Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

IF 6 1区 医学 Q1 OBSTETRICS & GYNECOLOGY
Itay Spector, Sanaz Derech-Haim, Ilana Boustanai, Myriam Safrai, Dror Meirow
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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.</p><p><strong>Main results and the role of chance: </strong>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. 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引用次数: 0

Abstract

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.

卵巢中的抗缪勒氏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提供了有关卵巢基质作用的新见解。
研究问题:卵巢基质如何参与抗缪勒氏管激素(AMH)信号传导途径,哪些基质细胞参与其中?小鼠和人类卵巢显示,卵泡周围的基质成纤维细胞中高表达AMH受体II(AMHR2),重组AMH激活了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,表明成纤维细胞被激活,启动了肌成纤维细胞分化: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)的募集和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,诱导靶基因表达。虽然AMHR2在卵泡单位内有表达,但有证据表明它也可能在基质中被发现:从 BALB/c 小鼠(6 周大;n = 12 和 21 天大;n = 56)中提取新鲜的小鼠卵巢。冷冻解冻的卵巢片段取自 10 名 18-35 岁的女性,她们接受了卵巢组织冷冻保存并捐赠了冷冻卵巢组织用于研究:对小鼠(6 周大)和人类捐献者的卵巢进行 AMHR2 和胶原 1α/αSMA/VCAM1 免疫染色,并对小鼠进行额外的波形蛋白染色。小鼠(21 天大)和人类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 对分离的成纤维细胞培养的影响之前,进行了纯度验证测试,以确保没有上皮细胞、免疫细胞、内皮细胞、颗粒细胞和卵巢癌细胞群。通过 RT-qPCR 和 Western-blot 检测验证了成纤维细胞培养物的均一性,确认 E-cadherin、CD31、芳香化酶、CYP17A1 为阴性,αSMA 和波形蛋白为阳性。然后对成纤维细胞进行体外 rAMH 处理(200 毫微克/毫升)0-72 小时,对人类样本进行 96 小时的额外时间点处理,然后进行 RT-qPCR、Western 印迹和免疫细胞化学(ICC)检测 AMHR2 的表达。AMHR2受体后信号转导通过pSMAD1,5,9水平进行Western印迹检测。活化的成纤维细胞标记物αSMA通过Western印迹和ICC进行评估:对小鼠和人类卵巢组织的免疫染色显示,处于各个发育阶段的卵泡周围的基质细胞均表现出较高的 AMHR2 表达,而生长卵泡的颗粒细胞则表现出较低的 AMHR2 表达水平。这些 AMHR2 阳性的基质细胞大部分被鉴定为成纤维细胞(小鼠和人的胶原 1α;小鼠的波形蛋白)。对培养的小鼠和人类成纤维细胞进行了 RT-qPCR、Western 印迹和免疫染色,证实它们由纯成纤维细胞组成(αSMA/波形蛋白阳性,其他细胞类型标志物阴性)。这些成纤维细胞中共有 99.81%(小鼠样本平均 28.94 ± 1.34 个细胞/场)和 100%(人类样本平均 19.20 ± 1.39 个细胞/场)表达 AMHR2(ICC)。经 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。此外,rAMH 处理增加了 AMHR2 蛋白表达,如在 ICC(人类)中观察到的那样:AMHR2 平均荧光强度 (MFI) 上调了 2.57 倍(96 小时,P = 0.00036);Western 印迹显示,小鼠的 AMHR2 蛋白表达随时间增加了 4.2 倍(48 小时,P = 0.026),而人类供体的 AMHR2 蛋白表达则增加了 2.4 倍(48 小时,P = 0.0003)。暴露于 rAMH 会影响 AMHR2 的转录上调,小鼠变化 6.48 倍(72 小时,P = 0.0137),人类变化 7.87 倍(72 小时,P 大比例数据:局限性,需谨慎的原因:在体外,成纤维细胞基因表达可能会因粘附在组织培养板上而发生变化。然而,培养的成纤维细胞(含有和不含 rAMH)处于相同的条件下。因此,可以认为观察结果或显著差异是可靠的。此外,rAMH对成纤维细胞的影响与已知的AMH对卵泡活化的抑制作用没有直接联系:阐明卵巢中AMH反应性细胞群为进一步研究整个卵巢中复杂的AMH信号转导奠定了基础。AMH释放和响应细胞的组成可以揭示卵泡与其环境之间的通讯网络,这可能会阐明AMH对PMF激活的抑制作用背后的机制:这项工作得到了卡恩基金会(Kahn Foundation)的资助。本研究不存在利益冲突。 试验登记号:不适用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Human reproduction
Human reproduction 医学-妇产科学
CiteScore
10.90
自引率
6.60%
发文量
1369
审稿时长
1 months
期刊介绍: Human Reproduction features full-length, peer-reviewed papers reporting original research, concise clinical case reports, as well as opinions and debates on topical issues. Papers published cover the clinical science and medical aspects of reproductive physiology, pathology and endocrinology; including andrology, gonad function, gametogenesis, fertilization, embryo development, implantation, early pregnancy, genetics, genetic diagnosis, oncology, infectious disease, surgery, contraception, infertility treatment, psychology, ethics and social issues.
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