睾丸“新”基因PRP在雄性生殖中作用研究

Proliferin related protein(PRP) was originally identified as a angiogenesis inhibitor in mouse placenta. The tissue expressions of PRP are mainly documented in placenta.We reported for the first time that PRP is expressed in male testis tissue.Immunocytochemical and in situ results showed that positive PRP immunostaining in the testis Leydig cells.Immunocytofluorescent staining of PRP in TM3 cells indicates that PRP is located within the cytoplasm.The expression pattern of PRP in the rat testis exhibit an age-related increase.Interestingly,ginsenosides could significantly down-regulate the expression level of PRP in aging rat testis.To elucidate its function, experiments were carried out to examine the consequences of lentiviral-mediated RNA interference (RNAi) of PRP on cell growth,cell cycle,testosterone production and expression of several leydig genes.Results indicated that PRP silenced cause a decrease in hCG-stimulated testosterone production.In addition,PRP silenced attenuate the effect of hCG-stimulated PRLR mRNA increased,moreover,the increased effect of PRL on hCG-induced testosterone production is also weakened for PRP silenced,indicating that PRP may be involved in PRL function through affecting its receptor expression in response to stimuli.Cell growth and cell cycle analysis show that PRP maybe play negative roles in cell proliferation.To test whether PRP has anti-proliferation activity in human tumor cells,PRP-eGFP-C1 vector were transfected into SGC-7901 cells.As opposed to underexpression of PRP, overexpression PRP prolongs the doubling time of SGC-7901 cell lines, and induces an arrest in G0/G1 phase.Taken together,these data suggest that PRP might play roles in male reproduction,including testosterone production and cell proliferation. . However, all the above research results of PRP were based on in vitro tests.To further investigate the role of PRP in male reproductive system, we plan to screen the proteins interacting with PRP by yeast two-hybrid system，and generate transgenic over-expression and knock-down of PRP mice by lentivirus-mediated gene transfer.Lentiviruses were subzonally injected into single cell fertilized eggs of ICR mice to infect fertilized eggs, subsequently, embryos were transplanted into the pseudopregnant mice to attain F0 mice，followed by screening PRP over-expression and knock-down mice under fluorescent stereo microscope，and subsequently confirmed by PCR-based genotyping．Then the expression of PRP in testis will be analysed by real-time PCR and westernblotting. Additionally, the levels of hormones and morphologic changes in reproductive organs of PRP over-expression and knock-down mice will be measured.

不育、性功能低下为男性常见病，认识相应机理有助于疾病预防与治疗。.我们新近发现PRP基因表达于雄性睾丸，此前研究认为PRP仅在雌性胎盘组织表达，研究范围局限于雌性。课题组前期实验表明：PRP定位于雄性睾丸Leydig细胞，参与细胞增殖和睾酮产生(in vitro)；其表达呈年龄依赖的方式增加，具改善生殖功能与抗衰老活性的人参皂甙可显著下调老年大鼠高表达状态。提示：PRP参与生殖功能，且与衰老过程有关，其可能是生殖功能低下和生殖衰老之间存在"共性"的分子基础之一，是研究生殖功能的一个较好切入点。.为了深入探讨PRP在雄性生殖中的作用，本研究拟筛选与PRP相互作用的蛋白，并制备PRP over-expression与knock-down小鼠，分析生殖表型等方面的变化和相关机制，明确其在生殖系统中的活性及是否与生殖衰老有关。研究结果将有助于认识雄性生殖功能低下的机制，对男性生殖健康具有重要意义

研究表明，表达于睾丸的某些基因可能在雄性生殖发育过程中扮演重要角色。在前期基础上，本课题对PRP基因体内功能进行了深入研究。通过TALEN技术制备了PRP敲除小鼠。PRP敲除不影响睾丸器官系数，对形态学未见明显影响。睾酮水平、LH水平无明显改变。此外，纯合雄性与野生雌性繁殖实验显示对每胎产仔数、性别比无显著影响。. 通过受精卵显微注射转基因片段成功建立间质细胞特异性过表达PRP小鼠。PRP小鼠生精小管结构完整，生精小管直径、周长无异常。PRP小鼠精子计数明显减少，提示PRP小鼠生精功能损伤。TUNEL分析表明，PRP小鼠睾丸精母细胞和精子细胞凋亡增加，促凋亡相关蛋白CASP3、BAX表达显著上调，表明PRP通过调节凋亡相关蛋白参与精细胞凋亡。血清和睾丸睾酮水平明显降低，表明PRP影响睾丸间质细胞睾酮产生功能。机制分析显示STAR 与HSD3B 表达明显下调，LHR, TSPO和 CYP11A1表达无明显变化，提示PRP 可能通过调节STAR 与HSD3B 而参与睾酮产生。为了分析PRP是否通过旁分泌方式参与调节睾丸支持细胞功能，用WB方式分析了支持细胞特异基因。结果显示TF表达下调，但不影响Claudin-11，TJP1，SHBG表达。. 基因表达谱初筛发现在PRP基因异常导致生精功能下降睾丸中，lncRNA Gas5、TXNIP、sat2、DPT与Bex4基因表达水平均显著上调。这些基因的共同表现均是在肿瘤组织中表达水平下降或缺失。为了证实这几个基因是否与生殖功能有关，我们对其在睾丸中表达进行了定位，并通过Cdcl2或busulfan导致的小鼠生精功能下降的模型与体外细胞模型，证明这几个基因的确参与生精功能。因此PRP可能通过影响睾丸生精微环境而参与生殖功能。. 总体而言，PRP表达敲除后未见明显可见的生殖表型改变，而其表达上调导致生精功能损伤。鉴于PRP表达水平随年龄增加而增加，表明PRP主要在生殖衰老中发挥效应。PRP表达上调可能促进睾丸精细胞凋亡，并通过下调STAR 与HSD3B表达参与睾酮产生；此外，还可能作为旁分泌调节因子参与调节睾丸支持细胞功能。另外，研究提示PRP上调可能介导睾丸生精微环境，使其处于抑制细胞生长状态。因此，调节生精微环境对防治生精功能下降、生殖衰老具有重要意义。