Functions of AKT1 kinase in testis and its effects on phosphorylation modification of LMNA protein

【Objective】This study aimed to investigate the functions of serine/threonine protein kinase 1 （AKT1） in testicular spermatogenesis and effects on phosphorylation modification of nuclear lamina protein A/C （LMNA）， thereby providing theoretical reference for elucidating male reproductive regulatory networks.【Method】Testicular tissues from mature buffaloes were collected for immunohistochemical staining to detect the distribution of AKT1 and AKT1 （pS473）. An in vitro culture model of testicular tissues was established， in which the inhibitor A-674563 and activator SC79 were added respectively to intervene AKT1 activity. The morphology of testicular tissues was observed by hematoxylin-eosin staining. The cell apoptosis rate and proliferation rate were determined using assay kits， while the relative expression of apoptosis-related proteins （Bax， Bcl2， and Caspase 3） and phosphorylation level of LMNA protein at Ser392 locus were detected by Western blotting.【Result】AKT1 and AKT1 （pS473） were widely distributed in types of spermatogenic cells at seminiferous tubules， mainly locating at cytoplasm. As the concentration of A-674563 increased， the structure of the seminiferous tubules began to be disorganized， the spermatogenic cell number was reduced， and intercellular spaces were widened. Compared with the control and after inhibition of AKT1 kinase， the 500 nmol/L A-674563 group showed an extremely significant increase in the apoptosis rate （P<0.01， the same below）， and the cell proliferation rate under treatments of 100， 500， and 1000 nmol/L A-674563 showed an extremely significant decrease. The relative expression of pro-apoptotic proteins BAX and Caspase 3 in the 500 and 1000 nmol/L A-674563 groups extremely significantly increased， and the relative expression of anti-apoptotic protein Bcl2 in the 500 and 1000 nmol/L A-674563 groups extremely significantly decreased. The relative expression of LMNA （pS392） protein in the 500 nmol/L A-674563 group significantly decreased （P<0.05）. The relative expression of LMNA （pS392） protein in the 1000 nmol/L A-674563 group was extremely significantly decreased. Compared with the control and after activating the AKT1 kinase， the cell apoptosis rate of SC79 group extremely significantly decreased， and the cell proliferation rate extremely significantly increased； the relative expression of AKT1 （pS473） and LMNA （pS392） proteins extremely significantly increased.【Conclusion】The AKT1 kinase is the key regulatory factor for survival and proliferation of testicular cells， which regulates the process of cell division by modulating the phosphorylation level of LMNA protein at Ser392 locus. The inhibition of AKT1 kinase can reduce phosphorylation level of LMNA protein， disrupt the morphological structure of testicular tissue structure， and induce apoptosis of spermatogenic cells. The activation of AKT1 kinase can promote phosphorylation modification of LMNA protein and increase proliferation activity of testicular tissue cells.