The Study Of Protective Effect Of CIRP Gene Transfer Into Mouse Cryptorchidism By In Vivo Electroporation On Germ Cell

BackgroundPhysiological scrotal hypothermia is necessary for normal spermatogenesis and fertility in mammals. In experimental animals, surgical induction of cryptorchidism or exposure to heat stress causes disruption of spermatogenesis, leading to infertility. Cold-inducible RNA-binding protein(CIRP) is a member of RNA-binding protein family. In the mouse testis, CIRP is constitutively expressed in the germ cells. CIRP expression was the highest when mouse testis was placed in normal scrotal temperature, and CIRP expression was decreased at the elevated scrotal temperature or when mouse testis was placed in abdominal environment. CIRP expression was down-regulation as elevated temperature and up-regulation in hypothermia, which is coincidence with the reaction of testis to heat stress. So we explore the effect of CIRP in spermatogenesis.ObjectiveTo explore the relation between the expression of CIRP in cryptorchid testis and the damage of testicular germ cells. Then we construct the eukaryotic expression vector of mouse pVAX1-CIRP, and observe the CIRP gene expression in cryptorchid testicular tissue, which was transfected with the vector by in vivo electroporation. In addition, we investigate the protective effect of overexpression of CIRP on testicular damage induced by cryptorchidism and its possible mechanism.MethodPart I:20 male BALB/c mice aged 7 weeks were made surgically left cryptorchidism. Seven days and 10 days after the model was made, mice were sacrificed and bilateral testes were removed and weighted. Histological changes of the testis was observed by light microscope. The expression of CIRP mRNA and protein were detected by reverse-transcription polymerase chain reaction and immunoblotting in tissues of testis, respectively. Meanwhile, Annexin V-FITC and propidium iodide (PI) double staining was used to detect germ cell apoptosis analysis by flow cytometry.Part II:The CIRP DNA fragment was obtained from the plasmid pBluescript SK(-)-CIRP by EcoR I and Pst I double enzymes digestion, and inserted into the polylinker of eukaryotic expression plasmid pVAXl to construct recombinant pVAX1-CIRP, sequence was proved by enzymes digestion and sequencing.32 male BALB/c mice aged 7 weeks were divided into 2 groups:experimental group, pVAXl-CIRP was transfered into mouse left testis by combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation; control group was transferred with empty vector plasmid pVAX1, then the mice were made surgically left cryptorchidism. Seven or ten days after electroporation, the expression of CIRP mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and immunoblotting, respectively.PartⅢ:48 male BALB/c mice aged 7 weeks were divided into 3 groups:Group 1, eukaryotic expression plasmid pVAX1-CIRP was transferred into left testis by in vivo electroporation; Group 2, empty vector plasmid pVAXl was transferred into left testis; Group 3, PBS was injected into left testis, then the mice were made surgically left cryptorchidism. Seven or ten days after electroporation, the expression of CIRP, p53 and Fas mRNA and protein were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Meanwhile, Histopathological changes were observed by light microscope, and flow cytometry was used to detect testicular cell apoptosis.ResultsPartⅠ:CIRP was strongly expressed in mouse normal testis. After the mice were made surgically cryptorchidism, the expression of CIRP mRNA and protein were both decreased (P<0.05), CIRP expression on day 10 was much lower than that on day 7 in cryptorchid testis. Meanwhile, cryptorchid testicular weight was significantly lighter than that of contralateral testis (P<0.05). Testicular weight of contralateral testis on day 10 was much heavier than that on day 7(P<0.05), but there was no difference in cryptorchid testis (P>0.05). Furthermore, testicular germ cells apoptosis was increased (P<0.05). Apoptotic rate of cryptorchid testis on day 10 was much higher than that on day 7(P<0.05), but there was no difference in contralateral testis(P>0.05). Cryptorchid testis exhibited disordered and sloughed germinal cells.PartⅡ:The recombinant plasmid pVAXl-CIRP was constructed and identified containing the target CIRP DNA fragment proved by enzymes digestion and sequencing. Seven days after pVAXl-CIRP was transferred into mouse testis by combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation, the expression levels of CIRP mRNA and protein were much higher than that in pVAX1 treated group (P<0.05),but there was no difference between that on day10 and day 7 after the electroporation(P>0.05).PartⅢ:The weight of testis transfected with pVAX1-CIRP was heavier than that in pVAXl or PBS-treated mice at each indicated time point(P<0.05), but there was no difference between pVAXl and PBS-treated mice (P>0.05).Testicular cells apoptotic rate transfected with pVAXl-CIRP was lowerer than that in pVAXl or PBS-treated mice (P< 0.05), but there was no difference between pVAXl and PBS-treated mice at each indicated time point (P>0.05). In addition, the expression of CIRP mRNA and protein in the testes transfected with pVAXl-CIRP were both increased (P<0.05) at each indicated time point. Meanwhile, the expression of p53 was decreased on day 7 (P<0.05) and Fas was decreased on day 10(P<0.05).Conclusions1. CIRP is strongly expressed in mouse normal testis;2. CIRP expression is significantly decreased in mouse cryptorchidism, it shows a down-regulation tendency in 7 days and 10 days after the mouse was made surgical unilateral cryptorchidism;3. Down-regulation of CIRP expression induces increasing of testicular cells apoptosis, then aggravate the demage of germ cell. So down-regulation of CIRP expression may play an important role in germ cell demage induced by cryptorchidism;4. The eukaryotic expression vector of mouse pVAXl-CIRP can be successfully constructed by gene cloning technique;5. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation can transfer CIRP gene into mouse testis, and CIRP gene is normally transcribed and translated in mouse testis, then the expression of CIRP is up-regulated in cryptorchidism;6. Overexpression of CIRP may to some extent reduce testicular germ cell damage induced by abdominal heat stress in cryptorchidism;7. Overexpression of CIRP can reduce the apoptosis of testicular cells in cryptorchidism;8. Overexpression of CIRP can down-regulate the levels of p53 and Fas expression in cryptorchidism.