摘要
目的
NDRG2与NDRG1,NDRG3和NDRG4共同组成NDRG家族,该家族成员可能与细胞的增殖和分化相关,但其具体的生物学功能有待于进一步研究。在前期的研究中我们通过免疫组织化学染色观察到NDRG2在成年人和小鼠睾丸内均表达定位于睾丸间质细胞,而在生精细胞内未见明显的阳性着色,推测其可能参与睾丸间质细胞睾酮的分泌与调节,进而调控精子的发生,因此本实验的目的在于探讨NDRG2在睾丸发育与精子发生过程中的作用及其机制。0
方法
1.利用RT-PCR、蛋白质免疫印迹杂交及免疫组织化学等方法对比研究出生后大鼠睾丸发育不同阶段(1、5、15、21、35和75 d)中NDRG2的表达与定位;并分离21、35和75 d大鼠睾丸间质细胞和生精细胞,利用RT-PCR和蛋白质免疫印迹杂交分析和验证NDRG2在大鼠睾丸不同发育阶段中表达的差异性。
2.建立成年大鼠单剂量MAA(650 mg/kg)腹腔注射诱导精母细胞凋亡模型,采用TUNEL、RT-PCR、蛋白质免疫印迹杂交、免疫组织化学、间接免疫荧光和激光显微切割技术分析NDRG2在生精细胞凋亡过程中表达的变化,探讨NDRG2与生精细胞凋亡之间的关系。
3.青春期大鼠睾丸内生精细胞自发性凋亡达到顶峰,建立青春期(21 d)大鼠隐睾模型,通过TUNEL染色验证该模型、运用免疫组织化学和间接免疫荧光等方法分析NDRG2在隐睾和对照侧睾丸表达的差异,并且分离术后不同时间点的间质细胞和生精细胞,运用免疫印迹杂交检测NDRG2和p53的表达变化。
结果
1.RT-PCR和Western blot结果均显示NDRG2在出生后1 d大鼠睾丸内即有表达,随后逐渐升高,至青春期达到顶峰(21和35 d),在成年期其表达反而下降。免疫组织化学结果显示NDRG2持续表达于睾丸间质细胞,但在睾丸发育不同阶段,其在生精小管内的表达却存在明显的差异。在新生期(1和5 d)主要表达于原始生殖细胞,而不表达于支持细胞;在15和21 d大鼠睾丸,NDRG2在生精小管内主要表达于精母细胞,而精原细胞和支持细胞未见阳性着色;在35 d大鼠睾丸,NDRG2在生精小管内主要表达于精母细胞和圆形精子细胞;在成年(75 d)大鼠,生精小管内几乎未见NDRG2阳性着色。分离纯化21,35和75 d大鼠睾丸生精细胞,采用RT-PCR和Western blot进一步验证了NDRG2在青春期大鼠生精细胞内高表达,而在成年大鼠生精细胞内几乎不表达。分离纯化21,35和75 d大鼠睾丸间质细胞,结果显示NDRG2在这三个时间点间质细胞内均有较强的表达,但相互间无明显差异。
2.MAA腹腔注射3和6 h与对照组相比未见显著性差异,但MAA诱导12 h后,成年大鼠睾丸内出现大量初级精母细胞的凋亡,主要集中在生精周期的第X-XIII期,通过分离生精细胞和激光显微切割技术均检测到NDRG2在MAA诱导12 h后在生精细胞的表达上调,免疫组织化学和间接免疫荧光结果均显示NDRG2在自发性和MAA诱导凋亡的生精细胞中均高表达。
3.建立21 d大鼠单侧隐睾模型,术后7 d和14 d,隐睾侧睾丸重量明显减轻,凋亡生精细胞数明显升高,免疫组织化学与间接免疫荧光结果显示NDRG2在自发性和热刺激诱导凋亡的生精细胞内表达均明显上调,但在一些正常的精母细胞和圆形精子内仍存在一定量的表达,并且NDRG2在隐睾侧间质细胞的表达强度明显降低。分离纯化术后不同时间点隐睾侧和对照侧睾丸生精细胞,发现NDRG2和p53在术后7 d隐睾侧生精细胞的表达量明显上调,而在隐睾后14 d未见明显差异,可能与生精细胞的凋亡与脱落有关;分离纯化术后不同时间点隐睾侧和对照侧睾丸间质细胞,进一步证实了NDRG2在术后7和14 d隐睾侧间质细胞的表达量减少。
结论
1. NDRG2在大鼠睾丸发育不同阶段持续表达于间质细胞,可能参与睾丸早期发育和睾酮的分泌与调节。
2. NDRG2在大鼠睾丸发育过程中在生精细胞的表达存在较大差异,在青春期前在生精细胞有较高水平的表达,但在成年期表达量很低,提示NDRG2可能参与睾丸发育早期生精细胞增殖与分化的调节。
3. NDRG2在MAA和热刺激诱导的生精细胞凋亡中表达均明显上调,并且在青春期和成年期大鼠睾丸自发性生精细胞凋亡中均存在较强的表达,提示NDRG2可能参与了生精细胞凋亡的调控。
Objective
N-myc downstream regulated gene 2 (NDRG2), together with NDRG1, NDRG3 and NDRG4, constitute the NDRG family. Although NDRG family is possibly related to cell proliferation and differentiation, the exactly biological function has not yet been completely elucidated. Our previous study has shown that NDRG2 was expressed in the interstitial cells of adult human and mouse testis, but not in the seminiferous tubule, which suggested the potential role of NDRG2 in the regulation and secretion of testosterone, thereby regulating spermatogenesis. Therefore, the aim of the present study was to explore the roles of NDRG2 play in testicular development and spermatogenesis
Methods
1. RT-PCR, Western blot and immunohistochemistry were used to observe the expression and location of NDRG2 in the rat testes at different stages of development (1, 5, 15, 21, 35 and 75 days). Then Leydig cells and spermatogenic cells were isolated from 21, 35 and 75 days old rats respectively, the relative levels of NDRG2 mRNA and protein were detected by RT-PCR and Western blot.
2. Groups of adult SD rats were treated intraperitoneally with MAA (650 mg/kg) in saline to induce spermatocyte apoptosis. To explore the relationship between NDRG2 and apoptosis, the expression of NDRG2 in apoptotic spermatogenic cells was observed by RT-PCR, Western blot, immunohistochemistry, indirect immunofluorescence and Laser Capture Microdissection (LCM).
3. The spontaneous apoptosis of spermatogenic cells reach the peak during adolescence in rat testis. Therefore, in this study, we generated the experimental model of cryptorchidism in pubertal male Sprague-Dawley rats, characterized the apoptotic testicular cells by the TUNEL assay and compared the expression of NDRG2 in the abdominal and contralateral scrotal testes longitudinally by immunohistochemistry and immunofluorescent analyses. Furthermore, we purified the Leydig and germ cells from the cryptorchid and control testes at different time points post surgery and analysed the expression of NDRG2 and p53 by western blot assays.
Results
1. The NDRG2 gene in the testis was transcribed at all rat ages. The level of NDRG2 mRNA and protein increased during the testicular development, reaching a peak in the adolescence period of rats occurring between 21 and 35 days of age. Levels then significantly decreased in adult rat testis. Anti-NDRG2 staining was positively detected in Leydig cells in the testis of rats at different ages. In the infantile period, the seminiferous tubule is mainly comprised of Sertoli cells and gonocytes. Positive staining of anti-NDRG2 was found predominately in the cytoplasm of gonocytes, but not in the Sertoli cells of the seminiferous tubule. On day 15 and 21, the expression of NDRG2 was detected as accumulated in the lumen of spermetocytes, but was not found in spermatogonial or Sertoli cells. On day 35, the expression of NDRG2 was found in the cytoplasma of spermatocytes and also in the round spermatids. In contrast, NDRG2 appeared to be expressed predominately by Leydig cells in the testis of 75 days old rats, but almost not observed in the seminiferous tubule. Analysis of the NDRG2 expression in the spermatogenic cells isolated from 21, 35, and 75 day old rats revealed high levels of NDRG2 mRNA transcripts and proteins in spermatogenic cells isolated from 21 and 35 days old rats, but little in spermatogenic cells from adult rats. The expression of NDRG2 in Leydig cells isolated from 21, 35, and 75 day old rats was observed by RT-PCR and Western blot. There was no significant difference in the levels of NDRG2 mRNA transcripts and proteins among these periods of Leydig cells.
2. Three and six hours post treatment with MAA, the number of apoptotic cells was slightly but insignificantly increased in the seminiferous tubules as compared with that in the control. However, dramatically increased number of apoptotic cells were observed 12 h after MAA treatment and identified as the pachytene spermatocytes. Interestingly, although apoptotic cells spread near all stages of the seminiferous epithelial cycle, apoptotic spermatocytes particularly displayed predominately at stages X-XIII of the seminiferous epithelial cycle in the rat testis. Up-regulated of NDRG2 expression was deteced in the isolated spermatogenic cells from 12 h after MAA treatment rats and in the seminiferous tubule of X-XIII stages obtained by LCM. Furthermore, high levels of NDRG2 expression were observed in both spontaneous and induced apoptotic spermatogenic cells in the testis of SD rats by immunohistochemistry and immunofluorescent analyses.
3. The experimental model of cryptorchidism was generated in pubertal male Sprague-Dawley rats (21 days). 7 and 14 days post surgery, the mean weight of cryptorchid testes was reduced significantly as compared with that of control, and significantly increased numbers of apoptotic cells were detected in the seminiferous tubules of cryptorchid testes, as compared with that of controls. A strong immunostaining of NDRG2 was observed in both spontaneous and heat-induced apoptotic spermatogenic cells by immunohistochemistry and immunofluorescent analyses. However, immunostaining of NDRG2 was still observed in many healthy spermatocytes and round spermids. Notably, lower density of NDRG2 staining in the cytoplasma of Leydig cells was obviously in the cryptorchid testes, as compared with that in control testes at 7 and 14 days post surgery. Further investigation of the expression of NDRG2 and p53 in spermatogenic cells purified from the cryptorchid and control testes showed that both levels of NDRG2 and p53 increased significantly in the cryptorchid testes at 7 days post surgery. In addition, significantly decreased levels of NDRG2 expression were detected in Leydig cells purified from the cryptorchid testes at 7 and 14 days post surgery, consistent with the observations of immunohistochemistry and immunofluorescence.
Conclusions
1. NDRG2 was expressed in the cytoplasm of Leydig cells in the testes of rats at all ages. It may be involved in the early testicular development as well as the secretion and regulation of testosterone.
2. NDRG2 was highly expressed in spermatogenic cells of immature rats, but not in spermatogenic cells of adult rats. It is possible that NDRG2 may be a regulatory factor for cellular proliferation and differentiation during spermatogenesis.
3. The expression of NDRG2 was up-regulated in both MAA-induced and heat-induced apoptosis of spermatogenic cells. Furthermore, it was also strong expressed in the spontaneous apoptosis of spermatogenic cells in both adolescent and adult rat testis. These results suggested that NDRG2 may be involved in the regulation of spermatogenic cell apoptosis.
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