{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T23:21:58Z","timestamp":1787008918507,"version":"build-2736575974"},"reference-count":60,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T00:00:00Z","timestamp":1626393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"DOI":"10.13039\/501100003510","name":"Estonian Ministry of Education and Research","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003510","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100002301","name":"Estonian Research Council","doi-asserted-by":"publisher","award":["PRG1076, PRG454, PSG230 and PSG608"],"award-info":[{"award-number":["PRG1076, PRG454, PSG230 and PSG608"]}],"id":[{"id":"10.13039\/501100002301","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006598","name":"Enterprise Estonia","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006598","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Horizon 2020 innovation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,7,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>STUDY QUESTION<\/jats:title>\n                    <jats:p>Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo?<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>SUMMARY ANSWER<\/jats:title>\n                    <jats:p>Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>WHAT IS KNOWN ALREADY<\/jats:title>\n                    <jats:p>Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)\/cAMP-dependent protein kinase (PKA)\/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>STUDY DESIGN, SIZE, DURATION<\/jats:title>\n                    <jats:p>In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N\u2009=\u200915) and PCOS patients (N\u2009=\u20099). In parallel, lysates of non-cultured ESCs isolated from proliferative (N\u2009=\u20094) or secretory (N\u2009=\u20094) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N\u2009=\u20093) or secretory endometrium (N\u2009=\u20093), and in proliferative or secretory full tissue samples from controls (N\u2009=\u20098 and N\u2009=\u20099, respectively) or PCOS patients (N\u2009=\u200910 for both phases).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>PARTICIPANTS\/MATERIALS, SETTING, METHODS<\/jats:title>\n                    <jats:p>The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>MAIN RESULTS AND THE ROLE OF CHANCE<\/jats:title>\n                    <jats:p>During in vitro decidualization, the activities of PKA, protein kinase B (Akt\/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P\u2009&amp;lt;\u20090.05) and are further augmented following the 9-day treatment (P\u2009&amp;lt;\u20090.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt\/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P\u2009&amp;lt;\u20090.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation &amp;lt; 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P\u2009&amp;lt;\u20090.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P\u2009&amp;lt;\u20090.001) as well as in vitro decidualized ESCs (P\u2009&amp;lt;\u20090.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P\u2009&amp;lt;\u20090.05).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>LARGE SCALE DATA<\/jats:title>\n                    <jats:p>Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>LIMITATIONS, REASONS FOR CAUTION<\/jats:title>\n                    <jats:p>The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>WIDER IMPLICATIONS OF THE FINDINGS<\/jats:title>\n                    <jats:p>The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies\u2014including the context of decidualization-related subfertility and infertility.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>STUDY FUNDING\/COMPETING INTEREST(S)<\/jats:title>\n                    <jats:p>This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/humrep\/deab161","type":"journal-article","created":{"date-parts":[[2021,6,7]],"date-time":"2021-06-07T23:15:57Z","timestamp":1623107757000},"page":"2230-2248","source":"Crossref","is-referenced-by-count":18,"title":["Progesterone triggers Rho kinase-cofilin axis during\n                    <i>in vitro<\/i>\n                    and\n                    <i>in vivo<\/i>\n                    endometrial decidualization"],"prefix":"10.1093","volume":"36","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5167-2817","authenticated-orcid":false,"given":"Darja","family":"Lavogina","sequence":"first","affiliation":[{"name":"Competence Centre on Health Technologies, Tartu, Estonia"},{"name":"Department of Bioorganic Chemistry, Institute of Chemistry, University of Tartu, Tartu, 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