Expression Of NDRG2 During Rat Testis Development And Its Roles In Apoptotic Spermatogenic Cells

ObjectiveN-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 spermatogenesisMethods1. 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.Results1. 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.Conclusions1. 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.