RESUMEN

Polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants and potent ovarian toxicants. The transcription factor NRF2 is an important regulator of the cellular response to electrophilic toxicants like BaP and to oxidative stress. NRF2 regulates transcription of genes involved in the detoxification of reactive metabolites of BaP and reactive oxygen species. We therefore hypothesized that Nrf2-/- mice have accelerated ovarian aging and increased sensitivity to the ovarian toxicity of BaP. A single injection of BaP dose-dependently depleted ovarian follicles in Nrf2+/+ and Nrf2-/- mice, but the effects of BaP were not enhanced in the absence of Nrf2. Similarly, Nrf2-/- mice did not have increased ovarian BaP DNA adduct formation compared to Nrf2+/+ mice. Ovarian follicle numbers did not differ between peripubertal Nrf2-/- and Nrf2+/+ mice, but by middle age, Nrf2-/- mice had significantly fewer primordial follicles than Nrf2+/+ mice, consistent with accelerated ovarian aging.

Asunto(s)

Benzo(a)pireno/toxicidad , Senescencia Celular/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Células Epiteliales/efectos de los fármacos , Eliminación de Gen , Factor 2 Relacionado con NF-E2/deficiencia , Folículo Ovárico/efectos de los fármacos , Ovario/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Aductos de ADN/metabolismo , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Reserva Ovárica/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Estrés Oxidativo/efectos de los fármacos , Fenotipo

RESUMEN

OBJECTIVE: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs). DESIGN: Ex vivo and in vitro experimental study. SETTING: University. ANIMAL(S): Immature female Sprague Dawley rats. INTERVENTION(S): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or ß-glycan antibody. MAIN OUTCOME MEASURE(S): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription-polymerase chain reaction. RESULT(S): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, ß-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent. CONCLUSION(S): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs by virtue of FSH seem to be required.

Asunto(s)

Andrógenos/metabolismo , Células de la Granulosa/metabolismo , Comunicación Paracrina/fisiología , Células Tecales/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Femenino , Hormona Folículo Estimulante/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Folículo Ovárico/fisiología , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/fisiología , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Células Tecales/efectos de los fármacos , Células Tecales/fisiología

RESUMEN

A single somatic FOXL2 mutation (FOXL2(C134W)) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2(C134W) has altered activity. We show that in primary granulosa cells, GDF-9 and activin increase Smad3-mediated follistatin transcription. In contrast to findings in the pituitary, FOXL2 negatively regulates GDF-9 and activin-stimulated follistatin transcription in the ovary. Knockdown of endogenous FOXL2 confirmed this inhibitory role. FOXL2(C134W) displayed enhanced inhibitory activity, completely ablating GDF-9 and activin-induced follistatin transcription. GDF-9 and activin activity was lost when either the smad binding element or the forkhead binding element were mutated, indicating that both sites are required for Smad3 actions. This study highlights that FOXL2 negatively regulates follistatin expression within the ovary, and that the pathogenesis of FOXL2(C134W) may involve an altered interaction with Smad3.

Asunto(s)

Activinas/fisiología , Folistatina/genética , Factores de Transcripción Forkhead/genética , Células de la Granulosa/metabolismo , Factor 9 de Diferenciación de Crecimiento/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Femenino , Folistatina/metabolismo , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Tumor de Células de la Granulosa , Humanos , Mutación Missense , Cultivo Primario de Células , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína smad3/metabolismo , Activación Transcripcional

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants formed by the incomplete combustion of organic materials. The tripeptide glutathione (GSH) is a major antioxidant and is important in detoxification of PAH metabolites. Mice null for the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations. We investigated the effects of Gclm deletion alone on male fertility and spermatogenesis and its effect on the sensitivity of male embryos to the transplacental testicular toxicity of BaP. Gclm-/- males had dramatically decreased testicular and epididymal GCL enzymatic activity and total GSH concentrations compared with Gclm+/+ littermates. Ratios of reduced to oxidized GSH were significantly increased in Gclm-/- testes. GSH reductase enzymatic activity was increased in Gclm-/- epididymides. We observed no changes in fertility, testicular weights, testicular sperm head counts, or testicular histology and subtle changes in cauda epididymal sperm counts, motility, and morphology in Gclm-/- compared with Gclm+/+ males. Prenatal exposure to BaP from gestational day 7 to 16 was dose dependently associated with significantly decreased testicular and epididymal weights, testicular and epididymal sperm counts, and with vacuolated seminiferous tubules at 10 weeks of age. Gclm-/- males exposed prenatally to BaP had greater decreases in testicular weights, testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility than Gclm+/+ littermates. These results show no effects of Gclm deletion alone on male fertility and testicular spermatogenesis and subtle epididymal effects but support increased sensitivity of Gclm-/- males to the transplacental testicular toxicity of BaP.

Asunto(s)

Benzo(a)pireno/toxicidad , Contaminantes Ambientales/toxicidad , Glutamato-Cisteína Ligasa/metabolismo , Efectos Tardíos de la Exposición Prenatal , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Benzo(a)pireno/administración & dosificación , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/administración & dosificación , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Epidídimo/patología , Femenino , Glutamato-Cisteína Ligasa/genética , Glutatión/metabolismo , Glutatión Reductasa/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Embarazo , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Testículo/metabolismo , Testículo/patología

RESUMEN

Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm-/- mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm-/- females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm-/- females. Prepubertal Gclm-/- and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm-/- females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/- females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm-/- females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm-/- females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.

Asunto(s)

Blastocisto/fisiología , Ectogénesis , Glutamato-Cisteína Ligasa/fisiología , Glutatión/metabolismo , Oocitos/metabolismo , Subunidades de Proteína/fisiología , Animales , Implantación del Embrión , Femenino , Fertilización In Vitro , Glutamato-Cisteína Ligasa/genética , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Infertilidad Femenina/fisiopatología , Tamaño de la Camada , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica , NADP Transhidrogenasas/genética , Subunidades de Proteína/genética , Interacciones Espermatozoide-Óvulo , Superovulación

RESUMEN

Oxidative stress occurs when generation of reactive oxygen species (ROS) overwhelms antioxidant defenses. Oxidative stress has been associated with male infertility. The transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) regulates basal and inducible transcription of genes encoding enzymes important for protection against ROS. We hypothesized that deletion of the Nrf2 gene causes testicular and epididymal oxidative stress, which disrupts spermatogenesis. Our results show that male Nrf2(-/-) mice have decreased fertility compared to wild-type and heterozygous littermates, due to accumulating seminiferous tubule damage with increasing age. Testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility in 2-month-old Nrf2(-/-) males did not differ from those of wild-type littermates; however, by age 6 months, Nrf2(-/-) males had 44% lower testicular sperm head counts, 65% lower epididymal sperm counts, and 66% lower epididymal sperm motility than wild-type males. Two- to 4-month-old Nrf2(-/-) males had elevated levels of testicular and epididymal lipid peroxidation and testicular germ cell apoptosis, and decreased levels of antioxidants, compared to wild-type males. These results provide evidence that oxidative stress has deleterious effects on the testis and epididymis and demonstrate a critical role for the transcription factor NRF2 in preventing oxidative disruption of spermatogenesis.

Asunto(s)

Envejecimiento/metabolismo , Infertilidad Masculina/metabolismo , Infertilidad Masculina/fisiopatología , Factor 2 Relacionado con NF-E2/metabolismo , Factores de Transcripción/metabolismo , Envejecimiento/genética , Animales , Progresión de la Enfermedad , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Recuento de Espermatozoides , Motilidad Espermática/genética , Espermatogénesis/genética , Factores de Transcripción/genética

RESUMEN

Glutathione (GSH), the most abundant intracellular nonprotein thiol, is critical for many cellular functions. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The tissue-specific regulation of GSH synthesis is poorly understood. We showed previously that gonadotropin hormones regulate ovarian GSH synthesis. In the present study, we sought to clarify the ovarian cell type-specific effects of follicle-stimulating hormone (FSH) and estradiol on GSH synthesis. Immature female rats were treated with estradiol to stimulate development of small antral follicles. Granulosa cells (GCs) from these follicles or whole follicles were cultured in serum-free media, with or without FSH and 17beta-estradiol. The GSH and GCLC protein and mRNA levels increased in GCs treated with FSH alone. The effects of FSH on GCLC and GCLM protein and mRNA levels, GCL enzymatic activity, and GSH concentrations in GCs were significantly enhanced by the addition of estradiol. Estradiol alone had no effects on GSH. Dibromo-cAMP mimicked and protein kinase A (PKA) inhibitors prevented FSH stimulation of GCL subunit protein levels. In cultured small antral follicles, FSH stimulated estradiol synthesis and robustly increased GCL subunit mRNA and protein levels and GSH concentrations. The GCL subunit mRNA expression increased in both the granulosa cells and theca cells of follicles with FSH stimulation. These data demonstrate that maximal stimulation of GSH synthesis by FSH in granulosa cells and follicles requires estradiol. Without estradiol, FSH causes lesser increases in GCL subunit expression via a PKA-dependent pathway.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estradiol/biosíntesis , Hormona Folículo Estimulante/metabolismo , Glutatión/biosíntesis , Células de la Granulosa/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Activación Enzimática , Estabilidad de Enzimas , Femenino , Glutamato-Cisteína Ligasa/metabolismo , Progesterona/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

The antioxidant tripeptide glutathione (GSH) protects ovarian follicles against oxidative damage that may lead to apoptotic death. The rate-limiting step in synthesis of GSH is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic subunit (GCLC), and a modifier subunit (GCLM). We hypothesized that GSH depletion in vivo or in vitro with buthionine sulfoximine (BSO), a specific inhibitor of GCL activity, would increase ovarian and granulosa cell GCL subunit expression. Ovarian glutathione levels are lowest on proestrous morning and increase to their highest levels on estrus and metestrus. Therefore, we treated rats on proestrous morning or on proestrous morning and again 12h later to prevent the normal increase in ovarian glutathione between proestrus and estrus. Ovarian Gclc and Gclm mRNA levels and GCLC protein levels increased transiently by 1.4-1.5-fold at 8 h, but not at 12 or 24 h, after a single dose of BSO administered to adult rats on the morning of proestrus. GCLC protein levels were also modestly increased 1.4-fold at 12 h after a second dose of BSO. GCLM protein levels increased 1.4-fold at 24 h after a single dose of BSO, but not at other time points. BSO treatment did not significantly alter ovarian GCL enzymatic activity or the intraovarian localization of either GCL subunit mRNA. Treatment of a human granulosa cell line or primary rat granulosa cells with BSO suppressed intracellular GSH; however, there was no compensatory upregulation of GCL subunit protein or mRNA levels. These results demonstrate that ovarian follicles and granulosa cells are minimally able to respond to acute GSH depletion by upregulating expression of GCL.

Asunto(s)

Butionina Sulfoximina/farmacología , Inhibidores Enzimáticos/farmacología , Glutamato-Cisteína Ligasa/biosíntesis , Glutatión/antagonistas & inhibidores , Ovario/efectos de los fármacos , Ovario/enzimología , Regulación hacia Arriba/efectos de los fármacos , Animales , Northern Blotting , Western Blotting , Células Cultivadas , Estradiol/metabolismo , Ciclo Estral/efectos de los fármacos , Femenino , Glutamato-Cisteína Ligasa/genética , Glutatión/farmacología , Células de la Granulosa/metabolismo , Hibridación in Situ , Tamaño de los Órganos/efectos de los fármacos , Progesterona/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa