| 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. |
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