STUDY QUESTION What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? SUMMARY ANSWER Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. WHAT IS KNOWN ALREADY AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. STUDY DESIGN, SIZE, DURATION Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18–35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. PARTICIPANTS/MATERIALS, SETTING, METHODS Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture’s homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0–72 h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. MAIN RESULTS AND THE ROLE OF CHANCE Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. WIDER IMPLICATIONS OF THE FINDINGS Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. STUDY FUNDING/COMPETING INTEREST(S) This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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卵巢中的抗苗勒管激素信号传导涉及基质成纤维细胞：一项对人类和小鼠的研究为卵巢基质的作用提供了新的见解


研究问题 卵巢基质在抗苗勒管激素 （AMH） 信号通路中有什么参与，涉及哪些基质细胞？总结答案小鼠和人卵巢在卵泡周围的基质成纤维细胞中显示 AMH 受体 II （AMHR2） 的高表达，重组 AMH 激活 AMHR2 后通路表现为 SMAD1,5 和 9 磷酸化增加、AMHR2 表达增加和 αSMA 上调，表明成纤维细胞激活启动肌成纤维细胞分化。已知的 AMH 由生长中的小卵泡分泌，调节卵巢活动。它抑制初始原始卵泡 （PMF） 募集和 FSH 依赖性生长。AMH 信号转导由 AMHR2 介导，激活细胞内 SMAD 蛋白和其他信号级联反应以诱导靶基因表达。尽管在卵泡单位内报道了 AMHR2 的表达，但有证据表明它也可能在基质中被发现。研究设计、大小、持续时间 从 BALB/c 小鼠 （6 周龄;n = 12 和 21 天;n = 56） 中提取新鲜小鼠卵巢。冻融卵巢碎片是从 10 名年龄在 18-35 岁之间的女性那里获得的，她们接受了卵巢组织冷冻保存并捐献了冷冻卵巢组织用于研究。参与者/材料、设置、方法 小鼠 （6 周龄） 和人供体卵巢对 AMHR2 和胶原蛋白 1α/αSMA/VCAM1 进行免疫染色，并在小鼠中进行额外的波形蛋白染色。小鼠 （21 天龄） 和人供体卵巢用于成纤维细胞分离和随后的 7 天培养。 在评估 AMH 对分离成纤维细胞培养物的影响之前，实施了纯度验证测试，以确保不存在上皮、免疫、内皮、颗粒和卵泡膜卵巢细胞群。通过 RT-qPCR 和 western blot 测定验证成纤维细胞培养物的均一性，证实 E-钙粘蛋白、CD31、芳香酶、CYP17A1 阴性以及 αSMA 和波形蛋白阳性。然后将成纤维细胞在体外 （200 ng/ml） 进行 rAMH 处理 0-72 小时，人样品的额外时间点为 96 小时，然后进行 RT-qPCR、蛋白质印迹和免疫细胞化学 （ICC） 检测 AMHR2 表达。通过 western blot 通过 pSMAD1,5,9 水平检查 AMHR2 受体后信号传导。活化成纤维细胞标志物 αSMA 通过 western blot 和 ICC 进行评估。主要结果和机会的作用小鼠和人卵巢组织的免疫染色表明，在所有发育阶段，卵泡周围的基质细胞都表现出高 AMHR2 表达，而生长卵泡的颗粒细胞表现出相当低的水平。这些 AMHR2 阳性基质细胞中的大多数被鉴定为成纤维细胞（小鼠和人中的 Collagen1α;小鼠中的 vimentin）。对培养的小鼠和人成纤维细胞进行 RT-qPCR、western blot 和免疫染色，确认它们由纯成纤维细胞群组成 （其他细胞类型标志物的 αSMA/波形蛋白阳性和阴性）。这些成纤维细胞总共 99.81% （小鼠平均 28.94 ± 1.34 个细胞/场） 和 100% （人类样本中平均 19.20 ± 1.39 个细胞/场） 表达 AMHR2 （ICC）。rAMH 处理的培养成纤维细胞显示 pSMAD1,5 和 9 水平升高，表明 AMH 对其下游信号通路的影响。pSMAD1,5 和 9 表达增加，通过 western blot 检测到： 1.在小鼠中为 92 倍 （48 小时，P = 0.026），在人样品中为 2.37 倍 （48 小时，P = 0.0002）。此外，rAMH 处理增加了 AMHR2 蛋白表达，如 ICC（人类）中观察到的那样：AMHR2 平均荧光强度 （MFI） 上调 2.57 倍（96 小时，P = 0.00036），蛋白质印迹显示小鼠的时间依赖性增加 4.2 倍（48 小时，P = 0.026），人类供体变化 2.4 倍（48 小时，P = 0.0003）。暴露于 rAMH 影响了 AMHR2 转录上调，小鼠变化 6.48 倍 （72 小时，P = 0.0137），人类变化 7.87 倍 （72 小时，P < 0.0001）。rAMH 处理诱导成纤维细胞活化 （αSMA 阳性），证明了 AMH 对成纤维细胞行为的动态影响。在 ICC 中检测到 αSMA 表达升高，人类 MFI 增加 2.28 倍 （96 小时，P = 0.000067），在蛋白质印迹中，小鼠 MFI 增加 5.12 倍 （48 小时，P = 0.0345），人类增加 2.69 倍 （48 小时，P ≤ 0.0001）。活化的 AMHR2 阳性染色成纤维细胞组分仅位于人类和小鼠中生长的卵泡周围。此外，观察到少量 AMHR2 阳性染色的膜细胞 （VCAM1 阳性）。大规模数据 N/A. 局限性，谨慎原因 离体，成纤维细胞基因表达可能会因粘附到组织培养板而改变。然而，培养的成纤维细胞（有和没有 rAMH）受到相同的条件。因此，观察结果或显著差异可以被认为是可靠的。此外，rAMH 对成纤维细胞的已知抑制作用与 AMH 对卵泡活化的已知抑制作用没有直接关系。 研究结果的更广泛意义阐明卵巢中 AMH 反应细胞的群体为进一步研究整个卵巢的复杂 AMH 信号传导提供了基础。AMH 释放和反应细胞的组成可以阐明卵泡与其环境之间的通信网络，这可能阐明 AMH 抑制作用对 PMF 激活的机制。研究资金/竞争利益 这项工作得到了 Kahn 基金会的资助。本研究中没有利益冲突。试验注册号 N/A。