Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype.

Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort (n = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region (P = 0.009) and aromatase gene methylation mediates (P = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention.

Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes.

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 µM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

Hsa_circ_0043532 contributes to PCOS through upregulation of CYP19A1 by acting as a ceRNA for hsa-miR-1270.

BACKGROUND: Polycystic ovarian syndrome (PCOS) accounts for about 75% of anovulatory infertility. The cause of PCOS is not clear. CircRNAs acting as miRNA sponges mediate the post-transcriptional regulation of multiple genes. CYP19A1 is a limiting enzyme in the ovarian steroidogenesis pathway. However, the mechanism of circRNAs regulating granulosa cell (GC) estradiol secretion in PCOS remains to be elucidated. METHODS: Bioinformatics was used to predict the potential target miRNAs of circ_0043532 and target genes of miR-1270. Target miRNAs and mRNA expression were verified by qRT-PCR in GCs from 45 women with PCOS and 65 non-PCOS. Western blot, ELISA and dual-luciferase reporter assays were applied to confirm the substrate of miR-1270. RESULTS: Circ_0043532 and CYP19A1 were significant up-regulation in GCs from patients with PCOS. The predicted target miRNAs of circ_0053432, miR-1270, miR-576-5p, miR-421 and miR-142-5p, were notably decreased in GCs from patients with PCOS. Mechanistic experiments showed that circ_0043532 specifically binds to miR-1270. MiR-1270 was negatively regulated by circ_0043532. Concomitantly, miR-1270 inhibited CYP19A1 expression and estradiol production, which could be reversed by circ_0043532 over-expression. CONCLUSION: We identified that circ_0043532/miR-1270/CYP19A1 axis contributes to the aberrant steroidogenesis of GCs from patients with PCOS. This study broadens the spectrum of pathogenic factors of PCOS, and circ_0043532 might be a potential therapeutic target for PCOS.

Inositol Restores Appropriate Steroidogenesis in PCOS Ovaries Both In Vitro and In Vivo Experimental Mouse Models.

Androgen excess is a key feature of several clinical phenotypes of polycystic ovary syndrome (PCOS). However, the presence of FSH receptor (FSHR) and aromatase (CYP19A1) activity responses to physiological endocrine stimuli play a critical role in the pathogenesis of PCOS. Preliminary data suggest that myo-Inositol (myo-Ins) and D-Chiro-Inositol (D-Chiro-Ins) may reactivate CYP19A1 activity. We investigated the steroidogenic pathway of Theca (TCs) and Granulosa cells (GCs) in an experimental model of murine PCOS induced in CD1 mice exposed for 10 weeks to a continuous light regimen. The effect of treatment with different combinations of myo-Ins and D-Chiro-Ins on the expression of Fshr, androgenic, and estrogenic enzymes was analyzed by real-time PCR in isolated TCs and GCs and in ovaries isolated from healthy and PCOS mice. Myo-Ins and D-Chiro-Ins, at a ratio of 40:1 at pharmacological and physiological concentrations, positively modulate the steroidogenic activity of TCs and the expression of Cyp19a1 and Fshr in GCs. Moreover, in vivo, inositols (40:1 ratio) significantly increase Cyp19a1 and Fshr. These changes in gene expression are mirrored by modifications in hormone levels in the serum of treated animals. Myo-Ins and D-Chiro-Ins in the 40:1 formula efficiently rescued PCOS features by up-regulating aromatase and FSHR levels while down-regulating androgen excesses produced by TCs.

Smad4 and FoxH1 potentially interact to regulate cyp19a1a promoter in the ovary of ricefield eel (Monopterus albus).

BACKGROUND: Cyp19a1a is a key enzyme in the pathway that converts androgens into estrogen and is regulated by TGF-ß signaling. Smad4 and FoxH1 are downstream effectors of TGF-ß signaling and may play important roles in ovarian development in M. albus. METHODS: We investigated the expression pattern of the Smad4 and FoxH1 using qRT‒PCR and immunofluorescence, then tested the changes of smad4 and foxh1 by qRT‒PCR after ovary incubation with FSH in vitro, and analysed the regulation of cyp19a1a transcription by Smad4 and FoxH1 by dual-luciferase reporter assays. RESULTS: We found that Smad4 encoded a putative protein of 449 amino acids and harbored the three conserved domains typical of this protein family. Smad4 and foxh1 exhibited similar expression patterns during ovarian development and after FSH incubation, with Pearson's coefficients of 0.873 and 0.63-0.81, respectively. Furthermore, Smad4, FoxH1 and Cyp19a1a colocalized in the granulosa cells and theca cells of ovaries during the mid-to-late vitellogenic stage. Smad4 repressed cyp19a1a activity via SBE1 (- 1372/-1364) and SBE2 (- 415/-407) in the cyp19a1a promoter, whereas mutating SBE1 or SBE2 restored cyp19a1a promoter activity. Co-overexpression of Smad4 and FoxH1 significantly reduced cyp19a1a promoter activity. CONCLUSIONS: This study provides new insights into the potential functions of transcription factors Smad4 and FoxH1 in ovarian development and the transcriptional regulation mechanism of cyp19a1a in M. albus, which will reveal Smad4/FoxH1-mediated TGF-ß signaling in reproduction and the regulation of the cyp19a1a. Aromatase, encoded by cyp19a1a, is involved in ovarian development and plays an important role in the quality of eggs, as well the sex ratio, of the teleost fish, M. albus. The research on the transcriptional regulation of cyp19a1a has contributed to the understanding of its role in ovarian development. In previous study, it was shown that FoxH1 inhibits cyp19a1a transcription. In the present study, Smad4 was confirmed as a cyp19a1a transcriptional repressor and Smad4 may also coordinate with FoxH1 to repress cyp19a1a transcription. At present, we provide a new perspective for the transcriptional regulation of cyp19a1a by transcription factors Smad4 and FoxH1 in teleost fish ovary. In the future, the regulatory networks of Smad4 and FoxH1 will be further studied and the gene editing technology will be applied to screen specific regulatory factors of cyp191a1a gene, so as to alter the female cycle and modulate the sex ratio of the eggs production.

Expression levels of the selenium-uptake receptor LRP8, the antioxidant selenoprotein GPX1 and steroidogenic enzymes correlate in granulosa cells.

Abstract: Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1-8, CAT, SODs 1 and 2, PRDXs 1-6, GSR, TXN, TXNRDs 1-3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones. Lay summary: In the ovary, eggs are housed in follicles which contain the cells that produce oestrogen in the days leading up to ovulation of the egg. Oestrogen is produced by the action of enzymes. However, some of these enzymes also produce by-products called reactive oxygen species (ROS). These are harmful to eggs. Fortunately, cells have protective antioxidant enzymes that can neutralise ROS. This study was interested in which particular antioxidant enzyme(s) might be involved in neutralising the ROS in follicle cells. It was found that only one antioxidant enzyme, GPX1, appeared to be co-regulated with the enzymes that produce oestrogen and progesterone in the follicular cells. GPX1 contains the essential mineral selenium. In summary, this study has identified which antioxidant appears to be involved in neutralising ROS in the days leading to ovulation. It highlights the importance of selenium in the diet.

Long-term exposure of metamifop affects sex differentiation and reproductive system of zebrafish (Danio rerio).

The extensive use of herbicide metamifop (MET) in rice fields for weeds control will inevitably lead to its entering into water environments and threaten the aquatic organisms. Previous researches have demonstrated that sublethal exposure of MET significantly affected zebrafish development. Yet the long-term toxicological impacts of MET on aquatic life remains unknown. Herein, we investigated the potential effects of MET (5 and 50 µg/L) on zebrafish during an entire life cycle. Since the expression level of male sex differentiation-related gene dmrt1 and sex hormone synthesis-related gene cyp19a1b were significantly changed after 50 µg/L MET exposure for only 7 days, indicators related to sex differentiation and reproductive system were further investigated. Results showed that the transcript of dmrt1 was inhibited, estradiol content increased and testosterone content decreased in zebrafish of both sexes after MET exposure at 45, 60 and 120 dpf. Histopathological sections showed that the proportions of mature germ cells in the gonads of male and female zebrafish (120 dpf) were significantly decreased. Moreover, males had elevated vitellogenin content while females did not after MET exposure; MET induced feminization in zebrafish, with the proportion of females significantly increased by 19.6% while that of males significantly decreased by 13.2% at 120 dpf. These results suggested that MET interfered with the expression levels of gonad development related-genes, disrupted sex hormone balance, and affected sex differentiation and reproductive system of female and male zebrafish, implying it might have potential endocrine disrupting effects after long-term exposure.

Embryonic exposure to water accommodated fraction of crude oil inhibits reproductive capability in adult female marine medaka (Oryzias melastigma).

The water accommodated fraction (WAF) of spilled crude oil is a severe threat to the health of marine fish. This study was conducted to investigate the effects of short-term embryonic exposure to the WAF on the ovarian development and reproductive capability of F0 adult female marine medaka (Oryzias melastigma). Following embryonic exposure to the WAF with nominal total petroleum hydrocarbon concentrations of 0.5, 5, 50, and 500 µg/L for 7 days, the number of spawned eggs and gonadosomatic indices of F0 adult females were significantly reduced at 130 days postfertilization. In these F0 adult females, the proportion of mature oocytes was significantly lower, the level of 17ß-estradiol was lower, and the level of testosterone was greater than those in control group. The mRNA levels of the follicle-stimulating hormone ß subunit, luteinizing hormone ß subunit, cytochrome P450 aromatase 19b, estrogen receptor α and ß, and androgen receptor α and ß genes were upregulated, while the mRNA level of the salmon-type gonadotropin-releasing hormone was downregulated in F0 adult females exposed to the WAF during the embryonic stage. Additionally, the methylation level of vitellogenin (vtg) in F0 adult females was significantly elevated, this might have, in turn, downregulated the mRNA level of vtg. The mortality rate of the unexposed F1 embryos was significantly increased and the hatching success was significantly reduced. These results collectively indicated the necessity of incorporating and evaluating the effects of short-term early-life exposure to crude oil in the assessment of risks to the reproductive health of marine fish.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

CYP19A1 Expression Is Controlled by mRNA Stability of the Upstream Transcription Factor AP-2γ in Placental JEG3 Cells.

CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C, which encodes AP-2γ, a transcription factor that regulates CYP19A1, was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression.

Luteolin and its analog luteolin-7-methylether from Leonurus japonicus Houtt suppress aromatase-mediated estrogen biosynthesis to alleviate polycystic ovary syndrome by the inhibition of tumor progression locus 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt (L. japonicus, Chinese motherwort), known as Yi Mu Cao which means "good for women", has long been widely used in China and other Asian countries to alleviate gynecological disorders, often characterized by estrogen dysregulation. It has been used for the treatment of polycystic ovary syndrome (PCOS), a common endocrine disorder in women but the underlying mechanism remains unknown. AIM OF THE STUDY: The present study was designed to investigate the effect and mechanism of flavonoid luteolin and its analog luteolin-7-methylether contained in L. japonicus on aromatase, a rate-limiting enzyme that catalyzes the conversion of androgens to estrogens and a drug target to induce ovulation in PCOS patients. MATERIALS AND METHODS: Estrogen biosynthesis in human ovarian granulosa cells was examined using ELISA. Western blots were used to explore the signaling pathways in the regulation of aromatase expression. Transcriptomic analysis was conducted to elucidate the potential mechanisms of action of compounds. Finally, animal models were used to assess the therapeutic potential of these compounds in PCOS. RESULTS: Luteolin potently inhibited estrogen biosynthesis in human ovarian granulosa cells stimulated by follicle-stimulating hormone. This effect was achieved by decreasing cAMP response element-binding protein (CREB)-mediated expression of aromatase. Mechanistically, luteolin and luteolin-7-methylether targeted tumor progression locus 2 (TPL2) to suppress mitogen-activated protein kinase 3/6 (MKK3/6)-p38 MAPK-CREB pathway signaling. Transcriptional analysis showed that these compounds regulated the expression of different genes, with the MAPK signaling pathway being the most significantly affected. Furthermore, luteolin and luteolin-7-methylether effectively alleviated the symptoms of PCOS in mice. CONCLUSIONS: This study demonstrates a previously unrecognized role of TPL2 in estrogen biosynthesis and suggests that luteolin and luteolin-7-methylether have potential as novel therapeutic agents for the treatment of PCOS. The results provide a foundation for further development of these compounds as effective and safe therapies for women with PCOS.

A Cohort Study to Evaluate Genetic Predictors of Aromatase Inhibitor Musculoskeletal Symptoms: Results from ECOG-ACRIN E1Z11.

PURPOSE: Aromatase inhibitor (AI)-associated musculoskeletal symptoms (AIMSS) are common and frequently lead to AI discontinuation. SNPs in candidate genes have been associated with AIMSS and AI discontinuation. E1Z11 is a prospective cohort study designed to validate the association between 10 SNPs and AI discontinuation due to AIMSS. PATIENTS AND METHODS: Postmenopausal women with stage I to III hormone receptor-positive breast cancer received anastrozole 1 mg daily and completed patient-reported outcome measures to assess AIMSS (Stanford Health Assessment Questionnaire) at baseline, 3, 6, 9, and 12 months. We estimated that 40% of participants would develop AIMSS and 25% would discontinue AI treatment within 12 months. Enrollment of 1,000 women with a fixed number per racial stratum provided 80% power to detect an effect size of 1.5 to 4. SNPs were found in ESR1 (rs2234693, rs2347868, and rs9340835), CYP19A1 (rs1062033 and rs4646), TCL1A (rs11849538, rs2369049, rs7158782, and rs7159713), and HTR2A (rs2296972). RESULTS: Of the 970 evaluable women, 43% developed AIMSS and 12% discontinued AI therapy within 12 months. Although more Black and Asian women developed AIMSS than White women (49% vs. 39%, P = 0.017; 50% vs. 39%, P = 0.004, respectively), the AI discontinuation rates were similar across groups. None of the SNPs were significantly associated with AIMSS or AI discontinuation in the overall population or in distinct cohorts. The OR for rs2296972 (HTR2A) approached significance for developing AIMSS. CONCLUSIONS: We were unable to prospectively validate candidate SNPs previously associated with AI discontinuation due to AIMSS. Future analyses will explore additional genetic markers, patient-reported outcome predictors of AIMSS, and differences by race.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

A Novel Investigation for Early Sex Determination in Alive Adult European Seabass (Dicentrarchus labrax) Using cyp19a1a, dmrt1a, and dmrt1b Genes Expression in Tail Fin tissues.

This study is the first investigation for using sex-related gene expression in tail fin tissues of seabass as early sex determination without killing the fish. The European seabass (Dicentrarchus labrax) is gonochoristic and lacks distinguishable sex chromosomes, so, sex determination is referred to molecular actions for some sex-related genes on autosomal chromosomes which are well known such as cyp19a1a, dmrt1a, and dmrt1b genes which play crucial role in gonads development and sex differentiation. cyp19a1a is expressed highly in females for ovarian development and dmrt1a and dmrt1b are for testis development in males. In this study, we evaluated the difference in the gene expression levels of studied genes by qPCR in tail fins and gonads. We then performed discriminant analysis (DA) using morphometric traits and studied gene expression parameters as predictor tools for fish sex. The results revealed that cyp19a1a gene expression was significantly higher in future females' gonads and tail fins (p ≥ 0.05). Statistically, cyp19a1a gene expression was the best parameter to discriminate sex even the hit rate of any other variable by itself could not correctly classify 100% of the fish sex except when it was used in combination with cyp19a1a. In contrast, Dmrt1a gene expression was higher in males than females but there were difficulties in analyzing dmrt1a and dmrt1b expressions in the tail because levels were low. So, it could be used in future research to differentiate and determine the sex of adult fish using the cyp19a1a gene expression marker without killing or sacrificing fish.

Aromatase deficiency in transplanted bone marrow cells improves vertebral trabecular bone quantity, connectivity, and mineralization and decreases cortical porosity in murine bone marrow transplant recipients.

Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17ß-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

Aromatase (CYP19) gene as a biomarker for detection of naphthalene and phenanthrene in Colombo to Mirissa coastal water in Sri Lanka.

Naphthalene (NAP) and phenanthrene (PHE) are prevalent Polycyclic Aromatic Hydrocarbons (PAHs) in the environment. High-Performance Liquid Chromatography (HPLC) analysis was performed on marine water samples (n = 57) collected from 19 locations. Molecular screening of the aromatase (CYP19) gene expression was examined using quantitative Reverse Transcriptase PCR (qRT-PCR). The findings of the study showed a significant range of naphthalene concentrations along the coastline, spanning from 1.70 to 15.05 mg/L, where phenanthrene concentrations varied from undetectable to a maximum of 5.36 mg/L. The relative expression of the CYP19 gene ranged from 0.5 to 13.9 in the sampling sites. The ANOVA analysis showed a significant positive correlation (p < 0.05) between the concentrations of PAHs and CYP19 gene expression. The study concluded that the CYP19 gene could be useful in detecting contaminants such as naphthalene and phenanthrene in water. This study may help develop effective strategies to detect and mitigate PAH pollution in coastal areas.

PPARγ antagonists induce aromatase transcription in adipose tissue cultures.

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens and a key risk factor for hormone receptor-positive breast cancer. In postmenopausal women, estrogens synthesized in adipose tissue promotes the growth of estrogen receptor positive breast cancers. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) in adipose stromal cells (ASCs) leads to decreased expression of aromatase and differentiation of ASCs into adipocytes. Environmental chemicals can act as antagonists of PPARγ and disrupt its function. This study aimed to test the hypothesis that PPARγ antagonists can promote breast cancer by stimulating aromatase expression in human adipose tissue. Primary cells and explants from human adipose tissue as well as A41hWAT, C3H10T1/2, and H295R cell lines were used to investigate PPARγ antagonist-stimulated effects on adipogenesis, aromatase expression, and estrogen biosynthesis. Selected antagonists inhibited adipocyte differentiation, preventing the adipogenesis-associated downregulation of aromatase. NMR spectroscopy confirmed direct interaction between the potent antagonist DEHPA and PPARγ, inhibiting agonist binding. Short-term exposure of ASCs to PPARγ antagonists upregulated aromatase only in differentiated cells, and a similar effect could be observed in human breast adipose tissue explants. Overexpression of PPARG with or without agonist treatment reduced aromatase expression in ASCs. The data suggest that environmental PPARγ antagonists regulate aromatase expression in adipose tissue through two mechanisms. The first is indirect and involves inhibition of adipogenesis, while the second occurs more acutely.

Transforming Growth Factor α Evokes Aromatase Expression in Gastric Parietal Cells during Rat Postnatal Development.

Estrogen, well known as a female hormone, is synthesized primarily by ovarian aromatase. However, extra-glandular tissues also express aromatase and produce estrogen. It is noteworthy that aromatase in gastric parietal cells begins expression around 20 days after birth and continues secreting considerable amounts of estrogen into the portal vein throughout life, supplying it to the liver. Estrogen, which is secreted from the stomach, is speculated to play a monitoring role in blood triglyceride, and its importance is expected to increase. Nevertheless, the regulatory mechanisms of the aromatase expression remain unclear. This study investigated the influence of transforming growth factor α (TGFα) on gastric aromatase expression during postnatal development. The administration of TGFα (50 µg/kg BW) to male Wistar rats in the weaning period resulted in enhanced aromatase expression and increased phosphorylated ERK1+2 in the gastric mucosa. By contrast, administration of AG1478 (5 mg/kg BW), a protein tyrosine kinase inhibitor with high selectivity for the epidermal growth factor receptor and acting as an antagonist of TGFα, led to the suppression of aromatase expression. In fact, TGFα expression in the gastric fundic gland isthmus began around 20 days after birth in normal rats as did that of aromatase, which indicates that TGFα might induce the expression of aromatase in the parietal cells concomitantly.