To explore the role and underlying mechanism of the transcriptional coactivator with PDZ-binding motif (TAZ) and a histone variant of acute histone H2A (H2A. Z) in the repair of hypoxia-induced lung injury.

A mouse model of hypoxic lung injury was established by being placed in a hypoxia chamber (simulating an altitude of 5800 m) for 4 d. HE staining was used to observe the severity of lung injury. A hypoxia model of murine alveolar epithelial cells (murine lung epithelial-12, MLE12) was constructed by treating the cells in a hypoxia workstation (1%O₂ concentration) for 24 h. Western blotting was employed to detect TAZ expression. The proliferation of alveolar epithelial cells (AECs) was evaluated by CCK-8 assay. Co-immunoprecipitation (Co-IP) assay was utilized to verify the interaction between TAZ and H2A. Z. CUT&Tag sequencing was performed to determine the effect of TAZ on the chromatin deposition of H2A. Z.

Hypoxia significantly induced alveolar atrophy and inflammatory infiltration in mouse lung tissues (P<0.01). Hypoxia significantly up-regulated the protein level of TAZ in MLE12 cells (P<0.05). CCK-8 assay showed that knockdown of TAZ significantly reduced the proliferative capacity of AECs (P<0.01). Co-IP assay confirmed the physical interaction between TAZ and H2A. Z. CUT&Tag sequencing revealed that hypoxia promoted the deposition of H2A. Z on chromatin (31817 peaks under normoxia, 44078 peaks under hypoxia), which was partially reversed by TAZ knockdown (37840 peaks).

Hypoxia significantly up-regulates the expression of TAZ, which combines with H2A. Z and promotes the deposition of H2A. Z on chromatin, thus enhancing the proliferation of AECs in response to hypoxic injury.

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.

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 3500, 4500 and 5500 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; ② Western 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.

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±0.0002 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 G2spermatocytes 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<0.0001), 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<0.0001), with a binding strength of 0.74±0.06 times of that of the control group, while CEP290mRNA level was significantly increased (P<0.0001), with an up-regulation of 3.35±0.37 times.

Hypoxic environment impair sperm flagellar microtubule stability via the RING 1A-H2AK119ub-CEP290 signaling axis, which affects spermatid differentiation and leads to spermatogenic dysfunction.