neurons to estradiol. By using letrozole, an inhibitor of aromatase, estradiol levels in hippocampal dispersion cultures as well as in hippocampal slice cultures were significantly suppressed which in turn led to a downregulation of estrogen receptor α. Letrozole treatment was followed by a significant decrease in the density of spines and spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a dose-dependent downregulation of spinophilin, a spine marker, and of synaptophysin, a presynaptic marker, and of growth-associated protein 43 after letrozole treatment. Our data provide strong evidence for estrogens being potent modulators of structural synaptic plasticity and point to a paracrine rather than endocrine mechanism of estrogen action in the hippocampus. [Copyright &y& Elsevier]

Abstract: Stroke-induced lesions of the insular cortex in the brain have been linked to autonomic dysfunction (sympathoexcitation) leading to arrhythmogenesis and sudden cardiac death. In experimental models, systemic estrogen administration in male rats has been shown to reduce stroke-induced cell death in the insular cortex as well as prevent sympathoexcitation. The central nucleus of the amygdala has been postulated to mediate sympathoexcitatory output from the insular cortex. We therefore set out to determine if endogenous estrogen levels within the central nucleus of the amygdala are altered following stroke and if microinjection of estrogen into the central nucleus of the amygdala modulates autonomic tone. Plasma estrogen concentrations were not altered by middle cerebral artery occlusion (22.86±0.14pg/ml vs. 21.24±0.33pg/ml; P>0.05). In contrast, estrogen concentrations in the central nucleus of the amygdala increased significantly following middle cerebral artery occlusion (from 20.83±0.54pg/ml to 76.67±1.59pg/ml; P<0.05). Local infusion of an aromatase inhibitor, letrozole, into the central nucleus of the amygdala at the time of middle cerebral artery occlusion prevented the increase in estrogen concentration suggesting that this increase was dependent on aromatization from testosterone. Furthermore, bilateral microinjection of estrogen (0.5 μM in 200nl) directly into the central nucleus of the amygdala significantly decreased arterial pressure and sympathetic tone and increased baroreflex sensitivity, and these effects were enhanced following co-injection with either an N-methyl-d-aspartate or non-N-methyl-d-aspartate receptor antagonist. Taken together, the results suggest that middle cerebral artery occlusion resulted in synthesis of estrogen within the central nucleus of the amygdala and that this enhanced estrogen level may act to attenuate overstimulation of central nucleus of the amygdala neurons to prevent middle cerebral artery occlusion-induced autonomic dysfunction. [Copyright &y& Elsevier]

Some investigators have suggested that the triazine herbicide atrazine can cause demasculinization of male amphibians via upregulation of the enzyme aromatase. Male adult African clawed frogs (Xenopus laevis) were exposed to three nominal concentrations of atrazine (1, 25, or 250μg atrazine/l) for 36 days, and testicular aromatase activity and CYP19 gene expression, as well as concentrations of the plasma sex steroids testosterone (T) and 17β-estradiol (E2), and gonad size (GSI) were measured. There were no effects on any of the parameters measured, with the exception of plasma T concentrations. Plasma T concentrations in X. laevis exposed to the greatest concentration of atrazine were significantly less (p = 0.034) than those in untreated frogs. Both CYP19 gene expression and aromatase activities were low regardless of treatment, and neither parameter correlated with the other. We conclude that aromatase enzyme activity and gene expression were at basal levels in X. laevis from all treatments, and that the tested concentrations of atrazine did not interfere with steroidogenesis through an aromatase-mediated mechanism of action. [ABSTRACT FROM AUTHOR]

Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 days post-fertilization). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function, and the transcriptome profiles will provide a valuable resource for future studies., Competing Interests: YL, MK, MM, LC, SS, SW, CK, SH, NA, ID, CJ, BD No competing interests declared, (© 2022, Liu et al.)

Pesticides are widely applied all over the world, and pesticide exposure can induce different biological effects posing a possible threat to human health. Due to their effects on the endocrine system, some pesticides are classified as endocrine disruptors. The aim of the study is to assess the interference of five pesticides on estrogen biosynthesis and estrogen signaling. Three neonicotinoid insecticides (Acetamiprid, Clothianidin, and Thiamethoxam), a carbamate insecticide (Methiocarb) and a herbicide (Oxadiazon) were tested. The effect of pesticides on estrogen biosynthesis was studied through an ELISA assay using a recombinant form of human aromatase, the enzyme that catalyzes the transformation of androgens to estrogens. Moreover, the effect of pesticides on estrogen signaling was assessed using a gene reporter assay on MELN cells, which measures estrogen receptor-mediated estrogenic activity. The results of the ELISA assay showed that the pesticides did not alter aromatase activity (no interference with estrogen biosynthesis), while the results of the gene reporter assay showed that only Methiocarb was able to alter estrogen signaling at high doses. The estrogenic activity of Methiocarb, expressed as 17β-estradiol equivalency factor (EEF), was equal to 8.0 × 10 -8 . In conclusion, this study suggested that Methiocarb should be considered a potential endocrine disruptor.