Fam71d基因在精子运动中的作用及其分子机制

Athenozoospermia or low sperm motility is one of the main causes of male infertility, yet the mechanisms involved in low sperm motility are still unclear. High through-put gene expression profiling of a diverse array of normal mouse tissues, organs, and cell lines at the website of BioGPS.org showed that Fam71d transcripts was detected in testis only. In our previous experiments, we confirmed that FAM71D was specifically expressed in mouse testis, and FAM71D was localized in the cytosol of round and elongated spermatids, as well as the sperm tail. Besides, FAM71D antibody could remarkably inhibit the motility of both human sperm and mouse sperm. These data suggested FAM71D played an important role in sperm motility.. In the present study, we will compare the fertility, sperm motility and ultrastructure of sperm tail between wild type and Fam71d knockout mice to confirm the role of FAM71D in sperm motility. With the methods of immunostaining, co-immunoprecipitation, Western blot and calcium level detection, we will elucidate the mechanism of FAM71D in the regulation of sperm motility through interacting with calmodulin. We will also analyze the polymorphism and expression of FAM71D with PCR-Sanger sequencing, RT-PCR and Western blot to explore the relationship between FAM71D and athenozoospermia. This study will demonstrate the mechanisms in regulating sperm motility, and provide new insights for the diagnosis and treatment of athenozoospermia.

弱精子症是男性不育的重要原因，但关于精子运动和精子活动力低下的分子机理尚未完全明了。我们前期研究发现，Fam71d为睾丸特异性基因，其蛋白定位于圆形精子和长形精子细胞的胞质及成熟精子尾部；FAM71D抗体可显著抑制人和小鼠的精子运动。上述结果表明，FAM71D在精子运动中具有重要作用。本项目将利用Fam71d基因敲除小鼠模型，检测小鼠生育力和精子运动能力的变化，进一步确定该基因在小鼠精子运动中的作用；通过免疫共沉淀、钙离子检测等方法，分析FAM71D与钙调蛋白的相互作用、探讨FAM71D通过钙调蛋白信号通路调控精子运动的分子机制；采用PCR-Sanger测序、RT-PCR等技术，比较正常生育男性与弱精子症患者FAM71D基因的多态性及FAM71D表达水平的差异，以确定FAM71D与弱精子症的相关性。本项目的实施将进一步阐明精子运动的调控机制，也为弱精子症的诊断和治疗提供新的依据。

弱精子症是男性不育的重要原因，但精子运动和精子活动力低下的原因尚未明了。我们通过分析正常小鼠组织、器官及各个细胞系的高通量基因表达谱芯片数据（http://biogps.org），以及表达序列标签（expressed sequence tags, ESTs）表达谱（http://www.ncbi.nlm.nih.gov/UniGene, Mm.56514），发现Fam71d (Family with sequence similarity 71, member B) 基因为睾丸特异性基因。进一步研究结果显示，FAM71D/Fam71d在人和小鼠中均为睾丸组织特异性表达，Fam71d在小鼠睾丸中的表达呈年龄依赖性增加；FAM71D蛋白在人和小鼠成熟精子中均定位于精子尾部中段；FAM71D抗体可显著抑制人和小鼠精子的运动能力；免疫荧光结合免疫共沉淀结果显示，FAM71D和钙调蛋白之间有相互作用；临床样本分析发现FAM71D表达水平与弱精子症有显著的相关性。以上研究为弱精子症的诊断提供了新的依据。