Regulation of Stem Spermatogonia in the Mature Testis

项目总结（见说明）： 维持雄性生殖力的一个基本要求是维持足够数量的精原细胞，这是精子发生的基础。为了实现这一点，当干细胞分裂时，一些后代必须保持干精原细胞，而其他后代则分化。很明显，干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力至关重要，并且有间接证据表明GDNF在正常成熟睾丸中维持这种平衡中起重要作用。然而，我们几乎不知道在体内调节这种平衡的成熟器官，GDNF在成年睾丸中的特定功能，或如果GDNF表达的生理变化显着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，该模型允许GDNF信号传导到干精原细胞， 在体内被ATP拮抗剂特异性地和可逆地抑制。使用这个模型，我们已经产生了第一个直接的证据，GDNF是需要在一个正常的成熟睾丸干精原细胞池的维护。此外，我们已经证明，当GDNF信号传导的抑制被逆转时，干细胞开始重建干细胞库。重要的是，我们的数据表明，当GDNF信号被抑制仅2天时，一些干精原细胞会丢失，而其他干细胞则存活长达11天。这表明干细胞的内在或外在因素调节对GDNF的反应。使用这种新的小鼠模型，我们在特定目标1中提出定义GDNF信号传导抑制后干精原细胞损失的机制，并确定为什么有些细胞损失迅速，而另一些细胞损失较慢。在这些结果的基础上，我们还建议与项目1合作， GDNF mRNA表达和/或干精原细胞数量显着减少，在一些少精子症男性。具体目标2研究干精原细胞数量部分耗尽后重新填充睾丸的能力。在这样做的过程中，我们将确定在何种程度上确定数量的干精原细胞可以恢复这个池和负责这种恢复的生物过程。 此外，与项目1合作，我们将研究接受内分泌治疗以增加精子产量的少精子男性，以确定对治疗有积极反应的男性是否存在GDNF表达的增加和/或干精原细胞数量的增加。据我们所知，这些特定的目的构成了第一个详细的体内分析干细胞或任何其他成体干细胞对抑制信号传导的反应，然后恢复由必需的生长因子。我们预期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.