ANALYSIS OF THE ROLE OF GCNF IN SPERMATOGENESIS

我们克隆了一种新的核受体超家族孤儿成员，其在成年小鼠中的表达仅限于卵母细胞和生精细胞，称为生殖细胞核因子（GCNF）。在雄性中，它在减数分裂后以圆形精子表达。迄今为止对其转录特性的分析表明，在没有添加配体的情况下，它是一个转录抑制因子，因此预测了对配体的转录激活需求。它与应答元件结合，应答元件是序列AGGTCA的直接重复序列，间隔为0 bp。这些元件已经在包括蛋白1和蛋白2在内的几个精细胞特异性基因的启动子中被鉴定出来。因此，我们的假设是，GCNF调节精子发生的某些方面，可能是终端分化，以响应一个尚未确定的信号通路。在结合配体后，它会激活一组特定的反应基因，如蛋白蛋白，从而为完整的精子发生设定一组特定的事件。本提案中详细的实验旨在回答关于GCNF在精子发生中的功能的关键问题。为了确定GCNF在精子发生中的生理功能，我们正在使用胚胎干细胞技术在生精细胞中产生GCNF的条件敲除。GCNF基因的零突变导致胚胎致死，表明它在胚胎发育中也起着关键作用。为了避免胚胎致死，我们将使用Cre/Lox系统在成年精子发生过程中特异性敲除GCNF基因。我们将使用仅在生精细胞中表达Cre的Sycp1/Cre小鼠来实现这一目标。本实验将告诉我们GCNF功能缺失对精子发生的影响。也有人提出建立功能获得的GCNF转基因小鼠模型，以弥补功能突变的缺失。此外，我们将研究GCNF在原始生殖细胞发育中的作用。我们已经证明GCNF可以调控OCT4转录因子基因。该因子是决定全能性生殖细胞谱系的一个决定因素，在GCNF敲除突变胚胎中被错误调控。因此，我们将研究GCNF在生殖细胞发育中的作用及其调控Oct4表达的机制。最后，我们将使用这些小鼠模型克隆GCNF应答基因并表征GCNF对其的调控。这些特定目标的最终结果将导致对GCNFs在精子发生和生殖细胞发育调节中的作用有更深入的了解。

We have cloned a novel orphan member of the nuclear receptor superfamily, whose expression in the adult mouse is restricted to the oocytes and spermatogenic cells, called Germ Cell Nuclear Factor (GCNF). In the male it is post-meiotically expressed in round spermatids. Analysis of its transcriptional properties to date shows that, in the absence of added ligands, it is a transcriptional repressor, thus predicting the requirement for a ligand for transactivation. It binds to response elements, which are direct repeats of the sequence AGGTCA with 0 bp spacing. Such elements have been identified in the promoters of several spermatid specific genes including the protamines 1 and 2. Thus, it is our hypothesis that GCNF regulates some aspect of spermatogenesis, probably terminal differentiation, in response to an as yet unidentified signaling pathway. It will activate a specific set of responsive genes, such as the protamines, upon binding its ligand, setting in train a specific set of events required for complete spermatogenesis. The experiments detailed in this proposal are designed to answer the key questions as to the function of GCNF in spermatogenesis. To determine the physiological function of GCNF in spermatogenesis we are generating a conditional knock-out of GCNF in spermatogenic cells using embryonic stem cell technology. The null mutation of the GCNF gene causes embryonic lethality indicating it also plays a critical role in embryonic development. To by-pass the embryonic lethality we will use the Cre/Lox system to specifically knock- out the GCNF gene during adult spermatogenesis. We will use the Sycp1/Cre mice, that express Cre only in spermatogenic cells to achieve this. This experiment will tell us the consequence of the lack of GCNF function on spermatogenesis. It is also proposed to generate gain-of-function GCNF transgenic mouse models as well, which will complement the loss of function mutations. In addition, we will examine the role of GCNF in primordial germ cell development. We have shown that GCNF can regulate the OCT4 transcription factor gene. This factor is a determinant of the totipotent germ cell lineage and is mis- regulated in the GCNF knock-out mutant embryos. Thus, we will examine the role of GCNF in germ cell development and its mechanism of regulation of Oct4 expression. Finally, we will use these mouse models to clone GCNF responsive genes and characterize their regulation by GCNF. The culmination of these specific aims will lead to a greater understanding of GCNFs role in the regulation of spermatogenesis and germ cell development.