Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules

WANG Xiao; ZHANG Mengjie; DENG Fang; YIN Jun; NI Bing

doi:10.16016/j.2097-0927.202502061

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Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules

Basic Medicine | 更新时间：2025-05-26

- Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules
- Journal of Army Medical University Vol. 47, Issue 10, Pages: 1059-1068(2025)
- 作者机构：
  
  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，全军高原医学重点实验室，教育部极端环境医学重点实验室，重庆
- 作者简介：
  
  NI Bing， E-mail： nibing@tmmu.edu.cn；
- 基金信息：
  
  the General Project of Natural Science Foundation of Chongqing(CSTB2023NSCQ-MSX0034)
- DOI：10.16016/j.2097-0927.202502061
  CLC： R339.2;Q254;R363.2
- Received：23 February 2025，
  
  Revised：12 April 2025，
  
  Published：30 May 2025
- 稿件说明：
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王潇,张梦洁,邓芳,等.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[J].陆军军医大学学报,2025,47(10):1059-1068.

WANG X，ZHANG M J，DENG F，et al.Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules［J］.J Army Med Univ，2025，47（10）：1059-1068.DOI：10.16016/j.2097-0927.202502061.
王潇,张梦洁,邓芳,等.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[J].陆军军医大学学报,2025,47(10):1059-1068. DOI：10.16016/j.2097-0927.202502061.

WANG X，ZHANG M J，DENG F，et al.Molecular mechanism of hypoxia-induced spermatogenesis impairment by inhibiting the stability of sperm cilial microtubules［J］.J Army Med Univ，2025，47（10）：1059-1068.DOI：10.16016/j.2097-0927.202502061. DOI：

摘要

目的

旨在探讨低氧环境对精细胞分化及精子纤毛微管稳定性的影响及其分子机制，以明确低氧条件对雄性生殖功能的潜在危害。

方法

采用48只8周龄SPF级健康雄性SD大鼠（体质量300~399 g），由陆军军医大学实验动物研究所提供。大鼠实验设计如下：①氧浓度梯度实验：设置21%常氧对照、13.5%、11.8%、10.4%氧浓度，分别模拟海拔3 500、4 500、5 500 m低氧环境，持续暴露2个月，每组样本量为6只；②氧暴露时间梯度实验：在10.4%氧浓度下暴露0、0.5、1.0、2.0个月，每组样本量为6只。采用流式分选技术分离圆形精细胞，并采用以下方法测量相关指标：①转录组测序，分析低氧胁迫下精子分化障碍及纤毛结构异常的基因表达谱变化；②Western blot，检测关键蛋白CEP290、RING 1A、H2AK119ub的表达水平；②荧光漂白恢复技术（fluorescence recovery after photobleaching/FRAP），监测微管组装动力学，评估低氧对微管稳定性的即时影响。

结果

实验结果显示，在氧浓度梯度实验中，10.4%氧浓度暴露2个月后，大鼠睾丸生精小管中精原细胞、次级精母细胞及圆形精细胞的比例显著增加（

0.05），分别为21%常氧组的（1.33±0.04）倍、（1.06±0.01）倍、（1.60±0.02）倍；而初级精母细胞和长形精细胞的比例显著降低（

0.05），分别为21%常氧组的（0.89±0.01）倍、（0.88±0.000 2）倍。这种变化呈现出明显的氧浓度依赖性。在时间梯度实验中，暴露于10.4%氧浓度0.5个月时，精原细胞、次级精母细胞及圆形精细胞的比例开始增加（

0.05），分别为0个月对照组的（1.11±0.03）倍、（1.04±0.01）倍、（1.29±0.003）倍；而初级精母细胞和长形精细胞的比例在暴露1个月后开始显著降低（

0.05），分别为0个月对照组的（0.94±0.03）倍、（0.95±0.008）倍。暴露于10.4%氧浓度2个月的大鼠，其附睾内精子尾部畸形率显著增加（

0.000 1），畸形率从21%常氧组的（12.1±1.7）%上升至（30.8±3.7）%。精母细胞系G2暴露于1%低氧环境中24 h后， FRAP显示微管组装速率降低，微管动态不稳定性增加，荧光恢复最大值从21%常氧组的（0.37±0.02）倍下降至（0.29±0.01）倍。转录组测序结果显示，低氧环境下纤毛基体关键分子CEP290的转录水平上调，上调倍数为21%常氧组的（1.81±0.11）倍；而PRC1复合体成员RING 1A、RING 1B、CBX2、PHC1及PCGF1的表达水平下调，分别为21%常氧组的（0.74±0.02）倍、（0.73±0.01）倍、（0.78±0.02）倍、（0.71±0.01）倍、（0.86±0.03）倍。Western blot验证结果显示，CEP290蛋白表达水平在低氧组上调，RING 1A蛋白表达水平下调。ChIP-qPCR实验显示，RING 1A及其产物H2AK119ub在CEP290启动子区的结合显著减少（

0.000 1），结合强度分别为21%常氧组的（0.38±0.02）倍、（0.52±0.06）倍。而在siRING 1A组G2细胞中，H2AK119ub在CEP290启动子区的结合显著减少（

0.000 1），结合强度为对照组的（0.74±0.06）倍，同时CEP290 mRNA水平显著上升（

0.000 1），上调倍数为（3.35±0.37）倍。

结论

低氧环境通过RING 1A-H2AK119ub-CEP290信号轴抑制精子纤毛微管稳定性，影响精细胞分化，导致精子发生障碍。

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 ①an oxygen concentration gradient experiment (

=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 (

=6): the rats were exposed to 10.4% oxygen for 0

0.5

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; ③fluorescence 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 (

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 (

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 (

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 (

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 (

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 mi

crotubule 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 (

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 (

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 (

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.

关键词

Keywords

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