Effects Of Body Temperature On The Expression And Localization Of Meiosis-related Proteins In Testes Of Boars,Dogs And Rats

Objective: High ambient temperature,local scrotal heating and cryptorchidism damaged spermatogenesis and sperm quality,which had been widely concerned by research.Body temperature could lead to interruption of spermatogenesis,but the molecular mechanism was still unclear.Cryptorchidism was defined as the failure of testes to enter the scrotum,which exposed the testes to body temperature.Cryptorchidism was one of the most typical model to study the effects of heat stress on spermatogenesis.Meiosis was a unique feature of germ cell development.Whether cryptorchidism damage the initiation of meiosis had not been reported.The aim of this study was to determine whether spermatogonia in the cryptorchid testes entered into meiosis by detecting meiosis-related markers Stimulated by retinoic acid gene 8(STRA8),Doublesex and mab-3 related transcription factor 1(DMRT1)and Synaptonemal complex protein 3(SCP3).The present study will provide the histological basis for cryptorchidism-induced infertility.Methods: The present study was performed using three boars with spontaneous unilateral abdominal cryptorchidism(Landrace,7 months of age).Scrotal testes(control group)and abdominal testicular tissues(cryptorchidism group)were collected after castration.The testicular tissues of each group were cut into small pieces.Part of boar testes was frozen in liquid nitrogen for extraction of total RNA and total protein.Another part of boar testicular tissues was fixed for immunohistochemistry in Bouin’s solution at 4 °C.Three male dogs(Akita,1 year old)with spontaneous unilateral abdominal cryptorchidism were present to a private animal clinic for treatment of cryptorchidism.Testes samples were obtained after cryptorchidectomy performed by veterinary surgeon.Scrotal testes(control group)and abdominal testes(cryptorchidism group)were placed in Bouin’s solution for histological and immunohistochemical examination.Three healthy male SD rats(20 days old)were purchased from Experimental Animal Center of Shanxi Medical University.The unilateral cryptorchidism was experimentally induced in the rats under ether anesthesia by cuting the distal gubernaculum on the right side and displacing the testis inside the abdominal cavity,then the inguinal canal was sutured.The left testis remained in the scrotum as the control which underwent a sham operation.53 days after operation,the cryptorchid rats were killed by cervical dislocation and scrotal testes(control group)and abdominal testes(cryptorchidism group)were harvested and fixed in Bouin’s solution for 24 h to make paraffin sections.Results:1.Effects of cryptorchidism on the expression and localization of meiosis-related proteins in boar testesqRT-PCR and Western Blot results showed that m RNA and protein levels of STRA8,DMRT1 and SCP3 were significantly reduced in cryptorchid boars.Immunohistochemical results showed that in the control testes of boars,STRA8 was localized to the nucleus of spermatogonia and spermatocytes,DMRT1 was localized to the cytoplasm of spermatogonia and Sertoli cell,and SCP3 was localized to the nucleus of spermatocytes;in cryptorchidism group,STRA8,DMRT1 and SCP3 immunopositive cells were also observed.2.Effects of cryptorchidism on the histological localization of meiosis-related proteins in testes of dogs and ratsImmunohistochemical results showed that in the control group of dogs,STRA8 was localized to the nucleus of spermatogonia and spermatocytes,DMRT1 was localized to the cytoplasm of spermatogonia and Sertoli cell,and SCP3 was localized to the nucleus of spermatocytes;in the control group of rats,STRA8 was localized to the nucleus of spermatogonia,DMRT1 was localized to the cytoplasm of spermatocytes,and SCP3 was localized to the nucleus of spermatocytes.In the cryptorchidism group of dogs and rats,STRA8,DMRT1 and SCP3 immunopositive cells were observed.Conclusion: Compared with the control,the cryptorchid testes of boars,dogs and rats showed a reduction in the number of germ cells in the seminiferous epithelium,which contained Sertoli cells,spermatogonia and a few spermatocytes.No spermatids were found in the seminiferous tubules.The expression of meiosis-related markers STRA8,DMRT1 and SCP3 in the testes of cryptorchid boars,dogs and rats suggested that spermatogonia could still enter into meiosis,which was a common phenomenon in cryptorchid animals,indicating that the mechanism of cryptorchidism-induced infertility did not block the initiation of meiosis.