The Changes Of Expression And Significance Of RNA-binding Protein Ptbp2 In Cryptorchidism Murine Model

Objective RNA binding protein polypyrimidine tract binding protein 2(Ptbp2)as a key alternative splicing regulator for male germ cell development is well established.However,its expression levels and role in cryptorchidism testes tissues has not been explored.Additionally,the molecular mechanism of heat stress impacts the correct proliferation and differentiation of germ cells is unclear.To investigate whether changes in Ptbp2 expression are correlated with heat stress-induced germ cell injury in testicular tissue,we used a murine model of intraperitoneal cryptorchidism with surgical operation.Methods 50 six-week-old male ICR mice were randomly divided into two groups,25 for sham-operated(SO)group,and 25 for unilateral cryptorchidism(UC)group.When the groups had reached 3,7,14,21 and 28 days post-operation(dpo),mice weresacrificed by cervical dislocation and the testes and epididymidestissues were obtained and weighed.To assess the degree of damage to the sperm cells,HE staining and sperm analysis were used.Western blot、IHC and QRT-PCR were performed to detect the expression levels of the Ptbp2 protein and Pgk2 m RNAin the testicular tissues.Results We report compelling evidence that the cryptorchidism murine model was successfully constructed.With the increase of postoperative time,the results of HE staining and semen analysis showed that sperm quality deteriorated and the sperm cells were severely damaged,in addition to non-obstructive azoospermia.In CRY testes,western blot and IHC showed that the Ptbp2 was significantly decreased by 7 dpo,and was barely detected thereafter.To investigate whether the decrease in Ptbp2 affected Pgk2 m RNA expression levels in vivo,quantitative real-time PCR was employed to detect the Pgk2 transcript levels in UC and SO testes.Pgk2 m RNA expression was significantly decreased at 14,21 and 28 dpo than the control-group,the difference was statistically significant(P<0.05).Conclusions We hypothesize that Ptbp2 is susceptible to heat stress and itsdisruption has resulted in stability decline of germ cell transcripts Pgk2 m RNA,whichconsequently lead to germ cell injury in cryptorchidism testes.Thus,we confirm that Ptbp2 is an essential factor in heat stress-induced sperm cell injury and non-obstructive azoospermia.Which provides a new way to study the basic research and clinical diagnosis of cryptorchidism.