Is there potential for estradiol receptor signaling in lophotrochozoans?

Estrogen receptors (ERs) are thought to be the ancestor of all steroid receptors and are present in most lophotrochozoans studied to date, including molluscs, annelids, and rotifers. A number of studies have investigated the functional role of estrogen receptors in invertebrate species, although most are in molluscs, where the receptor is constitutively active. In vitro experiments provided evidence for ligand-activated estrogen receptors in annelids, raising important questions about the role of estrogen signalling in lophotrochozoan lineages. Here, we review the concordant and discordant evidence of estradiol receptor signalling in lophotrochozoans, with a focus on annelids and rotifers. We explore the de novo synthesis of estrogens, the evolution and expression of estrogen receptors, and physiological responses to activation of estrogen receptors in the lophotrochozoan phyla Annelida and Rotifera. Key data are missing to determine if de novo biosynthesis of estradiol in non-molluscan lophotrochozoans is likely. For example, an ortholog for the CYP11 gene is present, but confirmation of substrate conversion and measured tissue products is lacking. Orthologs CYP17 and CYP19 are lacking, yet intermediates or products (e.g. estradiol) in tissues have been measured. Estrogen receptors are present in multiple species, and for a limited number, in vitro data show agonist binding of estradiol and/or transcriptional activation. The expression patterns of the lophotrochozoan ERs suggest developmental, reproductive, and digestive roles but are highly species dependent. E2 exposures suggest that lophotrochozoan ERs may play a role in reproduction, but no strong dose-response relationship has been established. Therefore, we expect most lophotrochozoan species, outside of perhaps platyhelminths, to have an ER but their physiological role remains elusive. Mining genomes for orthologs gene families responsible for steroidogenesis, coupled with in vitro and in vivo studies of the steroid pathway are needed to better assess whether lophotrochozoans are capable of estradiol biosynthesis. One major challenge is that much of the data are divided across a diversity of species. We propose that the polychaetes Capitella teleta or Platyneris dumerilii, and rotifer Brachionus manjavacas may be strong species choices for studies of estrogen receptor signalling, because of available genomic data, established laboratory culture techniques, and gene knockout potential.

Mitochondrial function and E2 synthesis are impaired following alteration of CLOCK gene expression in porcine ovarian granulosa cells.

Circadian locomotor output cycles kaput (CLOCK) is a critical component of the mammalian circadian clock system and regulates ovarian physiology. However, the functions and mechanisms of CLOCK in porcine granulosa cells (GCs) are poorly understood. The present study focused on CLOCK's effects on estradiol synthesis. Similarity analysis showed that CLOCK is highly conserved between pigs and other species. The phylogenetic tree analysis indicated that porcine CLOCK was most closely related to that in Arabian camels. CLOCK significantly reduced E2 synthesis in GCs. CLOCK reduced the expression of steroidogenesis-related genes at the mRNA and protein levels, including CYP19A1, CYP11A1, and StAR. CYP17A1 levels were significantly downregulated. We demonstrated that CLOCK dramatically decreased ATP content, mitochondrial copy number, and mitochondrial membrane potential (MMP) and increased reactive oxygen species levels in GCs. We observed that mitochondria were severely damaged with fuzzy and fractured cristae and swollen matrix. These findings suggest that mitochondrial function and E2 synthesis are impaired following the alteration of CLOCK gene expression in porcine ovarian GCs.

Immunoendocrine abnormalities in the female reproductive system, and lung steroidogenesis during experimental pulmonary tuberculosis.

INTRODUCTION: Tuberculosis (TB) caused by Mycobacterium tuberculosis mainly affects the lungs, but can spread to other organs. TB chronically activates the immune and endocrine systems producing remarkable functional changes.So far, it is unknown whether pulmonary non-disseminated TB cause changes in the female reproductive system and lung endocrinology. OBJECTIVE: To investigate whether pulmonary TB produces immunoendocrine alterations of the female mice reproductive organs, and lung estradiol synthesis. METHODS: BALB/c mice were infected intratracheally with Mycobacterium tuberculosis (Mtb) strain H37Rv. Groups of six non-infected and infected animals were euthanized on different days. Bacillary loads were determined in the lungs, ovaries and uterus. Immunohistochemistry and morphometry studies were performed in histological sections. Serum estradiol wasassayed, and supernatantfrom cultured lung cells was analyzed by Thin Layer Chromatography (TLC). RESULTS: Mtb only grew in lung tissue. Histopathology revealed abnormal folliculogenesis and decreased corpora lutea. Altered ovarian expression of IL-6, IL-1ß was found. The infection increased serum estradiol. Estradiol synthesis by infected lung cells triplicate after 30 pi days.Aromatase immunostaining was found in the alveolar and bronchial epithelium, being stronger in the infected lungs, mainly in macrophages. CONCLUSION: Pulmonary TB affects the histophysiology of the female reproductive system in absence of its local infection, and disturbslung endocrinology.

miR-27a-3p targets NR5A2 to regulate CYP19A1 expression and 17-ß estradiol synthesis in ovine granulosa cells.

Although 17-ß estradiol (E2) synthesis is important in regulating female fertility, we know little regarding the molecular mechanism of miRNA-regulated ovine E2 synthesis. Here, our experiments with granulosa cells (GCs) from Hu sheep revealed miR-27a-3p involvement in E2 synthesis and its association with ovine litter size. First, we showed that miR-27a-3p of sheep and other mammals share a high nucleotide identity. Next, gain- and loss-of-function assays indicated that miR-27a-3p inhibits CYP19A1 expression and E2 synthesis in GCs. Moreover, we demonstrated that NR5A2 is a direct target of miR-27a-3p. Ovine miR-27a-3p suppresses E2 synthesis via the NR5A2 and CYP19A1 axes. We also identified four single nucleotide polymorphisms in the ovine miR-27a gene, and g.-13 G>A and g 0.24 T > G were significantly associated with the first and the second parity litter size, respectively (P < 0.05). In summary, our findings reveal that miR-27a-3p is a novel regulator of E2 synthesis and may predict litter size of Hu sheep, providing insight into mechanisms underlying granulosa cell function and female fertility.

Long non­coding RNA NEAT1 promotes mouse granulosa cell proliferation and estradiol synthesis by sponging miR­874­3p.

It has been reported that long non-coding RNA nuclear-enriched abundant transcript 1 (NEAT1) is involved in follicular growth and multiple ovarian diseases, but not the physiological function of NEAT1 in mouse granulosa cells (mGCs). Therefore, the aim of the present study was to investigate the biological roles and regulatory mechanisms of NEAT1 in mGCs. The biological effects of NEAT1 on mGCs proliferation, apoptosis, production of 17ß-Estradiol (E2) and progesterone (P4) were investigated using MTS, flow cytometry and enzyme-linked immunosorbent assays, respectively. The association between NEAT1 and microRNA (miR)-874-3p was verified using luciferase reporter assay and RNA immunoprecipitation analysis. The results demonstrated that the knockdown of NEAT1 in mGC cells significantly promoted mGCs cell proliferation, inhibited apoptosis and increased the production of E2 and P4 in mGCs. The interference-mediated effect of NEAT1 on mGCs could be partially reversed by the downregulation of miR-874-3p. Overall, these results indicated that NEAT1 served as a competing endogenous RNA by competitively binding with miR-874-3p, thereby modulating mGCs proliferation and the production of E2 and P4 in mGCs.

Phthalate toxicity mechanisms: An update.

Phthalates are one of the most widely used plasticizers in polymer products, and they are increasingly being exposed to people all over the world, generating health concerns. Phthalates are often used as excipients in controlled-release capsules and enteric coatings, and patients taking these drugs may be at risk. In both animals and human, phthalates are mainly responsible for testicular dysfunction, ovarian toxicity, reduction in steroidogenesis. In this regard, for a better understanding of the health concerns corresponding to phthalates and their metabolites, still more research is required. Significantly, multifarious forms of phthalates and their biomedical effects are need to be beneficial to investigate in the various tissues or organs. Based on these investigations, researchers can decipher their toxicity concerns and related mechanisms in the body after phthalate's exposure. This review summarizes the chemical interactions, mechanisms, and their biomedical applications of phthalates in animals and human.

The masculinization steroid milieu caused by fluorene-9-bisphenol disrupts sex-typical courtship behavior in female zebrafish (Danio rerio).

In vertebrates, the behavior of congenital sex differences between males and females is highly dependent on steroid signals and hormonal milieu. The presence of endocrine disrupting chemicals (EDCs) in the environment generally plays a similar role to sex hormones, so its interference with aquatic organism population stability can not be ignored and is worth studying. Fluorene-9-bisphenol (BHPF) has been clarified as an endocrine disruptor on organisms by several studies but its mechanism in perturbation of courtship behavior of female zebrafish is not clear. Here, we proposed an automated multi-zebrafish tracking method quantifying the courtship process and reported that zebrafish females exposed to BHPF, are not receptive to males but rather court females, and lose normal ovarian function with an altered sex steroid milieu. Our results showed that BHPF damaged 17ß-estradiol synthesis by down-regulation of sox3 and cyp19a1a, linking apoptosis with ovary development and female fecundity. The down-regulated expression of estrogen signaling through an estrogen receptor, esr2b, caused the induction of masculinization of female courtship behavior and sexual preference in zebrafish females after BHPF treatment. This process might be mediated by inhibiting the transcription of a neuropeptide B (npb) in the brain. Our study reveals that the estrogen signaling pathway may play an important role in classical courtship behavior and sexual preference of zebrafish. This study provided evidence that anti-estrogenic chemical exposure caused adverse effects on the regulation of the brain-gonad-estrogen axis of aquatic organisms, which should be of concern and highlighted the importance of controlling environmental contamination.

Totipotency of miR-184 in porcine granulosa cells.

Estradiol (E2) synthesis, cell proliferation and the apoptosis of porcine granulosa cells (GCs) affect follicular growth and development. The miR-184 level in ovary tissues of Yorkshire × Landrace sows was significantly higher in high-yielding sows than that in low-yielding sows, which was the same as in Yorkshire sows. However, the roles of miR-184 on E2 granulosa cells (GCs) are still unclear. We found that miR-184 promoted E2 synthesis and proliferation but inhibited apoptosis in GCs by targeting nuclear receptor subfamily 1 group D member 1 (NR1D1), cyclin dependent kinase inhibitor 1A (P21,CDKN1A) and homeodomain interacting protein kinase 2 (HIPK2) respectively. These findings indicated that miR-184 is a novel key factor that regulates the physiological functions of GCs.

MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells.

BACKGROUND: Granulosa cells (GCs) proliferation and estradiol synthesis significantly affect follicular development. The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows, indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis. RESULTS: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B (Cyclin B), cell cycle protein D (Cyclin D), cell cycle protein E (Cyclin E), and cyclin-dependent kinase 4 (CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and steroidogenic acute regulatory protein (StAR) (i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively. CONCLUSIONS: Our findings suggest that miR-214-3p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.

US EPA's regulatory pesticide evaluations need clearer guidelines for considering mammary gland tumors and other mammary gland effects.

Breast cancer risk from pesticides may be missed if effects on mammary gland are not assessed in toxicology studies required for registration. Using US EPA's registration documents, we identified pesticides that cause mammary tumors or alter development, and evaluated how those findings were considered in risk assessment. Of 28 pesticides that produced mammary tumors, EPA's risk assessment acknowledges those tumors for nine and dismisses the remaining cases. For five pesticides that alter mammary gland development, the implications for lactation and cancer risk are not assessed. Many of the mammary-active pesticides activate pathways related to endocrine disruption: altering steroid synthesis in H295R cells, activating nuclear receptors, or affecting xenobiotic metabolizing enzymes. Clearer guidelines based on breast cancer biology would strengthen assessment of mammary gland effects, including sensitive histology and hormone measures. Potential cancer risks from several common pesticides should be re-evaluated, including: malathion, triclopyr, atrazine, propylene oxide, and 3-iodo-2-propynyl butylcarbamate (IPBC).

Decreased levels of H3K9ac and H3K27ac in the promotor region of ovarian P450 aromatase mediated low estradiol synthesis in female offspring rats induced by prenatal nicotine exposure as well as in human granulosa cells after nicotine treatment.

Prenatal nicotine exposure (PNE) could induce ovarian dysplasia in offspring. This study aimed to confirm its intrauterine origin and explore a programming mechanism of ovarian dysplasia caused by PNE. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestation day (GD) 9 to GD20. Serum of female offspring was obtained for hormone assays and ovarian tissues were collected. The results showed that PNE impaired ovarian development, and inhibited estradiol production and cytochrome P450 aromatase (P450arom) expression before and after birth. Moreover, the nicotinic acetylcholine receptors (nAChRs) expression was increased in utero, while histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac levels in the P450arom promoter region were decreased persistently in PNE group before and after birth. In vitro, nicotine decreased P450arom expression and estradiol production in human granulosa cell line KGN. Furthermore, nicotine treatment up-regulated nAChRα6 and α9 expression and down-regulated the H3K9ac and H3K27ac levels of the P450arom promoter region. Non-specific nAChRs inhibitor vecuronium bromide reversed these effects. These results suggest that PNE could induce ovarian dysplasia and inhibit estradiol synthesis in the female offspring rats, which was related to the decreased H3K9ac and H3K27ac levels in the promotor region of the P450arom via the nAChRs.

Food components and environmental chemicals of inhibiting human placental aromatase.

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy.

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1.

MicroRNA-205 (miR-205) is involved in various physiological and pathological processes, but its biological function in follicular atresia remains unclear. In this study, we investigated miR-205 expression in mouse granulosa cells (mGCs) and analyzed its functions in primary mGCs by performing a series of in vitro experiments. Quantitative real-time polymerase chain reaction showed that miR-205 expression was significantly higher in early atretic follicles and progressively atretic follicles than in healthy follicles. miR-205 overexpression in mGCs significantly promoted apoptosis and caspase-3/9 activities, as well as inhibited estrogen (E2) release and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene encoding cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a direct target of miR-205 in mGCs. CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production, and CYP19A1 expression on mGCs. These results indicate that miR-205 might play an important role in ovarian follicular development and provide new insights into follicular atresia.

Knockdown of CREB1 promotes apoptosis and decreases estradiol synthesis in mouse granulosa cells.

Cyclic AMP response element-binding protein 1 (CREB1), a member of the CREB family, is known to be involved in follicular growth, ovulation, and ovarian disease. However, the physiological function of CREB1 in mouse granulosa cells (mGCs) remains lagerly unknown. The aim of this study was to determine the role of CREB1 in mGCs by knocking down CREB1 expression. CREB1 knock-down in mGCs at the mRNA and protein levels, was confirmed by quantitative real-time polymerase chain reaction and western blot. Results of enzyme linked immunosorbent assay revealed that CREB1 knockdown significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in mGCs. Furthermore, the CREB1 knockdown in mGCs promoted cell proliferation and apoptosis, and arrested the cell cycle in S-phase. To elucidate the regulatory mechanism underlying the effects of CREB1 knockdown on steroid synthesis, cell cycle, and apoptosis, we measured the protein expression levels of several related genes in mGCs knocked down CREB1. When CREB1 was knocked down, the expression of Cyp1b1 and Cyp19a1, which encode steroidogenic enzymes, was down-regulated; the expression of the cell cycle factors CyclinA1, CyclinB1, and CyclinD2 were significantly decreased. Among apoptosis-related genes, Bcl-2 was down-regulated, whereas Bax and cleaved Caspase3 were upregulated. Moreover, CREB1 knockdown significantly decreased expression level of Has2, Ptgs2, and Igfbp4, which are essential genes for folliculogenesis in mGCs. Taken together, these findings suggested that CREB1 might be a key regulator of mGCs through regulating steroid synthesis, cell proliferation, cell cycle, apoptosis, and other regulators of folliculogenesis.

Effects of orexins on 17ß-estradiol synthesis and P450 aromatase modulation in the testis of alpaca (Vicugna pacos).

The steroidogenic enzyme P450 aromatase (ARO) has a key role in the conversion of testosterone (T) into estrogens (E), expressed as 17ß-estradiol. The presence and localization of this key enzyme have not been described before in the South American camelid alpaca (Vicugna pacos). In our previous studies of the expression and biological effects of orexin A (OxA) and OxB on the alpaca testis demonstrated that OxA, via its specific receptor 1 (OX1R), stimulated T synthesis. In order to extend these findings, we presently explored the presence and localization of ARO in the alpaca male gonad, and the possible correlation between ARO and the orexinergic complex. Western blotting and immunohistochemistry demonstrated the presence of ARO in tissue homogenates and its localization in the tubular and interstitial compartments of the alpaca testis, respectively. The addition of OxA to fresh testicular slices decreased the 17ß-estradiol E levels. This effect was annulled by the sequential addition of the selective OX1R antagonist, SB-408124. OxB incubation did not have any effect on the biosynthesis of E. Furthermore, the OxA-mediated down-regulation of E secretion could be ascribed to ARO inhibition by exogenous OxA, as indicated by measurement of ARO activity in tissue slices incubated with OxA. Overall, our findings suggest that locally secreted OxA interacting with OX1R could indirectly inhibit ARO activity, disabling the conversion of T to E, and consequently lowering E biosynthesis and increasing the production of T in mammalian testis.

miR-1275 controls granulosa cell apoptosis and estradiol synthesis by impairing LRH-1/CYP19A1 axis.

miR-1275 is one of the microRNAs (miRNAs) that are differentially expressed during follicular atresia in pig ovaries, as identified by a miRNA microarray assay in our previous study [1]. However, its functions in follicular atresia remain unknown. In this study, we showed that miR-1275 promotes early apoptosis of porcine granulosa cells (pGCs) and the initiation of follicular atresia, and inhibits E2 release and expression of CYP19A1, the key gene in E2 production. Bioinformatics and luciferase reporter assays revealed that liver receptor homolog (LRH)-1, not CYP19A1, is a direct functional target of miR-1275. In vitro overexpression and knockdown experiments showed that LRH-1 significantly repressed apoptosis and induced E2 secretion and CYP19A1 expression in pGCs. LRH-1, whose expression was regulated by miR-1275, prevented apoptosis in pGCs. Furthermore, luciferase and chromatin immunoprecipitation assays demonstrated that LRH-1 protein bound to the CYP19A1 promoter and increased its activity. Our findings suggest that miR-1275 attenuates LRH-1 expression by directly binding to its 3'UTR. This prevents the interaction of LRH-1 protein with the CYP19A1 promoter, represses E2 synthesis, promotes pGC apoptosis, and initiates follicular atresia in porcine ovaries.

Distinct effects of estrogen receptor antagonism on object recognition and spatial memory consolidation in ovariectomized mice.

Exogenous treatment with the potent estrogen 17ß-estradiol (E2) or selective estrogen receptor α/ß (ERα/ß) agonists enhances the consolidation of hippocampal-dependent object recognition (OR) and object placement (OP) memories in ovariectomized rodents. Although such data suggest that individual ERs are sufficient for memory consolidation, the necessity of a given ER for memory consolidation can only be demonstrated by blocking receptor function, for example with an ER antagonist. However, the effects on memory of antagonizing ERα or ERß function are not well understood. Moreover, ER antagonism in ovariectomized subjects also provides indirect information about the role of individual ERs in the memory-enhancing effects of local hippocampal E2 synthesis. Therefore, this study used pharmacological inhibition of ERα and ERß to elucidate the importance of each ER to memory consolidation. Specifically, we examined effects on OR and OP memory consolidation of immediate post-training dorsal hippocampal (DH) infusion of MPP and PHTPP, selective antagonists for ERα and ERß, respectively. Each drug exhibited a distinct effect on OR and OP. DH infusion of MPP (0.28 or 2.78ng/hemisphere) impaired memory in OP, but not OR. However, DH infusion of PHTPP (0.21 or 2.12ng/hemisphere) impaired memory in both OR and OP. Neither drug affected the elapsed time to accumulate object exploration in either task, suggesting a specific effect on memory. These results indicate that activation of either classical ER within the dorsal hippocampus is important for hippocampal memory consolidation in ovariectomized mice, but suggest that specific ER involvement is memory- or task-specific. The findings also indirectly support a role for ERα and ERß in mediating the memory-enhancing effects of hippocampally-synthesized E2.