Loss of MIG-6 results in endometrial progesterone resistance via ERBB2

Loss of MIG-6 results in endometrial progesterone resistance via ERBB2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Loss of MIG-6 results in endometrial progesterone resistance via ERBB2 Jung-Yoon Yoo, Tae Hoon Kim, Jung-Ho Shin, Ryan Marquardt, Ulrich Mueller, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-95903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2022 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Female subfertility is highly associated with endometriosis. Although the exact etiology of endometriosis-related infertility remains to be determined, endometrial progesterone resistance has recently been suggested as a crucial element in the development of endometrial diseases. Here, we report that MIG-6 , a progesterone-induced gene, is downregulated in the endometrium of infertile women with endometriosis and in a non-human primate model of endometriosis. In an endometriosis mouse model with a fluorescent reporter used to identify lesions, an increase of endometriosis development and implantation failure were observed in mice with Mig-6 deficient endometrium compared to controls. MIG-6 is known to inhibit ERBB2, which we found overexpressed in the endometrium from uterine-specific Mig-6 knock-out mice ( Pgr cre/+ Mig-6 f/f ; Mig-6 d/d ). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduced Erbb2 ablation in Mig-6 d/d mice ( Mig-6 d/d Erbb2 d/d mice). The additional knockout of Erbb2 rescued all phenotypes seen in Mig-6 d/d mice including endometrial progesterone resistance, infertility, and endometriosis lesion development. Transcriptomic analysis showed that genes differentially expressed in Mig-6 d/d mice reverted to their normal expression amounts in Mig-6 d/d Erbb2 d/d mice. Together, our results demonstrate that MIG-6-induced ERBB2 overexpression causes endometrial progesterone resistance and a nonreceptive endometrium in endometriosis-related infertility and that ERBB2 targeting reverses these effects. Endocrinology & Metabolism Sexual & Reproductive Medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Critical for fertility, the uterine endometrium’s epithelial and stromal compartments undergo dynamic hormonally controlled molecular and morphological changes to prepare for embryo implantation and development. Estrogen (E2) stimulates the proliferation of uterine epithelial cells, and progesterone (P4) suppresses E2-induced proliferation. Endometrial P4 resistance implies decreased responsiveness of target tissue to bioavailable P4 1, 2, 3 . Endometrial P4 resistance is seen in women with a nonreceptive endometrium, endometriosis, polycystic ovary syndrome (PCOS), and endometrial cancer 4 , 5 , 6 , 7 , 8 , 9 . Moreover, P4-induced molecular changes in the eutopic (intrauterine) endometrial tissue of women with endometriosis are either blunted or undetectable 10 , 11 , 12 . Endometriosis affects about 10% of all women of reproductive age, and the incidence increases to 50–60% of women with chronic pelvic pain and infertility 13 , 14 . While progestin-based therapies are commonly used to treat endometriosis and lead to disease regression in some women, other women with endometriosis and pelvic pain do not respond effectively to progestins 15 , 16 . Moreover, many P4-induced molecular changes in the eutopic endometrial tissue of women with endometriosis are either blunted or dysregulated 17 , 18 , but an impaired P4 response is seen in the endometrium of women with endometriosis 4 , 5 , 6 , 10 . Despite knowing the effects, the molecular mechanism responsible for endometrial P4 resistance and dysregulation remains unclear. Therefore, understanding the molecular mechanisms of endometrial P4 resistance is critical. The present study revealed that the amount of mitogen inducible gene 6 (MIG-6) was decreased in endometrium from infertile women with endometriosis. We used uterine-specific Mig-6 knock-out mice to demonstrate that MIG-6 loss results in endometrial progesterone resistance via ERBB2. Our findings provide new insight into the etiology of female infertility and provide a new molecular framework useful for the design of new therapeutic strategies. Results MIG-6 expression is decreased in endometrium from women with endometriosis. We previously identified Mig-6 as a P4-regulated gene that mediates the ability of P4 to repress E2 action in the mouse uterus 19 , 20 . During the menstrual cycle, P4 amounts rise at the early secretory phase. As measured by RT-qPCR, MIG-6 expression in the human endometrium was significantly higher in the early secretory phase of the menstrual cycle than in the proliferative phase (p < 0.001) (Fig. 1 A), suggesting that MIG-6 is a P4-induced gene in the human endometrium as has been demonstrated in the mouse 20 . Because many P4-induced endometrial molecular changes are either blunted or eliminated in women with endometriosis 1 , 5 , 8 , we examined MIG-6 expression in endometrial biopsies from infertile women with endometriosis. RT-qPCR and immunohistochemistry showed that amounts of MIG-6 mRNA (p < 0.01) and protein (p < 0.001) were significantly lower in the eutopic endometrium of infertile women with endometriosis compared to controls in the early secretory phase (Fig. 1 , A-C). To assess how MIG-6 expression is affected by endometriosis progression, we used a baboon model 21 . Intraperitoneal inoculation with autologous menstrual effluent in female non-human primates results in formation of endometriotic lesions highly similar in histomorphology to those seen in women 22 . We found that endometrial MIG-6 protein abundance was significantly reduced in baboons during the progression of endometriosis after experimental disease induction as compared to paired pre-inoculation control samples (Fig. 1 D; p < 0.001). These results demonstrate that reduced MIG-6 expression can be caused by the development of endometriotic lesions. Uncovering pathophysiological mechanisms of endometriosis-related infertility with animal models requires easy identification of lesions to distinguish them from the surrounding normal tissues. With this in mind, we developed a mouse model of endometriosis using mT/mG reporters. In Pgr cre/+ Rosa26 mTmG/+ mice, progesterone receptor ( Pgr )-positive uterine cells express mG, while Pgr -negative cells express mT (fig. S1 A and B). Using this model, we surgically induced endometriosis in Pgr cre/+ Rosa26 mTmG/+ mice by inoculating autologous endometrial tissue fragments into the peritoneal cavity after 3 days of E2 treatment (fig. S1C). This method leads to the development of endometriotic lesions similar to those in humans without the need for ovariectomy or unopposed E2 treatment (fig. S1 D-F). To examine the responsiveness of our endometriosis model to E2 and P4, Pgr cre/+ Rosa26 mTmG/+ mice induced with endometriosis were treated with vehicle, E2, or E2 + P4 for 2 weeks. While E2 treatment after endometriosis induction significantly increased the number of endometriotic lesions compared to the vehicle group, the addition of P4 suppressed the E2-induced increase in lesion number (fig. S1 G and H; p < 0.01). Our mouse model thus closely mirrors human endometriosis as an E2-dependent and P4-suppressed disorder. To determine whether MIG-6 expression is dysregulated after endometriosis development in a distinct mammalian system, we examined MIG-6 amount in the eutopic endometrium from Pgr cre/+ Rosa26 mTmG/+ mice with endometriosis. MIG-6 protein expression was significantly reduced in eutopic endometrium from the mice with endometriosis compared to the sham group (Fig. 1 E; p < 0.001). Mig-6 loss accelerated the development of endometriosis and endometriosis-related infertility. Next, we assessed whether endometriosis in mice causes infertility by assessing implantation and decidualization success. One month after endometriosis induction, the number of implantation sites in mice with endometriosis was not changed compared to the sham group. However, 63.6% (7 out of 11) of mice with endometriosis experienced implantation failure 3 months after endometriosis development (fig. S2A). We next examined the impact of endometriosis on decidualization using an artificial decidualization model 23 . One month after endometriosis induction, mice with endometriosis displayed a uterine horn that responded well to artificial decidualization; however, after 3 month of endometriosis development, the mice with endometriosis exhibited a significant defect in decidual response compared to control and sham mice (fig. S2B; p < 0.001). Our result suggests that endometriosis development causes implantation failure and a defect of decidualization, as has been hypothesized in humans 5 . Having established the link between endometriosis development and MIG-6 attenuation in the eutopic endometrium, we sought to determine if MIG-6 depletion is involved in endometriotic lesion development. In a comparison of MIG-6 expression in paired ectopic and eutopic endometrial biopsies taken from women with endometriosis, MIG-6 amounts were significantly reduced in the ectopic endometrial specimens (p < 0.01) (Fig. 2 A and B). To assess the effect of MIG-6 deficiency in endometriosis development, we induced endometriosis in control ( Pgr cre/+ Rosa26 mTmG/+ ) and Mig-6 d/d Rosa26 mTmG/+ mice and found that uterine MIG-6 attenuation significantly increased incidence (p < 0.01) and weight of endometriotic lesions (p < 0.05) (Fig. 2 C and D). To address the role of MIG-6 in endometriosis-related infertility, we surgically induced endometriosis in wild type females using endometrial fragments from donor control ( Pgr cre/+ Rosa26 mTmG/+ ) and Mig-6 d/d Rosa26 mTmG/+ mice (Fig. 2 E). One month after endometriosis induction, the number of implantation sites was significantly reduced in the mice with Mig-6 d/d Rosa26 mTmG/+ ectopic lesions compared to the mice with control ectopic lesions (p < 0.05). Furthermore, implantation sites were entirely absent from mice with Mig-6 d/d Rosa26 mTmG/+ ectopic lesions after 2 months of endometriosis development (Fig. 2 F and G). These results demonstrate that MIG-6 attenuation in ectopic lesions increased endometriosis development and accelerated implantation failure compared to controls. Cessation of epithelial E2-induced proliferation is essential for implantation in all eutherian mammal species studied 24 , 25 . In mice, abundant proliferation of epithelial and stromal cells is detectable at day 2.5 of gestation (GD 2.5). However, just before implantation, P4 inhibits epithelial proliferation and induces differentiation to an embryo receptive state 26 . Establishing uterine receptivity by sequential actions of E2 and P4 on endometrial cells is critical for successful embryo apposition, attachment, implantation, and pregnancy maintenance, and lack of sufficient E2 and P4 action can result in infertility and pregnancy loss in humans 5 , 7 , 10 , 27 and mice 28 , 29 , 30 , 31 . Mig-6 d/d mice are infertile due to P4 resistance and implantation failure 32 . To determine whether a defect of embryo implantation is caused by an alteration in endometrial cell proliferation, we examined expression of a proliferation marker (Ki67) at pre-implantation (GD 3.5). Epithelial proliferation was significantly increased in the Mig-6 d/d endometrium compared to controls (p < 0.001) ( Mig-6 f/f ; fig. S3 A and B). To identify the molecular explanation for the effect of MIG-6 loss on epithelial proliferation, we examined amounts of several E2 signaling molecules, including epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2; also known as CD340, proto-oncogene Neu, or HER2) and extracellular-signal-regulated kinase 1/2 (ERK1/2) at GD 3.5 in Mig-6 f/f and Mig-6 d/d mice. EGFR amounts were unchanged, but ERBB2 and phospho-ERK1/2 (pERK1/2) amounts were selectively increased in Mig-6 d/d mice (fig. S3 C and D). These results suggest MIG-6 is a negative regulator of ERBB2/ERK signaling in the pre-implantation endometrium. Erbb2 overexpression causes infertility seen in Mig-6 mutant mice. In order to investigate the effect of ERBB2 targeting on nonreceptive endometrium and endometriosis with Mig-6 deficiency, we introduced Erbb2 ablation in Mig-6 d/d mice ( Mig-6 d/d Erbb2 d/d ; fig. S4). To address the effect of conditional Erbb2 knockout on the infertility phenotype of Mig-6 d/d mice, we mated female control, Mig-6 d/d , and Mig-6 d/d Erbb2 d/d ( Pgr cre/+ Mig-6 f/f Erbb2 f/f ) mice with wild type male mice for 6 months to determine their overall fertility. As expected, Mig-6 d/d mice were infertile 20 , but surprisingly, Mig-6 d/d Erbb2 d/d exhibited normal fecundity compared to controls (6.40 ± 0.49 and 7.29 ± 0.29 average pups/litter, respectively; table S1). This is the first report of molecular targeting to correct infertility caused by endometrial P4 resistance. To further dissect the reversal of Mig-6 -related infertility by attenuation of Erbb2 , we examined implantation rates. Uterine horns of Mig-6 d/d mice had no grossly visible implantation sites at GD 5.5, whereas Mig-6 d/d Erbb2 d/d mice averaged 7.00 ± 0.41 implantation sites that appeared normally spaced (Fig. 3 A). Subsequent histology revealed all embryos in Mig-6 d/d Erbb2 d/d uteri were positioned as expected alongside the anti-mesometrial luminal epithelium, and the stromal cells had the normal decidual response surrounding the embryo (Fig. 3 B). To identify the effect of additional Erbb2 knockout on the aberrantly increased epithelial proliferation of GD 3.5 Mig-6 d/d mice, we assessed Ki67 and cyclin D1 expression in Mig-6 d/d Erbb2 d/d mice. In contrast to Mig-6 d/d mice, Mig-6 d/d Erbb2 d/d endometrial epithelial cells exhibited normal cyclin D1 and Ki67 (Fig. 3 C). Since the increase of epithelial proliferation in Mig-6 d/d mice is accompanied by increased E2 signaling, we investigated whether excess E2 signaling is abrogated by Erbb2 ablation. The expression of the E2-responsive genes mucin 1 ( Muc-1 ) (p < 0.001), chloride channel calcium activated 3 ( Clca3 )(p < 0.01), lactoferrin ( Ltf )(p < 0.001), and complement component 3 ( C3 ) (p < 0.05) were significantly increased in Mig-6 d/d mice but restored to normal amounts in Mig-6 d/d Erbb2 d/d mice (Fig. 3 D). The same pattern was apparent for MUC1 and LTF protein amounts (Fig. 3 E). These results imply that ERBB2 overexpression resulting from Mig-6 attenuation causes female infertility due to a nonreceptive endometrium, and this effect may be reversed by ablation of Erbb2. Erbb2 ablation overcomes P4 resistance in Mig-6 mutant mice. Mig-6 attenuation causes endometrial P4 resistance demonstrated by P4’s inability to inhibit E2-induced uterine weight gain in Mig-6 d/d mice 20 . In order to determine if Erbb2 ablation restores endometrial P4 responsiveness in Mig-6 d/d mice, ovariectomized control, Mig-6 d/d , and Mig-6 d/d Erbb2 d/d mice were treated with vehicle or E2 + P4 for 3 days. While Mig-6 d/d mice treated with E2 + P4 experienced significant increases in uterine weight (p < 0.05), vascularization, and expression of the E2 target genes Muc1 , Clca3 , Ltf , and C3 compared to E2 + P4 treated control mice (p < 0.001), Mig-6 d/d Erbb2 d/d mice exhibited normal P4 responsiveness and expression of E2 target genes (Fig. 4 , A-C). We then examined the effect of Erbb2 ablation in the endometriosis development of Mig-6 d/d mice and found the number and weight of endometriotic lesions were restored to control amounts by the additional ablation of Erbb2 (Fig. 4 D and E; fig. S5). Uterine Mig-6 ablation causes endometrial hyperplasia by 5 months of age 20 . To investigate the impact of additional Erbb2 knockout on endometrial hyperplasia development due to Mig-6 attenuation, we examined uterine weight and gross histological morphology in control, Mig-6 d/d , and Mig-6 d/d Erbb2 d/d mice at 5 months of age. Uterine weight was significantly decreased in Mig-6 d/d Erbb2 d/d mice when compared to Mig-6 d/d mice (p < 0.001), and histological analysis revealed that Mig-6 d/d Erbb2 d/d mice did not develop endometrial hyperplasia (fig. S6). These results demonstrate that all known female reproductive phenotypes caused by knocking out uterine Mig-6 are restored to baseline by also knocking out Erbb2 . To identify the signaling pathways that Mig-6 regulates at pre-implantation, we performed transcriptomic analysis on the uteri from control, Mig-6 d/d , and Mig-6 d/d Erbb2 d/d mice at GD 3.5. We found 1,022 and 771 increased or decreased transcripts, respectively, in the Mig-6 d/d uterus as compared with controls (Fig. 5 A and table S2). Remarkably, 1,722 of the altered genes (96.04%) in Mig-6 d/d mice reverted to their normal expression amounts in Mig-6 d/d Erbb2 d/d mice. Pathway analysis showed that major altered pathways in the Mig-6 d/d uterus included cell-cycle control and DNA replication. P4 blocks E2-induced DNA synthesis by inhibiting replication licensing including mini-chromosome maintenance (MCM) proteins 33 , 34 which have a role in both the initiation and elongation phases of eukaryotic DNA replication as part of the MCM complex 35 , 36 . Fifteen genes associated with cell cycle and DNA replication were significantly changed in the Mig-6 d/d uterus (table S3). RT-qPCR analysis confirmed that the additional knockout of Erbb2 in Mig-6 d/d mice restored dysregulated cell-cycle control and DNA-replication-related gene transcripts to normal (Fig. 5 B). IHC results showed that at the protein level as well, aberrant overexpression of MCM2 and MCM6 occurred in Mig-6 d/d mice at the pre-implantation stage but reverted to normal in Mig-6 d/d Erbb2 d/d mice (Fig. 5 C). A similar action can be ascribed to P4 and E2 in the human endometrial epithelium, since a loss of MCM proteins occurs in the secretory phase, and P4 dominates this phase of the menstrual cycle 37 . Additionally, aberrant overexpression of MCM2 and MCM6 may cause abnormal epithelial proliferation and nonreceptive endometrium in infertile women with endometriosis 38 . Two Kruppel-like transcription factors (KLFs) are implicated in E2 and P4 modulation of uterine proliferation 38 . Klf4 expression is increased by E2 and promotes DNA replication, whereas Klf15 is increased by P4 and inhibits growth via regulation of Mcm2 38 . The expression of KLF4 was significantly increased in Mig-6 d/d mice compared to control mice while the expression of KLF15 was decreased in Mig-6 d/d mice, and the amounts reverted to normal in Mig-6 d/d Erbb2 d/d mice (p < 0.001) (Fig. 5 , B and D). These results suggest that Erbb2 overexpression due to Mig-6 ablation causes E2-induced epithelial proliferation and P4 resistance by disrupting cell cycle regulation. Discussion This study reveals the attenuation of MIG-6 in eutopic endometrium from infertile women with endometriosis compared to controls. MIG-6 expression was higher in human endometrium from the early secretory phase than in endometrium from the proliferative phase. Because of the complexity and dynamic nature of implantation, the molecular processes underlying these changes are poorly understood. Improving fertility rates requires unraveling molecular mechanisms of implantation. However, how regulation occurs between P4 and E2 is still not fully understood 39 , 40 , which is a critical barrier to better therapies for infertility. Amounts of MIG-6 mRNA and protein were lower in the eutopic endometrium of infertile women with endometriosis compared to controls in the early secretory phase. These results suggest that MIG-6 is a P4-responsive gene in human endometrium as in the mouse 20 , and MIG-6 loss may result in a non-receptive endometrium in endometriosis-related infertility. Nonhuman primates are advantageous for studying endometriosis because they are phylogenetically similar to humans 41 , 42 , 43 . Intraperitoneal inoculation with autologous menstrual effluent results in formation of endometriotic lesions similar in histology and morphology to those seen in women 22 . Paired sequential analysis showed MIG-6 protein amounts were decreased in the eutopic endometrium of baboons during progression of endometriosis as compared to pre-inoculation control. Furthermore, MIG-6 protein expression was reduced in the eutopic endometrium from the mice with endometriosis compared to the sham group. This result demonstrated reduced MIG-6 expression is associated with endometriosis development. We developed a mouse model of endometriosis based on Pgr cre/+ and mT/mG reporters that produces endometriotic lesions highly similar to those in humans. A mouse model in which excised human endometrial fragments are introduced into the peritoneum of immunocompromised mice is widely used, but is limited by lack of a normal immune system, which is thought to be important in endometriosis pathophysiology 44 , 45 , 46 . In contrast, the mouse model of induced endometriosis is a versatile model that has been used to study how the immune system 47 , hormones 48 , 49 and environmental factors 50 , 51 affect endometriosis. The availability of a large number of transgenic mice in which specific genes can be either eliminated or overexpressed make this induced endometriosis model ideal for studying specific pathways in development and progression of endometriosis and other diseases 46 . However, current mouse models of endometriosis that involve ovariectomy and E2 treatment are impractical for studies of physiological functions that require natural fluctuations in ovarian steroid hormones, such as fertility. On the other hand, our mouse model alleviates the need to apply ovariectomy and E2 treatment to enlarge endometriotic lesions because fluorescence reporter genes allow us to visualize in vivo and in real-time endometriotic lesions like those found in humans. Moreover, similarities between our mouse model and human endometriosis include: 1) development and progression of disease; 2) steroid hormone regulation; 3) fertility defect with implantation failure; and 4) P4 resistance in endometrium with Mig-6 deficiency. Furthermore, the fluorescence reporters enable us to quantitatively examine endometriotic lesions in these mice more accurately and easily than in prior models. Because Mig-6 d/d mice have a fertility defect 20 , 32 , we applied a syngeneic mouse model to examine the effect of endometriotic lesions with Mig-6 ablation on the eutopic endometrium. Several groups have used syngeneic mouse models of endometriosis, in which the uterus of one mouse is removed, minced and injected intraperitoneally into recipient mice 46 . Syngeneic murine models have several potential advantages over the rodent surgical model: 1) peritoneal seeding of uterine fragments is more similar to retrograde menstruation in women; 2) either the donor or recipient animal can receive therapeutic intervention or be otherwise manipulated prior to induction of disease; and 3) a large number of transgenic mice in which specific genes can be either eliminated or overexpressed are available. These advantages make syngeneic murine models ideal for studying the role of specific pathways in development and progression of endometriosis and other diseases. P4 is absolutely required for uterine implantation, decidualization, and maintenance of pregnancy 8 , 52 . How endometriosis contributes to infertility remains elusive, although P4 resistance is likely involved 1 . P4 resistance is seen in the endometrium of infertile women with endometriosis, and Mig-6 d/d mice exhibit P4 resistance by the inability of P4 to inhibit E2-induced uterine weight gain 20 . We demonstrate that MIG-6 mediates P4 inhibition of E2-induced cell proliferation by inhibition of ErbB2-ERK signaling. MIG-6 plays an important role in inhibiting epithelial cell proliferation and facilitating implantation. Epithelial cell proliferation and cyclin D1 amounts were higher in the epithelial cells of Mig-6 d/d mice, whereas both Mig-6 d/d Erbb2 d/d and control mice lacked elevated cyclin D1 amounts and epithelial cell proliferation. These results suggest that MIG-6 is a negative regulator of ErbB2 and suppresses E2-induced epithelial cell proliferation at the pre-implantation stage. We evaluated the potential therapeutic value of ErbB2 as a target for correcting endometrial P4 resistance in infertility. In our transcriptomic analysis in Mig-6 d/d and Mig-6 d/d Erbb2 d/d mice at GD 3.5, altered genes in Mig-6 d/d mice reverted to their normal expression amounts in Mig-6 d/d Erbb2 d/d mice. Pathway analysis using Ingenuity Systems Software showed that major altered pathways in the Mig-6 d/d uterus included cell-cycle control and DNA replication. Dr. Pollard’s group showed that P4 blocks E2-induced DNA synthesis by inhibiting replication licensing including mini-chromosome maintenance (MCM) proteins 33 , 34 . The MCM complex has a role in both the initiation and elongation phases of eukaryotic DNA replication 35 , 36 , 53 . The overlap of genes associated with cell cycle and DNA replication between the Pollard group's microarray results and ours is striking. In the uterine epithelium, E2 stimulates expression of MCMs, while P4 decreases transcripts of MCM2 through MCM6 33, 54 . Our IHC results showed aberrant overexpression of MCM2 and MCM6 in Mig-6 d/d mice at the pre-implantation stage. A similar action can be ascribed to P4 and E2 in the human endometrial epithelium, since a loss of MCM proteins occurs in the secretory phase, and P4 dominates this phase of the menstrual cycle 7 , 37 . However, aberrant overexpression of MCM2 and MCM6 may cause abnormal epithelial proliferation and nonreceptive endometrium in infertile women with endometriosis 38 . Our results regarding MIG-6 and ERBB2/ESR1 signaling in regulating uterine function in response to hormonal signals will bring insight into uterine pathophysiology and likely lead to new therapies for endometrial diseases. Deeper inquiry into endometrial epithelial-stromal crosstalk between ErbB2/ERK/ESR1 and PGR/MIG-6 signaling pathways will be of major importance to understanding infertility and endometriosis. In summary, our findings reveal that attenuation of MIG-6 occurs both in endometriotic lesions and in the endometriosis-effected eutopic endometrium. Evidence from mice indicates that loss of Mig-6 in endometriotic lesions promotes their development and accelerates endometriosis-related infertility, while loss of Mig-6 in the eutopic endometrium causes infertility due to defects in implantation and endometrial receptivity. We found that increased epithelial proliferation caused by Mig-6 loss is caused by E2 through the ERBB2/ERK pathway (Fig. 6 ). However, targeting Erbb2 can reverse all apparent female reproductive defects caused by Mig-6 loss including endometrial hyperplasia, infertility, and endometriosis lesion development. Attenuation of Mig-6 causes the inability of P4 to properly control the cell cycle and inhibit E2-induced aberrant epithelial proliferation that results from increases in MCMs. However, counteracting the overexpression of Erbb2 restores normal gene expression patterns, providing a molecular explanation for the rescue of normal reproductive function. These findings not only elucidate a critical pathway for understanding the hormonal control of normal uterine physiology, but they also provide the potential for new treatment strategies for uterine disease. Methods Study design The main objective of this study was to evaluate the role of MIG-6 in endometrial P4 resistance. First, the expression of MIG-6 was assessed in eutopic endometrium of infertile women with endometriosis compared to fertile women. To determine whether endometriosis affects MIG-6 expression, we examined MIG-6 expression in a nonhuman primate and mouse model of endometriosis. Subsequently, we identified ERBB2 as a MIG-6 target and evaluate the impact of Erbb2 ablation on the infertility and endometrial P4 resistance of Mig-6 d/d mice. Finally, transcriptomic analysis was applied to dissect the molecular mechanisms of Mig-6 in the uterus. The control and treatment groups and the number of biological replicates (sample sizes) for each experiment are specified in the figure legends. Animal numbers for each study type were determined by the investigators on the basis of previous experience with the standard disease models that were used or from pilot studies. Animals were randomly allocated to the control and treatment groups and housed together to minimize environmental differences and experimental bias. Analysis of endpoint readouts was carried out in a blinded fashion. Ethics Statement The institutional review board of Michigan State University, Greenville Health System, and University of North Carolina approved this study. The Institutional Animal Care and Use Committee at Michigan State University approved all experiments relating to mice. The Institutional Animal Care and Use Committees of both the University of Illinois at Chicago and Michigan State University approved the endometriosis baboon animal model. Human Endometrium Samples The human endometrial samples used to examine MIG-6 expression patterns were obtained from Michigan State University’s Center for Women’s Health Research Female Reproductive Tract Biorepository, the University of North Carolina, and the Greenville Hospital System in accordance with the guidelines set by the Institutional Review Boards of Michigan State University (Grand Rapids, MI), the University of North Carolina (Chapel Hill, NC), and Greenville Health System (Greenville, SC), respectively. Written informed consent was obtained from all participants. For experiments examining MIG-6 mRNA expression throughout the menstrual cycle, endometrial samples were analyzed from 22 cycling premenopausal women without endometriosis (n = 6 proliferative, n = 7 early secretory, n = 3 mid secretory, and n = 6 late secretory) and from 20 cycling premenopausal women with endometriosis (n = 2 proliferative, n = 6 early secretory, n = 9 mid secretory, and n = 3 late secretory). Control endometrial tissues were laparoscopically negative for endometriosis and had not been on any hormonal therapies for at least three months prior to surgery. Endometrial menstrual staging was confirmed by an experienced pathologist familiar with female reproduction. To investigate MIG-6 amounts in the endometrium from women, 10 control and 10 eutopic endometrium with endometriosis were used. To compare MIG-6 amounts in the eutopic endometrium and ectopic lesions of women with endometriosis, each of 12 samples were used. All women with endometriosis were infertile. Samples used for immunohistochemistry were fixed in 10% buffered formalin prior to embedding in paraffin wax. Animals And Tissue Collection Animals were maintained in a designated animal care facility according to Michigan State University’s Institutional Guidelines for the care and use of laboratory animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. For all animal studies, animals were randomly distributed among different conditions by the investigator as the animals did not show any size or appearance differences at the onset of the experiments. No animals were excluded, and the investigator was not blinded to group allocation during the experiment. Erbb2 conditional knockout mice were generated by crossing Pgr cre/+ Mig-6 f/f with Erbb2 f/f mice ( Pgr cre/+ Mig-6 f/f Erbb2 f/f ; Mig-6 d/d Erbb2 d/d ). Pregnant uterine samples were obtained by mating control ( Mig-6 f/f or Mig-6 f/f Erbb2 f/f ), Mig-6 d/d and Mig-6 d/d Erbb2 d/d female mice with C57BL/6 male mice the morning of a vaginal plug designated as day 0.5 of gestation (GD 0.5). Mice were sacrificed at GD 3.5 and 5.5. For the study of steroid hormone regulation, control, Mig-6 d/d and Mig-6 d/d Erbb2 d/d mice at 6 weeks of age were ovariectomized. Two weeks postsurgery, ovariectomized mice were injected with vehicle (sesame oil; Veh) or estradiol (0.1 µg/mouse; E2) plus progesterone (1 mg/mouse; P4) for 3 days and euthanized at 6 hours after injection. For the fertility studies, adult female control, Mig-6 d/d and Mig-6 d/d Erbb2 d/d mice were placed with wild type C57BL/6 male mice. The mating cages were maintained for 6 months and the number of litters and pups born during that period was recorded. Uterine tissues were then immediately processed at the time of dissection and either fixed with 4% (vol/vol) paraformaldehyde for histology or immunohistochemistry or snap frozen and stored at -80 °C for RNA/protein extraction. Induction Of Endometriosis For baboon uterine samples, endometriosis was induced by intraperitoneal inoculation of menstrual endometrium on two consecutive menstrual cycles and harvested using laparotomy via endometriectomy from four female baboons as previously described 55 . For mouse uterine samples, 8-weeks-old female mice which have conditional double-fluorescent Cre reporter gene ( Pgr cre/+ Rosa26 mTmG , Pgr cre/+ Mig-6 f/f Rosa26 mTmG , and Pgr cre/+ Mig-6 f/f Erbb2 f/f Rosa26 mTmG were injected with 1 µg/ml of E2 per a day at three times and had a surgical procedure to induce endometriosis. Under anesthesia, a midline abdominal incision was made to expose the uterus in female mice, and one of uterine horn was removed. In a Petri dish containing phosphate-buffered saline (PBS; pH 7.5), the uterine horn was opened longitudinally with scissors. The excised uterine horn was cut into small fragments of about 1 mm 3 , and then injected back into the peritoneum of same mouse. The abdominal incision and wound were closed with sutures and skin was closed with surgical wound clips, respectively. After a designated time, the mice were euthanized, and endometriosis-like lesions were removed using a fluorescence microscope and counted. Endometriosis-related Infertility Analysis Endometriosis were induced in 8 week old control female mouse recipient (fertile) receiving endometrial fragments from donor control ( Pgr cre/+ Rosa26 mTmG ) or Mig-6 d/d Rosa26 mTmG endometrium. A sham surgery group was included as a control. After 1, 2, and 3 months of the endometriosis induction, the mice with endometriotic lesions of control or Mig-6 d/d were mated with wild-type male mice and then collected at GD 7.5. RNA Isolation And Microarray Analysis Total RNA was extracted from the uterine tissues using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). RNA was pooled from the uteri of more than three mice per genotype at GD 3.5 and microarray analysis was performed using GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix) as described previously 56 (Gene Expression Omnibus accession code GSE138185). Array data were analyzed using Bioconductor for quantile normalization. We selected aberrantly expressed genes in the uteri of control, Mig-6 d/d and Mig-6 d/d Erbb2 d/d mice at GD 3.5 using a two-sample comparison according to significant fold change greater than 1.5. Aberrantly expressed genes were classified with canonical pathway analyzed by Ingenuity System Software (Ingenuity Systems Inc.). Reverse Transcription - Quantitative PCR The complementary DNAs (cDNAs) were synthesized with MMLV Reverse Transcriptase (Invitrogen Crop) according to the manufacturer’s instructions. RT-qPCR was performed on cDNA to assess the expression of genes of interest with SYBR Green (Bio-Rad) or TaqMan primers (Applied Biosystems). Experimental gene expression data were normalized against the housekeeping gene, 18S ribosomal RNA. Analysis of mRNA expression was first undertaken by the standard curve method, and results were corroborated by cycle threshold values assessing gene expression. Primer sequences used in these studies are shown in table S4. Immunohistochemistry Analyses Immunohistochemistry and immunofluorescence analyses were performed as previously described 57 . Briefly, dewaxed hydrated paraffin-embedded tissue sections were pre-incubated with 10% normal goat (for anti-MIG-6, Ki67, Cyclin D1, ErbB2, pERK1/2, ERK1/2, MUC1, LTF, and KLF4 antibodies) or donkey (for anti-MCM2, MCM6, and KLF15 antibodies) serum in PBS and then incubated with anti-MIG-6 (1:200 dilution; Customized antibody by Dr. Jeong Lab), anti-Ki67 (1:1000 dilution; #ab15580; Abcam), anti-Cyclin D1 (1:1000 dilution; #eo-RB9041-p0; Thermo Fisher Scientific), anti-ErbB2 (1:200 dilution; #2165; Cell Signaling), anti-pERK1/2 (1:500 dilution; #4370; Cell Signaling), anti-ERK1/2 (1:1000 dilution; #4695; Cell Signaling), anti-MUC1 (1:1000 dilution; #ab15481, Abcam), anti-LTF (1:2000 dilution; #07-682, Millipore Corp.), anti-MCM2 (1:20000 dilution; #sc9839, Santa Cruz Biotechnology), anti-MCM6 (1:20000 dilution; #sc9843; Santa Cruz Biotechnology), anti-KLF4 (1:5000 dilution; #sc20691; Santa Cruz Biotechnology), and anti-KLF15 (1:5000 dilution; #ab2647; Abcam) antibodies in PBS supplemented with 10% normal serum overnight at 4 °C. For immunohistochemistry, the sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories) for one hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB-Vector Laboratories) and analyzed using microscopy software from NIS Elements, Inc. (Nikon). A semi-quantitative grading system (H-score) was calculated to compare the immunohistochemical staining intensities. The H-score was calculated using the following equation: H-score = ∑ Pi (i), where i = intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%. The overall score ranged from 0 to 300 58 . Western Blot Analysis Western blot analyses were performed as described previously 59 . Proteins were extracted using lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, and 0.125% Nonidet P-40 (vol/vol)) supplemented with both a protease inhibitor cocktail (Roche, Indianapolis, IN) and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein lysates were electrophoresed via SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Membrane was blocked with Casein (0.5% w/v) in PBS with 0.1% Tween 20 (v/v; Sigma-Aldrich) prior to exposure to anti-ErbB2 (#2165; Cell Signaling, Danvers, MA), anti-EGFR (#2646; Cell Signaling), anti-phospho-ERK1/2 (pERK1/2; #4370; Cell Signaling), anti-ERK1/2 (#4695; Cell Signaling), anti-MIG-6 (Customized antibody by Dr Jeong Lab) or anti-β-actin (#sc1616; Santa Cruz Biotechnology) antibodies diluted to 1:1000. Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked secondary antibody followed by exposure to Electrochemiluminescence reagents (ECL) according to manufacturer’s instructions (GE Healthcare Biosciences). Statistical Analysis No statistical method was used to predetermine sample size for in vivo studies. Based on prior experience, all experiments used 5 mice per group to achieve adequate statistical power. For all animal experiments, block randomization was used to ensure a balance in sample size across groups. The investigators were blinded during the evaluation of results variations in the group. For all animal experiments, over three biological replicates were analyzed for each condition, and results are presented as the mean ± SEM. For data with only two groups, Student’s t test was used. For data containing more than two groups, an analysis of variance (ANOVA) test was used, followed by Tukey test for pairwise t-test. p < 0.05 was considered statistically significant. All statistical analyses were performed using the Instat package from GraphPad. The original data are provided in table S5. Data Availability All data are available in the manuscript or the supplementary material. The accession number for microarray generated in this study is GSE138185. Declarations Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MIST) (No. 2018R1A5A2025079 to H.G.Y.), and SRI and Bayer Discovery/Innovation Grant (to T.H.K.), as well as by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number R01HD084478 and R01HD101243 (to J.W.J) and F31HD101207 and T32HD087166 (to R.M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author contributions: H.-G.Y. and J.-W.J. were responsible for the concept of the study; A.T.F. collected baboon samples; S.L.Y. and B.A.L. collected human samples; J.-Y.Y. and T.H.K. carried out experiments; J.-Y.Y., T.H.K. and J.-H.S. analyzed data; U.M. provided transgenic mice; R.M.M. contributed to write the manuscript. All authors contributed to the final manuscript version. 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The Journal of clinical endocrinology and metabolism 88 , 2309-2317 (2003). Kim TH , et al. ARID1A Is Essential for Endometrial Function during Early Pregnancy. PLoS genetics 11 , e1005537 (2015). Additional Declarations There is NO Competing Interest. Supplementary Files Mig6ErbB2FigureNatureCommunicationSupplementarydata.docx Supplementary Data Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2022 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-95903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":4312596,"identity":"a7f2f342-b15e-41cc-bb70-ccaff2b1ccdd","order_by":0,"name":"Jung-Yoon Yoo","email":"","orcid":"","institution":"Yonsei University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jung-Yoon","middleName":"","lastName":"Yoo","suffix":""},{"id":4312597,"identity":"460165f9-c98e-47f0-894f-d85c2d628dd8","order_by":1,"name":"Tae Hoon Kim","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Hoon","lastName":"Kim","suffix":""},{"id":4312598,"identity":"6dd727a9-7836-41c9-bb15-8bc5b59dc426","order_by":2,"name":"Jung-Ho Shin","email":"","orcid":"","institution":"Guro Hospital, Korea University Medical Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jung-Ho","middleName":"","lastName":"Shin","suffix":""},{"id":4312599,"identity":"7cdfe5d6-d9ca-448f-bf81-fecace36557f","order_by":3,"name":"Ryan Marquardt","email":"","orcid":"https://orcid.org/0000-0002-6597-4506","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Marquardt","suffix":""},{"id":4312600,"identity":"11409f27-178d-4edf-83de-5ff3ff1f78b2","order_by":4,"name":"Ulrich Mueller","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ulrich","middleName":"","lastName":"Mueller","suffix":""},{"id":4312601,"identity":"25844b30-6ece-4c63-a91a-68196b5565a2","order_by":5,"name":"Asgerally Fazleabas","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Asgerally","middleName":"","lastName":"Fazleabas","suffix":""},{"id":4312602,"identity":"8a766c97-68a4-4239-bfae-b1f162d8262a","order_by":6,"name":"Steven Young","email":"","orcid":"https://orcid.org/0000-0002-5205-4495","institution":"University of North Carolina","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Young","suffix":""},{"id":4312603,"identity":"5dfcb3e0-c858-4a1a-a5cd-9e36ac2f9747","order_by":7,"name":"Bruce Lessey","email":"","orcid":"","institution":"Wake Forest Baptist Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bruce","middleName":"","lastName":"Lessey","suffix":""},{"id":4312604,"identity":"cc70d47e-325f-4f6a-ab33-3f518537a00a","order_by":8,"name":"Ho-Geun Yoon","email":"","orcid":"https://orcid.org/0000-0003-2718-3372","institution":"Yonsei University College of Medicine,","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ho-Geun","middleName":"","lastName":"Yoon","suffix":""},{"id":4312605,"identity":"7f4da4f1-9bd4-44c2-b6c9-11454f554a15","order_by":9,"name":"Jae-Wook Jeong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACxgYQyWMD4fGQoCWNBC1QcJgELcztzccefpE5Hy0/I4Hxwds2YhzWcyzdWIbndu6GGwnMhnOJ0jIjx0xaAqRFIoFNmpcoLfPfgLScy50/I4H9N3FaZvCYSX7gOZDbcCOBjZk4LT1padIMPMm5G848bJacc44ILYbth49J/uyxy53fnnzww5syYrQ0AAOatwdsYQMR6oFAHqT2xw/iFI+CUTAKRsEIBQCiyzXHmaa/uAAAAABJRU5ErkJggg==","orcid":"","institution":"Michigan State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jae-Wook","middleName":"","lastName":"Jeong","suffix":""}],"badges":[],"createdAt":"2020-10-21 07:30:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-95903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-95903/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-022-28608-x","type":"published","date":"2022-03-01T11:44:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3477990,"identity":"c392281c-032f-4cef-aa5a-8154df5c776d","added_by":"auto","created_at":"2020-11-10 00:19:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218441,"visible":true,"origin":"","legend":"MIG-6 expression in the endometrium of women with endometriosis and nonhuman primate, baboon model. (A), RT-qPCR analysis of MIG-6 gene expression in endometrium from women with and without endometriosis during the menstrual cycle (n ≥ 3 for each group). (B), (C), Immunohistochemical H-score (B) and representative photomicrographs (C) of MIG-6 in the endometrium from women with endometriosis as compared to control (n = 10 for each group). (D), Immunohistochemical H-score and representative photomicrographs of MIG-6 in the endometriosis baboon model induced by intraperitoneal inoculation of menstrual endometrium during progression of endometriosis in pre-inoculation, 3, 6, and 9 months (n = 4 per period). €, Immunohistochemical H-score and representative photomicrographs of MIG-6 in the endometriosis mouse model (n = 5 for group). Mean ± SEM, ** P\u003c0.01 and *** P\u003c0.001, Student’s t test for data containing only two groups and ANOVA followed by Tukey test for pairwise t-test for data containing more than two groups. ","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/976559788e7695b8313d8502.jpg"},{"id":3477992,"identity":"6bb5c3b5-ec43-4694-b189-7a2d75d35ac2","added_by":"auto","created_at":"2020-11-10 00:19:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145749,"visible":true,"origin":"","legend":"Reduction of MIG-6 in ectopic lesions in endometriosis patients and the effect of ectopic lesions with MIG-6 deficiency on endometriosis development and embryo implantation. (A), (B), Decrease of MIG-6 expression in ectopic endometriotic lesions compared to eutopic endometrium from the same endometriosis patients. H-score (A) and representative photomicrographs (B) of immunofluorescence analysis of MIG-6 in eutopic endometrium and ectopic lesions from women with endometriosis (n=10). (C), (D), The effect of ectopic lesions with MIG-6 deficiency on endometriosis development. Endometriosis was surgically induced in control (Pgrcre/+Rosa26mTmG/+) and Mig-6d/dRosa26mTmG/+ mice. Fluorescence photomicrographs (C) and average total number (D) of endometriosis lesions in control and Mig-6d/dRosa26mTmG/+ mice (n=5). (E), (F), (G), The effect of ectopic lesions with MIG-6 deficiency on embryo implantation. (E) Experimental design to access the effect of ectopic lesions with MIG-6 deficiency on embryo implantation. Average number (F) and uterine images (G) of implantation sites at GD 7.5 in mice with endometriosis on 1 and 3 months after endometriosis induction (n=5 or more per period). Mean ± SEM, ** p\u003c0.01 and *** p\u003c0.001, Student’s t test for data containing only two groups and ANOVA followed by Tukey test for data containing more than two groups. ","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/e043e4e0ef1f1a6598b54e7b.jpg"},{"id":3477993,"identity":"c39d41ec-1158-43e5-92a8-aecc7771dc65","added_by":"auto","created_at":"2020-11-10 00:19:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233153,"visible":true,"origin":"","legend":"Rescue of implantation defect and recovery of aberrant activated epithelial cells proliferation and ESR signaling in Mig-6d/d mice by Erbb2 double ablation. (A), Uteri of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice and number of implantation sites at GD 5.5 (n=4 for each genotype). (B), Hematoxylin and eosin (H\u0026E) staining in paired endometrium of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 5.5. Arrowheads indicate embryos. (C), Immunohistochemistry analysis of Ki67 and Cyclin D1 in the endometrium of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5. (D), (E), RT-qPCR analysis of Muc1, Clca3, Ltf, and C3 (D) and immunohistochemistry analysis of MUC1 and LTF (E) as epithelial ESR1 target genes in the uterus of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5 (n = 6 for each genotype). Mean ± SEM, * P\u003c0.05, ** P\u003c0.01, and *** P\u003c0.001, ANOVA followed by Tukey test.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/4a98f2d638d0feb266042ccf.jpg"},{"id":3477994,"identity":"6cafdfc5-8c0a-415d-985f-a2fa92fc4c5f","added_by":"auto","created_at":"2020-11-10 00:19:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":115470,"visible":true,"origin":"","legend":"Rescue of steroid hormone dysregulation in Mig-6d/d mice by Erbb2 double ablation. (A), (B), Ratio of uterine weight to body weight (A) and uteri (B) of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice treated with vehicle or E2+P4 for 3 days (n = 3 or more for each group). (C), RT-qPCR analysis of epithelial ESR1 target genes expression, Muc1, Clca3, Ltf, and C3 in the uteri of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice treated with E2+P4 for 3 days (n = 5 per genotype). (D), €, Average total number (D) and representative fluorescence photomicrographs (E) of endometriotic sites in control, Mig-6d/d, and Mig-6d/dErbb2d/d mice by induced endometriosis based on mT/mG mice (n = 5 per genotype). Arrowheads indicate lesions attached to the outside of uterus. Mean ± SEM, * P\u003c0.05, ** P\u003c0.01, and *** P\u003c0.001, ANOVA followed by Tukey test. ","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/51e63a4642a60fd2f805c6ba.jpg"},{"id":3477995,"identity":"b1684ef2-3654-45c2-bbe7-f0e6414035f0","added_by":"auto","created_at":"2020-11-10 00:19:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":188315,"visible":true,"origin":"","legend":"Recovery of gene expression (Microarray) in Mig-6d/d mice by Erbb2 double ablation. (A), Clustering analysis of Mig-6 dependent regulated genes in uteri of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5. The extent of gene expression changes is represented by a green-red color scale (green: low expression and red: high expression). (B), RT-qPCR analysis of transcript amounts of Mig-6 dependent regulated genes in uteri of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5 (n = 5 or 6 per each genotype). Mean ± SEM, * P\u003c0.05, ** P\u003c0.01, and *** P\u003c0.001, ANOVA followed by Tukey test. (C), Immunohistochemistry analysis of MCM2 and MCM6 in the uterus of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5. (D), Immunohistochemistry analysis of KLF4 and KLF15 in the uterus of control, Mig-6d/d, and Mig-6d/dErbb2d/d mice at GD 3.5. ","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/19ea11bf7ff39a8d68f05baf.jpg"},{"id":3477996,"identity":"4092b091-5e1e-451e-a029-a729091e17b2","added_by":"auto","created_at":"2020-11-10 00:19:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52070,"visible":true,"origin":"","legend":"Molecular mechanisms of MIG-6 function in the uterus. MIG-6 mediates P4 inhibition of E2 signaling by inhibiting ErbB2-ERK signaling (Left side), and the attenuation of MIG-6 leads to ErbB2-ERK activation, proliferation of uterine epithelial cells, and eventually to endometriosis and infertility (Right side).","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/5a93ef45de0eb7f866153ae1.jpg"},{"id":18728928,"identity":"9c736365-caa6-4019-ace7-2fc1c7f6f83c","added_by":"auto","created_at":"2022-03-01 11:44:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":987244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/bb5c14b9-3925-4b27-8e29-7b652b9d7740.pdf"},{"id":3477991,"identity":"77063fcb-d1f2-48e7-8b11-8578850bc0c3","added_by":"auto","created_at":"2020-11-10 00:19:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4046054,"visible":true,"origin":"","legend":"Supplementary Data","description":"","filename":"Mig6ErbB2FigureNatureCommunicationSupplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-95903/v1/d3bf9d27e4839f7c9c2f2474.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Loss of MIG-6 results in endometrial progesterone resistance via ERBB2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCritical for fertility, the uterine endometrium\u0026rsquo;s epithelial and stromal compartments undergo dynamic hormonally controlled molecular and morphological changes to prepare for embryo implantation and development. Estrogen (E2) stimulates the proliferation of uterine epithelial cells, and progesterone (P4) suppresses E2-induced proliferation. Endometrial P4 resistance implies decreased responsiveness of target tissue to bioavailable P4 \u003csup\u003e1, 2, 3\u003c/sup\u003e. Endometrial P4 resistance is seen in women with a nonreceptive endometrium, endometriosis, polycystic ovary syndrome (PCOS), and endometrial cancer \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Moreover, P4-induced molecular changes in the eutopic (intrauterine) endometrial tissue of women with endometriosis are either blunted or undetectable \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEndometriosis affects about 10% of all women of reproductive age, and the incidence increases to 50\u0026ndash;60% of women with chronic pelvic pain and infertility \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While progestin-based therapies are commonly used to treat endometriosis and lead to disease regression in some women, other women with endometriosis and pelvic pain do not respond effectively to progestins \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, many P4-induced molecular changes in the eutopic endometrial tissue of women with endometriosis are either blunted or dysregulated \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, but an impaired P4 response is seen in the endometrium of women with endometriosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Despite knowing the effects, the molecular mechanism responsible for endometrial P4 resistance and dysregulation remains unclear. Therefore, understanding the molecular mechanisms of endometrial P4 resistance is critical.\u003c/p\u003e\n\u003cp\u003eThe present study revealed that the amount of mitogen inducible gene 6 (MIG-6) was decreased in endometrium from infertile women with endometriosis. We used uterine-specific \u003cem\u003eMig-6\u003c/em\u003e knock-out mice to demonstrate that MIG-6 loss results in endometrial progesterone resistance via ERBB2. Our findings provide new insight into the etiology of female infertility and provide a new molecular framework useful for the design of new therapeutic strategies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMIG-6\u003c/em\u003e expression is decreased in endometrium from women with endometriosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously identified \u003cem\u003eMig-6\u003c/em\u003e as a P4-regulated gene that mediates the ability of P4 to repress E2 action in the mouse uterus \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. During the menstrual cycle, P4 amounts rise at the early secretory phase. As measured by RT-qPCR, \u003cem\u003eMIG-6\u003c/em\u003e expression in the human endometrium was significantly higher in the early secretory phase of the menstrual cycle than in the proliferative phase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that \u003cem\u003eMIG-6\u003c/em\u003e is a P4-induced gene in the human endometrium as has been demonstrated in the mouse \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Because many P4-induced endometrial molecular changes are either blunted or eliminated in women with endometriosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we examined MIG-6 expression in endometrial biopsies from infertile women with endometriosis. RT-qPCR and immunohistochemistry showed that amounts of \u003cem\u003eMIG-6\u003c/em\u003e mRNA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and protein (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were significantly lower in the eutopic endometrium of infertile women with endometriosis compared to controls in the early secretory phase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, A-C). To assess how MIG-6 expression is affected by endometriosis progression, we used a baboon model \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Intraperitoneal inoculation with autologous menstrual effluent in female non-human primates results in formation of endometriotic lesions highly similar in histomorphology to those seen in women \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We found that endometrial MIG-6 protein abundance was significantly reduced in baboons during the progression of endometriosis after experimental disease induction as compared to paired pre-inoculation control samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results demonstrate that reduced MIG-6 expression can be caused by the development of endometriotic lesions.\u003c/p\u003e\n\u003cp\u003eUncovering pathophysiological mechanisms of endometriosis-related infertility with animal models requires easy identification of lesions to distinguish them from the surrounding normal tissues. With this in mind, we developed a mouse model of endometriosis using \u003cem\u003emT/mG\u003c/em\u003e reporters. In \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice, progesterone receptor (\u003cem\u003ePgr\u003c/em\u003e)-positive uterine cells express mG, while \u003cem\u003ePgr\u003c/em\u003e-negative cells express mT (fig. S1 A and B). Using this model, we surgically induced endometriosis in \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice by inoculating autologous endometrial tissue fragments into the peritoneal cavity after 3 days of E2 treatment (fig. S1C). This method leads to the development of endometriotic lesions similar to those in humans without the need for ovariectomy or unopposed E2 treatment (fig. S1 D-F). To examine the responsiveness of our endometriosis model to E2 and P4, \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice induced with endometriosis were treated with vehicle, E2, or E2\u0026thinsp;+\u0026thinsp;P4 for 2 weeks. While E2 treatment after endometriosis induction significantly increased the number of endometriotic lesions compared to the vehicle group, the addition of P4 suppressed the E2-induced increase in lesion number (fig. S1 G and H; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Our mouse model thus closely mirrors human endometriosis as an E2-dependent and P4-suppressed disorder. To determine whether MIG-6 expression is dysregulated after endometriosis development in a distinct mammalian system, we examined MIG-6 amount in the eutopic endometrium from \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice with endometriosis. MIG-6 protein expression was significantly reduced in eutopic endometrium from the mice with endometriosis compared to the sham group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan class=\"BoldItalic\"\u003eMig-6\u003c/span\u003e\u003c/em\u003e loss accelerated the development of endometriosis and endometriosis-related infertility.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we assessed whether endometriosis in mice causes infertility by assessing implantation and decidualization success. One month after endometriosis induction, the number of implantation sites in mice with endometriosis was not changed compared to the sham group. However, 63.6% (7 out of 11) of mice with endometriosis experienced implantation failure 3 months after endometriosis development (fig. S2A). We next examined the impact of endometriosis on decidualization using an artificial decidualization model \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. One month after endometriosis induction, mice with endometriosis displayed a uterine horn that responded well to artificial decidualization; however, after 3 month of endometriosis development, the mice with endometriosis exhibited a significant defect in decidual response compared to control and sham mice (fig. S2B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Our result suggests that endometriosis development causes implantation failure and a defect of decidualization, as has been hypothesized in humans \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHaving established the link between endometriosis development and MIG-6 attenuation in the eutopic endometrium, we sought to determine if MIG-6 depletion is involved in endometriotic lesion development. In a comparison of MIG-6 expression in paired ectopic and eutopic endometrial biopsies taken from women with endometriosis, MIG-6 amounts were significantly reduced in the ectopic endometrial specimens (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). To assess the effect of MIG-6 deficiency in endometriosis development, we induced endometriosis in control (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice and found that uterine MIG-6 attenuation significantly increased incidence (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and weight of endometriotic lesions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). To address the role of MIG-6 in endometriosis-related infertility, we surgically induced endometriosis in wild type females using endometrial fragments from donor control (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). One month after endometriosis induction, the number of implantation sites was significantly reduced in the mice with \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e ectopic lesions compared to the mice with control ectopic lesions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, implantation sites were entirely absent from mice with \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG/+\u003c/em\u003e\u003c/sup\u003e ectopic lesions after 2 months of endometriosis development (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and G). These results demonstrate that MIG-6 attenuation in ectopic lesions increased endometriosis development and accelerated implantation failure compared to controls.\u003c/p\u003e\n\u003cp\u003eCessation of epithelial E2-induced proliferation is essential for implantation in all eutherian mammal species studied \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In mice, abundant proliferation of epithelial and stromal cells is detectable at day 2.5 of gestation (GD 2.5). However, just before implantation, P4 inhibits epithelial proliferation and induces differentiation to an embryo receptive state \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Establishing uterine receptivity by sequential actions of E2 and P4 on endometrial cells is critical for successful embryo apposition, attachment, implantation, and pregnancy maintenance, and lack of sufficient E2 and P4 action can result in infertility and pregnancy loss in humans \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice are infertile due to P4 resistance and implantation failure \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To determine whether a defect of embryo implantation is caused by an alteration in endometrial cell proliferation, we examined expression of a proliferation marker (Ki67) at pre-implantation (GD 3.5). Epithelial proliferation was significantly increased in the \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e endometrium compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; fig. S3 A and B). To identify the molecular explanation for the effect of MIG-6 loss on epithelial proliferation, we examined amounts of several E2 signaling molecules, including epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2; also known as CD340, proto-oncogene Neu, or HER2) and extracellular-signal-regulated kinase 1/2 (ERK1/2) at GD 3.5 in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. EGFR amounts were unchanged, but ERBB2 and phospho-ERK1/2 (pERK1/2) amounts were selectively increased in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (fig. S3 C and D). These results suggest MIG-6 is a negative regulator of ERBB2/ERK signaling in the pre-implantation endometrium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eErbb2\u003c/em\u003e overexpression causes infertility seen in\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eMig-6\u003c/span\u003e \u003cstrong\u003emutant mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the effect of ERBB2 targeting on nonreceptive endometrium and endometriosis with \u003cem\u003eMig-6\u003c/em\u003e deficiency, we introduced \u003cem\u003eErbb2\u003c/em\u003e ablation in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e; fig. S4). To address the effect of conditional \u003cem\u003eErbb2\u003c/em\u003e knockout on the infertility phenotype of \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, we mated female control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mice with wild type male mice for 6 months to determine their overall fertility. As expected, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice were infertile \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, but surprisingly, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e exhibited normal fecundity compared to controls (6.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 and 7.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 average pups/litter, respectively; table S1). This is the first report of molecular targeting to correct infertility caused by endometrial P4 resistance.\u003c/p\u003e\n\u003cp\u003eTo further dissect the reversal of \u003cem\u003eMig-6\u003c/em\u003e-related infertility by attenuation of \u003cem\u003eErbb2\u003c/em\u003e, we examined implantation rates. Uterine horns of \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice had no grossly visible implantation sites at GD 5.5, whereas \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice averaged 7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 implantation sites that appeared normally spaced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Subsequent histology revealed all embryos in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e uteri were positioned as expected alongside the anti-mesometrial luminal epithelium, and the stromal cells had the normal decidual response surrounding the embryo (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). To identify the effect of additional \u003cem\u003eErbb2\u003c/em\u003e knockout on the aberrantly increased epithelial proliferation of GD 3.5 \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, we assessed Ki67 and cyclin D1 expression in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. In contrast to \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e endometrial epithelial cells exhibited normal cyclin D1 and Ki67 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Since the increase of epithelial proliferation in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice is accompanied by increased E2 signaling, we investigated whether excess E2 signaling is abrogated by \u003cem\u003eErbb2\u003c/em\u003e ablation. The expression of the E2-responsive genes mucin 1 (\u003cem\u003eMuc-1\u003c/em\u003e) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), chloride channel calcium activated 3 (\u003cem\u003eClca3\u003c/em\u003e)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), lactoferrin (\u003cem\u003eLtf\u003c/em\u003e)(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and complement component 3 (\u003cem\u003eC3\u003c/em\u003e) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly increased in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice but restored to normal amounts in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). The same pattern was apparent for MUC1 and LTF protein amounts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results imply that ERBB2 overexpression resulting from \u003cem\u003eMig-6\u003c/em\u003e attenuation causes female infertility due to a nonreceptive endometrium, and this effect may be reversed by ablation of \u003cem\u003eErbb2.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan class=\"BoldItalic\"\u003eErbb2\u003c/span\u003e \u003c/em\u003eablation overcomes P4 resistance in\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eMig-6\u003c/span\u003e \u003cstrong\u003emutant mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMig-6\u003c/em\u003e attenuation causes endometrial P4 resistance demonstrated by P4\u0026rsquo;s inability to inhibit E2-induced uterine weight gain in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In order to determine if \u003cem\u003eErbb2\u003c/em\u003e ablation restores endometrial P4 responsiveness in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, ovariectomized control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice were treated with vehicle or E2\u0026thinsp;+\u0026thinsp;P4 for 3 days. While \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice treated with E2\u0026thinsp;+\u0026thinsp;P4 experienced significant increases in uterine weight (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), vascularization, and expression of the E2 target genes \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eClca3\u003c/em\u003e, \u003cem\u003eLtf\u003c/em\u003e, and \u003cem\u003eC3\u003c/em\u003e compared to E2\u0026thinsp;+\u0026thinsp;P4 treated control mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice exhibited normal P4 responsiveness and expression of E2 target genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, A-C). We then examined the effect of \u003cem\u003eErbb2\u003c/em\u003e ablation in the endometriosis development of \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice and found the number and weight of endometriotic lesions were restored to control amounts by the additional ablation of \u003cem\u003eErbb2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD and E; fig. S5).\u003c/p\u003e\n\u003cp\u003eUterine \u003cem\u003eMig-6\u003c/em\u003e ablation causes endometrial hyperplasia by 5 months of age \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To investigate the impact of additional \u003cem\u003eErbb2\u003c/em\u003e knockout on endometrial hyperplasia development due to \u003cem\u003eMig-6\u003c/em\u003e attenuation, we examined uterine weight and gross histological morphology in control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at 5 months of age. Uterine weight was significantly decreased in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice when compared to \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and histological analysis revealed that \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice did not develop endometrial hyperplasia (fig. S6). These results demonstrate that all known female reproductive phenotypes caused by knocking out uterine \u003cem\u003eMig-6\u003c/em\u003e are restored to baseline by also knocking out \u003cem\u003eErbb2\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify the signaling pathways that \u003cem\u003eMig-6\u003c/em\u003e regulates at pre-implantation, we performed transcriptomic analysis on the uteri from control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at GD 3.5. We found 1,022 and 771 increased or decreased transcripts, respectively, in the \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e uterus as compared with controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and table S2). Remarkably, 1,722 of the altered genes (96.04%) in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice reverted to their normal expression amounts in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. Pathway analysis showed that major altered pathways in the \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e uterus included cell-cycle control and DNA replication. P4 blocks E2-induced DNA synthesis by inhibiting replication licensing including mini-chromosome maintenance (MCM) proteins \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e which have a role in both the initiation and elongation phases of eukaryotic DNA replication as part of the MCM complex \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Fifteen genes associated with cell cycle and DNA replication were significantly changed in the \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e uterus (table S3). RT-qPCR analysis confirmed that the additional knockout of \u003cem\u003eErbb2\u003c/em\u003e in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice restored dysregulated cell-cycle control and DNA-replication-related gene transcripts to normal (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). IHC results showed that at the protein level as well, aberrant overexpression of MCM2 and MCM6 occurred in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at the pre-implantation stage but reverted to normal in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). A similar action can be ascribed to P4 and E2 in the human endometrial epithelium, since a loss of MCM proteins occurs in the secretory phase, and P4 dominates this phase of the menstrual cycle \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Additionally, aberrant overexpression of MCM2 and MCM6 may cause abnormal epithelial proliferation and nonreceptive endometrium in infertile women with endometriosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Two Kruppel-like transcription factors (KLFs) are implicated in E2 and P4 modulation of uterine proliferation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eKlf4\u003c/em\u003e expression is increased by E2 and promotes DNA replication, whereas \u003cem\u003eKlf15\u003c/em\u003e is increased by P4 and inhibits growth via regulation of \u003cem\u003eMcm2\u003c/em\u003e \u003csup\u003e38\u003c/sup\u003e. The expression of KLF4 was significantly increased in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice compared to control mice while the expression of KLF15 was decreased in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, and the amounts reverted to normal in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, B and D). These results suggest that \u003cem\u003eErbb2\u003c/em\u003e overexpression due to \u003cem\u003eMig-6\u003c/em\u003e ablation causes E2-induced epithelial proliferation and P4 resistance by disrupting cell cycle regulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reveals the attenuation of MIG-6 in eutopic endometrium from infertile women with endometriosis compared to controls. MIG-6 expression was higher in human endometrium from the early secretory phase than in endometrium from the proliferative phase. Because of the complexity and dynamic nature of implantation, the molecular processes underlying these changes are poorly understood. Improving fertility rates requires unraveling molecular mechanisms of implantation. However, how regulation occurs between P4 and E2 is still not fully understood\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, which is a critical barrier to better therapies for infertility. Amounts of MIG-6 mRNA and protein were lower in the eutopic endometrium of infertile women with endometriosis compared to controls in the early secretory phase. These results suggest that MIG-6 is a P4-responsive gene in human endometrium as in the mouse\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and MIG-6 loss may result in a non-receptive endometrium in endometriosis-related infertility.\u003c/p\u003e\n\u003cp\u003eNonhuman primates are advantageous for studying endometriosis because they are phylogenetically similar to humans\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Intraperitoneal inoculation with autologous menstrual effluent results in formation of endometriotic lesions similar in histology and morphology to those seen in women\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Paired sequential analysis showed MIG-6 protein amounts were decreased in the eutopic endometrium of baboons during progression of endometriosis as compared to pre-inoculation control. Furthermore, MIG-6 protein expression was reduced in the eutopic endometrium from the mice with endometriosis compared to the sham group. This result demonstrated reduced MIG-6 expression is associated with endometriosis development.\u003c/p\u003e\n\u003cp\u003eWe developed a mouse model of endometriosis based on \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emT/mG\u003c/em\u003e reporters that produces endometriotic lesions highly similar to those in humans. A mouse model in which excised human endometrial fragments are introduced into the peritoneum of immunocompromised mice is widely used, but is limited by lack of a normal immune system, which is thought to be important in endometriosis pathophysiology\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In contrast, the mouse model of induced endometriosis is a versatile model that has been used to study how the immune system\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, hormones\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and environmental factors\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e affect endometriosis. The availability of a large number of transgenic mice in which specific genes can be either eliminated or overexpressed make this induced endometriosis model ideal for studying specific pathways in development and progression of endometriosis and other diseases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, current mouse models of endometriosis that involve ovariectomy and E2 treatment are impractical for studies of physiological functions that require natural fluctuations in ovarian steroid hormones, such as fertility. On the other hand, our mouse model alleviates the need to apply ovariectomy and E2 treatment to enlarge endometriotic lesions because fluorescence reporter genes allow us to visualize \u003cem\u003ein vivo\u003c/em\u003e and in real-time endometriotic lesions like those found in humans. Moreover, similarities between our mouse model and human endometriosis include: 1) development and progression of disease; 2) steroid hormone regulation; 3) fertility defect with implantation failure; and 4) P4 resistance in endometrium with \u003cem\u003eMig-6\u003c/em\u003e deficiency. Furthermore, the fluorescence reporters enable us to quantitatively examine endometriotic lesions in these mice more accurately and easily than in prior models.\u003c/p\u003e\n\u003cp\u003eBecause \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice have a fertility defect\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we applied a syngeneic mouse model to examine the effect of endometriotic lesions with \u003cem\u003eMig-6\u003c/em\u003e ablation on the eutopic endometrium. Several groups have used syngeneic mouse models of endometriosis, in which the uterus of one mouse is removed, minced and injected intraperitoneally into recipient mice\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Syngeneic murine models have several potential advantages over the rodent surgical model: 1) peritoneal seeding of uterine fragments is more similar to retrograde menstruation in women; 2) either the donor or recipient animal can receive therapeutic intervention or be otherwise manipulated prior to induction of disease; and 3) a large number of transgenic mice in which specific genes can be either eliminated or overexpressed are available. These advantages make syngeneic murine models ideal for studying the role of specific pathways in development and progression of endometriosis and other diseases.\u003c/p\u003e\n\u003cp\u003eP4 is absolutely required for uterine implantation, decidualization, and maintenance of pregnancy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. How endometriosis contributes to infertility remains elusive, although P4 resistance is likely involved\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. P4 resistance is seen in the endometrium of infertile women with endometriosis, and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice exhibit P4 resistance by the inability of P4 to inhibit E2-induced uterine weight gain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We demonstrate that MIG-6 mediates P4 inhibition of E2-induced cell proliferation by inhibition of ErbB2-ERK signaling. MIG-6 plays an important role in inhibiting epithelial cell proliferation and facilitating implantation. Epithelial cell proliferation and cyclin D1 amounts were higher in the epithelial cells of \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice, whereas both \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and control mice lacked elevated cyclin D1 amounts and epithelial cell proliferation. These results suggest that MIG-6 is a negative regulator of ErbB2 and suppresses E2-induced epithelial cell proliferation at the pre-implantation stage.\u003c/p\u003e\n\u003cp\u003eWe evaluated the potential therapeutic value of ErbB2 as a target for correcting endometrial P4 resistance in infertility. In our transcriptomic analysis in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at GD 3.5, altered genes in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice reverted to their normal expression amounts in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. Pathway analysis using Ingenuity Systems Software showed that major altered pathways in the \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e uterus included cell-cycle control and DNA replication. Dr. Pollard\u0026rsquo;s group showed that P4 blocks E2-induced DNA synthesis by inhibiting replication licensing including mini-chromosome maintenance (MCM) proteins\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The MCM complex has a role in both the initiation and elongation phases of eukaryotic DNA replication\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The overlap of genes associated with cell cycle and DNA replication between the Pollard group's microarray results and ours is striking. In the uterine epithelium, E2 stimulates expression of MCMs, while P4 decreases transcripts of MCM2 through MCM6\u003csup\u003e33, 54\u003c/sup\u003e. Our IHC results showed aberrant overexpression of MCM2 and MCM6 in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at the pre-implantation stage. A similar action can be ascribed to P4 and E2 in the human endometrial epithelium, since a loss of MCM proteins occurs in the secretory phase, and P4 dominates this phase of the menstrual cycle\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, aberrant overexpression of MCM2 and MCM6 may cause abnormal epithelial proliferation and nonreceptive endometrium in infertile women with endometriosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our results regarding MIG-6 and ERBB2/ESR1 signaling in regulating uterine function in response to hormonal signals will bring insight into uterine pathophysiology and likely lead to new therapies for endometrial diseases. Deeper inquiry into endometrial epithelial-stromal crosstalk between ErbB2/ERK/ESR1 and PGR/MIG-6 signaling pathways will be of major importance to understanding infertility and endometriosis.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings reveal that attenuation of MIG-6 occurs both in endometriotic lesions and in the endometriosis-effected eutopic endometrium. Evidence from mice indicates that loss of \u003cem\u003eMig-6\u003c/em\u003e in endometriotic lesions promotes their development and accelerates endometriosis-related infertility, while loss of \u003cem\u003eMig-6\u003c/em\u003e in the eutopic endometrium causes infertility due to defects in implantation and endometrial receptivity. We found that increased epithelial proliferation caused by \u003cem\u003eMig-6\u003c/em\u003e loss is caused by E2 through the ERBB2/ERK pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). However, targeting \u003cem\u003eErbb2\u003c/em\u003e can reverse all apparent female reproductive defects caused by \u003cem\u003eMig-6\u003c/em\u003e loss including endometrial hyperplasia, infertility, and endometriosis lesion development. Attenuation of \u003cem\u003eMig-6\u003c/em\u003e causes the inability of P4 to properly control the cell cycle and inhibit E2-induced aberrant epithelial proliferation that results from increases in MCMs. However, counteracting the overexpression of \u003cem\u003eErbb2\u003c/em\u003e restores normal gene expression patterns, providing a molecular explanation for the rescue of normal reproductive function. These findings not only elucidate a critical pathway for understanding the hormonal control of normal uterine physiology, but they also provide the potential for new treatment strategies for uterine disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main objective of this study was to evaluate the role of MIG-6 in endometrial P4 resistance. First, the expression of MIG-6 was assessed in eutopic endometrium of infertile women with endometriosis compared to fertile women. To determine whether endometriosis affects MIG-6 expression, we examined MIG-6 expression in a nonhuman primate and mouse model of endometriosis. Subsequently, we identified ERBB2 as a MIG-6 target and evaluate the impact of \u003cem\u003eErbb2\u003c/em\u003e ablation on the infertility and endometrial P4 resistance of \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. Finally, transcriptomic analysis was applied to dissect the molecular mechanisms of \u003cem\u003eMig-6\u003c/em\u003e in the uterus. The control and treatment groups and the number of biological replicates (sample sizes) for each experiment are specified in the figure legends. Animal numbers for each study type were determined by the investigators on the basis of previous experience with the standard disease models that were used or from pilot studies. Animals were randomly allocated to the control and treatment groups and housed together to minimize environmental differences and experimental bias. Analysis of endpoint readouts was carried out in a blinded fashion.\u003c/p\u003e\n\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eThe institutional review board of Michigan State University, Greenville Health System, and University of North Carolina approved this study. The Institutional Animal Care and Use Committee at Michigan State University approved all experiments relating to mice. The Institutional Animal Care and Use Committees of both the University of Illinois at Chicago and Michigan State University approved the endometriosis baboon animal model.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eHuman Endometrium Samples\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe human endometrial samples used to examine MIG-6 expression patterns were obtained from Michigan State University\u0026rsquo;s Center for Women\u0026rsquo;s Health Research Female Reproductive Tract Biorepository, the University of North Carolina, and the Greenville Hospital System in accordance with the guidelines set by the Institutional Review Boards of Michigan State University (Grand Rapids, MI), the University of North Carolina (Chapel Hill, NC), and Greenville Health System (Greenville, SC), respectively. Written informed consent was obtained from all participants. For experiments examining \u003cem\u003eMIG-6\u003c/em\u003e mRNA expression throughout the menstrual cycle, endometrial samples were analyzed from 22 cycling premenopausal women without endometriosis (n\u0026thinsp;=\u0026thinsp;6 proliferative, n\u0026thinsp;=\u0026thinsp;7 early secretory, n\u0026thinsp;=\u0026thinsp;3 mid secretory, and n\u0026thinsp;=\u0026thinsp;6 late secretory) and from 20 cycling premenopausal women with endometriosis (n\u0026thinsp;=\u0026thinsp;2 proliferative, n\u0026thinsp;=\u0026thinsp;6 early secretory, n\u0026thinsp;=\u0026thinsp;9 mid secretory, and n\u0026thinsp;=\u0026thinsp;3 late secretory). Control endometrial tissues were laparoscopically negative for endometriosis and had not been on any hormonal therapies for at least three months prior to surgery. Endometrial menstrual staging was confirmed by an experienced pathologist familiar with female reproduction. To investigate MIG-6 amounts in the endometrium from women, 10 control and 10 eutopic endometrium with endometriosis were used. To compare MIG-6 amounts in the eutopic endometrium and ectopic lesions of women with endometriosis, each of 12 samples were used. All women with endometriosis were infertile. Samples used for immunohistochemistry were fixed in 10% buffered formalin prior to embedding in paraffin wax.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAnimals And Tissue Collection\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAnimals were maintained in a designated animal care facility according to Michigan State University\u0026rsquo;s Institutional Guidelines for the care and use of laboratory animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. For all animal studies, animals were randomly distributed among different conditions by the investigator as the animals did not show any size or appearance differences at the onset of the experiments. No animals were excluded, and the investigator was not blinded to group allocation during the experiment. \u003cem\u003eErbb2\u003c/em\u003e conditional knockout mice were generated by crossing \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e with \u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e). Pregnant uterine samples were obtained by mating control (\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e female mice with C57BL/6 male mice the morning of a vaginal plug designated as day 0.5 of gestation (GD 0.5). Mice were sacrificed at GD 3.5 and 5.5. For the study of steroid hormone regulation, control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at 6 weeks of age were ovariectomized. Two weeks postsurgery, ovariectomized mice were injected with vehicle (sesame oil; Veh) or estradiol (0.1\u0026nbsp;\u0026micro;g/mouse; E2) plus progesterone (1\u0026nbsp;mg/mouse; P4) for 3 days and euthanized at 6 hours after injection. For the fertility studies, adult female control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice were placed with wild type C57BL/6 male mice. The mating cages were maintained for 6 months and the number of litters and pups born during that period was recorded. Uterine tissues were then immediately processed at the time of dissection and either fixed with 4% (vol/vol) paraformaldehyde for histology or immunohistochemistry or snap frozen and stored at -80\u0026nbsp;\u0026deg;C for RNA/protein extraction.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInduction Of Endometriosis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFor baboon uterine samples, endometriosis was induced by intraperitoneal inoculation of menstrual endometrium on two consecutive menstrual cycles and harvested using laparotomy via endometriectomy from four female baboons as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. For mouse uterine samples, 8-weeks-old female mice which have conditional double-fluorescent Cre reporter gene (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG\u003c/em\u003e\u003c/sup\u003e were injected with 1\u0026nbsp;\u0026micro;g/ml of E2 per a day at three times and had a surgical procedure to induce endometriosis. Under anesthesia, a midline abdominal incision was made to expose the uterus in female mice, and one of uterine horn was removed. In a Petri dish containing phosphate-buffered saline (PBS; pH 7.5), the uterine horn was opened longitudinally with scissors. The excised uterine horn was cut into small fragments of about 1 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and then injected back into the peritoneum of same mouse. The abdominal incision and wound were closed with sutures and skin was closed with surgical wound clips, respectively. After a designated time, the mice were euthanized, and endometriosis-like lesions were removed using a fluorescence microscope and counted.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEndometriosis-related Infertility Analysis\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eEndometriosis were induced in 8 week old control female mouse recipient (fertile) receiving endometrial fragments from donor control (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG\u003c/em\u003e\u003c/sup\u003e) or \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003e\u003cem\u003emTmG\u003c/em\u003e\u003c/sup\u003e endometrium. A sham surgery group was included as a control. After 1, 2, and 3 months of the endometriosis induction, the mice with endometriotic lesions of control or \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e were mated with wild-type male mice and then collected at GD 7.5.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eRNA Isolation And Microarray Analysis\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from the uterine tissues using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). RNA was pooled from the uteri of more than three mice per genotype at GD 3.5 and microarray analysis was performed using GeneChip\u0026reg; Mouse Genome 430 2.0 Arrays (Affymetrix) as described previously \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e(Gene Expression Omnibus accession code GSE138185). Array data were analyzed using Bioconductor for quantile normalization. We selected aberrantly expressed genes in the uteri of control, \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice at GD 3.5 using a two-sample comparison according to significant fold change greater than 1.5. Aberrantly expressed genes were classified with canonical pathway analyzed by Ingenuity System Software (Ingenuity Systems Inc.).\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eReverse Transcription - Quantitative PCR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe complementary DNAs (cDNAs) were synthesized with MMLV Reverse Transcriptase (Invitrogen Crop) according to the manufacturer\u0026rsquo;s instructions. RT-qPCR was performed on cDNA to assess the expression of genes of interest with SYBR Green (Bio-Rad) or TaqMan primers (Applied Biosystems). Experimental gene expression data were normalized against the housekeeping gene, 18S ribosomal RNA. Analysis of mRNA expression was first undertaken by the standard curve method, and results were corroborated by cycle threshold values assessing gene expression. Primer sequences used in these studies are shown in table S4.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eImmunohistochemistry Analyses\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eImmunohistochemistry and immunofluorescence analyses were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Briefly, dewaxed hydrated paraffin-embedded tissue sections were pre-incubated with 10% normal goat (for anti-MIG-6, Ki67, Cyclin D1, ErbB2, pERK1/2, ERK1/2, MUC1, LTF, and KLF4 antibodies) or donkey (for anti-MCM2, MCM6, and KLF15 antibodies) serum in PBS and then incubated with anti-MIG-6 (1:200 dilution; Customized antibody by Dr. Jeong Lab), anti-Ki67 (1:1000 dilution; #ab15580; Abcam), anti-Cyclin D1 (1:1000 dilution; #eo-RB9041-p0; Thermo Fisher Scientific), anti-ErbB2 (1:200 dilution; #2165; Cell Signaling), anti-pERK1/2 (1:500 dilution; #4370; Cell Signaling), anti-ERK1/2 (1:1000 dilution; #4695; Cell Signaling), anti-MUC1 (1:1000 dilution; #ab15481, Abcam), anti-LTF (1:2000 dilution; #07-682, Millipore Corp.), anti-MCM2 (1:20000 dilution; #sc9839, Santa Cruz Biotechnology), anti-MCM6 (1:20000 dilution; #sc9843; Santa Cruz Biotechnology), anti-KLF4 (1:5000 dilution; #sc20691; Santa Cruz Biotechnology), and anti-KLF15 (1:5000 dilution; #ab2647; Abcam) antibodies in PBS supplemented with 10% normal serum overnight at 4\u0026nbsp;\u0026deg;C. For immunohistochemistry, the sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories) for one hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB-Vector Laboratories) and analyzed using microscopy software from NIS Elements, Inc. (Nikon). A semi-quantitative grading system (H-score) was calculated to compare the immunohistochemical staining intensities. The H-score was calculated using the following equation: H-score = \u0026sum; Pi (i), where i\u0026thinsp;=\u0026thinsp;intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%. The overall score ranged from 0 to 300 \u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\u003cdiv class=\"Heading\"\u003eWestern blot analyses were performed as described previously \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Proteins were extracted using lysis buffer (10\u0026nbsp;mM Tris-HCl (pH 7.4), 150\u0026nbsp;mM NaCl, 2.5\u0026nbsp;mM EDTA, and 0.125% Nonidet P-40 (vol/vol)) supplemented with both a protease inhibitor cocktail (Roche, Indianapolis, IN) and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein lysates were electrophoresed via SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Membrane was blocked with Casein (0.5% w/v) in PBS with 0.1% Tween 20 (v/v; Sigma-Aldrich) prior to exposure to anti-ErbB2 (#2165; Cell Signaling, Danvers, MA), anti-EGFR (#2646; Cell Signaling), anti-phospho-ERK1/2 (pERK1/2; #4370; Cell Signaling), anti-ERK1/2 (#4695; Cell Signaling), anti-MIG-6 (Customized antibody by Dr Jeong Lab) or anti-\u0026beta;-actin (#sc1616; Santa Cruz Biotechnology) antibodies diluted to 1:1000. Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked secondary antibody followed by exposure to Electrochemiluminescence reagents (ECL) according to manufacturer\u0026rsquo;s instructions (GE Healthcare Biosciences).\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo statistical method was used to predetermine sample size for in vivo studies. Based on prior experience, all experiments used 5 mice per group to achieve adequate statistical power. For all animal experiments, block randomization was used to ensure a balance in sample size across groups. The investigators were blinded during the evaluation of results variations in the group. For all animal experiments, over three biological replicates were analyzed for each condition, and results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For data with only two groups, Student\u0026rsquo;s t test was used. For data containing more than two groups, an analysis of variance (ANOVA) test was used, followed by Tukey test for pairwise t-test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using the Instat package from GraphPad. The original data are provided in table S5.\u003c/p\u003e\n\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eAll data are available in the manuscript or the supplementary material. The accession number for microarray generated in this study is GSE138185.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MIST) (No. 2018R1A5A2025079 to H.G.Y.), and SRI and Bayer Discovery/Innovation Grant (to T.H.K.), as well as by the Eunice Kennedy Shriver National Institute of Child Health \u0026amp; Human Development of the National Institutes of Health under Award Number R01HD084478 and R01HD101243 (to J.W.J) and F31HD101207 and T32HD087166 (to R.M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.-G.Y. and J.-W.J. were responsible for the concept of the study; A.T.F. collected baboon samples; S.L.Y. and B.A.L. collected human samples; J.-Y.Y. and T.H.K. carried out experiments; J.-Y.Y., T.H.K. and J.-H.S. analyzed data; U.M. provided transgenic mice; R.M.M. contributed to write the manuscript. All authors contributed to the final manuscript version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcKinnon B, Mueller M, Montgomery G. 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Mig-6 regulates endometrial genes involved in cell cycle and progesterone signaling. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e \u003cstrong\u003e462\u003c/strong\u003e, 409-414 (2015).\u003c/li\u003e\n\u003cli\u003eKim BG\u003cem\u003e, et al.\u003c/em\u003e Aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling in endometriosis. \u003cem\u003eHuman reproduction\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1069-1078 (2015).\u003c/li\u003e\n\u003cli\u003eIshibashi H\u003cem\u003e, et al.\u003c/em\u003e Sex steroid hormone receptors in human thymoma. \u003cem\u003eThe Journal of clinical endocrinology and metabolism\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 2309-2317 (2003).\u003c/li\u003e\n\u003cli\u003eKim TH\u003cem\u003e, et al.\u003c/em\u003e ARID1A Is Essential for Endometrial Function during Early Pregnancy. \u003cem\u003ePLoS genetics\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e1005537 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-95903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-95903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFemale subfertility is highly associated with endometriosis. Although the exact etiology of endometriosis-related infertility remains to be determined, endometrial progesterone resistance has recently been suggested as a crucial element in the development of endometrial diseases. Here, we report that \u003cem\u003eMIG-6\u003c/em\u003e, a progesterone-induced gene, is downregulated in the endometrium of infertile women with endometriosis and in a non-human primate model of endometriosis. In an endometriosis mouse model with a fluorescent reporter used to identify lesions, an increase of endometriosis development and implantation failure were observed in mice with \u003cem\u003eMig-6\u003c/em\u003e deficient endometrium compared to controls. MIG-6 is known to inhibit ERBB2, which we found overexpressed in the endometrium from uterine-specific \u003cem\u003eMig-6\u003c/em\u003e knock-out mice (\u003cem\u003ePgr\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduced \u003cem\u003eErbb2\u003c/em\u003e ablation in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice). The additional knockout of \u003cem\u003eErbb2\u003c/em\u003e rescued all phenotypes seen in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice including endometrial progesterone resistance, infertility, and endometriosis lesion development. Transcriptomic analysis showed that genes differentially expressed in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice reverted to their normal expression amounts in \u003cem\u003eMig-6\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eErbb2\u003c/em\u003e\u003csup\u003e\u003cem\u003ed/d\u003c/em\u003e\u003c/sup\u003e mice. Together, our results demonstrate that MIG-6-induced ERBB2 overexpression causes endometrial progesterone resistance and a nonreceptive endometrium in endometriosis-related infertility and that ERBB2 targeting reverses these effects.\u003c/p\u003e","manuscriptTitle":"Loss of MIG-6 results in endometrial progesterone resistance via ERBB2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-10 00:18:59","doi":"10.21203/rs.3.rs-95903/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6709d096-186d-4884-a012-762b2c899584","owner":[],"postedDate":"November 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1023315,"name":"Endocrinology \u0026 Metabolism"},{"id":1023316,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2022-03-01T11:44:24+00:00","versionOfRecord":{"articleIdentity":"rs-95903","link":"https://doi.org/10.1038/s41467-022-28608-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2022-03-01 11:44:24","publishedOnDateReadable":"March 1st, 2022"},"versionCreatedAt":"2020-11-10 00:18:59","video":"","vorDoi":"10.1038/s41467-022-28608-x","vorDoiUrl":"https://doi.org/10.1038/s41467-022-28608-x","workflowStages":[]},"version":"v1","identity":"rs-95903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-95903","identity":"rs-95903","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}