RESUMEN
The aims of this study were to evaluate the localization, by immunohistochemistry, of the anti-Müllerian hormone (AMH) in goat ovaries and to investigate its effects on the in vitro survival and development of caprine pre-antral follicles enclosed in fragments of ovarian tissue. Pre-antral follicles were cultured in vitro for 1 or 7 days in α-MEM(+) in the absence or presence of kit ligand (KL; 50 ng/ml, positive control) or AMH (50 or 150 ng/ml). The results showed that AMH was localized in oocytes and granulosa cells from the primordial follicle to antral follicle stages. Addition of AMH maintained the percentage of developing follicles, similar to that in the uncultured control; however, the percentage of developing follicles was significantly lower than that in the cultured control and KL. Nonetheless, addition of AMH to the culture medium did not affect survival rates and follicular growth. In conclusion, this study demonstrated that the expression of AMH varies according to the compartment and stage of follicular development. Furthermore, AMH inhibits the activation of caprine primordial follicles.
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Hormona Antimülleriana/metabolismo , Cabras , Folículo Ovárico/metabolismo , Animales , Hormona Antimülleriana/genética , Proliferación Celular , Fragmentación del ADN , Femenino , Oocitos/metabolismo , Transporte de Proteínas , Técnicas de Cultivo de TejidosRESUMEN
This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption.
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Alginatos/farmacología , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Folículo Ovárico/crecimiento & desarrollo , 3-Hidroxiesteroide Deshidrogenasas/análisis , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Alginatos/química , Animales , Aromatasa/análisis , Aromatasa/genética , Aromatasa/metabolismo , Técnicas de Cultivo de Célula , Femenino , Cabras , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Folículo Ovárico/efectos de los fármacosRESUMEN
We aimed in this study to assess whether serum-decreased bovine cumulus-oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 µM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17ß (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.
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Androstenodiona/farmacología , Estradiol/metabolismo , Oocitos/metabolismo , Suero , Animales , Bovinos , Células Cultivadas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Estradiol/biosíntesis , Femenino , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/efectos de los fármacos , Folículo Ovárico , Progesterona/metabolismoRESUMEN
The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.
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Animales , Estradiol/análisis , Oocitos/citología , Progesterona/análisis , Cabras/clasificaciónRESUMEN
The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.(AU)
Asunto(s)
Animales , Oocitos/citología , Estradiol/análisis , Progesterona/análisis , Cabras/clasificaciónRESUMEN
Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.
RESUMEN
OBJECTIVE: Vasoactive intestinal peptide (VIP) is a neuropeptide with elevated expression in regions that control urogenital functions. Estrogen appears to modulate VIP expression in various organs, but this effect has not been demonstrated in the vaginal wall. The aim of this study was to evaluate the influence of estrogen status on VIP expression in vessels of the vaginal wall. METHODS: Surgical specimens were removed from the vaginal walls of 18 premenopausal women and 12 postmenopausal women who were given surgery for genital prolapse grade I or II. Vaginal specimens were stained with estrogen receptor-alpha (ER-α) and VIP antibodies. Levels of follicle stimulating hormone (FSH), estradiol, prolactin, fasting glucose and serum thyroxine stimulating hormone were also measured. Estrogen status was assessed on the basis of FSH and ER-α scores. RESULTS: The vaginal walls of premenopausal women had significantly higher ER-α scores than those of menopausal women (premenopausal group, 3.6 ± 2.2; menopausal group, 1.4 ± 1.8; p = 0.01). Premenopausal women also had significantly higher levels of VIP in the vaginal wall than menopausal women (p = 0.02). Increasing age was associated with lower level of VIP staining (odds ratio 0.88; 95% confidence interval 0.78-0.99). CONCLUSION: Levels of ER-α and VIP expression in the posterior vaginal wall were higher in premenopausal than in menopausal women, but VIP expression was not associated with estrogen status. Age was an independent predictor of VIP staining in vaginal wall biopsies.
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Receptor alfa de Estrógeno/metabolismo , Menopausia/metabolismo , Vagina/irrigación sanguínea , Vagina/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Adulto , Factores de Edad , Glucemia/metabolismo , Vasos Sanguíneos/metabolismo , Índice de Masa Corporal , Estudios Transversales , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Menopausia/sangre , Persona de Mediana Edad , Análisis Multivariante , Oportunidad Relativa , Premenopausia/sangre , Premenopausia/metabolismo , Prolactina/sangre , Estadísticas no Paramétricas , Tirotropina/sangre , Adulto JovenRESUMEN
BACKGROUND: There is evidence that intrauterine growth restriction, resulting in newborn girls that are small for gestational age (SGA), may be related to the onset of polycystic ovary syndrome (PCOS). Thus, we studied whether women born SGA have a higher prevalence of PCOS than women born appropriate for gestational age (AGA). METHODS: This was a prospective birth cohort study of 384 women born at term between June 1, 1978, and May 31, 1979, in Ribeirão Preto, Brazil. After exclusion, 165 women effectively participated in this study, of whom 43 were SGA and 122 were AGA. The prevalence of PCOS was analysed. At a mean age of 29 years, the women agreed to follow the study protocol, which included: anamnesis, physical examination, serum tests [follicle stimulating hormone, luteinizing hormone, total and free testosterone, dehydroepiandrostenedione sulphate, 17-OH-progesterone, fasting insulin, sex steroid-binding globulin (SHBG) and fasting glucose] and pelvic ultrasound. Data regarding gestational age, birthweight, age at menarche and maternal data were obtained from the files of the cohort. The adjusted relative risk (RR) values of the SGA, insulin resistance, body mass index, maternal smoking and parity variables were analysed using Poisson regression with robust adjustment of variance for the prediction of PCOS. RESULTS: The prevalence of PCOS was higher in the SGA group than in the AGA group [adjusted RR = 2.44, 95% CI (1.39-4.28)]. Hyperandrogenism was more prevalent in the SGA women than in the AGA women (P = 0.011). Circulating SHBG was lower in the SGA women than in the AGA women (P = 0.041), but fasting insulinemia was similar in both groups. CONCLUSIONS: The prevalence of PCOS in SGA women was twice as high as in AGA women in our study population.
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Peso al Nacer , Síndrome del Ovario Poliquístico/epidemiología , Adulto , Femenino , Edad Gestacional , Humanos , Distribución de Poisson , Síndrome del Ovario Poliquístico/sangre , Prevalencia , Estudios Prospectivos , Análisis de Regresión , Factores de RiesgoRESUMEN
INTRODUCTION: Endometriosis is a benign, estrogen-dependent, chronic gynecological disorder associated with pelvic pain and infertility. The disease most commonly affects women during the reproductive age, although postmenopausal patients do rarely present it. These rare occurrences are generally associated with hormonal use. MATERIAL AND METHODS: We present three cases of endometriosis in postmenopausal patients who have no history of hormone therapy and no previous history of endometriosis or infertility. CASE REPORTS: In case 1, a 62-year-old woman presented with acyclic pelvic pain and a left ovarian homogeneous cystic mass. After laparoscopic salpingoophorectomy and histological analysis, an ovarian endometriotic cyst was confirmed. In case 2, a 78-year-old woman presented with a painful abdominal wall mass that was confirmed by ultrasound and tomography. Her past medical history included an abdominal hysterectomy 20 years prior to the discovery of this mass. The lesion was surgically excised and histological analysis showed areas of endometrial stroma and glands surrounded by fibrosis, compatible with endometriosis. In case 3, a 54-year-old woman presented with chronic pelvic pain and a nodule in the rectovaginal septum was noted during gynecological examination. Menopause occurred at 48 years of age. She had no previous dysmenorrhea. Ultrasound confirmed the nodule in the rectovaginal septum. The patient was submitted to a diagnostic colonoscopy that revealed a friable lesion, which was subsequently biopsied. The histological diagnosis was endometriosis. CONCLUSIONS: These three cases of postmenopausal endometriosis support the celomic metaplasia theory for the genesis of this disease.