MOLECULAR AND CELLULAR CONTROL OF SPERMATOGENESIS

精子发生的分子和细胞控制

• 批准号: 3329979
• 负责人: JOSE LUIS MILLAN
• 金额: $ 16.96万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3329979
• 关键词: Leydig cells; Sertoli cells; alternatives to animals in research; androstenedione; cell line; chorionic gonadotropin; clone cells; cyclic AMP; dehydroepiandrosterone; estradiol; follicle stimulating hormone; germ cells; hormone receptor; laboratory mouse; mixed tissue /cell culture; phenotype; progesterone; progesterone analog; radioimmunoassay; spermatogenesis; steroid biosynthesis; testis; testosterone; tissue /cell culture

In the mammalian testis, interactions between the different somatic cell types (Sertoli, peritubular and Leydig) provide a unique microenvironment controlling spermatogenesis. In the past years, in vitro cultures of somatic testicular cells have become increasingly used in investigating the role of these cell types in supporting germ cell differentiation. However, these investigations are limited by the fact that none of the cell populations used are pure lines and the viability of these primary cultures seldom exceeds fifteen days, thus limiting the time of observation that is necessary for germ cell adaptation and differentiation.We have recently immortalized (using the SV40 large T antigen) all the cell types contributing to a developing seminiferous tubule in the mouse testis. Eight Sertoli, 16 peritubular and 22 Leydig cell lines have been established, cloned to purity by repeated limited dilutions, and cultured successfully for 90 generations in a period of 2.0 years. Under defined culture conditions 2 of the Sertoli lines and 3 out of 7 tested Leydig lines were found to express FSH and LH receptors respectively. More significantly, one germ cell line has been established that, based on its morphology, electronmicroscopic characteristics and its expression of the testicular isoform of cytochrome c and of the testis-specific lactate dehydrogenase (LDH-C) isozyme, has been characterized as corresponding to a transition between a spermatogonia B and a primary spermatocyte stage. Furthermore, these four immortalized cell types, when plated together, are able to reaggregate and reconstitute spermatogenic tubule-like structures in vitro. The availability of these cell lines represents a major breakthrough in our ability to study spermatogenesis in vitro, to identify testis-specific factors that control gene expression and to characterize the molecules that control gametogenesis. The overall aim of this project is to establish a co-culture system of functional Sertoli, Leydig and peritubular cell lines able to support germ cell differentiation in vitro. The specific aims of this proposal are: I. to characterize the functionality, hormonal responsiveness and steroidogenic potential, of the Sertoli, peritubular, and Leydig cell lines. II. to investigate the ability of the in vitro spermatogenic tubule to support normal germ cell differentiation. III. to investigate the ability of our immortalized germ cell line to differentiate in vitro. IV. to immortalize primordial germ cells and spermatogonia following the above or improved immortalization strategies and investigate their ability to differentiate in vitro.

在哺乳动物睾丸中，不同的体细胞之间的相互作用 类型（Sertoli，周围和Leydig）提供独特的微环境 控制精子发生。 在过去的几年中，体外文化 体细胞细胞已越来越多地用于研究 这些细胞类型在支持生殖细胞分化中的作用。 但是，这些调查受到以下事实的限制 所使用的细胞群是纯线，这些主要的生存能力 培养很少超过15天，因此限制了 生殖细胞适应和 分化。我们最近永生了（使用SV40大t 抗原）所有促成开发生精的细胞类型 小管中的小管。 八个Sertoli，16个周围和22 Leydig 已经建立了细胞系，通过重复限制将克隆到纯度 在一段时间内，稀释并成功培养了90代 2.0年。 在确定的培养条件下，Sertoli线的2和3 在7个测试的Leydig线中，发现表达FSH和LH受体 分别。 更重要的是，一条生殖细胞系已经 确定，根据其形态，电子显微镜 特征及其表达的睾丸同工型 细胞色素C和睾丸特异性乳酸脱氢酶（LDH-C）的 同工酶的特征是对应于 精子B和原发性精子阶段。 此外，这些 将四种永生的细胞类型（当镀在一起时）能够 重新构建和重建类似小管的生理性小管状结构 体外。 这些细胞系的可用性代表了主要 我们在体外研究精子发生的能力的突破 识别控制基因表达和至的睾丸特异性因素 表征控制配子发生的分子。 总体目标 这个项目的是建立功能性的共文化系统 Sertoli，Leydig和周围细胞系能够支持生殖细胞 体外分化。 该提案的具体目的是： I.表征功能，激素反应性和 Sertoli，周围和Leydig细胞的类固醇生成潜力 线。 ii。研究体外精子小管的能力 支持正常的生殖细胞分化。 iii。研究我们永生的生殖细胞系的能力 在体外区分。 iv。在此之后使原始生殖细胞和精子永生 上面或改善了永生策略并调查其 在体外区分的能力。