Molecular control of male reproduction

描述(申请人提供)：尽管超过16种不同的下丘脑转录因子与控制生育有关，但生殖的分子机制仍不清楚。Hlh2碱性螺旋-环-螺旋(BHLH)转录因子在发育中的神经系统中表达。在成人中，表达仅限于下丘脑、视前区和丘脑中的特定神经元。Nhlh2基因敲除小鼠(N2KO)肥胖且性腺功能低下。此外，她们的卵泡刺激素和睾酮水平降低，没有表现出正常的男性性行为。N2KO小鼠尾部附睾处的精子密度也有所降低，但通过体外受精，我们发现N2KO小鼠的精子完全能够使卵母细胞受精。因此，雄性N2KO小鼠的不育症很大程度上是由于缺乏性行为，部分原因是精子产生减少。在其他研究中，我们发现在N2KO小鼠中，两种神经肽处理酶PC1和PC2的水平降低了90%。这些处理酶被认为处理促性腺激素释放激素以及其他与生育和肥胖有关的下丘脑神经肽。 基于这些初步发现，我们认为Nhlh2转录因子通过上调PC1和PC2加工酶的表达来调节男性的性行为和生育能力，PC1和PC2加工酶是在大脑的视前区产生成熟的GnRH肽所必需的。我们将使用基于动物和分子的方法来验证这一假设。在第一个目的中，我们将询问Nhlh2是否与GnRH、PC1和PC2共表达。我们还将检测N2KO和正常小鼠视前区神经元中PC1和PC2mRNA的水平，以及成熟GnRH肽的水平。在第二个目标中，我们将向雄性N2KO小鼠提供睾酮，并询问这种激素是否足以恢复男性的性行为和精子生成。 到目前为止，N2KO小鼠是唯一一只缺失神经元bHLH转录因子导致肥胖、性腺功能低下和缺乏男性性行为的基因敲除小鼠。对这些动物的研究是了解男性生育能力的分子控制的关键。

DESCRIPTION (provided by applicant): Although over 16 different hypothalamic transcription factors are implicated in controlling fertility, the molecular mechanisms of reproduction remain unclear. The hlh2 basic helix-loop-helix (bHLH) transcription factor is expressed throughout the developing nervous system. In adults, expression is limited to specific neurons in the hypothalamus, pre-optic area and hebenula. Nhlh2 knockout mice (N2KO) are obese and hypogonadal. In addition, they have reduced levels of follicle stimulating hormone and testosterone, and do not show normal male sexual behaviors. N2KO mice also have reduced sperm concentration in their caudal epididymus, but using in vitro fertilization, we have found that sperm from N2KO mice are fully capable of fertilizing oocytes. Thus, infertility in male N2KO mice is largely due to lack of sexual behavior and partially due to reduced sperm production. In other studies, we have found that levels of two neuropeptide processing enzymes, PC1 and PC2, are reduced up to 90% in N2KO mice. These processing enzymes are thought to process GnRH as well as other hypothalamic neuropeptides involved in both fertility and obesity. Based on these preliminary findings, we propose that the Nhlh2 transcription factor regulates male sexual behavior and fertility by up-regulating the expression of PC1and PC2 processing enzymes which are necessary for production of mature GnRH peptide in the pre-optic area of the brain. We will use both animal-based and molecular approaches to test this hypothesis. In the first aim, we will ask if Nhlh2 is co-expressed with GnRH, PC1 and PC2. We will also measure levels of PC1 and PC2 mRNA in pre-optic area neurons, and levels of mature GnRH peptide in N2KO and normal mice. In the second aim, we will supply male N2KO mice with testosterone and ask if this hormone is sufficient to restore male sexual behavior and spermatogenesis. To date, N2KO mice are the only knockout mice in which deletion of a neuronal bHLH transcription factor results in obesity, hypogonadism, and lack of male sexual behavior. Studies of these animals are key to understanding molecular control of male fertility.