{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T23:21:59Z","timestamp":1787008919807,"version":"build-2736575974"},"reference-count":57,"publisher":"Oxford University Press (OUP)","issue":"11","license":[{"start":{"date-parts":[[2024,10,3]],"date-time":"2024-10-03T00:00:00Z","timestamp":1727913600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"funder":[{"name":"Kahn Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2024,11,1]]},"abstract":"Abstract<\/jats:title>\n \n STUDY QUESTION<\/jats:title>\n What is the involvement of ovarian stroma in the anti-M\u00fcllerian hormone (AMH) signaling pathway and which stromal cells are involved?<\/jats:p>\n <\/jats:sec>\n \n SUMMARY ANSWER<\/jats:title>\n 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 \u03b1SMA, suggesting fibroblast activation to initiate myofibroblast differentiation.<\/jats:p>\n <\/jats:sec>\n \n WHAT IS KNOWN ALREADY<\/jats:title>\n 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.<\/jats:p>\n <\/jats:sec>\n \n STUDY DESIGN, SIZE, DURATION<\/jats:title>\n Fresh murine ovaries were extracted from BALB\/c mice (6\u2009weeks old; n\u2009=\u200912 and 21\u2009days old; n\u2009=\u200956). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18\u201335, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research.<\/jats:p>\n <\/jats:sec>\n \n PARTICIPANTS\/MATERIALS, SETTING, METHODS<\/jats:title>\n Murine (6\u2009weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1\u03b1\/\u03b1SMA\/VCAM1, with additional vimentin staining in mice. Murine (21\u2009days 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\u2019s homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for \u03b1SMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200\u2009ng\/ml) for 0\u201372\u00a0h, with an additional time point of 96\u2009h 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, \u03b1SMA, was assessed via western blot and ICC.<\/jats:p>\n <\/jats:sec>\n \n MAIN RESULTS AND THE ROLE OF CHANCE<\/jats:title>\n 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 (Collagen1\u03b1 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 (\u03b1SMA\/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94\u2009\u00b1\u20091.34 cells\/field in mice) and 100% (average 19.20\u2009\u00b1\u20091.39 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\u2009h, P\u2009=\u20090.026) and 2.37-fold in human samples (48\u2009h, P\u2009=\u20090.0002). 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\u2009h, P\u2009=\u20090.00036), and western blot, showing a 4.2-fold time-dependent increase (48\u2009h, P\u2009=\u20090.026) in mice and 2.4-fold change (48\u2009h, P\u2009=\u20090.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72\u2009h, P\u2009=\u20090.0137) in mice and a 7.87-fold change (72\u2009h, P\u2009&lt;\u20090.0001) in humans. rAMH treatment induced fibroblast activation (\u03b1SMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. \u03b1SMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96\u2009h, P\u2009=\u20090.000067), and in western blot with a 5.12-fold increase in mice (48\u2009h, P\u2009=\u20090.0345) and a 2.69-fold increase in humans (48\u2009h, P\u2009\u2264\u20090.0001). 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.<\/jats:p>\n <\/jats:sec>\n \n LARGE SCALE DATA<\/jats:title>\n N\/A.<\/jats:p>\n <\/jats:sec>\n \n LIMITATIONS, REASONS FOR CAUTION<\/jats:title>\n 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.<\/jats:p>\n <\/jats:sec>\n \n WIDER IMPLICATIONS OF THE FINDINGS<\/jats:title>\n 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.<\/jats:p>\n <\/jats:sec>\n \n STUDY FUNDING\/COMPETING INTEREST(S)<\/jats:title>\n This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study.<\/jats:p>\n <\/jats:sec>\n \n TRIAL REGISTRATION NUMBER<\/jats:title>\n N\/A.<\/jats:p>\n <\/jats:sec>","DOI":"10.1093\/humrep\/deae221","type":"journal-article","created":{"date-parts":[[2024,9,26]],"date-time":"2024-09-26T07:17:48Z","timestamp":1727335068000},"page":"2551-2564","source":"Crossref","is-referenced-by-count":18,"title":["Anti-M\u00fcllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma"],"prefix":"10.1093","volume":"39","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-7802-4245","authenticated-orcid":false,"given":"Itay","family":"Spector","sequence":"first","affiliation":[{"name":"Fertility Preservation Laboratory, Sheba Medical Center , Tel Hashomer,","place":["Israel"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanaz","family":"Derech-Haim","sequence":"additional","affiliation":[{"name":"Fertility 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