Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were involved in spermatogenic damages in response to heat-stress in cryptorchidism. Hematoxylin and eosin (HE) staining and DNA end labeling (TUNEL) were used to observe morphological and apoptotic characteristics of spermatogenic cells; Immunohistochemical analysis was used to detect changes of Uchl1 and its associated proteins Jab1 and p27(kip1) in response to heat-stress from cryptorchidism leading to spermatocyte losses; And protein affinity analysis (pull-down) and immunofluorescence co-localization were used to verify the relevance among the three proteins in spermatocytes. The results showed that, Jab1 and p27(kip1), in parallel to Uchl1, increased in spermatocytes of apoptotic appearances in response to heat-stress, but not in multinucleated giant cells; Jab1 bound to Uchl1 in testis protein extracts, and co-localized with Uchl1 and p27(kip1) specifically in spermatocytes with apoptotic appearances. These results suggest that the accumulation of Uchl1 protein is involved in the heat-stress-induced spermatocyte apoptosis through a new pathway related with Jab1 and p27(kip1), but not the formation of multinucleated giant cells.
Animals ; Apoptosis ; COP9 Signalosome Complex ; Cryptorchidism ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Hot Temperature ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Peptide Hydrolases ; metabolism ; Spermatocytes ; cytology ; metabolism ; Stress, Physiological ; Ubiquitin Thiolesterase ; metabolism

The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.