RESUMEN
STUDY QUESTION: Do alterations in pro- and anti-angiogenic estrogen metabolites in follicular fluid (FF) contribute to the follicular growth arrest and anovulation associated with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: FF of PCOS women with anovulation have reduced levels of pro-angiogenic estrogen metabolites (EMs) and vascular endothelial growth factor (VEGF) compared to that of fertile women with regular menstrual cycles, but exogenous gonadotropins increase the pro-angiogenic EMs and VEGF levels in PCOS women. WHAT IS KNOWN ALREADY: PCOS is characterized by the arrest of follicular development that leads to chronic anovulation. Follicular arrest is generally associated with elevated plasma levels of luteinizing hormone (LH), androgens and anti-Mullerian hormone (AMH). There is also reduced angiogenesis in the follicles of PCOS women compared to those of normal cycling women. It is known that angiogenesis is a critical factor during follicular development. We and other investigators have explored the role of EMs in ovarian angiogenesis, particularly in human corpus luteum function, showing that 4-hydroxyestrone (4-OHE1) and 16-ketoestradiol (16-kE2) have pro-angiogenic effects while 2-methoxyestradiol (2-ME2) and 2-methoxyestrone (2-ME1) have anti-angiogenic effects. Additionally, 2-hydroxyestradiol (2-OHE2), which is produced in the ovary, has proliferative and pro-angiogenic properties. We hypothesized that EMs could be involved in angiogenesis necessary for ovarian follicular development in fertile women, and that dysregulation of these factors may contribute to follicular arrest in PCOS. The relationship between EMs, VEGF and AMH in the pathophysiology of follicular arrest in PCOS has not been previously studied at a follicular level in anovulatory women without ovulation induction. STUDY DESIGN, SIZE, DURATION: This is a comparative experimental study of serum and FF collected from different sized follicles (antral Ë10 mm and dominant Ë16 mm) of women with and without ovarian stimulation. The study included women with regular menstrual cycles who were proven to be fertile (n = 20) and PCOS women with follicular arrest who were candidates for ovarian drilling (n = 17), as well as other patients requiring ovarian stimulation, i.e. control women undergoing IVF for male factor infertility (n = 12) and PCOS women undergoing IVF (n = 17). In vitro studies were carried out on granulosa-lutein cells (GCs) obtained from subsets of women undergoing IVF for male factor infertility (n = 6) and PCOS women undergoing IVF (n = 6). GCs were maintained in culture for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Intrafollicular estradiol, estrone and EMs concentrations were determined by high performance liquid chromatography-mass spectrometry. Testosterone in serum was measured by RIA, and LH, FSH and sex hormone-binding globulin in serum were measured with IRMA kits. AMH was determined in serum and FF by enzyme linked immunosorbant assay (ELISA). VEGF levels were measured in FF and conditioned medium by ELISA. Conditioned medium were obtained from cultured GCs. The angiogenic potential was assessed by in vitro angiogenic assays. MAIN RESULTS AND THE ROLE OF CHANCE: Pro-angiogenic EMs (4-OHE1, 16-kE2 and 2-OHE2) and VEGF were lower in FF of antral follicles of PCOS women with follicular arrest compared those of fertile women with ovulatory cycles (P < 0.05). In contrast, higher concentrations of AMH were found in FF of antral follicles from PCOS women with follicular arrest compared to those of fertile women with ovulatory cycles (P < 0.05). Exogenous gonadotropins used in IVF increased pro-angiogenic EMs and VEGF production in PCOS women, reaching similar profiles compared to control women receiving gonadotropins in their IVF treatment for male factor infertility. The pro-angiogenic EM 2-OHE2 increased the angiogenic potential and VEGF levels of GCs from PCOS women compared to the basal condition (P < 0.05). These findings suggest that there is a role for pro-angiogenic EMs in the control of follicular VEGF production. LIMITATIONS, REASONS FOR CAUTION: The limitations include the possibility that in vitro analysis of GCs might not reflect the in vivo mechanisms involved in the pro-angiogenic action of 2-OHE2 since GCs obtained at the time of oocyte retrieval belong to a very early stage of the luteal phase and might not be representative of GCs during follicular growth. Therefore, our findings do not conclusively rule out the possibility that other in vivo mechanisms also account for defective angiogenesis observed in PCOS. WIDER IMPLICATIONS OF THE FINDINGS: The present study highlights the significance of EMs, angiogenic factors and AMH and their interaction in the pathophysiology of follicular development in PCOS. This study provides new insights into the role of pro-angiogenic factors in follicular arrest in PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.
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Síndrome del Ovario Poliquístico , Hormona Antimülleriana , Estrógenos , Femenino , Líquido Folicular , Humanos , Masculino , Factor A de Crecimiento Endotelial VascularRESUMEN
An estradiol metabolite, 2-methoxyestradiol (2ME), has emerged as an important regulator of ovarian physiology. 2ME is recognized as a potent anti-angiogenic agent in clinical trials and laboratory studies. However, little is known about its molecular actions and its endogenous targets. 2ME is produced by human ovarian cells during the normal menstrual cycle, being higher during regression of the corpus luteum, and is postulated to be involved in the anti-angiogenic process that plays out during luteolysis. We utilized cell biology techniques to understand the molecular mechanism of 2ME anti-angiogenic effects on human granulosa luteal cells. The principal effect of 2ME was to alter Hypoxia Inducible Factor 1A (HIF1A) sub-cellular localization. Molecular modelling and multiple bioinformatics tools indicated that 2ME impairs Hypoxia Inducible Factor complex (HIF) nuclear translocation by binding to a buried pocket in the HIF1A Per Arnt Sim (PAS)-B domain. Binding of 2ME to HIF1A protein is predicted to perturb HIF1A-Hypoxia Inducible Factor B (HIFB) interaction, a key step in HIF nuclear translocation, preventing the transcriptional actions of HIF, including Vascular Endotelial Growth Factor (VEGF) gene activation. To our knowledge, 2ME is the first putative HIF endogenous ligand characterized with anti-angiogenic activity. This postulate has important implications for reproduction, because angiogenic processes are critical for ovarian follicular development, ovulation and corpus luteum regression. The present research could contribute to the development of novel pharmacological approaches for controlling HIF activity in human reproductive diseases.
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2-Metoxiestradiol/metabolismo , 2-Metoxiestradiol/farmacología , Biología Computacional , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Simulación de Dinámica Molecular , Línea Celular , Femenino , Humanos , Células Lúteas/efectos de los fármacos , Células Lúteas/metabolismo , Unión Proteica , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de ProteínaRESUMEN
In humans, data on gonadotrophin-activated (LH, HCG and FSH) progesterone receptor expression and signalling pathways involved in matrix metalloproteinases (MMPs) expression presumably linked to the follicle rupture, are limited. Our hypothesis is LH, HCG and FSH increase progesterone receptor expression in granulosa cells through different signalling pathways, leading to an increased expression of ADAMTS-1 and MMP3/10, which may mediate follicular rupture through the transcription factor, HIF1A. Human granulosa cells were isolated from follicular aspirates obtained from 22 healthy women participating in our IVF programme for male-factor infertility. Progesterone receptor and HIF1A expression was assessed by immunofluorescence, and PKA-PKC-PI3K- ERK1/2, ADAMTS-1 and MMP3/10 expression by Western blot in pre-ovulatory and in cultured granulosa cells. Results show that HCG, LH and FSH regulate progesterone receptor expression and activate PKA, PKC, PI3K and ERK1/2 signalling pathways in granulosa cells but progesterone receptor expression is only mediated by PKA, PKC and ERK pathways. HCG, FSH and LH regulated MMPs expression through progesterone receptors. Moreover, HCG-progesterone-receptor-dependent HIF1A expression stimulated MMP3/10 expression but not that of ADAMTS-1. These results suggest differential downstream progesterone receptor signalling, as progesterone receptor regulates MMP3/10 expression via HIF1A, which is not involved in ADAMTS-1 expression.
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Gonadotropinas Hipofisarias/metabolismo , Células de la Granulosa/metabolismo , Receptores de Progesterona/metabolismo , Transducción de Señal , Proteínas ADAMTS/metabolismo , Adulto , Células Cultivadas , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Infertilidad Masculina/terapia , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Inducción de la Ovulación , Adulto JovenRESUMEN
OBJECTIVE: To determine tissue concentrations of E2, estrone, P, and estrogens metabolites (EMs) 2-methoxyestradiol, 2-methoxyestrone, 4-hydroxyestrone, and 16-ketoestradiol in corpus luteum (CL) of different ages, and after hCG administration; and to examine the effects of EMs on vascular endothelial growth factor (VEGF) secretion and angiogenic activity released by cultured luteinizing granulosa cells in the presence and absence of hCG. DESIGN: Experimental study. SETTING: University. PATIENT(S): Thirty-two healthy women of reproductive age. INTERVENTION(S): Corpus luteum was collected at the time of minilaparotomy for tubal sterilization, at varying stages of the luteal phase (LP). Late-LP CL was collected 24 hours after IM administration of 10,000 IU hCG. Granulosa cells were isolated from follicular aspirates obtained from healthy women participating in our IVF program for male factor infertility. MAIN OUTCOMES MEASURE(S): Estrogen metabolite concentrations were determined in CL tissue, and VEGF was assessed in conditioned medium. The angiogenic activity was analyzed by bioassay. RESULT(S): Concentrations of EMs with proangiogenic activity (16-ketoestradiol and 4-hydroxyestrone) were higher in early and mid-LP CL vs. late-LP CL. These EMs and hCG increased VEGF production and angiogenic activity. Conversely, late-LP CL had significantly higher levels of 2-methoxyestrone and 2-methoxyestradiol, which have antiangiogenic activity. Administration of hCG reduced the production of these EMs. CONCLUSION(S): Our findings suggest that the EMs are important paracrine modulators of CL function. Administration of hCG increases the production of EMs with proangiogenic activity and reduces the secretion of those EMs with antiangiogenic action, suggesting a novel mechanism by which the late-LP CL is rescued in conception cycles.
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Cuerpo Lúteo/irrigación sanguínea , Cuerpo Lúteo/metabolismo , Estrógenos/metabolismo , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismo , 2-Metoxiestradiol , Biotransformación , Línea Celular , Gonadotropina Coriónica/farmacología , Cuerpo Lúteo/efectos de los fármacos , Células Endoteliales/metabolismo , Estradiol/análogos & derivados , Estradiol/metabolismo , Estrona/metabolismo , Femenino , Células de la Granulosa/metabolismo , Voluntarios Sanos , Humanos , Hidroxiestronas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Progesterona/metabolismoRESUMEN
Single nucleotide polymorphisms (SNPs) in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene are associated with preeclampsia (PE) in different populations. rs2549782, a coding variant (N392K) that significantly affects substrate specificity, is in linkage disequilibrium (LD) with rs2248374, a marker SNP associated with ERAP2 protein expression in previously studied populations. As a result of non-sense mediated RNA decay, ERAP2 protein is not expressed from the rs2248374 G allele. We previously reported that the fetal rs2549782 minor G allele is associated with PE in African-Americans, but not Chileans. In this study, we found that rs2549782 was in LD with rs2248374 in African-Americans, but not in Chileans. The unexpected lack of strong LD in Chileans raised the possibility that rs2248374 could be associated with PE in the absence of an association with rs2549782. However, we found no significant association for this allele with PE in Chileans. Chileans homozygous for the rs2248374 G allele did not express 110 kDa ERAP2 protein, consistent with non-sense mediated RNA decay, and carriers of the rs2248374 A allele did. We conclude that the Chilean ERAP2 haplotype structure allows for the expression of the major T allele of rs2549782 encoding 392N, which could impact peptide trimming and antigen presentation. Our discovery of racial differences in genetic structure and association with PE reveal here-to-fore unrecognized complexity of the ERAP2 locus.
RESUMEN
OBJECTIVE: To quantitate 2-methoxyestradiol (2-ME) in human corpus luteum (CL) of different ages and to determine the expression of cytochrome-P450-1A1 (CYP1A1) and catechol-O-methyl transferase (COMT) in CL and the action of 2-ME on P, vascular endothelial growth factor (VEGF) secretion, and luteal angiogenesis. DESIGN: Experimental study. SETTING: University division of reproductive endocrinology. PATIENT(S): Twenty-four women of reproductive age. INTERVENTION(S): CL was collected from 15 women during the minilaparotomy for tubal sterilization. Granulosa lutein cells were harvested 36 hours after hCG administration in patients undergoing IVF. MAIN OUTCOMES MEASURE(S): Levels of 2-ME were determined by high-performance liquid chromatography in CL. CYP1A1 and COMT were assessed by immunohistochemistry and Western blot. P and VEGF were measured by radioimmunoassay and ELISA. The angiogenic potential was analyzed using EA.hy926 cells. RESULT(S): Plasma levels of E2 decreased in the late luteal phase in association with an increase in luteal tissue of 2-ME concentrations. Concomitantly, there was a significant reduction of angiogenic activity in late CL. There was no significant variation in CYP1A1 and COMT expression in all CL. In physiological doses, 2-ME inhibited basal VEGF by granulosa lutein cells and diminished the angiogenic activity in conditioned media but did not prevent P and VEGF production stimulated by hCG. CONCLUSION(S): These data suggest the participation of 2-ME in physiological luteolysis by reducing angiogenesis. However, 2-ME did not prevent in vitro hCG stimulation of P biosynthesis, providing a mechanism for CL rescue in the cycle of conception.
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Cuerpo Lúteo/irrigación sanguínea , Cuerpo Lúteo/metabolismo , Estradiol/análogos & derivados , Células Lúteas/metabolismo , Fase Luteínica/metabolismo , Neovascularización Fisiológica , Progesterona/biosíntesis , 2-Metoxiestradiol , Adulto , Catecol O-Metiltransferasa/metabolismo , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Estradiol/sangre , Estradiol/metabolismo , Femenino , Humanos , Fase Luteínica/sangre , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. DESIGN: Experimental study. SETTING: University research unit. PATIENT(S): Twenty-five women of reproductive age. INTERVENTION(S): Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. MAIN OUTCOME MEASURE(S): To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. RESULT(S): The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. CONCLUSION(S): The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation.
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Catepsina L/metabolismo , Células de la Granulosa/enzimología , Ovulación , Receptores de Progesterona/metabolismo , Adulto , Western Blotting , Catepsina L/genética , Células Cultivadas , Gonadotropina Coriónica/administración & dosificación , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Femenino , Fármacos para la Fertilidad Femenina/administración & dosificación , Líquido Folicular/metabolismo , Células de la Granulosa/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Humanos , Ovulación/efectos de los fármacos , Inducción de la Ovulación , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/genética , Factores de TiempoRESUMEN
BACKGROUND: Preeclampsia affects 3-8% of pregnancies and is a major cause of maternal and perinatal morbidity and mortality worldwide. This complex disorder is characterized by alterations in the immune and vascular systems and involves multiple organs. There is strong evidence for a genetic contribution to preeclampsia. Two different single nucleotide polymorphisms (SNPs) in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene were recently reported to be associated with increased risk for preeclampsia in two different populations. ERAP2 is expressed in placental tissue and it is involved in immune responses, inflammation, and blood pressure regulation; making it is an attractive preeclampsia candidate gene. Furthermore, ERAP2 expression is altered in first trimester placentas of women destined to develop preeclampsia. METHODS: A case-control design was used to test for associations between two SNPs in ERAP2, rs2549782 and rs17408150, and preeclampsia status in 1103 Chilean maternal-fetal dyads and 1637 unpaired African American samples (836 maternal, 837 fetal). RESULTS: We found that the fetal minor allele (G) of rs2549782 was associated with an increased risk for preeclampsia in the African American population (P = 0.009), but not in the Chilean population. We found no association between rs17408150 and risk for preeclampsia in the Chilean population. Association between rs17408150 and risk for preeclampsia was not tested in the African American population due to the absence of the minor allele in this population. CONCLUSIONS: We report an association between fetal ERAP2 and preeclampsia in an African American population. In conjunction with previous studies, which have found maternal associations with this gene in an Australian/New Zealand population and a Norwegian population, ERAP2 has now been associated with preeclampsia in three populations. This provides strong evidence that ERAP2 plays a role in the development of preeclampsia.
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Aminopeptidasas/genética , Negro o Afroamericano/genética , Feto/enzimología , Polimorfismo de Nucleótido Simple/genética , Preeclampsia/genética , Adulto , Estudios de Casos y Controles , Chile , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , EmbarazoRESUMEN
The human corpus luteum is a temporary endocrine gland that develops after ovulation from the ruptured follicle during the luteal phase. It is an important contributor of steroid hormones, particularly progesterone, and is critical for the maintenance of early pregnancy. Luteal-phase dysfunction can result in premature regression of the gland, with a subsequent shift to an infertile cycle. Understanding the mechanism of steroidogenesis during corpus luteum growth and regression is crucial for evaluating the normal physiology and pathophysiology of reproductive cycles. The rate-limiting step in corpus luteum steroidogenesis is the transport of cholesterol to the site of steroid production. Steroidogenic acute regulatory protein is a key player in this process and is positively correlated with progesterone concentrations throughout the early and mid-luteal phase. Changes in the endocrine environment brought on by the gonadotrophins used for ovarian stimulation are thought to underlie the corpus luteum dysfunction associated with IVF cycles. While ovarian hyperstimulation syndrome is associated with human chorionic gonadotrophin (HCG), studies suggest that exogenous progesterone provides necessary luteal support in patients undergoing IVF. The current trend towards simple stimulation protocols and the use of single-embryo transfers provide further opportunity to revisit HCG administration as luteal support.
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Cuerpo Lúteo/fisiopatología , Fase Luteínica/fisiología , Inducción de la Ovulación , Gonadotropina Coriónica/uso terapéutico , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/patología , Femenino , Fertilización In Vitro , Hormonas Esteroides Gonadales/biosíntesis , Humanos , Infertilidad Femenina/patología , Fase Luteínica/efectos de los fármacos , Luteinización/efectos de los fármacos , Luteinización/fisiología , Luteólisis/fisiología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Folículo Ovárico/fisiopatología , Sustancias para el Control de la Reproducción/uso terapéuticoRESUMEN
CONTEXT: The natural process of luteolysis and luteal regression is induced by withdrawal of gonadotropin support. OBJECTIVE: The objectives of this study were: 1) to compare the functional changes and apoptotic features of natural human luteal regression and induced luteal regression; 2) to define the ultrastructural characteristics of the corpus luteum at the time of natural luteal regression and induced luteal regression; and 3) to examine the effect of human chorionic gonadotropin (hCG) on the steroidogenic response and apoptotic markers within the regressing corpus luteum. DESIGN: Twenty-three women with normal menstrual cycles undergoing tubal ligation donated corpus luteum at specific stages in the luteal phase. Some women received a GnRH antagonist prior to collection of corpus luteum, others received an injection of hCG with or without prior treatment with a GnRH antagonist. MAIN OUTCOME MEASURE: Main outcome measures were plasma hormone levels and analysis of excised luteal tissue for markers of apoptosis, histology, and ultrastructure. RESULTS: The progesterone and estradiol levels, corpus luteum DNA, and protein contents in induced luteal regression resembled those of natural luteal regression. hCG treatment raised progesterone and estradiol in both natural luteal regression and induced luteal regression. The increase in apoptosis detected in induced luteal regression by cytochrome c in the cytosol, activated caspase-3, and nuclear DNA fragmentation, was similar to that observed in natural luteal regression. The antiapoptotic protein Bcl-2 was significantly lower during natural luteal regression. The proapoptotic proteins Bax and Bak were at a constant level. Apoptotic and nonapoptotic death of luteal cells was observed in natural luteal regression and induced luteal regression at the ultrastructural level. hCG prevented apoptotic cell death, but not autophagy. CONCLUSION: The low number of apoptotic cells disclosed and the frequent autophagocytic suggest that multiple mechanisms are involved in cell death at luteal regression. hCG restores steroidogenic function and restrains the apoptotic process, but not autophagy.
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Gonadotropina Coriónica/farmacología , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Liberadora de Gonadotropina/sangre , Luteólisis/efectos de los fármacos , Adulto , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Células Cultivadas , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/ultraestructura , Citocromos c/biosíntesis , Citocromos c/metabolismo , ADN/biosíntesis , ADN/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Etiquetado Corte-Fin in Situ , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genéticaRESUMEN
The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.
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Citoplasma/metabolismo , Citoplasma/ultraestructura , Células Lúteas/citología , Células Lúteas/metabolismo , Fosfoproteínas/metabolismo , Western Blotting , Proteínas Portadoras/metabolismo , Citocromos c/metabolismo , Citoplasma/efectos de los fármacos , Femenino , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Liberadora de Gonadotropina/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Células Lúteas/efectos de los fármacos , Células Lúteas/ultraestructura , Fase Luteínica , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Mitocondrias/metabolismo , Fosfoproteínas/ultraestructuraRESUMEN
To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, porin, and adenosine 5(')-triphosphate-diphosphohydrolase activity showed the capacity to synthesize progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.
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Mitocondrias/metabolismo , Placenta/metabolismo , Progesterona/metabolismo , Adenilato Quinasa/metabolismo , Apirasa/metabolismo , Sitios de Unión , Western Blotting , Colesterol/metabolismo , Citocromos/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Isocitrato Deshidrogenasa/metabolismo , Monoaminooxidasa/metabolismo , Placenta/enzimología , Porinas/metabolismo , Estructura Terciaria de Proteína , Fracciones Subcelulares , Succinato Deshidrogenasa/metabolismo , Factores de TiempoRESUMEN
The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.