RESUMEN
The migratory and matrix-invading capacities of the cumulus oocyte complex (COC) have been shown to be important for the ovulatory process. In metastatic cancers, these capacities are due to increased expression of proteases, however, there is limited information on protease expression in the COCs. The present study examined COC expression of plasmins, matrix metalloproteases (MMP) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family members in the rat and human. In the rat, hCG administration increased COC expression of Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, Adamts1, and the protease inhibitors Timp1, Timp3 and Serpine1 by 8-12 hours. This ovulatory induction of proteases in vivo could be mimicked by forskolin and ampiregulin treatment of cultured rat COCs with increases observed in Mmp2, Mmp13, Mmp14, Mmp16, Mmp19, Plat, and the protease inhibitors Timp1, Timp3 and Serpine1. Comparison of expression between rat COCs and granulosa cells at the time of ovulation showed decreased Mmp9 and increased Mmp13, Mmp14, Mmp16, Adamts1, Timp1 and Timp3 expression in the COCs. In human, comparison of expression between cumulus and granulosa cells at the time of IVF retrieval showed decreased MMP1, MMP2, MMP9, and ADAMTS1, while expression of MMP16, TIMP1, and TIMP3 were increased. Treatment of expanding rat COCs with a broad spectrum MMP inhibitor, GM6001, significantly reduced the migration of cumulus cells in vitro. These data provide evidence that multiple proteases and their inhibitors are expressed in the COCs and play an important role in imparting the migratory phenotype of the COCs at the time of ovulation.
RESUMEN
Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3', 5'-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1-500 µg/ml; dimethylsulfoxide = vehicle control) for 0.5-36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 µg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 µg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.
Asunto(s)
Progestinas , Receptores de Progesterona , Humanos , Femenino , Receptores de Progesterona/metabolismo , Progestinas/farmacología , Células de la Granulosa/metabolismo , Progesterona/farmacología , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/metabolismo , ARN Mensajero/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células CultivadasRESUMEN
The luteinizing hormone (LH) surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day-old pregnant mare serum gonadotropin primed mice before (0 h) and after administration of human chorionic gonadotropin (hCG; an LH analog) across the periovulatory period (4, 8, 12, and 24 h). In response to hCG, Nts expression rapidly increased 250-fold at 4 h, remained elevated until 8 h, and decreased until 24 h. Expression of Nts receptors for Ntsr1 remained unchanged across the periovulatory period, Ntsr2 was undetectable, whereas Sort1 expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Nts regulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Nts expression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Nts induction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Nts silencing approach (si-Nts) followed by RNA-sequencing (RNA-seq). RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a, and Smtnl2 as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Nts and that Nts expression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.
Asunto(s)
Neurotensina , Ovario , Ovulación , Animales , Femenino , Ratones , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/metabolismo , Células de la Granulosa/metabolismo , Caballos , Hormona Luteinizante/metabolismo , Neurotensina/genética , Neurotensina/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Ovulación/genética , Ovulación/fisiología , Factores de Elongación Transcripcional/metabolismoRESUMEN
Exposure to endocrine-disrupting chemicals (EDCs) is unavoidable, which represents a public health concern given the ability of EDCs to target the ovary. However, there is a large gap in the knowledge about the impact of EDCs on ovarian function, including the process of ovulation. Defects in ovulation are the leading cause of infertility in women, and EDC exposures are contributing to the prevalence of infertility. Thus, investigating the effects of EDCs on the ovary and ovulation is an emerging area for research and is the focus of this review. The effects of EDCs on gametogenesis, uterine function, embryonic development, and other aspects of fertility are not addressed to focus on ovarian- and ovulation-related fertility issues. Herein, findings from epidemiological and basic science studies are summarized for several EDCs, including phthalates, bisphenols, per- and poly-fluoroalkyl substances, flame retardants, parabens, and triclosan. Epidemiological literature suggests that exposure is associated with impaired fecundity and in vitro fertilization outcomes (decreased egg yield, pregnancies, and births), while basic science literature reports altered ovarian follicle and corpora lutea numbers, altered hormone levels, and impaired ovulatory processes. Future directions include identification of the mechanisms by which EDCs disrupt ovulation leading to infertility, especially in women.
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Disruptores Endocrinos , Infertilidad , Embarazo , Humanos , Femenino , Ovario , Disruptores Endocrinos/toxicidad , Fertilidad , OvulaciónRESUMEN
Phthalates are solvents and plasticizers found in consumer products including cosmetics, food/beverage containers, housing materials, etc. Phthalates are known endocrine-disrupting chemicals that can directly target the ovary, potentially causing defects in ovulation and fertility. Women are exposed to multiple different phthalates daily, therefore this study investigated the effects of an environmentally relevant phthalate mixture (PHTmix) on ovulation. Ovulation is initiated by the luteinizing hormone (LH) surge, which induces prostaglandin (PG) production, progesterone (P4)/progesterone receptor (PGR) signaling, and extracellular matrix (ECM) remodeling. We hypothesized that the PHTmix would directly inhibit ovulation by altering the levels of PGs, P4/PGR, and enzymes involved in ECM remodeling. Antral follicles from CD-1 mice were treated with vehicle control alone (dimethylsulfoxide, DMSO), hCG alone (LH analog), and hCG+PHTmix (1-500µg/ml), and samples were collected across the ovulatory period. The PHTmix decreased ovulation rates at all doses tested in a dose-dependent manner when compared to hCG. PG levels were decreased by the PHTmix when compared to hCG, which was potentially mediated by altered levels of PG synthesis (Ptgs2) and transport (Slco2a1) genes. The PHTmix altered P4 and Pgr levels when compared to hCG, leading to decreases in downstream PGR-mediated genes (Edn2, Il6, Adamts1). ECM remodeling was potentially dysregulated by altered levels of ovulatory mediators belonging to the matrix metalloproteases and plasminogen activator families. These data suggest that phthalate exposure inhibits ovulation by altering PG levels, P4/PGR action, and ECM remodeling.
Asunto(s)
Ovulación , Ácidos Ftálicos , Animales , Femenino , Hormona Luteinizante , Ratones , Folículo Ovárico , Ácidos Ftálicos/toxicidad , ProgesteronaRESUMEN
The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.
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Células de la Granulosa/metabolismo , Neovascularización Fisiológica/genética , Ovario/irrigación sanguínea , Ovulación/genética , Secretogranina II/genética , Adulto , Animales , Células Cultivadas , Femenino , Humanos , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Ovario/metabolismo , Ratas , Ratas Sprague-Dawley , Secretogranina II/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that has been shown to impair normal reproductive function in males and females. This study investigated whether adult exposure to environmental and occupational doses of DEHP alters homeostasis of uterine proliferation, morphology, and number of uterine glands. Adult female CD1 mice were orally dosed with DEHP (0, 20⯵g/kg/day, 200⯵g/kg/day, 20â¯mg/kg/day or 200â¯mg/kg/day) for 30â¯days. Results indicated that DEHP at 200⯵g/kg/day caused a reduction in epithelial cell proliferation in the uterus (pâ¯<â¯.05). We also observed an increase (pâ¯<â¯.05) in the number of uterine glands in mice dosed with 200â¯mg/kg/day DEHP. Results showed that DEHP caused an increase (pâ¯<â¯.05) in dilated blood vessels in the endometrium at 200⯵g/kg/day, 20â¯mg/kg/day and 200â¯mg/kg/day. DEHP also increased proliferation of endometrial stromal cells at 200⯵g/kg/day DEHP (pâ¯<â¯.0010), 20â¯mg/kg/day DEHP (pâ¯<â¯.0001) and 200â¯mg/kg/day DEHP (pâ¯<â¯.0186). Results suggest that, exposure to specific doses of DEHP for 30â¯days can have adverse effects on reproductive function.
Asunto(s)
Dietilhexil Ftalato/toxicidad , Disruptores Endocrinos/toxicidad , Plastificantes/toxicidad , Útero/efectos de los fármacos , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Estradiol/sangre , Femenino , Ratones , Progesterona/sangre , Células del Estroma/efectos de los fármacos , Útero/irrigación sanguínea , Vasodilatación/efectos de los fármacosRESUMEN
Ovulation is dependent upon numerous factors mediating follicular growth, vascularization, and ultimately oocyte release via follicle rupture. Endothelin-2 (EDN2) is a potent vasoconstrictor that is transiently produced prior to follicle rupture by granulosa cells of periovulatory follicles and induces ovarian contraction. To determine the role of Edn2 expression, surgical transplant and novel conditional knockout mice were super-ovulated and analyzed. Conditional knockout mice utilized a new iCre driven by the Esr2 promoter to selectively remove Edn2. Follicle rupture and fertility were significantly impaired in the absence of ovarian Edn2 expression. When ovaries of Edn2KO mice were transplanted in wild type recipients, significantly more corpora lutea containing un-ovulated oocytes were present after hormonal stimulation (1.0 vs. 5.4, p = 0.010). Following selective ablation of Edn2 in granulosa cells, Esr2-Edn2KO dams had reduced oocytes ovulated (3.8 vs. 16.4 oocytes/ovary) and smaller litters (4.29 ± l.02 vs. 8.50 pups/dam). However, the number of pregnancies per pairing was not different and the reproductive axis remained intact. Esr2-Edn2KO ovaries had a higher percentage of antral follicles and fewer corpora lutea; follicles progressed to the antral stage but many were unable to rupture. Conditional loss of endothelin receptor A in granulosa cells also decreased ovulation but did not affect fecundity. These data demonstrate that EDN2-induced intraovarian contraction is a critical trigger of normal ovulation and subsequent fecundity.
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Endotelina-2/genética , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Ovulación , Animales , Endotelina-2/metabolismo , Femenino , Fertilidad , Ratones , Ratones Endogámicos C57BL , Folículo Ovárico/citología , Folículo Ovárico/fisiología , Receptor de Endotelina A/metabolismoRESUMEN
Context: In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. Objective: To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Design and Participants: Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures: The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. Results: PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. Conclusions: This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.
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Anfirregulina/genética , Ciclooxigenasa 2/genética , Transportadores de Anión Orgánico/genética , Folículo Ovárico/metabolismo , Progesterona/metabolismo , Prostaglandinas/metabolismo , Receptores de Progesterona/genética , Adulto , Anfirregulina/metabolismo , Western Blotting , Células Cultivadas , Gonadotropina Coriónica/farmacología , Ciclooxigenasa 2/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Fertilización In Vitro , Perfilación de la Expresión Génica , Células de la Granulosa , Humanos , Inmunohistoquímica , Células Lúteas , Hormona Luteinizante , Transportadores de Anión Orgánico/efectos de los fármacos , Transportadores de Anión Orgánico/metabolismo , Ovulación , Reacción en Cadena de la Polimerasa , Prostaglandina-E Sintasas/efectos de los fármacos , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , ARN Mensajero/metabolismo , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/metabolismoRESUMEN
Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the Pgr gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the Pgr deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.
Asunto(s)
Fertilidad , Kisspeptinas/genética , Neuronas/metabolismo , Receptores de Progesterona/metabolismo , Animales , Estradiol/farmacología , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/metabolismo , Ciclo Estral/efectos de los fármacos , Femenino , Fertilidad/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo Anterior/efectos de los fármacos , Hipotálamo Anterior/metabolismo , Inmunohistoquímica , Kisspeptinas/metabolismo , Hormona Luteinizante/sangre , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Humans are ubiquitously exposed to di(2-ethylhexyl) phthalate (DEHP), which is an environmental toxicant incorporated in consumer products. Studies have shown that DEHP targets the ovary to disrupt essential processes required for reproductive and nonreproductive health. Specifically, 10-day exposure to DEHP accelerates primordial follicle recruitment and disrupts estrous cyclicity in adult mice. However, it is unknown if these effects on folliculogenesis and cyclicity following acute DEHP exposure can have permanent effects on reproductive outcomes. Further, the premature depletion of primordial follicles can cause early reproductive senescence, and it is unknown if acute DEHP exposure accelerates reproductive aging. This study tested the hypothesis that acute DEHP exposure causes infertility, disrupts estrous cyclicity, alters hormone levels, and depletes follicle numbers by inducing atresia later in life, leading to accelerated reproductive aging. Adult CD-1 mice were orally dosed with vehicle or DEHP (20 µg/kg/day-500 mg/kg/day) daily for 10 days, and reproductive outcomes were assessed at 6 and 9 months postdosing. Acute DEHP exposure significantly altered estrous cyclicity compared to controls at 6 and 9 months postdosing by increasing the percentage of days the mice were in estrus and metestrus/diestrus, respectively. DEHP also significantly decreased inhibin B levels compared to controls at 9 months postdosing. Further, DEHP significantly increased the BAX/BCL2 ratio in primordial follicles leading to a significant decrease in primordial and total follicle numbers compared to controls at 9 months postdosing. Collectively, the adverse effects present following acute DEHP exposure persist later in life and are consistent with accelerated reproductive aging.
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Envejecimiento/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Contaminantes Ambientales/toxicidad , Ciclo Estral/efectos de los fármacos , Fertilidad/efectos de los fármacos , Infertilidad Femenina/inducido químicamente , Animales , Relación Dosis-Respuesta a Droga , Femenino , Atresia Folicular/efectos de los fármacos , Infertilidad Femenina/metabolismo , Masculino , Ratones Endogámicos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/patología , EmbarazoRESUMEN
Humans are ubiquitously exposed to di(2-ethylhexyl) phthalate (DEHP), which is an environmental toxicant present in common consumer products. DEHP potentially targets the ovary through its metabolite mono(2-ethylhexyl) phthalate (MEHP). However, the direct effects of MEHP on ovarian folliculogenesis and steroidogenesis, two processes essential for reproductive and nonreproductive health, are unknown. The present study tested the hypotheses that MEHP directly accelerates early folliculogenesis via overactivation of phosphatidylinositol 3-kinase (PI3K) signaling, a pathway that regulates primordial follicle quiescence and activation, and inhibits the synthesis of steroid hormones by decreasing steroidogenic enzyme levels. Neonatal ovaries from CD-1 mice were cultured for 6 days with vehicle control, DEHP, or MEHP (0.2-20 µg/ml) to assess the direct effects on folliculogenesis and PI3K signaling. Further, antral follicles from adult CD-1 mice were cultured with vehicle control or MEHP (0.1-10 µg/ml) for 24-96 h to establish the temporal effects of MEHP on steroid hormones and steroidogenic enzymes. In the neonatal ovaries, MEHP, but not DEHP, decreased phosphatase and tensin homolog levels and increased phosphorylated protein kinase B levels, leading to a decrease in the percentage of germ cells and an increase in the percentage of primary follicles. In the antral follicles, MEHP decreased the mRNA levels of 17alpha-hydroxylase-17,20-desmolase, 17beta-hydroxysteroid dehydrogenase, and aromatase leading to a decrease in testosterone, estrone, and estradiol levels. Collectively, MEHP mediates the effect of DEHP on accelerated folliculogenesis via overactivating PI3K signaling and inhibits steroidogenesis by decreasing steroidogenic enzyme levels.
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Dietilhexil Ftalato/análogos & derivados , Dietilhexil Ftalato/farmacología , Folículo Ovárico/fisiología , Animales , Animales Recién Nacidos , Dietilhexil Ftalato/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/genética , Hormonas Esteroides Gonadales/metabolismo , RatonesRESUMEN
Phthalates are commonly used as plasticizers in the manufacturing of flexible polyvinyl chloride products. Large production volumes of phthalates and their widespread use in common consumer, medical, building, and personal care products lead to ubiquitous human exposure via oral ingestion, inhalation, and dermal contact. Recently, several phthalates have been classified as reproductive toxicants and endocrine-disrupting chemicals based on their ability to interfere with normal reproductive function and hormone signaling. Therefore, exposure to phthalates represents a public health concern. Currently, the effects of phthalates on male reproduction are better understood than the effects on female reproduction. This is of concern because women are often exposed to higher levels of phthalates than men through their extensive use of personal care and cosmetic products. In the female, a primary regulator of reproductive and endocrine function is the ovary. Specifically, the ovary is responsible for folliculogenesis, the proper maturation of gametes for fertilization, and steroidogenesis, and the synthesis of necessary sex steroid hormones. Any defect in the regulation of these processes can cause complications for reproductive and non-reproductive health. For instance, phthalate-induced defects in folliculogenesis and steroidogenesis can cause infertility, premature ovarian failure, and non-reproductive disorders. Presently, there is a paucity of knowledge on the effects of phthalates on normal ovarian function; however, recent work has established the ovary as a target of phthalate toxicity. This review summarizes what is currently known about the effects of phthalates on the ovary and the mechanisms by which phthalates exert ovarian toxicity, with a particular focus on the effects on folliculogenesis and steroidogenesis. Further, this review outlines future directions, including the necessity of examining the effects of phthalates at doses that mimic human exposure.
RESUMEN
Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant found in consumer products that causes ovarian toxicity. Antral follicles are the functional ovarian units and must undergo growth, survival from atresia, and proper regulation of steroidogenesis to ovulate and produce hormones. Previous studies have determined that DEHP inhibits antral follicle growth and decreases estradiol levels in vitro; however, the mechanism by which DEHP elicits these effects is unknown. The present study tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and steroidogenesis to inhibit antral follicle functionality. Antral follicles from adult CD-1 mice were cultured with vehicle control or DEHP (1-100 µg/ml) for 24-96 h to establish the temporal effects of DEHP on the follicle. Following 24-96 h of culture, antral follicles were subjected to gene expression analysis, and media were subjected to measurements of hormone levels. DEHP increased the mRNA levels of cyclin D2, cyclin dependent kinase 4, cyclin E1, cyclin A2, and cyclin B1 and decreased the levels of cyclin-dependent kinase inhibitor 1A prior to growth inhibition. Additionally, DEHP increased the mRNA levels of BCL2-associated agonist of cell death, BCL2-associated X protein, BCL2-related ovarian killer protein, B-cell leukemia/lymphoma 2, and Bcl2-like 10, leading to an increase in atresia. Further, DEHP decreased the levels of progesterone, androstenedione, and testosterone prior to the decrease in estradiol levels, with decreased mRNA levels of side-chain cleavage, 17α-hydroxylase-17,20-desmolase, 17ß-hydroxysteroid dehydrogenase, and aromatase. Collectively, DEHP directly alters antral follicle functionality by inhibiting growth, inducing atresia, and inhibiting steroidogenesis.
Asunto(s)
Dietilhexil Ftalato/toxicidad , Disruptores Endocrinos/toxicidad , Atresia Folicular , Hormonas Esteroides Gonadales/metabolismo , Folículo Ovárico/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Medios de Cultivo Condicionados/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica , Ratones , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Folículo Ovárico/fisiopatología , ARN Mensajero/metabolismo , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Humans are exposed daily to di(2-ethylhexyl) phthalate (DEHP), a plasticizer found in many consumer, medical, and building products containing polyvinyl chloride. Large doses of DEHP disrupt normal ovarian function; however, the effects of DEHP at environmentally relevant levels, the effects of DEHP on folliculogenesis, and the mechanisms by which DEHP disrupts ovarian function are unclear. The present study tested the hypothesis that relatively low levels of DEHP disrupt estrous cyclicity as well as accelerate primordial follicle recruitment by dysregulating phosphatidylinositol 3-kinase (PI3K) signaling. Adult CD-1 mice were orally dosed with DEHP (20 µg/kg/day-750 mg/kg/day) daily for 10 and 30 days. Following dosing, the effects on estrous cyclicity were examined, and follicle numbers were histologically quantified. Further, the ovarian mRNA and protein levels of PI3K signaling factors that are associated with early folliculogenesis were quantified. The data indicate that 10- and 30-day exposure to DEHP prolonged the duration of estrus and accelerated primordial follicle recruitment. Specifically, DEHP exposure decreased the percentage of primordial follicles and increased the percentage of primary follicles counted following 10-day exposure and increased the percentage of primary follicles counted following 30-day exposure. DEHP exposure, at doses that accelerate folliculogenesis, increased the levels of 3-phosphoinositide-dependent protein kinase-1, mammalian target of rapamycin complex 1, and protein kinase B and decreased the levels of phosphatase and tensin homolog, potentially driving PI3K signaling. Collectively, relatively low levels of DEHP disrupt estrous cyclicity and accelerate primordial follicle recruitment potentially via a mechanism involving dysregulation of PI3K signaling.
Asunto(s)
Dietilhexil Ftalato/toxicidad , Ciclo Estral/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Transducción de Señal/fisiología , Factores de Edad , Animales , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/toxicidad , Femenino , Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos , Folículo Ovárico/metabolismo , Plastificantes/toxicidadRESUMEN
Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P4) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 µg/mL), pregnenolone (1 µg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P4, androstenedione (A), testosterone (T), estrone (E1), and 17ß-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.
Asunto(s)
Atresia Folicular/efectos de los fármacos , Inhibidores de Crecimiento/toxicidad , Insecticidas/toxicidad , Metoxicloro/análogos & derivados , Pregnenolona/administración & dosificación , Animales , Células Cultivadas , Femenino , Atresia Folicular/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/antagonistas & inhibidores , Humanos , Insecticidas/administración & dosificación , Metoxicloro/administración & dosificación , Metoxicloro/toxicidad , Ratones , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Resultado del TratamientoRESUMEN
Di-n-butyl phthalate (DBP) is present in many consumer products, such as infant, beauty, and medical products. Several studies have shown that DBP causes reproductive toxicity in rodents, but no studies have evaluated its effects on ovarian follicles. Therefore, we used a follicle culture system to evaluate the effects of DBP on antral follicle growth, cell cycle and apoptosis gene expression, cell cycle staging, atresia, and 17ß-estradiol (E(2)) production. Antral follicles were isolated from adult CD-1 mice and exposed to DBP at 1, 10, 100, and 1000 µg/ml for 24 or 168 h. Follicles treated with vehicle or DBP at 1-100 µg/ml grew over time, but DBP at 1000 µg/ml significantly suppressed follicle growth. Regardless of effect on follicle growth, DBP-treated follicles had decreased mRNA for cyclins D2, E1, A2, and B1 and increased p21. Levels of the proapoptotic genes Bax, Bad, and Bok were not altered by DBP treatment, but DBP 1000 µg/ml increased levels of Bid and decreased levels of the antiapoptotic gene Bcl2. DBP-treated follicles contained significantly more cells in G(1) phase, significantly less cells in S, and exhibited a trend for fewer cells in G(2). Although DBP did not affect E(2) production and atresia at 24 h, follicles treated with DBP had reduced levels of E(2) at 96 h and underwent atresia at 168 h. These data suggest that DBP targets antral follicles and alters the expression of cell cycle and apoptosis factors, causes cell cycle arrest, decreases E(2), and triggers atresia, depending on dose.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Dibutil Ftalato/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/toxicidad , Femenino , Ratones , Folículo Ovárico/citología , Plastificantes/toxicidadRESUMEN
Methoxychlor (MXC) and its metabolites bind to estrogen receptors (ESRs) and increase ovarian atresia. To test whether ESR alpha (ESR1) overexpressing (ESR1 OE) antral follicles are more sensitive to atresia compared to controls, we cultured antral follicles with vehicle, MXC (1-100 µg/ml) or metabolites (0.1-10 µg/ml). Results indicate that MXC and its metabolites significantly increase atresia in ESR1 OE antral follicles at lower doses compared to controls. Activity of pro-apoptotic factor caspase-3/7 was significantly higher in ESR1 OE treated antral follicles compared to controls. ESR1 OE mice dosed with MXC 64 mg/kg/day had an increased percentage of atretic antral follicles compared to controls. Furthermore, pro-caspase-3 levels were found to be significantly lower in ESR1 OE ovaries than controls dosed with MXC 64 mg/kg/day. These data suggest that ESR1 OE ovaries are more sensitive to atresia induced by MXC and its metabolites in vitro and in vivo compared to controls.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Atresia Folicular/metabolismo , Insecticidas/toxicidad , Metoxicloro/toxicidad , Folículo Ovárico/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Femenino , Atresia Folicular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Folículo Ovárico/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
The purpose of this study was to determine the proportion of available passive frontal plane rearfoot motion that is used during the stance phase of walking. Data were collected from 40 healthy, asymptomatic volunteer subjects (20 men and 20 women) aged 23 to 44 years. Passive inversion and eversion motion was measured in a non-weightbearing position by manually moving the calcaneus. Dynamic rearfoot motion was referenced to a vertical calcaneus and tibia and was measured using a three-dimensional electromagnetic motion-analysis system. The results indicated that individuals used 68.1% of their available passive eversion range of motion and 13.2% of their available passive inversion range of motion during walking. The clinical implication of individuals' regularly operating at or near the end point of their available rearfoot eversion range of motion is discussed.