Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules
10.16016/j.2097-0927.202502061
- VernacularTitle:低氧抑制精子纤毛微管稳定性致精子发生障碍的分子机制研究
- Author:
Xiao WANG
1
;
Mengjie ZHANG
;
Fang DENG
;
Jun YIN
;
Bing NI
Author Information
1. 陆军军医大学(第三军医大学):高原军事医学系病理生理学教研室
- Keywords:
hypoxia;
spermatogenesis;
cilia;
microtubule stability;
RING 1A
- From:
Journal of Army Medical University
2025;47(10):1059-1068
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effects of hypoxia on spermatid differentiation and stability of sperm flagellar microtubule,and investigate the underlying molecular mechanisms in order to clarify the potential adverse effects of hypoxia on male reproductive function.Methods Forty-eight 8-week-old healthy male SD rats(weighing 300～399 g)were subjected in this study.The experiments included ① an oxygen concentration gradient experiment(n=6):21％oxygen was regarded as normoxia(control),and 13.5％,11.8％,and 10.4％ oxygen were used to simulate hypoxic environments at altitudes of 3 500,4 500 and 5 500 m,respectively,for a continuous exposure of 2 months;② a time gradient experiment(n=6):the rats were exposed to 10.4％ oxygen for 0,0.5,1,and 2 months,respectively.Flow cytometry was employed to isolate round spermatids,and the following methods were employed to measure relevant indicators:① RNA sequencing to analyze gene expression profile changes related to impaired spermatogenesis and abnormal flagellar structure under hypoxic stress;②Western blotting to detect the expression levels of key proteins CEP290,RING 1A,and H2AK119ub;③ fluorescence recovery after photobleaching(FRAP)to monitor microtubule assembly dynamics and assess the immediate impact of hypoxia on microtubule stability.Results In the oxygen concentration gradient experiment,after 2 months of exposure to 10.4％ oxygen,the proportions of spermatogonia,secondary spermatocytes,and round spermatids in rat seminiferous tubules were significantly increased(P＜0.05),reaching 1.33±0.04,1.06±0.01 and 1.60±0.02 times higher,respectively than that of the 21％ normoxia group.Conversely,the proportions of primary spermatocytes and elongated spermatids were obviously decreased(P＜0.05),taking 0.89±0.01 and 0.88±0.000 2 times respectively when compared with that of the 21％ normoxia group,in a oxygen concentration-depended manner.In the time gradient experiment,after 0.5 months of exposure to 10.4％oxygen,the proportions of spermatogonia,secondary spermatocytes,and round spermatids began to increase(P＜0.05),reaching 1.11±0.03,1.04±0.01 and 1.29±0.003 times higher,respectively than that of the 0-month control group.The proportions of primary spermatocytes and elongated spermatids started to significantly decrease(P＜0.05)after 1 month of exposure,only 0.94±0.03 and 0.95±0.008 times,respectively than that of the 0-month control group.After 2 months of exposure to 10.4％ oxygen,the rate of sperm tail abnormalities in the epididymis of rats was significantly increased(P＜0.05),rising from(12.1±1.7)％ in the 21％ normoxia group to(30.8±3.7)％.In G2 spermatocytes exposed to 1％ hypoxia for 24 h,FRAP revealed a decrease in microtubule assembly rate and enhanced microtubule dynamic instability,with the maximum fluorescence recovery value decreasing from 0.37±0.02 in the normoxia group to 0.29±0.01.The results of RNA sequencing showed that under hypoxic condition,the transcription level of the key cilium basal body molecule CEP290 was increased,with an upregulation of 1.81±0.11 times than that of the 21％ normoxia group.In contrast,the expression levels of PRC1 complex members RING 1A,RING 1B,CBX2,PHC1,and PCGF1 were decreased,to 0.74±0.02,0.73±0.01,0.78±0.02,0.71±0.01 and 0.86±0.03 times of that of the 21％ normoxia group,respectively.Western blotting indicated that the protein level of CEP290 was up-regulated in the hypoxia group,while that of RING 1A was down-regulated.ChIP-qPCR experiments showed that the binding of RING 1A and its product H2AK119ub to the CEP290 promoter were significantly decreased(P＜0.000 1),with binding strengths of 0.38±0.02 and 0.52±0.06 times of that of the 21％ normoxia group,respectively.In siRING 1A-treated G2 cells,the binding of H2AK119ub to the CEP290 promoter was significantly decreased(P＜0.000 1),with a binding strength of 0.74±0.06 times of that of the control group,while CEP290 mRNA level was significantly increased(P＜0.000 1),with an up-regulation of 3.35±0.37 times.Conclusion Hypoxic environment impair sperm flagellar microtubule stability via the RING 1A-H2AK119ub-CEP290 signaling axis,which affects spermatid differentiation and leads to spermatogenic dysfunction.
