Regulation of Stem Spermatogonia in the Mature Testis

项目总结(见说明)： 维持男性生育能力的一个基本要求是保持足够数量的干细胞精原细胞，这是生殖细胞发生的基础。为了实现这一点，当干细胞分裂时，一些后代必须保持干细胞精原细胞，而另一些后代则分化。显然，干细胞精原细胞的自我更新复制和分化之间的正确平衡对男性生育至关重要，有间接证据表明，GDNF在维持正常成熟睾丸的这种平衡中发挥了重要作用。然而，我们几乎不知道成熟器官中这种平衡的体内调节，成体睾丸中GDNF的特定功能，或者GDNF表达的生理性变化是否显著影响干细胞的复制或分化。为了解决这些关键问题，我们开发了一种独特的小鼠模型，允许GDNF信号传递到干细胞精原细胞以 在体内被三磷酸腺苷拮抗剂特异性和可逆地抑制。使用这个模型，我们已经产生了第一个直接的证据，证明在正常成熟的睾丸中，GDNF是维持干细胞精原细胞池所必需的。此外，我们已经证明，当GDNF信号的抑制被逆转时，干细胞开始重建干细胞库。重要的是，我们的数据表明，当GDNF信号被抑制短短2天时，一些干细胞精原细胞丢失，而另一些干细胞存活长达11天。这表明干细胞的内在或外在因素调节了对GDNF的反应。利用这个新的小鼠模型，我们在特定的目标1中提出，以确定在GDNF信号被抑制时导致干细胞精原细胞丢失的机制，并确定为什么一些细胞丢失得很快，而另一些细胞丢失得更慢。在这些结果的基础上，我们还建议与项目1合作，以确定是否 在一些少精子症患者中，GDNF基因表达和/或干细胞精原细胞数量显著减少。特定目的2研究干细胞精原细胞在其数量部分耗尽后重新填充睾丸的能力。在这样做的过程中，我们将确定一定数量的干细胞精原细胞可以在多大程度上恢复这个池，以及负责这种恢复的生物过程。 此外，与项目1合作，我们将研究正在接受内分泌治疗以增加精子产量的少精子症男性，以确定在治疗呈阳性反应的男性中，是否存在GDNF表达增加和/或干细胞精原细胞数量增加。据了解，这些特定的目标构成了对干细胞或任何其他成体干细胞对必要的生长因子抑制和恢复信号的反应的第一次详细的活体分析。我们预计，这些研究的结果将对不育男性的评估和最终治疗做出重要贡献。

PROJECT SUMMARY (See instructions): An essential requirement for sustaining male fertility is maintaining an adequate number of stem spermatogonia, the foundation of spennatogenesis. To achieve this, when the stem cells divide, some progeny must remain stem spermatogonia while other progeny differentiate. It is obvious that the correct balance between self-renewing replication and differentiation of stem spermatogonia is crucial to male fertility, and there is indirect evidence that GDNF plays an important role in maintaining this balance in the normal mature testis. However, we known almost nothing about the in vivo regulation of this balance in the mature organ, of the specific function of GDNF in the adult testis, or if physiological changes in GDNF expression significantly affect the replication or differentiation of the stem cells. To address these critical issues, we have developed a unique mouse model that allows GDNF signaling to the stem spermatogonia to be specifically and reversibly inhibited in vivo by an ATP antagonist. Using this model, we have generated the first direct evidence that GDNF is required for maintaining the stem spermatogonial pool in a normal mature testis. Additionally, we have shown that when inhibition of GDNF signaling is reversed, the stem cells begin to rebuild the stem cell pool. Importantly, our data demonstrate that some stem spermatogonia are lost when GDNF signaling is inhibited for as little as 2 days, while other stem cells survive for up to 11 days. This suggests that factors intrinsic or extrinsic to the stem cells modulate the response to GDNF. Using this new mouse model we propose in Specific Aim 1 to define the mechanisms'responsible for the loss of stem spermatogonia upon inhibition of GDNF signaling and to detenmine why some cells are lost rapidly while others more slowly. Building on these results, we also propose to collaborate with Project 1 to determine if GDNF mRNA expression and/or stem spermatogonial numbers are significantly reduced in some oligospermic men. Specific Aim 2 studies the capacity of stem spermatogonia to repopulate the testis once their numbers are partially depleted. In doing so, we will identify the extent to which a defined number of stem spermatogonia can restore this pool and the biological processes responsible for this restoration. Additionally, in collaboration with Project 1 we will study oligospermic men who are receiving endocrine therapy to increase} sperm production in order to determine if in men who have a positive response to therapy, there is bath an increase in GDNF expression and/or an increase in numbers of stem spermatogonia. To jour knowledge, these specific aims constitute the first detailed in vivo analysis of theresponse of stem sjaermatogonia or any other adult stem cell to the inhibition and then restoration of signaling by an essbntial growth factor. We anticipate that the results from these studies will make an important contribution to the evaluation and eventually the treatment of infertile men.