[Frontiers in Bioscience 11, 1077-1089, January 1, 2006]
Germ cell apoptosis induced by experimental cryptorchidism is mediated by multiple molecular pathways in Cynomolgus Macaque
S-X Tao, J Guo, X-S Zhang, Y-C Li, Z-Y Hu, C-S Han and Y-X Liu
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuanxi Rd, Beijing 100080, China
Figure 1. Weight Changes in cryptorchid testis during treatment. N, testes before treatment; d1, d3, d5, d7, d10, d15 represent day 1, day 3, day 5, day 7, day 10 and day 15 after the surgery respectively. Each time point represents the average weight from three monkeys.
Figure 2. Histo-morphological analysis of scrotal and cryptorchid testes by HE stain. a: scrotal testis; b-e: cryptorchid testes on day 1, 5 ,10 and 15 after surgery respectively. f: a magnification of a (×400 ). The arrows show the multinucleated giant cell. The seminiferous epithelium of cryptorchid testis was obviously disturbed from day 5 (d) after the surgery, multinucleated giant cells were observed in the cryptorchid testis on day 10 (Figure 2d). Only Sertoli cells and a few spermatogonia were left in the cryptorchid seminiferous epithelium on day 15. Original magnificationx 200.
Figure 3. Apoptotic germ cells in scrotal (a) and cryptochid testes (b-f) identified by in situ 3'-end labeling (TUNEL). ST: seminiferous tubule. The arrows indicate the TUNEL positively labeled germ cells which were darkly stained. The scrotal testis (a) shows a few apoptotic germ cells in the seminiferous tubules; the cryptorchid testis on day 3 (c) shows increased TUNEL positively and reached the strongest on day 7 (e) after the surgery; On day 15 (f) after surgery, the number of apoptotic cells obviously reduced. Original magnificationx 200.
Figure 4. Changes in serum testosterone, LH, and FSH levels in cryptorchid cynomolgus monkeys. (a) Serum testosterone level significantly decreased from day 1 after cryptorchid operation, and remained low thereafter. (b) LH was significantly decreased on day 3, but no significant difference was observed after day five. (c) FSH level in serum was not obviously changed. The RIA data was obtained from the serum samples of three individual monkeys at each point. Data were shown as means ± SD (n=3).
Figure 5. Immunohistochemical staining for vimentin in cryptorchid testis. a, negative control; b, scrotal testis; c-e, cryptorchid testis on day 1, 7, and 15 after surgery; f, a magnification of b; arrows show a perinuclear expression of vimentin in scrotal testis. Vimentin staining in scrotal tseminiferous tubules was observed mainly in the perinuclear region of Sertoli cells; Appearance of increased and disorganized vimentin staining in cryptorchid testis on day 7 and 15 was observed. Original magnificationx 200.
Figure 6. Immunohistochemical staining of HSP60 in testes of Cynomolgus macaca. a, negative control; b, scrotal testis; c-e, cryptochid testis on day 1, 7 and 15 respectively after surgery. f, a magnification of b. HSP60 expression could be observed in spermatogonia, spermotocytes and Sertoli cells. A weak expression of this protein could be also observed in Leydig cells. Arrows show a perinuclear exression of HSP60 protein in Sertoli cells. Increased HSP60 expression was observed in cryptorchid testis on day 7 and day 15. Original magnificationx 200.
Figure 7. Western blot analysis of HSP60, Bcl-2 and LRH-1 expression in cryptorchid testes. (A) Proteins in the testicular extracts were separated by SDS-PAGE and immunoblotted with anti-HSP60, Bcl-2 and LRH-1 polyclonal antibody respectively. (B) Quantitative analysis for HSP60 (B), Bcl-2 (C) and LRH-1(D). These protein expression was significantly increased in cryptorchid testis. N, scrotal normal testis; d1 to d15: cryptorchid testis on day 1, 3, 5, 7, 10 and 15 respectively after surgery. Experiments from three individual monkeys and repeated three times. Data were shown as means ± SD (n=3). O.D. represents optical density.
Figure 8. RT-PCR analysis for changes in HSP60 mRNA in cryptorchid testis. (A) RT-PCR of HSP60 : Using total RNA extracted from cryptorchid testis; RT-PCR of actin mRNA as a control; (B) Analysis of HSP60 mRNA by scanning the RT-PCR blot density. The results show that HSP60 mRNA was increased in cryptorchid testis. N, scrotal normal testis; d1 to d15 represent cryptorchid testis on day 1, 3, 5, 7, 10 and day 15 after surgery respectively. Experiments were repeated three times. Data were shown as means ± SD (n=3). O.D., optical density.
Figure 9. A schematic representation of germ cell apoptosis induced by cryptorchidism. Two major, the extrinsic and the intrinsic, pathways for caspase activation in germ cells are presented. The extrinsic pathway can be induced by members of the TNF family of cytokine receptors, such as TNFR and Fas. The intrinsic pathway can also be activated by cryptorchidism, resulting in release of cytochrome C from mitochondria and the subsequent apoptosome formation. Both apoptotic pathways activate the common downstream effector protease, caspase-3, to execute apoptosis. p53 can result in increase of Fas expression and hence induce killing by the Fas/Fas ligand pathway. It can also interfere with Bcl-2 family proteins to regulate the fate of germ cell, including elevations in the levels of pro-apoptotic members such as Bax or down-regulation of anti-apoptotic Bcl-2 expression level. On the other hand, up-regulation of mitochondrial Hsp60 expression by cryptorchidism indirectly increase free Bcl-2 protein and decrease free Bax protein in Sertoli cells and spermatogonia to help the inhibition of activating caspase-3, thus inhibiting gem cell apoptosis. The increased LRH-1 production in the cryptorchid testis might accelerate transformation of testosterone into estrogen leading to the decrease in the serum testosterone concentration, subsequently enhancing germ cell apoptosis by activating testosterone-dependent Fas/FasL death receptor pathway.