RESUMEN

BACKGROUND: Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis. METHODS: Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique. RESULTS: MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage. CONCLUSIONS: We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.

Asunto(s)

Estradiol/farmacología , Profase Meiótica I/efectos de los fármacos , Oocitos/efectos de los fármacos , Ovario/efectos de los fármacos , Progesterona/farmacología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones Endogámicos C57BL , Oocitos/citología , Oocitos/metabolismo , Técnicas de Cultivo de Órganos , Ovario/embriología , Ovario/metabolismo , Fase Paquiteno/efectos de los fármacos , Embarazo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo

RESUMEN

Primordial follicle assembly is essential for reproduction in mammalian females. Oocytes develop in germ cell cysts that in late fetal development begin break down into individual oocytes and become surrounded by pregranulosa cells, forming primordial follicles. As they separate, many oocytes are lost by apoptosis. Exposure to steroid hormones delays cyst breakdown, follicle formation, and associated oocyte loss in some species. One model for regulation of follicle formation is that steroid hormones in the maternal circulation keep cells in cysts and prevent oocyte death during fetal development but that late in pregnancy hormone levels drop, triggering cyst breakdown and associated oocyte loss. However, herein we found that, while maternal circulating levels of progesterone drop during late fetal development, maternal estradiol levels remain high. We hypothesized that fetal ovaries were the source of hormones and that late in fetal development their production stops. To test this, mRNA and protein levels of steroidogenic enzymes required for estradiol and progesterone synthesis were measured. We found that aromatase and 3-beta-hydroxysteroid dehydrogenase mRNA levels drop before cyst breakdown. The 3-beta-hydroxysteroid dehydrogenase protein levels also dropped, but we did not detect a change in aromatase protein levels. The steroid content of perinatal ovaries was assayed, and both estradiol and progesterone were detected in fetal ovaries before cyst breakdown. To determine the role of steroid hormones in oocyte development, we examined the effects of blocking steroid hormone production in organ culture and found that the number of oocytes was reduced, supporting our model that steroid hormones are important for fetal oocyte survival.

Asunto(s)

Apoptosis , Células Madre Embrionarias/citología , Estradiol/metabolismo , Oogénesis , Folículo Ovárico/citología , Progesterona/metabolismo , Células Madre/citología , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Animales Recién Nacidos , Animales no Consanguíneos , Aromatasa/genética , Aromatasa/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/metabolismo , Estradiol/sangre , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Técnicas de Cultivo de Órganos , Folículo Ovárico/embriología , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/citología , Ovario/embriología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Progesterona/sangre , Células Madre/enzimología , Células Madre/metabolismo

RESUMEN

OBJECTIVE: This study aimed to assess the in vivo effects of estradiol treatment on arterial gene expression in atherosclerotic postmenopausal female monkeys. METHODS: Eight ovariectomized cynomolgus monkeys were fed atherogenic diets for 6.5 years. The left iliac artery was biopsied before randomization to the estradiol group (human equivalent dose of 1 mg/d, n = 4) or the vehicle group (n = 4) for 8 months. The right iliac artery was obtained at necropsy. Transcriptional profiles in pretreatment versus posttreatment iliac arteries were compared to assess the responses of atherosclerotic arteries to estradiol. RESULTS: Iliac artery plaque size did not differ between the estradiol group and the placebo group at baseline or during the treatment period. Nevertheless, estradiol treatment was associated with increased expression of 106 genes and decreased expression of 26 genes in the iliac arteries. Estradiol treatment increased the expression of extracellular matrix genes, including the α1 chain of type I collagen, the α2 chain of type VI collagen, and fibulin 2, suggestive of an increase in the proportion or phenotype of smooth muscles or fibroblasts in lesions. Also increased were components of the insulin-like growth factor pathway (insulin-like growth factor 1, insulin-like growth factor binding protein 4, and insulin-like growth factor binding protein 5) and the Wnt signaling pathway (secreted frizzled-related protein 2, secreted frizzled-related protein 4, low-density lipoprotein receptor-related protein 6, and Wnt1-inducible signaling pathway protein 2). CONCLUSIONS: Estradiol treatment of monkeys with established atherosclerosis affected iliac artery gene expression, suggesting changes in the cellular composition of lesions. Moreover, it is probable that the presence of atherosclerotic plaque affected the gene expression responses of arteries to estrogen.

Asunto(s)

Aterosclerosis/metabolismo , Estradiol/farmacología , Arteria Ilíaca/metabolismo , Ovariectomía , Posmenopausia , Transcriptoma/efectos de los fármacos , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Dieta Aterogénica , Modelos Animales de Enfermedad , Estradiol/uso terapéutico , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Arteria Ilíaca/química , Arteria Ilíaca/patología , Lípidos/sangre , Macaca fascicularis , Análisis de Secuencia por Matrices de Oligonucleótidos , Somatomedinas/genética

RESUMEN

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 µM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 µg/ml) or ACK4 (80 µg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.

Asunto(s)

Ciclohexenos/toxicidad , Contaminantes Ambientales/toxicidad , Ovario/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Compuestos de Vinilo/toxicidad , Animales , Animales Recién Nacidos , Anticuerpos Bloqueadores/metabolismo , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Ciclohexenos/antagonistas & inhibidores , Contaminantes Ambientales/antagonistas & inhibidores , Femenino , Atresia Folicular/efectos de los fármacos , Ligandos , Terapia Molecular Dirigida , Peso Molecular , Técnicas de Cultivo de Órganos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/agonistas , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Ratas Endogámicas F344 , Compuestos de Vinilo/antagonistas & inhibidores

RESUMEN

Women are born with a finite population of ovarian follicles, which are slowly depleted during their reproductive years until reproductive failure (menopause) occurs. The rate of loss of primordial follicles is determined by genetic and environmental influences, but certain toxic exposures can accelerate this process. Ionizing radiation reduces preantral follicle numbers in rodents and humans in a dose-dependent manner. Cigarette smoking is linked to menopause occurring 1-4 yr earlier than with nonsmokers, and components of smoke, polycyclic aromatic hydrocarbons, can cause follicle depletion in rodents or in ovaries in vitro. Chemotherapeutic agents, such as alkylating drugs and cisplatin, also cause loss of preantral ovarian follicles. Effects depend on dose, type, and reactivity of the drug, and the age of the individual. Evidence suggests DNA damage may underlie follicle loss induced by one common alkylating drug, cyclophosphamide. Occupational exposures have also been linked to ovarian damage. In an industrial setting, 2-bromopropane caused infertility in men and women, and it can induce ovarian follicle depletion in rats. Solvents, such as butadiene, 4-vinylcyclohexene, and their diepoxides, can also cause specific preantral follicle depletion. The mechanism(s) underlying effects of the latter compound may involve alterations in apoptosis, survival factors such as KIT/Kit Ligand, and/or the cellular signaling that maintains primordial follicle dormancy. Estrogenic endocrine disruptors may alter follicle formation/development and impair fertility or normal development of offspring. Thus, specific exposures are known or suspected of detrimentally impacting preantral ovarian follicles, leading to early ovarian failure.

Asunto(s)

Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Xenobióticos/farmacología , Animales , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Humanos , Ciclo Menstrual/efectos de los fármacos , Ciclo Menstrual/fisiología , Modelos Animales , Exposición Profesional/efectos adversos , Ovario/efectos de los fármacos , Ovario/fisiología , Ratas

RESUMEN

OBJECTIVE: The aim of this study was to evaluate global gene expression patterns in the common iliac arteries of monkeys with a varied extent of atherosclerosis. METHODS: The left common iliac artery was removed from ovariectomized cynomolgus monkeys (n = 12) after 6.5 years of consuming a diet containing fat and cholesterol at levels comparable with those consumed in Western populations. Arterial gene expression was analyzed using DNA microarray and real-time reverse transcription-polymerase chain reaction. RESULTS: Significant differential expression of 986 genes was observed in iliac arteries containing moderate to large atherosclerotic plaques compared with normal/minimally affected reference group arteries. Atherosclerosis-associated genes included cytokines, chemokines, components of signal transduction pathways, and transcriptional activators and repressors, as well as other functional categories. Real-time reverse transcription-polymerase chain reaction confirmed a differential expression of genes chosen from a variety of functional categories. Specifically, the expression of genes for estrogen receptor-1, claudin 11, and brain heart protocadherin 7 was reduced, whereas the expression of genes for apolipoprotein E, growth differentiation factor 15, superoxide dismutase-2, SET domain bifurcated 2, phospholipase A2 group IIA, phospholipase A2 group VII, and ring finger protein 149 was increased in atherosclerotic arteries. CONCLUSIONS: The gene expression environment in arteries containing atherosclerotic plaques is profoundly different from that of relatively unaffected arteries and reflects the cellular and molecular complexity of atherosclerosis and associated arterial remodeling processes.

Asunto(s)

Aterosclerosis/genética , Perfilación de la Expresión Génica , Arteria Ilíaca/metabolismo , Animales , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/genética , Aterosclerosis/fisiopatología , Cadherinas/biosíntesis , Cadherinas/genética , Claudinas/biosíntesis , Claudinas/genética , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Factor 15 de Diferenciación de Crecimiento/genética , N-Metiltransferasa de Histona-Lisina/biosíntesis , N-Metiltransferasa de Histona-Lisina/genética , Arteria Ilíaca/fisiopatología , Macaca fascicularis , Ovariectomía , Fosfolipasas A2/biosíntesis , Fosfolipasas A2/genética , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética

RESUMEN

Estrogen has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Gene targets of estrogen and SERMs in the vasculature are not well-known. Thus, the present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation.

Asunto(s)

Estrógenos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Arterias Mesentéricas/fisiología , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Arterias Mesentéricas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biosíntesis de Proteínas , Clorhidrato de Raloxifeno/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tamoxifeno/farmacología

RESUMEN

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 µM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.

Asunto(s)

Ciclohexenos/toxicidad , Ovario/efectos de los fármacos , Ovario/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Compuestos de Vinilo/toxicidad , Animales , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Ovario/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Transducción de Señal/efectos de los fármacos

RESUMEN

Anti-Müllerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30 µM, 2-8 days) decreased (P<0.05) AMH mRNA (d4-8) and protein (d6-8). Recombinant AMH (100-400 mg/ml) in PND4 ovaries cultured 8 days±VCD (30 µM) caused an increase (P<0.05) in primordial, and a decrease (P<0.05) in small primary follicles, supporting that AMH retarded primordial follicle recruitment. However, no concentration of AMH had an effect on VCD-induced ovotoxicity. Whereas, VCD caused a reduction in expression of AMH (d4-d8), it followed previously reported initial disruptions in Kit signaling induced by VCD (d2). Thus, collectively, these results do not support a mechanism whereby VCD causes ovotoxicity via generalized activation of primordial follicle recruitment, but instead provide further support for the specificity of other intracellular mechanisms involved in VCD-induced ovotoxicity.

Asunto(s)

Hormona Antimülleriana/metabolismo , Ciclohexenos/toxicidad , Ovario/efectos de los fármacos , Ovario/crecimiento & desarrollo , Compuestos de Vinilo/toxicidad , Animales , Animales Recién Nacidos , Hormona Antimülleriana/análisis , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Femenino , Técnicas de Cultivo de Órganos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Ratas , Ratas Endogámicas F344 , Distribución Tisular

RESUMEN

This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication may contribute to the pathology of endometriosis. An endometrial stromal cell line (telomerase-immortalized human endometrial stromal cell [T-HESC]) was treated with macrophage-conditioned medium (CM) +/- estradiol + progesterone. Macrophages were treated without or with T-HESC CM. DNA microarray identified 716 differentially expressed genes in T-HESCs in response to factors secreted by macrophages. Upregulated genes in T-HESC included interleukin 8 (IL-8)/chemokine (C-X-C motif) ligand 8 (CXCL8), matrix metalloproteinase 3 (MMP3), phospholamban, cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), tenascin C, and nicotinamide N-methyltransferase (NNMT), whereas integrin alpha-6 was downregulated. In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by endometrial stromal cells. The data document reciprocal communication between macrophages and endometrial stromal cells and suggest that interaction with macrophages stimulates the expression of genes in endometrial stromal cells that may support the establishment of endometriosis.

Asunto(s)

Endometriosis/metabolismo , Endometrio/metabolismo , Macrófagos/metabolismo , Comunicación Paracrina , Células del Estroma/metabolismo , Medios de Cultivo Condicionados/metabolismo , Endometriosis/genética , Endometriosis/patología , Endometrio/efectos de los fármacos , Endometrio/patología , Estradiol/farmacología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Acetato de Medroxiprogesterona/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Prolactina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Células U937