睾丸去神经支配对雄性生殖细胞的影响及其机制

发布时间：2018-01-05 00:20

本文关键词：睾丸去神经支配对雄性生殖细胞的影响及其机制　出处：《南京医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 目的研究睾丸支配神经(精索神经)在睾丸组织内的分布特征及去神经支配后对雄性生殖细胞凋亡与分化的影响,以及对性激素分泌的影响。方法 1)免疫组织化学ABC法,以特异性的抗体标记人睾丸组织内蛋白基因产物9.5(PGP9.5)、C末端神经多肽Y(CPON)、神经肽Y(NPY)和血管活性肠肽(VIP)等肽能神经,了解其在人睾丸组织内的分布特点 2)通过显微外科技术,同时切除SD大鼠(300-350g)精索上神经和精索下神经,分别于1、4、8天采取睾九组织,采用脱氧核搪核酸末端转移酶介导的dUTP的缺口末端标记技术(TUNEL)观察精原细胞和Leydig细胞的凋亡情况。 3)切除精索神经对性激素的影响,解剖显微镜下建立睾丸去神经支配大鼠模型,分别于术前7天、术后1、7、14、21天和术后28天进行麻醉下大鼠眼眶后静脉丛处取血,放射性免疫的方法检测血清中睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)、泌乳素(PRL)和雌二醇(E2)的浓度。了解性激素分泌的变化。 4)以EDS建立Leydig细胞特异性敲除模型,不同剂量EDS腹腔内注射给药,注射后3天、7、14天处死大鼠,取出睾丸组织,行HE染色光镜下组织学观察和P450scc免疫组化观察及灰度分析。观察Leydig细胞特异性敲除情况。结果 1)PGP9.5肽能神经和CPON肽能神经主要分布在睾丸间质内,与曲细小管相邻,CPON阳性纤维尚见于血管周围,其分布密度低于PGP9.5;NPY肽能神经在人睾丸实质内主要见于睾丸血管周围;VIP肽能神经在睾丸实质内未见分布。 2)术后1、4、8天,精原细胞和间质细胞均发生显著凋亡。其中精原细胞的凋亡峰值出现在术后第一天,间质细胞的凋亡在这3个时点没有明显差异 3)血清中睾酮测定:去精索神经组术后睾酮持续下降,与手术前相比差异有显著性(P＜0.05),去精索上下神经组较去精索上神经组和去精索下神经组睾酮下降更为明显,于28天时有显著差异性(P＜0.05);血清中的黄体生成素测定:去精索神经组术后黄体生成素持续上升,与手术前相比差异有显著性(P＜0.05),去精索上下神经组较去精索上神经组和去精索下神经组上升更为明显,于28天时有显著差异性(P＜0.05);血清中的卵泡刺激素测定:均于手术后1天迅速升高(P＜0.05),此后逐渐降低至手术前的水平;血清中泌乳素测定:去精索神经各组血清中泌乳素于手术后逐渐升高,术后28天达最高,其中去精索下神经组术后21天和术后28天与术前比差异有显著性(P＜0.05),去精索上神经组术后14天、术后21天和术后28天与术前比差异有显著性(P＜0.05),去精索上下神经组术后1天、术后7天、术后14天、术后21天和术后28天与术前比差异有显著性(P＜0.05);血清中雌二醇测定:去精索神经各组血清中雌二醇均于术后1天急剧下降并于术后14天降至最低(P＜0.05),而后稍有回升并保持稳定,但是仍低于手术前(P＜0.05)。 4)EDS处理3天后,均观察到Leydig细胞的减少,其中40mg/kg剂量组的减少程度不明显;EDS处理后7天,曲细精管内生精细胞出现排列紊乱的现象,以90mg/kg剂量组更为明显;EDS处理后14天,60mg/kg剂量组出现一种核圆形、核染色淡、体积较大的新形成Leydig细胞,而在75mg/kg剂量组没有发现此细胞。P450scc免疫组化与光镜结果一致。灰度分析结果显示,EDS处理后3天及7天,各剂量组与正常对照相比差异均有显著性(P＜0.05);60mg/kg剂量组14天与3天、7天相比差异有显著性(P＜0.05),且与正常对照差异无显著性(P＞0.05)。结论 1)人睾丸间质内分布着PGP9.5、CPON和NPY肽能神经纤维,为精索神经参与精子发生过程提供形态学基础。 2)在睾丸去神经早期阶段,精原细胞和间质细胞即有大量凋亡,这可能是去神经支配后期睾丸组织发生病理改变的重要原因。 3)睾丸去精索神经支配对大鼠性激素的产生有明显的影响,精索神经对精子的发生具有重要的调控作用。 4)EDS确实能特异性地敲除成年大鼠Leydig细胞,60mg/kg的剂量可以更好的模拟青春期Leydig细胞增殖分化过程。
[Abstract]:objective
The distribution characteristics of testicular innervation (spermatic cord nerve) in testicular tissue and the effect of denervation on the apoptosis and differentiation of male germ cells and the effects of sex hormones secretion were investigated.
Method
1) immunohistochemical ABC method was used to label the peptidergic nerve of human testicular tissue, protein gene product 9.5 (PGP9.5), C terminal neuropeptide Y (CPON), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) with specific antibodies, and to understand its distribution in human testicular tissue.
2) by microsurgical technique, simultaneous resection of SD rats (300-350g) spermatic nerve and spermatic nerve, take nine testis tissue on 1,4,8 days respectively, TUNEL using DNA RNA TDT mediated dUTP (TUNEL) to observe the apoptosis of spermatogenic cells and Leydig cells.
3) resection of spermatic nerve effect on sex hormone, under a dissecting microscope to establish the rat model of testicular denervation, respectively in the 7 days before surgery, 28 1,7,14,21 days after operation and postoperative anesthesia in rats after orbital venous plexus at the blood testosterone in serum of the radioactive immune method (T) (FSH), follicle stimulating hormone, luteinizing hormone (LH), prolactin (PRL) and estradiol (E2) concentrations. Understand the change of sex hormone secretion.
4) to establish EDS cell specific Leydig knockout model, different dose of EDS intraperitoneal injection, 3 days 7,14 days after injection, the rats were sacrificed, removal of testicular tissue by HE staining, light microscope observation and P450scc immunohistochemical observation and gray analysis. Observation of Leydig cell specific knockout situation.
Result
1) PGP9.5 peptidergic nerves and CPON peptidergic nerves are mainly distributed in the testicular interstitium, and seminiferous tubules adjacent to CPON positive fibers are found in the blood vessels around the density of less than PGP9.5; NPY peptidergic nerves in human testicular parenchyma mainly in the testicular vessels around; VIP peptide was not found in the testicular parenchyma of nerve.
2) 1,4,8 days after operation, spermatogonia and stromal cells all had significant apoptosis. The apoptosis peak of spermatogonia appeared on the first day after operation. There was no significant difference in the apoptosis of interstitial cells at these 3 points.
3) determination of testosterone in serum: to spermatic nerve group after testosterone declined compared with before surgery, there was significant difference (P < 0.05), to the cord nerve group than in denervation group and denervation nerve cord testosterone group decreased more significantly, in 28 days when there were significantly differences (P < 0.05); luteinizing hormone determination in serum: spermatic nerve group after LH continued to rise, compared with before surgery, there was significant difference (P < 0.05), to the cord nerve group than in denervation group and denervation nerve cord group increased more significantly, in 28 the day when there are significant differences (P < 0.05); serum levels of FSH were measured: 1 days after surgery increased rapidly (P < 0.05), then decreased gradually to the preoperative level of serum hormone determination; lactation: go to the spermatic nerves of serum prolactin after operation gradually increased. After 28 the highest in Tianda, among them Go to the spermatic nerve group after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05), go to the spermatic nerve group after 14 days, after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05), to the cord the nerve group after 1 days, 7 days after operation, 14 days after the operation, after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05); Determination of estradiol in serum: serum estradiol in the spermatic nerves were on the 1 postoperative day and decreased dramatically on the 14 day after surgery to the lowest (P < 0.05), and then recovered slightly and remained steady, but still lower than that before operation (P < 0.05).
4) EDS after 3 days of treatment, were observed in Leydig cells decreased, the 40mg/kg dose reduction degree is not obvious; 7 days after EDS treatment, the seminiferous tubules spermatogenic cells disordered phenomenon in 90mg/kg dosage group was more obvious; 14 days after treatment with EDS, a nuclear round appearance 60mg/kg dose group, nuclear staining, a larger volume of newly formed Leydig cells, while in the 75mg/kg group did not find this.P450scc cells by immunohistochemistry and light microscopy results. The gray analysis results showed that EDS treatment after 3 days and 7 days, each dose group and normal controls were significantly different (P < 0.05); 60mg/kg group 14 days and 3 days, there was significant difference between the 7 days (P < 0.05), and no significant difference with normal control (P > 0.05).
conclusion
1) PGP9.5, CPON and NPY peptide can provide a morphological basis for the spermatogenesis of the spermatic cord.
2) in the early stage of testicular denervation, there is a large number of apoptosis in spermatogonia and interstitial cells, which may be an important reason for pathological changes in testicular tissue at the late stage of denervation.
3) testicular denervation of spermatic cord has an obvious effect on the production of sex hormones in rats. The spermatic nerve plays an important role in regulating the occurrence of sperm.
4) EDS can specifically knock out Leydig cells in adult rats. The dose of 60mg/kg can better simulate the proliferation and differentiation process of Leydig cells in puberty.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R321.1

【引证文献】