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Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.
Assuntos
Receptor beta de Estrogênio , Proteínas Hedgehog , Animais , Feminino , Ratos , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , RNA Mensageiro/metabolismoRESUMO
Loss of ERß increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERß in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erß knockout (Erßnull) mouse ovaries, and their transcriptomes were compared with those of control Erßfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erßnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERß has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erßnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erßfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erß-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erßnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erßnull PrFs were downregulated. Such deregulation of genes in Erßnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erßnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERß either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.
Assuntos
Receptor beta de Estrogênio , Folículo Ovariano , Feminino , Camundongos , Animais , Receptor beta de Estrogênio/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Regulação da Expressão Gênica , Transcriptoma , Camundongos KnockoutRESUMO
Adenosine deaminases acting on RNA-(ADAR) comprise one family of RNA editing enzymes that specifically catalyze adenosine to inosine (A-to-I) editing. A granulosa cell (GC) specific Adar depleted mouse model [Adar flox/flox:Cyp19a1-Cre/+ (gcAdarKO)] was used to evaluate the role of ADAR1 during the periovulatory period. Loss of Adar in GCs led to failure to ovulate at 16 h post-hCG, delayed oocyte germinal vesicle breakdown and severe infertility. RNAseq analysis of GC collected from gcAdarKO and littermate control mice at 0 and 4 h post-hCG following a super-ovulatory dose of eCG (48 h), revealed minimal differences after eCG treatment alone (0 h), consistent with normal folliculogenesis observed histologically and uterine estrogenic responses. In contrast, 300 differential expressed genes (DEGs; >1.5-fold change and FDRP < 0.1) were altered at 4 h post-hCG. Ingenuity pathway analysis identified many downstream targets of estrogen and progesterone pathways, while multiple genes involved in inflammatory responses were upregulated in the gcAdarKO GCs. Temporal expression analysis of GCs at 0, 4, 8, and 12 h post-hCG of Ifi44, Ifit1, Ifit3b, and Oas1g and Ovgp1 confirmed upregulation of these inflammatory and interferon genes and downregulation of Ovgp1 a glycoprotein involved in oocyte zona pellucida stability. Thus, loss of ADAR1 in GCs leads to increased expression of inflammatory and interferon response genes which are temporally linked to ovulation failure, alterations in oocyte developmental progression and infertility.
Assuntos
Infertilidade , Ovulação , Feminino , Animais , Camundongos , Ovulação/genética , Células da Granulosa , Interferons , Infertilidade/genética , Oócitos , AdenosinaRESUMO
Kisspeptins (KPs) secreted from the hypothalamic KP neurons act on KP receptors (KPRs) in gonadotropin (GPN) releasing hormone (GnRH) neurons to produce GnRH. GnRH acts on pituitary gonadotrophs to induce secretion of GPNs, namely follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are essential for ovarian follicle development, oocyte maturation and ovulation. Thus, hypothalamic KPs regulate oocyte maturation indirectly through GPNs. KPs and KPRs are also expressed in the ovarian follicles across species. Recent studies demonstrated that intraovarian KPs also act directly on the KPRs expressed in oocytes to promote oocyte maturation and ovulation. In this review article, we have summarized published reports on the role of hypothalamic and ovarian KP-signaling in oocyte maturation. Gonadal steroid hormones regulate KP secretion from hypothalamic KP neurons, which in turn induces GPN secretion from the hypothalamic-pituitary (HP) axis. On the other hand, GPNs secreted from the HP axis act on the granulosa cells (GCs) and upregulate the expression of ovarian KPs. While KPs are expressed predominantly in the GCs, the KPRs are in the oocytes. Expression of KPs in the ovaries increases with the progression of the estrous cycle and peaks during the preovulatory GPN surge. Intrafollicular KP levels in the ovaries rise with the advancement of developmental stages. Moreover, loss of KPRs in oocytes in mice leads to failure of oocyte maturation and ovulation similar to that of premature ovarian insufficiency (POI). These findings suggest that GC-derived KPs may act on the KPRs in oocytes during their preovulatory maturation. In addition to the intraovarian role of KP-signaling in oocyte maturation, in vivo, a direct role of KP has been identified during in vitro maturation of sheep, porcine, and rat oocytes. KP-stimulation of rat oocytes, in vitro, resulted in Ca2+ release and activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1 and 2. In vitro treatment of rat or porcine oocytes with KPs upregulated messenger RNA levels of the factors that favor oocyte maturation. In clinical trials, human KP-54 has also been administered successfully to patients undergoing assisted reproductive technologies (ARTs) for increasing oocyte maturation. Exogenous KPs can induce GPN secretion from hypothalamus; however, the possibility of direct KP action on the oocytes cannot be excluded. Understanding the direct in vivo and in vitro roles of KP-signaling in oocyte maturation will help in developing novel KP-based ARTs.
Assuntos
Kisspeptinas , Oogênese , Animais , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Camundongos , Oócitos/fisiologia , Ratos , Ovinos , SuínosRESUMO
DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.
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Gonadotropins are essential for regulating ovarian development, steroidogenesis, and gametogenesis. While follicle stimulating hormone (FSH) promotes the development of ovarian follicles, luteinizing hormone (LH) regulates preovulatory maturation of oocytes, ovulation, and formation of corpus luteum. Cognate receptors of FSH and LH are G-protein coupled receptors that predominantly signal through cAMP-dependent and cAMP-independent mechanisms that activate protein kinases. Subsequent vital steps in response to gonadotropins are mediated through activation or inhibition of transcription factors required for follicular gene expression. Estrogen receptors, classical ligand-activated transcriptional regulators, play crucial roles in regulating gonadotropin secretion from the hypothalamic-pituitary axis as well as gonadotropin function in the target organs. In this review, we discuss the role of estrogen receptor ß (ERß) regulating gonadotropin response during folliculogenesis. Ovarian follicles in Erß knockout (ErßKO) mutant female mice and rats cannot develop beyond the antral state, lack oocyte maturation, and fail to ovulate. Theca cells (TCs) in ovarian follicles express LH receptor, whereas granulosa cells (GCs) express both FSH receptor (FSHR) and LH receptor (LHCGR). As oocytes do not express the gonadotropin receptors, the somatic cells play a crucial role during gonadotropin induced oocyte maturation. Somatic cells also express high levels of estrogen receptors; while TCs express ERα and are involved in steroidogenesis, GCs express ERß and are involved in both steroidogenesis and folliculogenesis. GCs are the primary site of ERß-regulated gene expression. We observed that a subset of gonadotropin-induced genes in GCs, which are essential for ovarian follicle development, oocyte maturation and ovulation, are dependent on ERß. Thus, ERß plays a vital role in regulating the gonadotropin responses in ovary.
Assuntos
Gonadotropina Coriônica/metabolismo , Receptor beta de Estrogênio/metabolismo , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/metabolismo , Células Tecais/metabolismo , Animais , Gonadotropina Coriônica/genética , Receptor beta de Estrogênio/genética , Feminino , Hormônio Foliculoestimulante/genética , Humanos , Camundongos , Camundongos Knockout , RatosRESUMO
Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erßnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERß is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erßnull mutant model that fails to show follicle maturation was also included in order to identify the ERß-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erßnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erßnull GCs. These latter ERß-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERß-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erßnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERß.
Assuntos
Gonadotropina Coriônica/administração & dosagem , Receptor beta de Estrogênio/genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/química , Células da Granulosa/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação com Perda de Função , Folículo Ovariano/química , Folículo Ovariano/efeitos dos fármacos , Ratos , Análise de Sequência de RNARESUMO
Kisspeptin (KISS1) signaling in the hypothalamic-pituitary (H-P) axis plays an essential role in regulating gonadotropin secretion. KISS1 and KISS1 receptor (KISS1R) are also expressed in the ovary; however, the role of intraovarian KISS1 signaling remains unclear. Granulosa cell (GC)-specific expression of KISS1, and oocyte-specific expression of KISS1R indicate that GC-derived KISS1 may act on oocytes. Expression of KISS1 in GCs is induced by gonadotropins but it is absent in estrogen receptor ß knockout (Erßnull) rat ovaries. We also observed that gonadotropin stimulation failed to induce maturation of Erßnull oocytes. Interestingly, KISS1 treatment of cumulus oocyte complexes (COCs) isolated from antral follicles promotes in vitro maturation of oocytes. Treatment of oocytes with KISS1 induced intracellular Ca2+ release, and increased activation of MAP kinase ERK1/2. KISS1 treatment also induced the expression of oocyte genes that are crucial for differentiation of GCs, and maturation of oocytes. Our findings suggest that ovarian KISS1-signaling plays an important role in gonadotropin induced follicle development and oocyte maturation.
Assuntos
Receptor beta de Estrogênio/metabolismo , Células da Granulosa/metabolismo , Kisspeptinas/metabolismo , Sistema de Sinalização das MAP Quinases , Oócitos/metabolismo , Animais , Receptor beta de Estrogênio/genética , Feminino , Kisspeptinas/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos TransgênicosRESUMO
Disruption of estrogen receptor beta (ESR2) dysregulates granulosa cell genes essential for follicle maturation and ovulation. The datasets presented in this article depict gonadotropin-induced genes, which are differentially expressed in Esr2-null rat granulosa cells. Synchronized follicle development was initiated in four-week-old wildtype and Esr2-null female rats by administration of PMSG. Forty-eight hours after PMSG injection, further maturation of ovarian follicles was induced by hCG treatment. Granulosa cells were collected from the ovaries before gonadotropin administration, 48â¯h after PMSG treatment, and 4â¯h after hCG injection to the PMSG-treated rats. Total RNA was purified from granulosa cells and whole transcriptome was assessed by RNA-sequencing on an Illumina HiSeq X platform. RNA-seq data of wildtype and Esr2-null granulosa cells were analyzed and differentially expressed genes were identified by CLC Genomics Workbench. Gonadotropin-induced genes were identified by comparing the transcriptome data of PMSG- or hCG-induced wildtype granulosa cells with those without gonadotropin treatment. Furthermore, differentially expressed genes in Esr2-null granulosa cells were determined by comparing the transcriptome data with that of wildtype granulosa cells. These datasets can be used to recognize the gonadotropin-induced genes in granulosa cells that are Esr2-regulated and important for ovarian follicle maturation.
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Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor ß (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.
Assuntos
Hormônio Antimülleriano/sangue , Estradiol/sangue , Receptor beta de Estrogênio/genética , Folículo Ovariano/metabolismo , Reserva Ovariana/fisiologia , Animais , Moduladores de Receptor Estrogênico/farmacologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina , Nitrilas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Disruption of estrogen receptor beta (ESR2) dysregulates oocyte maturation, which leads to failure of ovulation. We investigated ESR2-regulated genes during gonadotropin-induced oocyte maturation using RNA-sequencing. Through the administration of pregnant mare's serum gonadotropin (PMSG), synchronized follicle development was initiated in four-week-old wildtype and Esr2-null female rats. Forty-eight hours after the PMSG injection, human chorionic gonadotropin (hCG) was used for further maturation. Oocytes were collected from the ovaries 4 h after hCG injection. The total RNA was isolated from the oocytes and the whole oocyte transcriptome was determined by RNA-sequencing on the Illumina HiSeq4000 sequencer. RNA-sequencing data of wildtype and Esr2-null oocytes were analyzed, and differentially expressed genes were identified using the CLC Genomics Workbench. Whole oocyte transcriptome data of wildtype and Esr2-null oocytes were compared to identify the differentially expressed genes. Raw data are deposited to the NCBI Sequence Read Archive (SRA) and analyzed data are presented in this data article. These datasets can be utilized to identify the gonadotropin-induced genes in oocytes that are ESR2-regulated and important to oocyte maturation.
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Notch pathway activation plays a central role in the pathogenesis of many glomerular diseases. We have previously shown that Notch4 expression was upregulated in various renal cells in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) patients and rodent models of HIVAN. In this study, we examined whether the Notch pathway can be distinctly activated by HIV-1 gene products and whether Notch4, in particular, can influence disease progression. Using luciferase reporter assays, we did not observe activation of the NOTCH4 promoter with the HIV protein Nef in podocytes. Further, we observed upregulated expression of a gamma secretase complex protein, presenilin 1, but not Notch4, in podocytes infected with an HIV-1 expression construct. To assess the effects of Notch4 on HIVAN disease progression, we engineered Tg26 mice with global deletion of the Notch4 intracellular domain (Notch4dl ), which is required for signaling function. These mice (Notch4d1/Tg26+ ) showed a significant improvement in renal function and a significant decrease in mortality compared to Tg26 mice. Histological examination of kidneys showed that Notch4d1/Tg26+ mice had overall glomerular, tubulointerstitial injury and a marked decrease in interstitial inflammation. A significant decrease in the proliferating cells was observed in the tubulointerstitial compartments of Notch4d1/Tg26+ mice. Consistent with the diminished inflammation, kidneys from Notch4d1/Tg26+ mice also showed a significant decrease in expression of the inflammatory cytokine transcripts Il-6 and Ccl2, as well as the master inflammatory transcription factor NF-κB (Nfkb1 transcripts and p65 protein). These data identify Notch4 as an important mediator of tubulointerstitial injury and inflammation in HIVAN and a potential therapeutic target.
Assuntos
Nefropatia Associada a AIDS/metabolismo , Inflamação/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Receptor Notch4/metabolismo , Animais , Proliferação de Células , Cruzamentos Genéticos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Deleção de Genes , Células HEK293 , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Podócitos/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Resultado do Tratamento , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The objective of this study was to test the hypothesis that Leptin induced in vitro growth in preantral follicles in sheep involves modulation of P450 aromatase expression and steroidogenesis. Accordingly, the expression of P450 aromatase gene was studied in the cumulus cells and oocytes isolated from different stages of preantral follicles (PFs') grown in vivo, cultured in TCM 199B, TCM 199B + Leptin (10 ng/ml) (TCM199BL) or a standard PF culture medium supplemented with Leptin (10 ng/ml) (SML). Ovarian follicles grown in vivo or in SML expressed P450 aromatase both in cumulus cells and oocytes at all the development stages. In the oocytes from PFs' grown in vitro, P450 expression was consistently lower than in those from in vivo grown follicles at all except the preantral stage. The patterns of expression of aromatase gene in the cumulus cells from in vivo grown and the PFs' cultured in TCM 199BL were similar. Significantly higher levels of progesterone production were supported by SML at all the development stages than the other two media. Oestradiol concentration in the spent TCM 199B and SML showed a significant increase as the development progressed from preantral to large antral stage. However, such increase was not sustained beyond early antral stage in the PFs' cultured in TCM199BL. It is concluded that Leptin modulates the expression P450 aromatase while supporting the in vitro development of the ovarian follicles in sheep.
Assuntos
Aromatase/metabolismo , Estrogênios/biossíntese , Leptina/farmacologia , Folículo Ovariano/efeitos dos fármacos , Progesterona/biossíntese , Ovinos , Animais , Aromatase/genética , Meios de Cultura , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Cultura de Tecidos/veterináriaRESUMO
The liver helps maintain energy homeostasis by synthesizing and storing glucose and lipids. Gonadal steroids, particularly estrogens, play an important role in regulating metabolism. As estrogens are considered female hormones, metabolic disorders related to the disruption of estrogen signaling have mostly been studied in females. Estrogen receptor alpha (ESR1) is the predominant receptor in both the male and female liver, and it mediates the hepatic response to estrogens. Loss of ESR1 increases weight gain and obesity in female rats, while reducing the normal growth in males. Although Esr1-/- male rats have a reduced body weight, they exhibit increased adipose deposition and impaired glucose tolerance. We further investigated whether these metabolic disorders in Esr1-/- male rats were linked with the loss of transcriptional regulation by ESR1 in the liver. To identify the ESR-regulated genes, RNA-sequencing was performed on liver mRNAs from wildtype and Esr1-/- male rats. Based on an absolute fold change of ≥2 with a p-value ≤ 0.05, a total of 706 differentially expressed genes were identified in the Esr1-/- male liver: 478 downregulated, and 228 upregulated. Pathway analyses demonstrate that the differentially expressed genes include transcriptional regulators (Cry1, Nr1d1, Nr0b2), transporters (Slc1a2), and regulators of biosynthesis (Cyp7b1, Cyp8b1), and hormone metabolism (Hsd17b2, Sult1e1). Many of these genes are also integral parts of the lipid and carbohydrate metabolism pathways in the liver. Interestingly, certain critical regulators of the metabolic pathways displayed a sexual dimorphism in expression, which may explain the divergent weight gain in Esr1-/- male and female rats despite common metabolic dysfunctions.
Assuntos
Metabolismo dos Carboidratos/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Adiposidade , Animais , Feminino , Ontologia Genética , Glucose/metabolismo , Insulina/metabolismo , Lipídeos/sangue , Masculino , Modelos Biológicos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Aumento de PesoRESUMO
Estrogen signaling plays an important role in the pathophysiology of prostatic hyperplasia. While signaling through estrogen receptor alpha (ESR1) increases proliferation of stromal cells, estrogen receptor beta (ESR2) plays an anti-proliferative and differentiating role in glandular epithelium. Disruption of ESR2 signaling resulted in prostatic glandular hyperplasia in the rat. To identify the ESR2-target genes, and the molecular mechanisms involved, we performed RNA-seq analyses in prostate glands of Esr2 knockout (Esr2-/-) and age matched wildtype rats. The raw data were analyzed using CLC genomics workbench. High quality RNA-seq reads were aligned to the Rattus norvegicus genome. Differentially expressed genes were identified based on an absolute fold change of 2 with pValue ≤0.05. Of the total 32,623 genes detected, 824 were differentially expressed in Esr2-/- prostate glands, 550 downregulated and 274 upregulated. Pathway analyses identified altered expression of genes involved in epithelial proliferation and benign tumor formation.
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Estrogens are traditionally considered to be female sex steroid hormones and most of the studies examining estrogen regulation of metabolic function in the liver have been conducted in females. However, the liver expresses high levels of estrogen receptor alpha (ESR1) in both males and females, which mediates the hepatic response to estrogens. In this data article, we investigated whether metabolic disorders in Esr1 knockout (Esr1-/-) male rats were linked with loss of transcriptional regulation by ESR1 in liver. To identify the ESR1 regulated genes in the mutant liver, RNA-sequencing was performed on liver RNAs purified from young male rats. The raw data were analyzed using the CLC Genomics Workbench and high-quality RNA-sequencing reads were aligned to the Rattus norvegicus genome. Transcriptome data obtained from Esr1-/- liver RNAs were compared to that of wild type rats. Based on an absolute fold change of 2 with a p-valueâ¯≤â¯0.05, a total of 618 differentially expressed genes were identified in the Esr1-/- male liver. Pathway analyses demonstrated that the majority of differentially expressed genes are regulators of carbohydrate and lipid metabolism in the liver. These differentially expressed genes and their potential roles were further examined in a companion manuscript, "Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats" (Khristi et al., 2018).
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Hypothalamic expression of Kiss1 plays an essential role in the onset of puberty, gonadal development, and ovulation. Estrogens regulate the expression of Kiss1 in the hypothalamus through estrogen receptor-α. Kiss1 is also expressed in the ovary, where its expression correlates with the onset of puberty and progression of the estrous cycle. To date, estrogen regulation of Kiss1 expression in the ovary has not been investigated. We recently observed that gonadotropin-induced Kiss1 expression was absent in Esr2-null rat ovaries even though Esr1 was present. Wild-type granulosa cells abundantly expressed Kiss1 and oocytes expressed the Kiss1 receptor. We characterized estrogen receptor-ß (ESR2) regulation of Kiss1 expression in granulosa cells by identifying granulosa cell-specific transcript variants and potential regulatory regions. The Kiss1 promoter, an upstream enhancer, and a downstream enhancer all possessed conserved estrogen response elements (EREs) and showed active histone marks in gonadotropin-stimulated granulosa cells. The transcriptionally active Kiss1 promoter, as well as the enhancers, also revealed enrichment for ESR2 binding. Furthermore, activity of a Kiss1 promoter construct was induced after overexpression of ESR2 and was blocked upon mutation of an ERE within the promoter. Finally, pregnant mare serum gonadotropin and human chorionic gonadotropin administration induced phosphorylation of ESR2 and upregulated the AP-1 proteins FOSL2 and JUNB in granulosa cells. Activated MAPK ERK2 was associated with the ESR2 phosphorylation in granulosa cells, and AP-1 factors could synergistically activate the Kiss1 promoter activity. These gonadotropin-induced changes paralleled Kiss1 expression in granulosa cells. We conclude that gonadotropin-stimulated Kiss1 expression in granulosa cells is dependent on both the activation of ESR2 and the upregulation of AP-1.
Assuntos
Receptor beta de Estrogênio/genética , Células da Granulosa/metabolismo , Kisspeptinas/genética , Fator de Transcrição AP-1/genética , Animais , Gonadotropina Coriônica/farmacologia , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Feminino , Antígeno 2 Relacionado a Fos/efeitos dos fármacos , Antígeno 2 Relacionado a Fos/metabolismo , Técnicas de Inativação de Genes , Gonadotropinas/farmacologia , Gonadotropinas Equinas/farmacologia , Células da Granulosa/efeitos dos fármacos , Histonas , Kisspeptinas/efeitos dos fármacos , Kisspeptinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Ovário/efeitos dos fármacos , Ovário/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Substâncias para o Controle da Reprodução/farmacologia , Elementos de Resposta/genética , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Estrogen receptor 2 (ESR2) plays a critical role in folliculogenesis and ovulation. Disruption of ESR2-function in the rats results in female infertility due to failure of ovulation. Ovulation failure occurred in two distinct rat models, a null mutant and a DNA binding domain (DBD) mutant of ESR2, indicating that transcriptional regulation by ESR2 is indispensable for ovulation. To define the regulatory role of ESR2 in preovulatory follicular maturation and ovulation, we investigated ovarian responsiveness to exogenous gonadotropins in prepubertal females. Granulosa cells (GCs) play a vital role in follicle maturation and ovulation, and ESR2-dependent estrogen signaling is predominant in GCs, therefore, we examined the differential expression of gonadotropin-induced genes in GCs. Of 32,623 genes detected by RNA-sequencing, 1696 were differentially expressed in Esr2-mutant rats (789 downregulated, and 907 upregulated, absolute fold change 2, FDR pâ¯<â¯0.05). Molecular pathway analyses indicated that these differentially expressed genes are involved in steroidogenesis, follicle maturation, and ovulation. Many of these genes are known regulators of ovarian function and a subset were also disrupted in Esr2-mutant mice. Interestingly, Kiss1 was identified as one of the differentially expressed genes implicating a potential role within the follicle and its regulation by ESR2. Our findings indicate that ESR2 regulates key genes in GCs that are essential for follicle maturation and ovulation in the rat.