Germ cell apoptosis induced by experimental cryptorchidism is mediated by multiple molecular pathways in Cynomolgus Macaque

Abstract

Experimental cryptorchidism is a common model for examining the expression and function of heat-sensitive spermatogenesis-related genes in testis. Previous studies have shown that germ cells in cryptorchid testis die mainly in an apoptotic way. The molecular mechanism, however, is still unclear. We have established unilateral cryptorchid monkey model (Cynomolgus Macaque) to identify possible molecules involved in the germ cell apoptosis. The degree of germ cell apoptosis, the morphology of the cryptorchid testis, and the changes in the serum concentration of FSH, LH and testosterone after cryptorchid surgery were analyzed. Sertoli cell marker molecule vimentin, the orphan receptor LRH-1, as well as the mitochondria-related protein HSP60 and Bcl-2 were examined. Our results showed that the weight of the cryptorchid testis decreased in a time-dependent manner started from day 7 after the surgery, while the weight of the scrotal testis had no obvious change. HE staining showed that from day 5, some germ cells were detached from the epithelium. A massive degeneration of the seminiferous epithelium characteristic of epithelial structural disorganization and the formation of multinucleated giant cells as well as vacuoles was observed on day 10 and 15. The cryptorchidism induced a marked germ cell apoptosis on day 3 after the operation, reaching a peak level on day 7. The apoptotic germ cells were mainly primary spermatocytes. Radioimmunoassay results showed that serum testosterone level was significantly decreased (p<0.01) in the unilateral cryptorchid monkeys on day 1 and the low level was maintained to the end of the experiment. LH concentration in the serum decreased significantly on day 3 (p<0.05) and subsequently recovered to the normal level. In contrast, no obvious change in the serum FSH concentration was detected. Immunohistochemistry data showed that the pattern of HSP60 expression was mainly perinuclear in the spermatogonia, spermatocytes and Sertoli cells. Weaker staining was also observed in the Leydig cells. In the cryptorchid testis the staining for HSP60 was obviously stronger in these cells. Vimentin staining was observed mainly in cytoplasm of Sertoli cells in the scrotal testis. The expression of vimentin was collapsed and obviously increased in a time-dependent manner in the cryptorchid testis. Western blotting results indicated that HSP60, Bcl-2, and LRH-1 expression increased significantly in the cryptorchid testis as compared to the scrotal testis. RT-PCR data further verified the increase of hsp60 mRNA in the cryptorchid testis. These observations suggest that multiple molecular pathways participate in the germ cell apoptosis induced by cryptorchidism.