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Background <p>Primary ovarian insufficiency (POI) is a complex endocrine disorder with impairment of ovarian function before the age of 40 that can associate with mutations in TAp63alpha (TAp63α), a transcription factor essential for maintaining genome integrity in the female germline. Variants located in the C-terminal can constitutively self-activate TAp63α and induce oocyte apoptosis, resulting in POI. This research examined the involvement of the R643* truncating variant of TAp63α in the development of POI.</p> Methods <p>Experiments were performed in the human osteosarcoma-derived SAOS-2 cell line, a non-germline in vitro model selected due to its lack of endogenous TAp63α expression. This cell line cannot fully recapitulate the ovarian follicle microenvironment; however, the R643* variant was investigated alongside the previously validated R594* POI-related variant to confirm the robustness of the methodology. DNA damage was induced using Doxorubicin treatment, and subsequent analyses were performed using Western immunoblotting, luciferase assays, quantitative PCR, and flow cytometry.</p> Results <p>The R643* variant of the TAp63α protein was expressed at minimal RNA and protein levels in the transfected SAOS-2 cells (<i>p</i> &lt; 0.001). The overexpression of the TAp63α mutant R643* in SAOS-2 cells resulted in a significant alteration in the expression of target genes. Specifically, BAX expression was decreased (adjusted <i>p</i> = 0.0008), whereas the expression of IRF6, K14, WNT4, and MASPIN was significantly elevated (adjusted <i>p</i> &lt; 0.0001). MDM2 expression also increased (adjusted <i>p</i> &lt; 0.05). Notably, stable R643* variant expression increased cell survival following Doxorubicin treatment (adjusted <i>p</i> &lt; 0.05).</p> Conclusions <p>We propose a new molecular mechanism for the induction of POI. Our in vitro characterization indicated that the R643* variant of TAp63α may increase the susceptibility of germ cells to DNA damage accumulation during the extended period of meiotic arrest, likely due to a decrease in apoptosis. Concurrently, it appears to stimulate pathways and genes driving uncontrolled follicular development and proliferation. The consequence of these combined effects would be the persistence of poorly competent oocytes, which ultimately leads to premature depletion of the follicular reserve.</p> Clinical trial number <p>not applicable.</p>

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A TAp63alpha truncating variant associated with primary ovarian insufficiency lowers the cellular apoptotic rate

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月9日

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  • Silvia Moleri,
  • Sara Casafina,
  • Maria Orietta Borghi,
  • Luca Persani,
  • Raffaella Rossetti

摘要

Background

Primary ovarian insufficiency (POI) is a complex endocrine disorder with impairment of ovarian function before the age of 40 that can associate with mutations in TAp63alpha (TAp63α), a transcription factor essential for maintaining genome integrity in the female germline. Variants located in the C-terminal can constitutively self-activate TAp63α and induce oocyte apoptosis, resulting in POI. This research examined the involvement of the R643* truncating variant of TAp63α in the development of POI.

Methods

Experiments were performed in the human osteosarcoma-derived SAOS-2 cell line, a non-germline in vitro model selected due to its lack of endogenous TAp63α expression. This cell line cannot fully recapitulate the ovarian follicle microenvironment; however, the R643* variant was investigated alongside the previously validated R594* POI-related variant to confirm the robustness of the methodology. DNA damage was induced using Doxorubicin treatment, and subsequent analyses were performed using Western immunoblotting, luciferase assays, quantitative PCR, and flow cytometry.

Results

The R643* variant of the TAp63α protein was expressed at minimal RNA and protein levels in the transfected SAOS-2 cells (p < 0.001). The overexpression of the TAp63α mutant R643* in SAOS-2 cells resulted in a significant alteration in the expression of target genes. Specifically, BAX expression was decreased (adjusted p = 0.0008), whereas the expression of IRF6, K14, WNT4, and MASPIN was significantly elevated (adjusted p < 0.0001). MDM2 expression also increased (adjusted p < 0.05). Notably, stable R643* variant expression increased cell survival following Doxorubicin treatment (adjusted p < 0.05).

Conclusions

We propose a new molecular mechanism for the induction of POI. Our in vitro characterization indicated that the R643* variant of TAp63α may increase the susceptibility of germ cells to DNA damage accumulation during the extended period of meiotic arrest, likely due to a decrease in apoptosis. Concurrently, it appears to stimulate pathways and genes driving uncontrolled follicular development and proliferation. The consequence of these combined effects would be the persistence of poorly competent oocytes, which ultimately leads to premature depletion of the follicular reserve.

Clinical trial number

not applicable.