【作者】 郑小波；

【导师】 张家骅；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 两性配子的生成是动物繁衍的前提和基础,睾酮在精子生成过程中具有十分重要的作用。睾酮不仅在精子生成过程中具有十分重要的作用,而且睾酮还参与维持雄性动物的第二性征,对精子的活力、授精的成功率也有重要的影响。间质细胞是雄性动物体内产生睾酮最主要的细胞,研究间质细胞睾酮合成的调控机制对于深入认识精子生成的分子机制,预防和治疗雄性不育均有十分重要的意义。关于睾酮合成机制的研究以往多集中在下丘脑-垂体-睾丸轴内分泌调节和促性腺激素对睾酮合成的影响上,但近年来的研究表明,原癌基因的表达在睾酮合成中起着十分重要的作用。本研究以荣昌猪为实验动物,利用体内体外模型探讨原癌基因c-jun对睾酮合成的调节机制,为进一步认识原癌基因在精子生成中的作用提供基础数据。大量的研究表明,睾丸间质细胞的睾酮分泌也伴有某些原癌基因的表达变化,特别是即时早期基因的表达变化。这些即时早期基因主要包括fos成员和jun家族。我们以c-jun为目的基因,以1、3、5周龄的荣昌仔猪睾丸为研究对象,采用实时荧光定量RT-PCR方法和放射免疫法研究了c-jun mRNA在仔猪睾丸中的表达及表达与血浆睾酮水平的关系,并切片观察睾丸组织结构的变化。实验结果表明：c-jun mRNA在1、3、5周龄仔猪睾丸组织中均有表达。其中3周龄表达较高,5周龄次之,1周龄较低,3周龄与5周龄和1周龄相比存在显著差异(P<0.05)。血浆睾酮水平在第3周最高,在其他时期较低,3周龄与1周龄、5周龄比较差异显著(P<0.01),3周龄仔猪睾丸间质细胞结构逐渐完整,5周龄数量有所下降。c-jun mRNA的表达与血浆睾酮水平及间质细胞结构变化可能存在一致性。3周龄荣昌仔猪睾丸作为研究睾酮分泌机制的材料是适宜的。通过不同时间仔猪睾丸组织c-jun mRNA表达分析发现,其表达水平与血浆睾酮浓度检测结果有一致性。为了进一步了解c-jun mRNA表达和睾酮分泌的关系,采用了体外培养的间质细胞进行研究,目的是排除其他细胞(如支持细胞、生精细胞)的干扰。通过锥虫蓝试验和3β-HSD酶活性检测,用酶解法结合差速离心分离的荣昌仔猪间质细胞数量较多,纯度较高,其活力为(93.0±4.9)%,纯化后的细胞百分率为(85.1±3.6)%。睾酮基础分泌量随培养的时间延长而增高,两者呈线性关系(r=0.826,P<0.01),培养至4h后增长峰趋于平缓。随着hCG刺激浓度的增加,睾酮分泌量增加,两者呈明显线性关系(r=0.893,P<0.01)。当hCG浓度达到50IU/mL时,诱导睾丸间质细胞睾酮分泌量最高。本实验中均采用50IU/mL为hCG诱导浓度。为了进一步准确了解c-jun mRNA表达与睾酮分泌的关系,我们采用了反义核酸技术,即将c-jun基因制备成反义c-junASODNs与体外培养的3周龄睾丸间质细胞共育,了解c-jun表达与睾酮分泌的量效关系。同时,通过MTT和TUNEL法了解睾丸间质细胞睾酮分泌过程中细胞增殖和凋亡的作用。结果显示,c-jun ASODNs以剂量依赖性方式抑制基础状态下睾酮分泌(P<0.01)及睾丸间质细胞增殖(P<0.01),当加入1μmol/L c-junASODNs时,显著抑制睾丸间质细胞的凋亡(P<0.05)。这表明c-jun可促进基础状态下仔猪睾丸间质细胞睾酮的分泌,这种作用与c-jun促进睾丸间质细胞增殖和凋亡有关。睾丸间质细胞的分泌有基础性分泌和促性腺激素诱导的分泌,后者受下丘脑—垂体—性腺轴调节。绒毛膜促性腺激素hCG可与间质细胞表面受体结合,使ATP(?)→cAMP,而cAMP可动员胆固醇经过一系列中间步骤,最终生成睾酮。本试验通过c-jun ASODNs诱导观察c-jun在调节hCG诱导的仔猪睾丸间质细胞分泌的作用,采用c-jun ASODNs拮抗c-jun,维拉帕米阻断钙通道,添加cAMP观察其对体外培养睾丸间质细胞睾酮分泌的影响。结果发现hCG可刺激荣昌仔猪睾丸间质细胞的睾酮分泌,c-jun ASODNs(0.125-2μmol/L)呈剂量依赖性抑制hCG诱导离体睾丸间质细胞的分泌(P<0.01),加用cAMP后睾酮分泌增加,可逆转c-jun ASODNs抑制hCG诱导离体睾丸间质细胞的睾酮分泌。维拉帕米(10-5mol/L)可增加c-jun ASODNs抑制睾酮分泌作用。因此,推测c-jun在促进hCG诱导间质细胞分泌睾酮过程中,主要通过以下信号途径传递信息：(1)hCG作用于仔猪睾丸间质细胞膜特异性受体,通过G蛋白介导,激活腺苷酸环化酶,从而使cAMP生成增多,然后以cAMP作为第二信使,通过cAMP-PKA途径导致核内原癌基因c-jun转录和翻译,其表达产物间或产物与c-fos蛋白在亮氨酸拉链区(1eucne zipper)形成同源或异源二聚体复合物,与靶基因的AP-1 (activator protein 1)位点结合,直接调节转录活性,激活一些晚期基因转录,如P450c17mRNA,后经过一系列酶作用,促进睾酮的合成和分泌。(2)hCG作用于睾丸间质细胞膜上特异性受体,使细胞内Ca2+浓度增高,然后以Ca2+作为第二信使,通过IP3-DAG-钙调蛋白激酶途径继而使核内原癌基因c-jun转录激活。其表达产物间或产物与c-fos蛋白在亮氨酸拉链区形成同源或异源二聚体复合物,与靶基因的AP-1位点结合,直接调节转录活性,激活一些晚期基因转录,如P450c17 mRNA,然后经过一系列酶作用,促进睾酮的合成和分泌。综上所述,我们可以得到以下结论：1、c-jun mRNA在1、3、5周龄荣昌仔猪睾丸中均有表达,3周龄较高,其变化趋势与血浆中睾酮水平变化有一致性。2、酶解法结合差速离心分离的荣昌仔猪间质细胞活力强,纯度较高,数量较多。随着培养时间延长,睾酮分泌量增加,4h达峰值。随着hCG的浓度增加,睾酮分泌增加,50IU/mL的hCG刺激的睾酮分泌达峰值。3、c-jun能剂量依赖性促进基础状态下的睾酮分泌,可能与促进间质细胞增殖和凋亡有关。4、c-jun能剂量依赖性影响hCG诱导的睾酮分泌。5、c-jun在调节hCG诱导睾酮的分泌过程中,可能的机制是通过hCG→激活受体→激活第二信使(cAMP,Ca2+)→诱导c-jun基因转录mRNA→在胞浆内表达产物c-Jun进入核内其表达产物间或产物与c-fos蛋白在亮氨酸拉链区(1eucine zipper)形成同源或异源二聚体复合物,与靶基因的AP-1 (activator protein-1)位点结合→激活一些晚期基因转录,如P450c17mRNA,后经过一系列酶促作用→促进睾酮的合成和分泌。更多还原

【Abstract】 Sexual reproduction is the prerequisite and foundation of animal breeding. Testosterone not only plays a very important role on gonepoiesis, but also maintains the secondary sexual characteristics in male and effects a lot on motility of sperm and fertilization rate. Leydig cells are the main cells to synthesize testosterone in male. It has vital significance to research the regulatory mechanism of testosterone synthesis in leydig cells for understanding the molecular mechanism of sperm production and prevention and treatment of male infertility. The researches of testosterone synthesis mechanism before often focus on the influence of the hypothalamic-pituitary-testicular axis and gonadotropin. However, recent studies suggest that the proto-oncogene gene expression plays a very important role on testosterone synthesis. So this study was performed to investigate the regulatory mechanism of proto-oncogene c-jun in Rongchang piglets’ testosterone synthesis utilizing in vivo and in vitro model. This will provide evidence for further study on proto-oncogene’s effect on gonepoiesis.A number of researches indicate that there are some proto-oncogene expressions when leydig cells secrete testosterone, especially the expression of EGs (Immediate-early genes), which mainly contains members of fos family and the jun family. One week, three weeks and five weeks old Rongchang pigs’ testis were selected to monitor the level of the c-jun mRNA expression by using real-time fluorescence quantitative RT-PCR and radioimmunoassay, and to clear the relationship between its expression and serum testosterone level. Meanwhile, the structure of testis tissues was observed by histological section. The results indicated that c-jun mRNA expressed at one weeks, three weeks and five weeks old. Its expression peak appeared at three weeks old, which had difference with one weeks old (P<0.01) and five weeks old (P<0.05). The level of serum testosterone also gained the peak at three weeks old, this significantly higher than that of one week old (P<0.01) and five week old (P<0.01). At 3rd week, the structure of leydig cells was integrity, it decreased at 5th week. The above suggested that c-jun mRNA expression may keep consistent with the level of serum testosterone and the change of leydig cells’structure. So three weeks old piglet’s testis could be the suitable material to study the testosterone secretion.It was found that the expression of c-jun mRNA in piglet testis tissue at different time kept consistent with the concentration of serum testosterone. To exclude the interference of other cells (sertoli cells and spermatogenic cells), the leydig cells were cultured in vitro to learn more about the dose-effect relationship between c-jun mRNA expression and testosterone secretion. The trypan blue test and 3β-HSD activity detection showed the activity of leydig cells was (93.0±4.9)% through separated by combining enzymatic method and differential centrifugation, and the percentage of purified cells was (85.1±3.6)%. The basic secretion of testosterone increased with the incubation time, which existed a linear relationship(r=0.826, P<0.01). It leveled off the growth peak from 4th h. With concentration of hCG increasing, testosterone secretion gone up and there was a linear relationship between them(r=0.893, P<0.01). Because the testosterone secretion reached the highest when the concentration of hCG was added to 50 IU/ml, so this concentration of hCG was selected to induce the testosterone secretion in subsequent test. In order to accurately understand the relationship between the c-jun mRNA expression and testosterone secretion, antisense technology were used. It’s that the antisense ASODNs which were prepared from c-jun gene were co-cultured with leydig cells in vitro for learning the dose-effect relationship between c-jun expression and testosterone secretion. Also, the proliferation and apoptosis of leydig cells were detected through MTT and TUNEL method during testosterone synthesis. The results indicated c-jun ASODNs inhibited testosterone secretion (P<0.01) in basic state and leydig cells’proliferation (P<0.01) in a dose-dependent manner. The apoptosis of leydig cells was significantly inhibited (P<0.05) under 1μmol/L c-jun ASODNs. This showed that c-jun can promote the testosterone secretion of the basic state on piglets and this effect was related to c-jun’s promotion of proliferation and apoptosis in leydig cells.Leydig cells’ testosterone secretion contains basic secretion and gonadotropin-induced secretion, the latter one is regulated by the hypothalamus-pituitary-gonadal axis. Human chorionic gonadotropin can bind to receptor of cell surface, this makes ATP to cAMP with the help of adenylyl cyclase, then cAMP can mobilize cholesterol, and finally testosterone is produced through a series of intermediate steps. In vitro, c-jun ASODNs were antagonized by c-jun, verapamil was used to prevent calcium channel and cAMP was added to observe the regulatory effect of c-jun in hCG-induced testosterone secretion at leydig cells. As a result, the secretion could be stimulated by hCG in Rongchang piglets and c-jun ASODNs (from 0.125μmol/L to 2μmol/L) dose-dependently inhibited hCG-induced testosterone secretion in vitro (P<0.01). However, this inhibition was reversed after cAMP was added. But verapamil (10-5 mol/L) could strengthen the inhibition. So it could be speculated that c-jun promoted hCG-induced testosterone secretion through the following signal pathway.First, as hCG binded with the specific receptor on membrane, the adenylyl cyclase was activated through G mediated protein which caused an increase in cAMP. Then the intranuclear proto-oncogene c-jun was transcribed and translated in the direction of cAMP by the way of cAMP-PKA. The expression products or the expression product and the c-fos protein formed homologous or heterogenetic dimer at leucine zipper. Later, these composites combined with the AP-1 on target gene, this can regulate the transcriptional activity directly and activate the transcription of some advanced stage gene such as P450c17 mRNA. For a series of enzyme actions, the synthesis and secretion of testosterone was promoted.Second, hCG binded with the specific receptor on membrane as the same, but this raised the calcium concentration of intracellular. Then the intranuclear proto-oncogene c-jun was activated to be transcribed in the direction of calcium by IP3-DAG-Calmodulin kinase pathway. Besides the above, the backward procedures were the same as the first signal pathway.In summary, conclusions were made in the following.1 c-jun mRNA expresses in 1st,3rd,5th week of Rongchang piglets’ testes and 3rd week has the highest expression. Furthermore, the variation of c-jun expression and the serum testosterone are the same.2 The leydig cells of Rongchang piglets which abstracted by enzymatic method and differential centrifugation has stronger activity, higher purity and larger quantity. Along with the time of cultivation, the testosterone volume increases and achieves the peak at 4th. As the hCG concentration increased, the testosterone volume goes up and reached the peak at 50 IU/mL.3 c-jun can dose-dependently promote testosterone secretion under basic state which may concern with the proliferation and apoptosis of leydig cells.4 c-jun has a dose-dependent influence on hCG-induced testosterone secretion.5 In the testosterone secretion process, the regulation mechanism of c-jun is possible that:hCG activates receptor, this causes a raise in the second messengers(cAMP, Ca2+), then the second messengers induce the transcription of c-jun mRNA and the intracellular expression products enter the intranuclear. In the nuclear, the expression products or the expression product and the c-fos protein formed homologous or heterogenetic dimer at leucine zipper. Later, these composites combined with the AP-1 on target gene, this can activate the transcription of some advanced stage gene such as P450c17 mRNA. For a series of enzyme actions, the synthesis and secretion of testosterone was promoted.更多还原