Study On The Molecular Mechanism Of Spermatogenesis Disorder In Cryptorchidism Boars

Objective: Spontaneous unilateral cryptorchid boars have one testis in the abdomen or inguinal canal,which causes its temperature to be at or near body temperature.The prevalence of porcine litters of different breeds with unilateral cryptorchidism is 2.The major concerns of cryptorchidism in pigs are the interruption of normal spermatogenesis due to the aberrant high temperature of the undescended testis in the abdomen.The establishment and maintenance of germ cells are critical that ensure the survival of a species,which is affected by many factors,temperature is the major factor in this process.The male germ cells(sperm)are produced by the complex process of spermatogenesis,which is carried out in scrotum,where the temperature is lower than the core body temperature in most male mammals.Testicular exposure to the body temperature leaded to spermatogenesis arrest,but the detailed molecular mechanism remained unclear.Spontaneous unilateral cryptorchidism was a good animal model to study the effect of body temperature on spermatogenesis.Methods: The present study was performed using four unilateral cryptorchid boars(7-9 months of age,140-150kg)with undescended testes,considered as the cryptorchidism group(cryptorchid),and with descended testes,considered as the scrotal group(contrascrotal),and four preweaning piglets collected from healthy boars(20 days of age,15kg),considered as a preweaning piglet group(preweaning),were castrated and testes were harvested.Testicular tissue samples were divided into parts.One parts were snap-frozen in liquid nitrogen and used to extract total RNA and total protein,was extracted for RNA-seq and i TARQ analysis,respectively.The other parts was fixed in the Bouin’s solution for in situ terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling of free DNA ends(TUNEL),hematoxylin and eosin(HE),and immunohistochemistry assays.Sex healthy male SD rats(30 days of age,90-100g)were induced for the unilateral cryptorchidism.Three rats were castrated and testes were harvested,and fixed in the Bouin’s solution for immunohistochemistry assays after 30d for the unilateral cryptorchidism.Three rats were used for the detection of biotin.Results:1.Changes of Sertoli and germ cells in spontaneous unilateral cryptorchidism boar testes The HE results showed that the seminiferous tubule contained only a few spermatogonia,but did not contain post-meiotic germ cells in cryptorchid compared to contrascrotal.The histomorphometrical analysis results showed that the seminiferous tubule contained only Sertoli cells and a few spermatogonia,but did not contain post-meiotic germ cells in cryptorchid testes.The total number of spermatogonia markedly decreased,and Sertoli cells did not change remarkably in cryptorchid testes.2.Effects of spontaneous unilateral cryptorchidism on the expression and localization of proliferation and apoptosis related proteins in boar testes TUNEL assay results showed that apoptosis signals was observed predominantly in spermatogonia.qRT-PCR and Western Blot results showed that levels of PCNA and Bax significantly decreased,but Bcl-2was significant increased in cryptorchid testes.Immunohistochemistry results showed that in cryptorchid and contrascrotal testes,PCNA were located in the nucleus of spermatogonia.Cryptorchidism results in the redistribution of Bax in spermatogonia and transfer from cytoplasm to nucleus.Bcl-2 was preferentially expressed in cytoplasm and nucleus of germ cells near lumen in all experimental groups.The number of PCNA-positive,TUNEL-positive and LC3-positive cells were decreased in cryptorchid testes.3.Effects of spontaneous unilateral cryptorchidism on the expression and localization of autophagy related proteins in boar testes qRT-PCR and Western Blot results showed that levels of LC3 and Beclin1 significantly decreased,and p62 significantly increased in cryptorchid testes.Immunohistochemistry results showed that in cryptorchid and contrascrotal testes,LC3 was located in the cytoplasm of spermatogonia.LC3-positive cells were decreased in cryptorchid testes.4.Effects of spontaneous unilateral cryptorchidism on the expression and localization of tight junction molecules of blood testis barrier in boar testis qRT-PCR and Western Blot results showed that levels of Claudin-11 significantly increased,andOccludin and ZO-1 significantly decreased in cryptorchid testes.Immunohistochemistry results showed that cryptorchidism leads to the transfer expression of Claudin-11,Occludin and ZO-1 from cell membrane to cytoplasm.5.The effect of unilateral cryptorchidism on the permeability of testicular blood testis barrier in rats Immunohistochemical results showed that cryptorchidism the transfer expression of Claudin-11 and ZO-1 from cell membrane to cytoplasm.The trace biotin showed that biotin entered the testicular lumen of cryptorchidism.6.Effect of spontaneous unilateral cryptorchidism on gene expression in boar testis by RNA-seq analysis RNA-seq results showed that total 6901 differential genes were identified in the preweaning and cryptorchid,including 4603 up-regulated and 2298 down regulated genes.Total 13873 differential genes were identified in the cryptorchid and contrascrotal,including 8216 up-regulated and 2298 down regulated genes.GO enrichment analysis found that the differences between the preweaning and cryptorchid of genes were mainly concentrated in cell adhesion,cell development and spermatogenesis,and the differences between the cryptorchid and contrascrotal of genes were mainly concentrated in spermatogenesis,cell differentiation and cell migration.KEGG pathway pathway enrichiment analysis found that the differences between the preweaning and cryptorchid of genes were mainly concentrated in metabolism,ECM receptor interaction and cell adhesion pathway.The differences between the cryptorchid and contrascrotal of genes were mainly concentrated in metabolism,cell adhesion molecules(CAMs)and signal transduction pathways(apoptosis,phagocytosis,RAS and MAPK pathways).And most of genes were up-regulated in CAMs,including CLDN 11(claudin-11),and the down regulated genes,actin-3,played an imported role in cell adhesion,migration and polarization.7.Effect of spontaneous unilateral cryptorchidism on protain expression in boar testis by i TRAQ analysis iTRAQ results showed that total 1459 differential proteins were identified in the preweaning and cryptorchid,including 773 up-regulated and 686 down-regulated proteins.Total 1424 differential genes were identified in the cryptorchid and contrascrotal,including 656 up-regulated and 768 down-regulated proteins.GO enrichment analysis found that the differences between the preweaning and cryptorchid of proteins were mainly concentrated in cell adhesion,metabolism,and extracellular matrix,and the differences between the cryptorchid and contrascrotal of proteins were mainly concentrated in spermatogenesis,reproduction and energy metabolism.KEGG pathway pathway enrichiment analysis found that the differences between the preweaning and cryptorchid of proteins mainly concentrated in focal adhesion,DNA replication and retinol metabolism pathway.The differences between the cryptorchid and contrascrotal of proteins were mainly concentrated in DNA replication,proteasome,gap junction and adhesion junction pathway.The down-regulation of STR5A1 and CYP11A proteins suggested that the synthesis and metabolism of testosterone were blocked.Conclusion: Insufficient proliferation,excessive apoptosis,and autophagy were involved in the regulation of the decrease of spermatophore in cryptorchid boar testes.The damage of the structure and function of Sertoli cells,the increase of BTB permeability and the insufficiency of supporting cells,which eventually leaded to spermatogenesis disorder of in cryptorchid boar testes.