RESUMO
Previous studies have proposed the involvement of caspase-3, a downstream executioner enzyme common to many paradigms of programmed cell death (PCD), in mediating the apoptosis of both germ and somatic cells in the ovary. Herein we used caspase-3 gene knockout mice to directly test for the functional requirement of this protease in oocyte and/or granulosa cell demise. Using both in vivo and in vitro approaches, we determined that oocyte death initiated as a result of either developmental cues or pathological insults was unaffected by the absence of caspase-3. However, granulosa cells of degenerating antral follicles in both mouse and human ovaries showed a strong immunoreaction using an antibody raised against the cleaved (activated) form of caspase-3. Furthermore, caspase-3 mutant female mice possessed aberrant atretic follicles containing granulosa cells that failed to be eliminated by apoptosis, as confirmed by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling) analysis of DNA cleavage and 4',6-diamidino-2-phenylindole staining of nuclear morphology (pyknosis). These in vivo results were supported by findings from in vitro cultures of wild-type and caspase-3-deficient antral follicles or isolated granulosa cells. Contrasting the serum starvation-induced occurrence of apoptosis in wild-type granulosa cells, caspase-3-null granulosa cells deprived of hormonal support were TUNEL-negative, showed attenuated chromatin condensation by 4',6-diamidino-2-phenylindole staining and exhibited delayed internucleosomal DNA cleavage. Such ex vivo findings underscore the existence of a cell autonomous (granulosa cell intrinsic) defect in apoptosis execution resulting from caspase-3 deficiency. We conclude that caspase-3 is functionally required for granulosa cell apoptosis during follicular atresia, but that the enzyme is dispensable for germ cell apoptosis in the female.
Assuntos
Apoptose , Caspases/genética , Ovário/citologia , Transdução de Sinais , Animais , Animais Recém-Nascidos , Caspase 3 , Caspase 7 , Caspases/análise , Caspases/deficiência , Caspases/metabolismo , Técnicas de Cultura , Fragmentação do DNA , Ativação Enzimática , Feminino , Atresia Folicular , Células da Granulosa/enzimologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/anatomia & histologia , Ovário/enzimologiaRESUMO
OBJECTIVE: To compare the ability of four chromotubation techniques to generate and maintain intrauterine pressures in the diagnosis of proximal tubal obstruction. METHODS: Sixteen extirpated uteri were used for this study. A pressure catheter was placed through the fundus into the endometrial cavity. Three cannulas were evaluated: 1) the Cohen cannula with hold and no-hold techniques, 2) the BARD cervical cannula (dual intrauterine and intracervical balloons), and 3) the Harris-Kronner uterine manipulator-injector catheter with an intrauterine balloon. Intrauterine pressures were monitored while warm saline was infused. The studies were performed with the tubes obstructed, and measurements of peak attainable intrauterine pressures were recorded. Data were analyzed by t test, with significance set at P < .05. RESULTS: Peak intrauterine pressures for the four groups were as follows: 1) Cohen cannula, not holding, 40.7 +/- 5.1 mmHg; 2) Cohen cannula, holding in place, 63.6 +/- 5.3 mmHg; 3) BARD cannula, 112.4 +/- 3.5 mmHg; and 4) Harris-Kronner cannula, 106.3 +/- 4.3 mmHg. The BARD and Harris-Kronner cannulas achieved significantly higher intrauterine pressures than either method of using the Cohen cannula (P < .001). There was no statistically significant difference between the BARD and Harris-Kronner cannulas. CONCLUSION: Significant differences in achievable intrauterine pressures were demonstrated among catheters in our in vitro model. Based on these findings, we believe that the BARD, Harris-Kronner, or other intrauterine balloon-type cannula should be used before diagnosing proximal tubal obstruction.
Assuntos
Doenças das Tubas Uterinas/diagnóstico , Testes de Obstrução das Tubas Uterinas/métodos , Cateterismo/instrumentação , Cateterismo/métodos , Constrição Patológica/diagnóstico , Testes de Obstrução das Tubas Uterinas/instrumentação , Feminino , Humanos , Técnicas In Vitro , Pressão , Útero/fisiologiaRESUMO
OBJECTIVES: We sought to measure the characteristics of a quantitative human papillomavirus deoxyribonucleic acid assay and repeated cervical cytologic examination in screening for cervical intraepithelial neoplasia among human immunodeficiency virus-infected women. STUDY DESIGN: Human immunodeficiency virus-infected women with screening CD4+ lymphocyte counts of < or = 500 cells/mm3 (n = 103) were examined by quantitative human papillomavirus deoxyribonucleic acid assay and serial cervical cytologic examination and by colposcopy with biopsy and endocervical curettage during the course of 1 year. RESULTS: Quantitative measures of total human papillomavirus deoxyribonucleic acid and high-risk human papillomavirus deoxyribonucleic acid were strongly associated with any cervical intraepithelial neoplasia (P = .005) and high-grade cervical intraepithelial neoplasia (P = .0006), but they improved the sensitivity and negative predictive value of baseline screening only slightly when combined with cervical cytologic examination. Incident cervical intraepithelial neoplasia occurred frequently (20%) during 1 year of follow-up and was more common among human papillomavirus-infected women. Repeated cytologic examination identified 60% of women with new cervical intraepithelial neoplasia. CONCLUSION: Human immunodeficiency virus-infected women with at least mild immunosuppression have a high incidence of cervical intraepithelial neoplasia, which warrants close follow-up. Those with high baseline human papillomavirus deoxyribonucleic acid levels may be at the highest risk for incident cervical intraepithelial neoplasia.