RESUMEN
Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were treated with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development, followed 48h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.
Asunto(s)
Luteinización/metabolismo , Ovario/metabolismo , Ovulación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Femenino , Gonadotropinas Equinas/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Luteinización/efectos de los fármacos , Luteinización/genética , Ovario/efectos de los fármacos , Ovulación/efectos de los fármacos , Ovulación/genética , Proteínas Serina-Treonina Quinasas/genética , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation-sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.
Asunto(s)
Células de la Granulosa/metabolismo , Histonas/metabolismo , Ovulación/fisiología , Transcriptoma , Animales , Gonadotropina Coriónica/farmacología , Femenino , Perfilación de la Expresión Génica , Células de la Granulosa/efectos de los fármacos , Código de Histonas/efectos de los fármacos , Código de Histonas/genética , Luteinización/efectos de los fármacos , Luteinización/genética , Luteinización/metabolismo , Hormona Luteinizante/metabolismo , Ratones , Ratones Endogámicos C57BL , Ovulación/genética , Ovulación/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Integración de Sistemas , Transcriptoma/efectos de los fármacosRESUMEN
Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.
Asunto(s)
Búfalos/genética , Técnicas de Cultivo de Célula/métodos , Células de la Granulosa/metabolismo , RNA-Seq/métodos , Transcriptoma/genética , Animales , Búfalos/metabolismo , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Luteinización/genética , Mapas de Interacción de Proteínas/genética , ARN Largo no Codificante/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/aislamiento & purificación , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
WNT proteins are widely expressed in the murine ovaries. WNTLESS is a regulator essential for all WNTs secretion. However, the complexity and overlapping expression of WNT signaling cascades have prevented researchers from elucidating their function in the ovary. Therefore, to determine the overall effect of WNT on ovarian development, we depleted the Wntless gene in oocytes and granulosa cells. Our results indicated no apparent defect in fertility in oocyte-specific Wntless knockout mice. However, granulosa cell (GC) specific Wntless deletion mice were subfertile and recurred miscarriages. Further analysis found that GC-specific Wntless knockout mice had noticeably smaller corpus luteum (CL) in the ovaries than control mice, which is consistent with a significant reduction in luteal cell marker gene expression and a noticeable increase in apoptotic gene expression. Also, the deletion of Wntless in GCs led to a significant decrease in ovarian HCGR and ß-Catenin protein levels. In conclusion, Wntless deficient oocytes had no discernible impact on mouse fertility. In contrast, the loss of Wntless in GCs caused subfertility and impaired CL formation due to reduced LHCGR and ß-Catenin protein levels, triggering GC apoptosis.
Asunto(s)
Aborto Espontáneo/genética , Cuerpo Lúteo/metabolismo , Células de la Granulosa/metabolismo , Infertilidad Femenina/genética , Luteinización/genética , Oocitos/metabolismo , Receptores Acoplados a Proteínas G/genética , Aborto Espontáneo/metabolismo , Animales , Apoptosis/genética , Cuerpo Lúteo/patología , Femenino , Infertilidad Femenina/metabolismo , Células Lúteas/metabolismo , Células Lúteas/patología , Ratones , Ratones Noqueados , Ovario/metabolismo , Progesterona/metabolismo , Receptores de HL/metabolismo , beta Catenina/metabolismoRESUMEN
Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to -715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.
Asunto(s)
Células de la Granulosa/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Luteinización/metabolismo , Hormona Luteinizante/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Regulación de la Expresión Génica , Factor 4 Similar a Kruppel , Luteinización/genética , Hormona Luteinizante/fisiología , RatasRESUMEN
The pathophysiology of polycystic ovary syndrome (PCOS) is characterized by granulosa cell (GC) dysfunction. m6 A modification affects GC function in patients with premature ovarian insufficiency (POI), but the role of m6 A modification in PCOS is unknown. The purpose of the prospective comparative study was to analyse the m6 A profile of the luteinized GCs from normovulatory women and non-obese PCOS patients following controlled ovarian hyperstimulation. RNA m6 A methylation levels were measured by m6 A quantification assay in the luteinized GCs of the controls and PCOS patients. Then, m6 A profiles were analysed by methylated RNA immunoprecipitation sequencing (MeRIP-seq). We reported that the m6 A level was increased in the luteinized GCs of PCOS patients. Comparative analysis revealed differences between the m6 A profiles from the luteinized GC of the controls and PCOS patients. We identified FOXO3 mRNA with reduced m6 A modification in the luteinized GCs of PCOS patients. Selectively knocking down m6 A methyltransferases or demethylases altered expression of FOXO3 in the luteinized GCs from the controls, but did not in PCOS patients. These suggested an absence of m6 A-mediated transcription of FOXO3 in the luteinized GCs of PCOS patients. Furthermore, we demonstrated that the involvement of m6 A in the stability of the FOXO3 mRNA that is regulated via a putative methylation site in the 3'-UTR only in the luteinized GCs of the controls. In summary, our findings showed that altered m6 A modification was involved in up-regulated expression of FOXO3 mRNA in the luteinized GCs from non-obese PCOS patients following controlled ovarian hyperstimulation.
Asunto(s)
Adenosina/análogos & derivados , Proteína Forkhead Box O3/genética , Células de la Granulosa/metabolismo , Luteinización/genética , Síndrome del Ovario Poliquístico/genética , Regulación hacia Arriba/genética , Adenosina/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Proteína Forkhead Box O3/metabolismo , Células de la Granulosa/patología , Humanos , Síndrome del Ovario Poliquístico/patología , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.
Asunto(s)
Permeabilidad Capilar/fisiología , Cuerpo Lúteo/irrigación sanguínea , Neovascularización Fisiológica , Serina Endopeptidasas/deficiencia , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/genética , Células Cultivadas , Cuerpo Lúteo/patología , Cuerpo Lúteo/fisiopatología , Femenino , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Técnicas de Silenciamiento del Gen , Hemorragia/etiología , Hemorragia/genética , Hemorragia/fisiopatología , Humanos , Luteinización/genética , Luteinización/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/genética , Fenotipo , Serina Endopeptidasas/genética , Serina Endopeptidasas/fisiologíaRESUMEN
High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22-23 days old) were treated with 10IU, s.c., pregnant mare's serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4-12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.
Asunto(s)
Diferenciación Celular/fisiología , Proteína HMGA1a/metabolismo , Luteinización/metabolismo , Ovario/metabolismo , Ovulación/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Femenino , Regulación de la Expresión Génica , Gonadotropinas Equinas/farmacología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Proteína HMGA1a/genética , Luteinización/efectos de los fármacos , Luteinización/genética , Ovulación/efectos de los fármacos , Ovulación/genética , Ratas , Ratas Sprague-Dawley , Células Tecales/efectos de los fármacos , Células Tecales/metabolismoRESUMEN
The balances of mitochondrial dynamic changes, mitochondrial morphology, and mitochondrial number are critical in cell metabolism. Once they are disturbed, disorders in these processes generally cause diseases or even death in animals. We performed large-scale genetic screenings in fruit flies and discovered the mitoguardin gene (Miga) that encodes for a mitochondrial outer membrane protein. To examine the physiological functions of its mammalian homologs Miga1 and Miga2, we generated Miga1 and Miga2 single- and double-knockout mouse strains and found that the knockout mice were viable, but the females were subfertile. The ovarian phenotypes of these mice suggested that the MIGA1/2 proteins play an important role in ovulation and ovarian steroidogenesis. In vivo and in vitro analyses of Miga1/2-knockout granulosa cells showed severe defects in luteinization and steroidogenesis and disordered mitochondrial morphology and function in response to gonadotropins. This is a report of genes involved in mitochondrial fusion and morphology-regulating mitochondrial functions during ovulation and luteinization. These results suggest a mechanism of gonadotropin-regulated ovarian endocrine functions and provide clues for therapeutic treatments of infertile females.
Asunto(s)
Hormonas Gonadales/metabolismo , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Proteínas Mitocondriales/fisiología , Ovario/metabolismo , Ovulación/genética , Animales , Células Cultivadas , Femenino , Gonadotropinas/metabolismo , Células de la Granulosa/metabolismo , Luteinización/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Ovulación/metabolismoRESUMEN
After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells.
Asunto(s)
Perfilación de la Expresión Génica , Células Lúteas/metabolismo , Folículo Ovárico/metabolismo , Animales , Biomarcadores/metabolismo , Bovinos , Linaje de la Célula/genética , Análisis por Conglomerados , Femenino , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Luteinización/genética , Análisis por Micromatrices , Análisis de Componente Principal , Reproducibilidad de los Resultados , Células Tecales/metabolismo , Transcriptoma/genéticaRESUMEN
Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.
Asunto(s)
Células de la Granulosa/metabolismo , Ovulación/genética , Progesterona/metabolismo , Receptores de Progesterona/genética , Animales , Células Cultivadas , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Células Lúteas/efectos de los fármacos , Células Lúteas/metabolismo , Luteinización/efectos de los fármacos , Luteinización/genética , Macaca mulatta , Progesterona/farmacología , TransfecciónRESUMEN
Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1(flox/flox) mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12-16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Ciclo Celular/genética , Cuerpo Lúteo/metabolismo , Ovulación/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células del Cúmulo/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células de la Granulosa/metabolismo , Luteinización/genética , Luteinización/metabolismo , Ratones , Ratones Noqueados , Ovulación/metabolismoRESUMEN
The surge of luteinizing hormone triggers the genomic reprogramming, cell differentiation, and tissue remodeling of the ovulated follicle, leading to the formation of the corpus luteum. During this process, called luteinization, follicular granulosa cells begin expressing a new set of genes that allow the resulting luteal cells to survive in a vastly different hormonal environment and to produce the extremely high amounts of progesterone (P4) needed to sustain pregnancy. To better understand the molecular mechanisms involved in the regulation of luteal P4 production in vivo, the transcription factors GATA4 and GATA6 were knocked down in the corpus luteum by crossing mice carrying Gata4 and Gata6 floxed genes with mice carrying Cre recombinase fused to the progesterone receptor. This receptor is expressed exclusively in granulosa cells after the luteinizing hormone surge, leading to recombination of floxed genes during follicle luteinization. The findings demonstrated that GATA4 and GATA6 are essential for female fertility, whereas targeting either factor alone causes subfertility. When compared to control mice, serum P4 levels and luteal expression of key steroidogenic genes were significantly lower in conditional knockdown mice. The results also showed that GATA4 and GATA6 are required for the expression of the receptors for prolactin and luteinizing hormone, the main luteotropic hormones in mice. The findings demonstrate that GATA4 and GATA6 are crucial regulators of luteal steroidogenesis and are required for the normal response of luteal cells to luteotropins.
Asunto(s)
Cuerpo Lúteo/metabolismo , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA6/genética , Infertilidad Femenina/genética , Luteinización/genética , Progesterona/biosíntesis , Animales , Gonadotropina Coriónica/farmacología , Cuerpo Lúteo/efectos de los fármacos , Femenino , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA6/metabolismo , Técnicas de Silenciamiento del Gen , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Infertilidad Femenina/metabolismo , Luteinización/efectos de los fármacos , Luteinización/metabolismo , Ratones , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMEN
Variations in mRNA levels and sources of metastin/kisspeptin, neurokinin B (NKB), dynorphin, and kisspeptin receptor GPR54 were examined in the ovaries of cycling rats. Kisspeptin and dynorphin mRNAs dramatically increased at 2000 h of the proestrous day. NKB mRNA also increased, but the peak was delayed by 6 h. GPR54 mRNA declined inversely with kisspeptin. Whole-ovary expressions of kisspeptin and dynorphin mRNAs, but not of NKB mRNA, were augmented by the administration of human chorionic gonadotropin (hCG). By means of laser-capture microdissection, kisspeptin mRNA was shown mostly in follicles at 2000 h of proestrus, whereas NKB and dynorphin were expressed mainly in interstitial tissues. GPR54 mRNA was detected equally in follicles, corpora lutea, and interstitial tissues. The hCG stimulated the follicular expression of kisspeptin and interstitial tissue expression of dynorphin mRNA. In primary cultures of granulosa cells prepared from equine chorionic gonadotropin-pretreated immature rats, hCG stimulated the expression of kisspeptin, dynorphin, and NKB mRNAs. Distortion of the corpus luteum and surrounding tissue borders was sometimes seen after intra-ovarian bursa administration of kisspeptin antagonist p234 for 3 days from proestrus. Progesterone production stimulated by hCG in granulosa cell culture was suppressed by p234. These data demonstrate that significant amounts of kisspeptin are synthesized in granulosa cells and dynorphin in interstitial tissues, in response to the proestrous luteinizing hormone surge, whereas granulosa cells also contain dynorphin and NKB, suggesting at least a role for kisspeptin in the luteinization of granulosa cells.
Asunto(s)
Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Kisspeptinas/metabolismo , Luteinización/metabolismo , Hormona Luteinizante/metabolismo , Proestro/metabolismo , Animales , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/metabolismo , Dinorfinas/genética , Dinorfinas/metabolismo , Femenino , Células de la Granulosa/efectos de los fármacos , Kisspeptinas/genética , Luteinización/genética , Péptidos/farmacología , Proestro/efectos de los fármacos , Proestro/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1RESUMEN
Luteal-phase insufficiency is one of the major causes of female infertility, but the molecular mechanisms are still largely unknown. Here we found that disruption of Lgr4/Gpr48, the newly identified receptor for R-spondins, greatly reduced female fertility in mice. The expression of Lgr4 was induced specifically in granulosa-lutein cells during luteinization. In Lgr4-deficient female mice, the estrous cycle was prolonged and serum progesterone levels were dramatically downregulated. In Lgr4(-/-) corpora lutea, the expression of key enzymes for steroidogenesis as well as common luteal marker genes was significantly decreased. Additionally, the activity of epidermal growth factor receptor (EGFR)-ERK signaling was attenuated in Lgr4(-/-) granulosa-lutein cells. We found that the maturation of Lgr4(-/-) cells was impaired in cultured primary granulosa cells, but the defect was partially rescued by reactivation of EGFR signaling by heparin-binding EGF-like growth factor treatment. We found that the expression of wingless-type MMTV integration site family (WNT)/catenin (cadherin associated protein), beta 1 (CTNNB1) downstream targets, including matrix metalloproteinase 9, which is a critical matrix metalloproteinase for activation of EGF-like factors, was significantly downregulated in Lgr4(-/-) ovaries. Matrix metalloproteinase 9 inhibitor treatment attenuated human chorionic gonadotropin- but not heparin-binding EGF-like growth factor-induced ERK activation and luteinization in primary granulosa cells. Together, we report that Lgr4 modulates WNT-mediated EGFR-ERK signaling to facilitate corpus luteum maturation and ovarian steroidogenesis to maintain female reproduction.
Asunto(s)
Cuerpo Lúteo/crecimiento & desarrollo , Cuerpo Lúteo/metabolismo , Receptores ErbB/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Receptores ErbB/genética , Femenino , Células de la Granulosa/metabolismo , Humanos , Luteinización/genética , Luteinización/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Receptores Acoplados a Proteínas G/genética , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
Cell culture techniques of human mural granulosa cells (MGCs) serve as a major in vitro tool. However, the use of luteinized MGCs has major limitations due to their luteinized state. Our aim was to establish a standardized protocol for the culture of MGCs as a model for different stages of folliculogenesis. We showed that early-non-luteinized, preovulatory-non-luteinized and luteal-MGCs have distinct gene expression pattern. After 4 days of incubation of luteinized-MGCs, ovulatory genes mRNA's achieve expression levels similar to the early non-luteinized follicles. FSH stimulation for 48 h of these 4 days cultured MGCs showed ovulatory genes mRNA's expression similar to the pre-ovulatory non-luteinized follicles. These FSH-stimulated cells responded to hCG stimulation in a pattern similar to the response of pre-ovulatory follicles. This novel model may provide a standardized research tool for delineation of the molecular processes occurring during the latter stages of follicular development in the human ovary.
Asunto(s)
Células de la Granulosa/citología , Luteinización/genética , ARN Mensajero/genética , Adulto , Aromatasa/genética , Aromatasa/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Femenino , Hormona Folículo Estimulante/farmacología , Expresión Génica , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Modelos Biológicos , ARN Mensajero/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismoRESUMEN
The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat.
Asunto(s)
Aromatasa/genética , Células de la Granulosa/enzimología , Modelos Biológicos , Regiones Promotoras Genéticas , Animales , Aromatasa/metabolismo , Bovinos , Colforsina/farmacología , Cuerpo Lúteo/metabolismo , Estradiol/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Factores de Transcripción Forkhead/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cabras , Células de la Granulosa/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Luteinización/efectos de los fármacos , Luteinización/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de HFE/metabolismo , Especificidad de la Especie , TransfecciónRESUMEN
Progesterone, the main steroid synthesized by the corpus luteum (CL), prepares the uterus for implantation, maintains the CL survival, and induces progesterone auto-secretion. However, the molecular mechanisms involving the progesterone auto-secretion pathways at the luteal phase are not fully understood, especially in humans. We aim to study the molecular mechanism of the progesterone pathway in human granulosa cells. Our model system consists of luteinized human-mural-granulosa-cells (hmGCs) obtained from follicles aspirated during in vitro fertilization (IVF) procedures. hmGCs were seeded in culture and were subjected to different hormonal treatments. mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). Progesterone levels were measured by enzyme immunoassay (EIA). We show that exposure of luteinized hmGCs to the progesterone receptor antagonist, RU486 (mifepristone), resulted in inhibition of LHCGR, LH/hCG target genes and progesterone secretion. Exposure of hmGCs to medium that was incubated with hmGCs for 4 d - conditioned medium (CM), which contain 150 ± 7.5 nM progesterone, resulted in induction of LHCGR and LH/hCG target genes, which was blocked by RU486. In addition, RU486 inhibited some of the progesterone biosynthesis pathway genes. Our results revealed a novel mechanism of the progesterone antagonist pathway in the luteal granulosa cells and emphasis the fundamental role of progesterone in the early luteal phase.
Asunto(s)
Gonadotropina Coriónica/metabolismo , Células de la Granulosa/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Hormona Luteinizante/metabolismo , Mifepristona/farmacología , Receptores de HL/genética , Adulto , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Células de la Granulosa/metabolismo , Humanos , Luteinización/genética , Luteinización/metabolismo , Progesterona/antagonistas & inhibidores , Receptores de HL/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein ß to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.
Asunto(s)
Aromatasa/genética , Metilación de ADN/genética , Células de la Granulosa/metabolismo , Histonas/metabolismo , Fosfoproteínas/genética , Regiones Promotoras Genéticas/genética , Animales , Gonadotropina Coriónica/farmacología , Metilación de ADN/efectos de los fármacos , Femenino , Células de la Granulosa/efectos de los fármacos , Histonas/efectos de los fármacos , Caballos , Luteinización/efectos de los fármacos , Luteinización/genética , Ovulación/efectos de los fármacos , Ovulación/genética , Regiones Promotoras Genéticas/efectos de los fármacos , RatasRESUMEN
Neovascularization is necessary for formation of the corpus luteum (CL) and includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. Here we investigated whether vasculogenesis occurs in neovascularization during CL formation. Mice transplanted with bone marrow from transgenic mice expressing green fluorescent protein (GFP) were injected with equine chorionic gonadotropin and human chorionic gonadotropin (hCG) to induce ovulation and subsequent CL formation. Immunohistochemistry was performed on the ovaries obtained before hCG injection and at 6, 12, and 24 h after hCG injection using antibodies for CD34 or CD31 (an endothelial cell marker), platelet-derived growth factor receptor beta (PDGFR-beta, a pericyte marker), F4/80 (a macrophage marker), and GFP (a bone marrow-derived cell marker). Cells immunostained for CD34, PDGFR-beta, F4/80, and GFP were present in the theca cell layer of the preovulatory follicle before hCG injection. Each of these cell types invaded the granulosa cell layer after hCG injection, and a number of them were observed in the CL 24 h after hCG injection. Fluorescence-based immunohistochemistry or double immunohistochemical staining revealed that a few CD34/CD31-positive cells and PDGFR-beta-positive cells were also positive for GFP in the preovulatory follicle and CL, and that many of the GFP-positive cells recruited to the CL during CL formation were F4/80-positive macrophages. In conclusion, bone marrow-derived vascular progenitor cells and macrophages contribute to neovascularization during CL formation.