SERTOLI-SPERMATOGENIC CELL INTERACTION IN CULTURE

培养中支持细胞与生精细胞的相互作用

• 批准号: 3315358
• 负责人: LAURA TRES
• 金额: $ 11.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3315358
• 关键词: Sertoli cells; all trans retinol; cell differentiation; cell type; epidermal growth factor; follicle stimulating hormone; gel electrophoresis; growth media; immunofluorescence technique; insulin; meiosis; radioimmunoassay; somatotropin; sperm; spermatogenesis; testis; tissue /cell culture; transferrin

The long term objective of this research is to develop a serum-free, hormone/growth factor-supplemented medium that will support the completion of spermatogenesis in co-cultures of rat spermatogenic cells with Sertoli cells. We will attempt to determine whether various components of this culture medium interact synergistically with follicle-stimulating hormone (FSH) and androgens to stimulate secretion of Sertoli cell regulatory proteins or have direct actions on the developing spermatogenic cells. Development of an in vitro system for the study of spermatogenic cell development is necessary to fully understand the molecular basis for the regulation of spermatogenesis and causes of male infertility. We will study the effects of FSH and growth hormone on the accumulation of somatomedin-C (SM-C), a growth factor secreted by Sertoli cells, in media of Sertoli cells alone and Sertoli-spermatogeneic cell co-cultures. SM-C will be measured by radioimmunoassay. Newly-synthesized SM-C will be examined by immunoprecipitation of [35S]methionine-labeled protein using a specific anti-SM-C serum. SM-C immunoreactivity in cells will be studied by indirect immunofluorescence. To determine dose-dependent effects of FSH, growth hormone, insulin, SM-C, epidermal growth factor, transferrin and retinol on spermatogonia proliferation and differentiation of pre-leptotene spermatocytes into advanced stages of spermatogenesis, we will use [3H] thymidine combined with autoradiography in short- and long-term co-culture experiments. Studies of the effect of alternating high/low androgen concentrations in the presence or absence of FSH will determine the role of androgens in facilitating completion of spermatogenesis in vitro. Finally, two-dimensional polyacrylamide gel electrophoresis will be used to study newly-synthesized [35S]methionine-labeled proteins resulting from the interaction of Sertoli cells with specific types of spermatogenic cells co-cultured with Sertoli cells and during Sertoli-spermatogenic cell reaggregation experiments.

这项研究的长期目标是开发一种无血清、 补充激素/生长因子的培养基将支持完成 大鼠生精细胞与支持细胞共培养中精子发生的影响 细胞。 我们将尝试确定该组件的各个组件是否 培养基与卵泡刺激素协同相互作用 (FSH) 和雄激素刺激支持细胞调节分泌 蛋白质或对发育中的生精细胞有直接作用。 生精细胞研究体外系统的开发 为了充分了解其分子基础，开发是必要的 精子发生的调节和男性不育的原因。 我们将 研究FSH和生长激素对积累的影响 生长调节素-C (SM-C)，一种由支持细胞分泌的生长因子，位于培养基中 单独支持细胞和支持生精细胞共培养物。 SM-C 将通过放射免疫测定法进行测量。 新合成的SM-C将 使用 [35S] 蛋氨酸标记的蛋白质进行免疫沉淀检查 特异性抗 SM-C 血清。 将研究细胞中的 SM-C 免疫反应性 通过间接免疫荧光。 确定剂量依赖性效应 FSH、生长激素、胰岛素、SM-C、表皮生长因子、转铁蛋白 和视黄醇对精原细胞增殖和分化的影响 前细线精母细胞进入精子发生的高级阶段，我们 将使用[3H]胸苷结合放射自显影术进行短期和 长期共培养实验。 交替效应的研究 在存在或不存在 FSH 的情况下，高/低雄激素浓度将 确定雄激素在促进完成中的作用 体外精子发生。 最后，二维聚丙烯酰胺凝胶 电泳将用于研究新合成的 Sertoli 相互作用产生的[35S]蛋氨酸标记蛋白 具有特定类型生精细胞与 Sertoli 共培养的细胞 细胞和支持生精细胞再聚集实验期间。