Molecular cloning of novel temperature-induced expressed sequence tags related to apoptosis in spermatogenic cells of mouse

Abstract

Objective: Spermatogenesis needs the relative cool environment of the scrotum and Cryptorchidism could cause human male infertility .It is most likely due to the cease of spermatogenesis and induced apoptosis of spermatogenic cells when the testes were exposed to the mild hyperthermia environment of abdomen. In this study, we have established a unilateral operative cryptorchid mouse model and used the method of suppression subtractive hybridization (SSH) to screen temperature-induced genes related to apoptosis in Spermatogenic Cells and to investigate the mechanism of spermatogenesis arrest in experimental cryptorchidism. Design: Scrotal testis was removed surgically into abdomen to established unilateral operative cryptorchid mouse model. On day 3 after surgery, SSH was performed between scrotal testis (‘driver’) and cryptorchid testis (‘tester’) to isolate expressed sequence tags (ESTs) which represent genes overexpressed in cryptorchid testis. Novel ESTs will be used to clone full-length cDNA in the future. Materials and Methods: Male C57BL/6 mice at 8–10 wk of age were anesthetized and a small incision was made in the abdomen. The testes on right side were displaced into the abdomen and the inguinal canals on right side were suturing to prevent testes decent. On day 3 after surgery, three mice were killed and testes were removed. mRNA from cryptorchid testes and scrotal testes were extracted and pooled each. SSH was performed between scrotal testes (‘driver’) and cryptorchid testes (‘tester’) using PCR-SelectTM cDNA Substraction Kit (Clontech) according to the manufacturer’s recommendations. The substracted library cDNA was cloned into pGEM-T vector (Promega) and total 168 individual recombinant clones were picked. After screening by reversed northern blots, total 54 recombinant clones were sequenced to analyze insert sequences. Results: 4 novel ESTs were isolated. Homology searches revealed that among them 3 ESTs were unknown cDNA and one EST was identical to a hypothetical protein whose function had not been reported. Conclusion: Experimental unilateral cryptorchidism provide us suitable model for studying temperature-induced alteration of gene expression in the process of hypospermatogenesis and spermatogenic cell apoptosis, because the environmental temperature was the only difference between cryptorchid and scrotal testes in experimental cryptorchidism mice. By using SSH technique, we have identified 4 novel ESTs overexpressed in cryptorchid testis. In further study, we can begin from these ESTs to clone full-length cDNA represent novel genes which may play important roles in temperature-induced germ cell apoptosis.