{"paper_id":"a8b17b55-94c1-4424-91c3-29d236a12b0a","body_text":"Major functions of the uterus include receiving the embryo, sheltering the fetus during pregnancy, and delivering the newborn at term. The uterine endometrium consists of glandular and luminal epithelium, and stroma. During pregnancy, the uterus undergoes dynamic molecular and morphological changes to allow for embryo implantation and development. These changes of uterine components are tightly regulated by two ovarian steroid hormones, estrogen (E2) and progesterone (P4) 1 . The success of fertility is dependent on the balanced interaction of E2 and P4 acting through their receptors, E2 receptor (ESR) and P4 receptor (PGR). E2 is known to stimulate uterine epithelial cell proliferation while P4 is inhibitory to E2-mediated effects 2 ,  3 .\nP4-PGR signaling is essential in the uterus for successful implantation, decidualization, and glandular development 4 ,  5 . P4 is critical for the development of decidual tissues, and if fertilization occurs, high circulating P4 levels are important not only for facilitating implantation, but also for maintaining pregnancy by stimulating uterine growth and opposing the actions of factors involved in myometrial contraction. Previous research using a transgenic mouse model with a null mutation in the  Pgr  gene (PRKO) demonstrates the critical role for PGR in P4-mediated uterine responses 5 ,  6 , and have led to the identification of several P4-PGR signaling pathways within the uterus 7 .\nDuring early pregnancy, the uterine stromal cells undergo a process called decidualization. P4-PGR signaling is critical in the process of decidualization 8 . Decidualization is unique to species with hemochorial placenta, such as human, primates and rodents and serves to protect the maternal uterus during trophoblast invasion as well as providing nourishment to the embryo 9 . Endometrial stromal cells undergoing decidualization become plumper, acquire a secretory epithelioid-like morphology, and secrete a variety of factors, including prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1) 10 . Moreover, this transformation results in extensive changes in cellular gene expression, including alterations in steroid hormone receptor, extracellular matrix (ECM) and cytoskeletal gene profiles 11 – 13 . Multiple transgenic mouse models demonstrate that the decidualization process is important for the maintenance of pregnancy 5 ,  14 – 18 .\nDecidualization is the P4 mediated differentiation of small stromal fibroblast into large epithelioid decidual cells. In humans, decidualization of the stromal compartment occurs in the mid-secretory phase of the menstrual cycle, independently of pregnancy 10 . The decidual reaction is inhibited in PGR-A knock-out mice, but not PGR-B knock-out mice, suggesting a critical of PGR-A in this process 19 – 21 . In humans, decidualization process occurs in stromal cells surrounding the spiral arties approximately 10 days after the postovulatory rise in ovarian P4 level, indicating that the expression of the decidua-specific genes is under the direct control of activated PGR. Therefore, the identification of P4-PGR regulated genes is crucial in understanding the causes of impairments in fertility.\nG-protein coupled receptor 64 (GPR64) is also known as Adhesion g protein-coupled receptor G2 (ADGRG2) and Human Epididymis-specific protein 6 (HE6), and a member of the G protein-coupled receptor (GPCR) family described as an epididymis-specific transmembrane protein 22 ,  23 . GPCRs have a pivotal role in cancer development and progression 24 ,  25 . The levels of  GPR64  are significantly overexpressed in the Wnt signaling-dependent subgroup of medulloblastoma 26  and higher in Ewing sarcomas 27 . GPR64 promotes tumor invasion and metastasis through induction of the placental growth factor (PGF) and metalloproteinase (MMP1) expression 27 .  GPR64  also suggests a novel target gene candidate in ovarian endometrioid adenocarcinoma caused by dysregulation of β-catenin/T-cell factor (TCF) signaling by using oligonucleotide microarrays 28 .\nFurthermore, GPR64 is crucial for male fertility 29 .  Gpr64  knockout male mice result in infertility due to sperm stasis and duct obstruction by abnormal fluid reabsorption. Additionally, hemizygous knockout males and homozygous knockout females show no apparent developmental or behavioral abnormalities compared with wild-type littermates 29 . However, the role of  Gpr64  in the female reproductive tract is unclear. In this study, we explored the spatiotemporal expression profile and regulation of  Gpr64  in the response to P4-PGR and during early pregnancy in mice uteri. To investigate the function of  GPR64 , we used the well-characterized  in vitro  primary human endometrial stromal cell (hESC) decidualization model.\n\nTo investigate the expression profile of  Gpr64  during early pregnancy, we examined levels of  Gpr64  in wild-type female uteri during early pregnancy. The initiation of pregnancy was marked by the presence of a postcoital vaginal plug (0.5 dpc). The levels of  Gpr64  mRNA were detected on 0.5 dpc, which gradually increased until 7.5 dpc, reaching statistical significance after 1.5 dpc in the uterus (Fig.  1a ). To further investigate the spatiotemporal expression profile of GPR64 protein during early pregnancy, we performed immunohistochemistry analysis for GPR64 (Fig.  1b ). GPR64 proteins were very weak in the epithelium and stroma at 0.5 dpc. Consistent with the real-time PCR results, the levels of GPR64 were significantly higher detected in the glandular and luminal epithelium at 2.5 and 3.5 dpc. The GPR64 proteins were also weakly detected in the stromal cells at 2.5 and 3.5 dpc and then markedly increased in the stromal cells at 4.5 dpc. Interestingly, GPR64 was remarkably strong in primary decidual cells at implantation sites of 5.5 dpc. The expression of GPR64 proteins in primary decidual cells was changed to the secondary decidual zone (further from the embryo) from the primary decidual zone (closer to the embryo) at 7.5 dpc. These results suggest that GPR64 may have an important role for implantation and decidualization during early pregnancy. To confirm the antibody specificity, we performed immunohistochemistry analysis of GPR64 as a positive control 30 ,  31  in mouse epididymal tissue. The expression of GPR64 was detected in apical membranes as well as in some nuclei of epididymal duct epithelial cells. Additionally, the IgG antibody was used as a negative control for immunohistochemistry analysis in the mouse epididymal tissue (Fig.  S1 ). Figure 1 The expression patterns of  Gpr64  during early pregnancy. ( a ) The expression levels of  Gpr64  were measured in uteri during early pregnancy. Total RNA used for the quantitative real-time PCR assays was prepared from early pregnancy uteri. The results represent the mean ± SEM of three independent RNA sets. *p < 0.05 and **p < 0.01. ( b ) The immunohistochemistry analysis of GPR64 during natural pregnancy was investigated at 0.5 dpc, 2.5 dpc, 3.5 dpc, 4.5 dpc, 5.5 dpc, and 7.5 dpc. Black arrow head indicates embryo. Nuclei were counterstained with hematoxylin.\nThe expression patterns of  Gpr64  during early pregnancy. ( a ) The expression levels of  Gpr64  were measured in uteri during early pregnancy. Total RNA used for the quantitative real-time PCR assays was prepared from early pregnancy uteri. The results represent the mean ± SEM of three independent RNA sets. *p < 0.05 and **p < 0.01. ( b ) The immunohistochemistry analysis of GPR64 during natural pregnancy was investigated at 0.5 dpc, 2.5 dpc, 3.5 dpc, 4.5 dpc, 5.5 dpc, and 7.5 dpc. Black arrow head indicates embryo. Nuclei were counterstained with hematoxylin.\nDuring early pregnancy, P4 and temporal E2 induction are important for embryo implantation 32 . On the basis of Fig.  1  results, we postulated that GRP64 expression is hormonally regulated in the uterus. To investigate whether steroid hormone regulates the expression of  Gpr64 , we treated ovariectomized C57BL/6 female mice with vehicle (sesame oil), P4 (1 mg/mouse), or E2 (0.1 μg/mouse). After 6 hours, we analyzed the expression levels of  Gpr64  mRNA in these murine uteri by real-time PCR. Our results showed that  Gpr64  mRNA expression was significantly increased in the uteri of mice treated with P4 as compared with vehicle and E2 (Fig.  2a ). Next, we performed immunohistochemical analysis to identify the spatial expression of the GPR64 expression. These results showed that GPR64 was highly expressed in both stromal and epithelial cells of the uteri treated with P4 for 6 hours compared with other groups (Fig.  2b ). Taken together, these finding suggest that P4 induces the  Gpr64  expression but E2 cannot regulate  Gpr64  expression in uteri. Figure 2 The regulation of  Gpr64  by steroid hormones. ( a ) The expression levels of  Gpr64  from vehicle, progesterone (P4), or estrogen (E2) treated uteri by quantitative real-time PCR. Total RNA used for the real-time PCR assays was prepared from ovariectomized wild-type C57BL/6 mice treated with vehicle, P4, or E2 for 6 hours. The results represent the mean ± SEM of three independent RNA sets. *p < 0.5 and **p < 0.01. ( b ) The immunohistochemistry analysis of GPR64 in mice uteri treated with vehicle, P4, or E2 for 6 hours. Nuclei were counterstained with hematoxylin.\nThe regulation of  Gpr64  by steroid hormones. ( a ) The expression levels of  Gpr64  from vehicle, progesterone (P4), or estrogen (E2) treated uteri by quantitative real-time PCR. Total RNA used for the real-time PCR assays was prepared from ovariectomized wild-type C57BL/6 mice treated with vehicle, P4, or E2 for 6 hours. The results represent the mean ± SEM of three independent RNA sets. *p < 0.5 and **p < 0.01. ( b ) The immunohistochemistry analysis of GPR64 in mice uteri treated with vehicle, P4, or E2 for 6 hours. Nuclei were counterstained with hematoxylin.\nTo determine whether  Gpr64  is a P4-PGR signaling targeted gene, we performed real-time PCR in the uterine samples of ovariectomized wild-type and progesterone receptor knock-out (PRKO) female mice treated with vehicle or P4 for 6 hours. As shown in Fig.  3a , levels of  Gpr64  mRNA were significantly increased in the wild-type mice uteri treated with P4 compared with vehicle. However,  Gpr64  mRNA was not increased by P4 treatment in the PRKO mice. To analyze the spatial expression of GPR64 by P4 treatment in the uterus, we performed immunohistochemistry analysis in the vehicle or P4-treated wild-type and PRKO mice. Consistent with the real-time PCR outcomes, we observed GPR64 expression in the stromal and epithelial cells of the uterus section obtained from P4-treated wild-type uterus (Fig.  3b ). The GPR64 protein was not detected in the PRKO uterus treated with vehicle or P4. Figure 3 The expression of  Gpr64  in PRKO mice uteri by P4 signal. ( a ) The expression levels of  Gpr64  from P4 treated wild-type or PRKO uteri by quantitative real-time PCR. Total RNA used for the quantitative real-time PCR was prepared from wild-type or PRKO uteri treated with vehicle or P4 for 6 hours. The results represent the mean ± SEM of three independent RNA sets. ***p < 0.001. ( b ) The immunohistochemistry analysis of GPR64 in vehicle or P4-treated uteri. Uterine sections were collected from vehicle or P4 treated wild-type and PRKO mice for 6 hours. Nuclei were counterstained with hematoxylin.\nThe expression of  Gpr64  in PRKO mice uteri by P4 signal. ( a ) The expression levels of  Gpr64  from P4 treated wild-type or PRKO uteri by quantitative real-time PCR. Total RNA used for the quantitative real-time PCR was prepared from wild-type or PRKO uteri treated with vehicle or P4 for 6 hours. The results represent the mean ± SEM of three independent RNA sets. ***p < 0.001. ( b ) The immunohistochemistry analysis of GPR64 in vehicle or P4-treated uteri. Uterine sections were collected from vehicle or P4 treated wild-type and PRKO mice for 6 hours. Nuclei were counterstained with hematoxylin.\nTo determine whether PGR directly regulates transcriptional activation of  Gpr64 , we performed ChIP analysis on mice uterine chromatin treated with vehicle or P4 for 2 hours based on previous PGR ChIP-seq data 33 . Recruitments of PGR on progesterone response element (PRE; GAATAAAATGATC) were significantly increased by treatment of P4 compared to vehicle (Fig.  4 ). However, PGR binding was not changed on the negative control region of exon 15 by P4 treatment. These results indicate that  Gpr64  is a direct transcriptional PGR target gene in the uterus. Figure 4 Recruitment of PGR on PRE of  Gpr64  gene. ChIP assay performed with uterine chromatin isolated from C57BL/6 female mice treated with vehicle or P4 for 2 hours using PGR antibody followed by quantitative real-time PCR. A normal rabbit IgG antibody was used as the negative control. NC region on the  Gpr64  gene was used as negative control of ChIP assay. The results represent the mean ± SEM. ***p < 0.001.\nRecruitment of PGR on PRE of  Gpr64  gene. ChIP assay performed with uterine chromatin isolated from C57BL/6 female mice treated with vehicle or P4 for 2 hours using PGR antibody followed by quantitative real-time PCR. A normal rabbit IgG antibody was used as the negative control. NC region on the  Gpr64  gene was used as negative control of ChIP assay. The results represent the mean ± SEM. ***p < 0.001.\nTo examine the role of GPR64 in decidualization, we used a well-characterized  in vitro  decidualization model in hESCs 34 . The hESCs were treated with E2, MPA and cAMP to induce decidualization. Prior to treatment, hESCs possessed a fibroblast-like morphology. After  in vitro  decidualization treatment, hESCs enlarged and became round in shape, typical of the decidual transformation (Fig.  5a ). Quantitative PCR analysis revealed significantly increased expression levels of the decidualization marker genes ( IGFBP1 and  PRL ) after the decidualization induction (Fig.  5b ). To examine GPR64 levels during decidualization process, qPCR analysis was performed on hormone-treated hESCs on day 0, 1, 3 and 6 of  in vitro  decidualization. The expression of GPR64 was increased on day 6 compared to day 1 and day 3. Figure 5 Effect of  GPR64  knock-down during  in vitro  decidualization of hESCs. ( a ) Morphological change of hESCs treated with or without GPR64 siRNA after  in vitro  decidualization treatment for 6 days. Control is non-treatment with  in vitro  decidualization to hESCs. ( b ) The expression levels of  GPR64  were examined on hESCs treated with or without  GPR64  siRNA during  in vitro  decidualization using quantitative real-time PCR. Expression of decidualization marker genes,  IGFPB1  and  PRL  were examined during  in vitro  decidualization of hESCs treated with or without  GPR64  siRNA. The results represent the mean ± SEM. *p < 0.05. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\nEffect of  GPR64  knock-down during  in vitro  decidualization of hESCs. ( a ) Morphological change of hESCs treated with or without GPR64 siRNA after  in vitro  decidualization treatment for 6 days. Control is non-treatment with  in vitro  decidualization to hESCs. ( b ) The expression levels of  GPR64  were examined on hESCs treated with or without  GPR64  siRNA during  in vitro  decidualization using quantitative real-time PCR. Expression of decidualization marker genes,  IGFPB1  and  PRL  were examined during  in vitro  decidualization of hESCs treated with or without  GPR64  siRNA. The results represent the mean ± SEM. *p < 0.05. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\nTo further analyze the role of GPR64 in hESC decidualization, we performed siRNA-mediated knockdown of  GPR64  expression. GPR64 attenuation was confirmed that the  GPR64  mRNA levels were significantly decreased in hESCs treated with  GPR64  siRNA compared with non-targeting pool siRNA during  in vitro  decidualization by quantitative real-time PCR (Fig.  5b ). hESCs treated with  GPR64  siRNA showed fibroblast-like morphology (Fig.  5a ). Quantitative PCR analysis showed that the expression of decidualization marker genes, insulin-like growth factor-binding protein 1 ( IGFBP1 ) and prolactin ( PRL ), were significantly reduced in hESCs treated with  GPR64  siRNA as compared to controls (Fig.  5b ).\nPGR signaling is a critical regulator of reproductive events associated with endometrial stromal cell decidualization and the maintenance of pregnancy. Therefore, we examined the expression of PGR target genes in decidualized hESC transfected with or without  GPR64  siRNA by real time PCR. The levels of  PGR , Forkhead box protein O1 ( FOXO1 ), cysteine-rich secretory protein LCCL domain-containing 2 ( CRISPLD2 ), patched-1 ( PTCH1 ), and chicken ovalbumin upstream promoter-transcription factor II ( COUP-TFII ) were increased in hESCs during  in vitro  decidualization. However, the transcriptional induction of  PGR ,  FOXO1 , CRISPLD2,  PTCH1 , and  COUP-TFII  were significantly decreased in hESCs treated with  GPR64  siRNA (Fig.  6 ). These results suggest that GPR64 plays an important role as a PGR target gene in decidualization. Figure 6 The expression of PGR target genes by reduction of  GPR64  during  in vitro  decidualization of hESCs. The expression levels of  PGR  ( a ),  FOXO1  ( b ),  CRISPLD2  ( c ),  PTCH1  ( d ), and  COUP-TFII  ( e ) during  in vitro  decidualization of hESCs treated with or without  GPR64  siRNA by quantitative real-time PCR. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\nThe expression of PGR target genes by reduction of  GPR64  during  in vitro  decidualization of hESCs. The expression levels of  PGR  ( a ),  FOXO1  ( b ),  CRISPLD2  ( c ),  PTCH1  ( d ), and  COUP-TFII  ( e ) during  in vitro  decidualization of hESCs treated with or without  GPR64  siRNA by quantitative real-time PCR. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\nGPR64 activates serum response element (SRE) for cell adhesion and migration in HEK293 cells 35 . To examine the activity of GPR64 in hESC, we performed SRE-luciferase assay on hESC with and without  GPR64  siRNA. The SRE activation level was significantly decreased in hESCs treated with  GPR64  siRNA compared with non-targeting pool siRNA (Fig.  7a ). Serum response factor (SRF) is a transcription factors and binds to SRE in the promoter region of target genes. Therefore, we examined the expression of SRF signaling genes in hESCs with GPR64 deficiency. Our qPCR results revealed that the expression of activating transcription factor-6 ( ATF-6 ), G-protein subunit alpha 12 ( GNA12 ),  SRC , and CREB binding protein ( CBP ) were significantly reduced in hESC transfected with  GPR64  siRNA compared to the control (Fig.  7b–e ). These results suggest that GPR64 induces SRF signaling molecules to regulate SRE activation. Figure 7 The SRE activation and SRE activated related gene expression is dependent on GPR64 during  in vitro  decidualization of hESCs. ( a ) The activation level of SRE during  in vitro  decidualization of hESCs transfected with or without  GPR64  siRNA. The expression level of related SRE activation genes,  ATF-6  ( b ),  GNA12  ( c ),  SRC  ( d ), and  CBP  ( d ) in decidulized hESCs with or without  GPR64  siRNA transfection. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\nThe SRE activation and SRE activated related gene expression is dependent on GPR64 during  in vitro  decidualization of hESCs. ( a ) The activation level of SRE during  in vitro  decidualization of hESCs transfected with or without  GPR64  siRNA. The expression level of related SRE activation genes,  ATF-6  ( b ),  GNA12  ( c ),  SRC  ( d ), and  CBP  ( d ) in decidulized hESCs with or without  GPR64  siRNA transfection. The results represent the mean ± SEM. *p < 0.05 and **p < 0.01.\n\nIn this study, we have identified  Gpr64  as a P4-PGR target gene in the mouse uterus. P4 is a well-known critical regulator of the female reproductive system associated with embryo implantation, decidualization and the maintenance of pregnancy 36 – 39 . The binding between P4 and PGR results in nuclear translocation of PGR and subsequent regulation of P4 target gene transcription. To identify uterine PGR-regulated mechanisms and downstream targets, Jeong  et al . and Rubel  et al . performed high density DNA microarray analysis 7  and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) 33 , respectively. These microarray results also showed a  Gpr64  as a potential P4-PGR target gene and ChIP-Seq results also revealed that PGR directly binds to the in gene region of  Gpr64  gene in uteri of ovariectomized mice treated with P4. However, the results of microarray and ChIP-seq have not been examined by qPCR and ChIP analysis.\nP4 is required throughout pregnancy associated with embryo implantation, decidualization and the maintenance of pregnancy. In previous studies,  Pgr  gene null mutated female mice show several reproductive defects such as infertility and malfunction of ovulation, implantation, and decidualization 5 – 7 . The expression levels of PGR increase from 0.5 days post coitum (dpc) and reach peak on 2.5 dpc in the epithelium of mouse uterus 40 ,  41 . Then, PGR expressions are higher in the primary decidual stromal cells surrounding the embryo at 5.5 dpc of mouse uterus and its expressions move outward into the secondary decidual stromal cells at 7.5 dpc 42 – 44 . The expression of downstream P4 target genes are important for embryo implantation and decidualization of the uterus such as indian hedgehog ( IHH ), patched-1 ( PTCH1 ), hedgehog interacting protein-1 ( HIP-1 ), chicken ovalbumin upstream promoter-transcription factor II ( COUP-TFll ), GATA Binding Protein 2 ( GATA2 ), cysteine-rich secretory protein LCCL domain-containing 2 ( CRISPLD2 ) and are triggered in turn according to the expression pattern of PGR 44 – 46 . As our results suggest, the mRNA expression of  Gpr64  was gradually increased until 7.5 dpc. GPR64 protein levels were highly expressed in the glandular epithelium at 2.5 dpc, which correlates with elevated P4 levels 42 ,  43 . Levels of GPR64 were highly expressed in the stromal cells at 4.5 dpc. Those are the time points during which the uterine epithelium prepares for and permits the embryo to implant. GPR64 was highly detected in fully differentiated decidual cells at 5.5 dpc and in the secondary decidual zone (further from the embryo) at 7.5 dpc. Therefore, these results suggest that GPR64 may have an important role both for receptivity in the epithelium and for endometrial decidualization in the stromal cells.\nAccording to previous studies, an acute dose of intraperitoneally administered P4 induces up- and down regulation of P4 target genes in the uterus of ovariectomized mice, and that this induction is lacking in PRKO mice 7 ,  44 ,  46 ,  47 . Our study herein has shown that the mRNA expression levels of  Gpr64  were significantly increased in the ovariectomized mice uteri treated with P4 as compared to vehicle for 6 hours. Additionally, GPR64 was detected highly in the epithelium compartment of the mouse uterus. E2 as well as P4 is important for maintenance of the female reproductive system 48 . However, our results showed that mRNA and protein levels of GPR64 were not affected by E2 stimulation. Additionally, the mRNA and protein levels of GPR64 were significantly upregulated by P4 in wild-type mice, but not in PRKO. Furthermore, our ChIP analysis shows that PGR directly bind to the PRE of  Gpr64  gene. Thus, our results suggest that  Gpr64  may play an important role during early pregnancy as a PGR target.\nWe showed that the inhibition of  GPR64  by siRNA treatment impaired decidualization capacity in human endometrial stromal cells (hESCs) as evidenced by suppression of  PRL  and  IGFBP1  expression which are known decidualization marker genes.  Gpr64  knockout mice results in male infertility 29 . Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts 29 . However, the  Gpr64  knockout female mice are fertile and do not have apparent developmental or behavioral abnormalities. These result suggests that ablation of GPR64 is not enough to abolish decidualization due to functional redundancy or compensation. We postulated that the identification of the regulatory pathways mediated by  Gpr64  would shed light on how these proteins mediate decidualization in the uterus.\nP4 is closely associated with endometrial stromal cell decidualization 49 . hESC from patients with endometriosis, with P4-resistance 50  and PRKO mice demonstrate a decidualization defect, supporting a serious role for P4-PGR signaling in decidualization in both humans and mice 5 . Previous studies have shown that P4 activates IHH and PTCH1 signaling to induce expression of COUP-TFII during decidualization in endometrial stromal cells of mice uteri 51 ,  52 . COUP-TFII, has been shown to promote decidualization of hESCs via induction of bone morphogenetic protein 2 (BMP2) and inhibition of ESR1 activation 51 . FOXO1 is critical for interferon regulatory factor member 4 (IRF4) expression via binding with PGR on IRF4 gene and a novel transcriptional regulator of endometrial stromal decidualization 53 . Additionally,  CRISPLD2  is a target gene regulated by P4-PGR response and it has critical role during  in vitro  decidualization of hESCs 46 . Our study shows that expression of  PGR ,  FOXO1 ,  CRISPLD2 ,  PTCH1 , and  COUP-TFII  were significantly decreased in  GPR64- deficient hESCs.\nGPR64 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation 29 . It is co-localized apical and subapical F-actin in male excurrent duct epithelia 31 . Our results showed that GPR64 is critical as a PGR target gene for decidualization. These functional differences suggested that GPR64 has tissue-specific roles in male and female reproductive function.\nGPRs are the largest family of membrane protein involved in signal transduction with heterotrimeric G protein. Heterotrimeric G proteins are classified into four G alpha subunit, Gs, Gi, Gq, and G12 54 . This G alpha subunit dissociates from βγ dimeric subunit, and initiates signal transduction for target gene transcription by various response element such as cAMP response element (CRE), serum response element (SRE), nuclear factor of activated T-cell response element (NFAT-RE), and serum response factor response element (SRF-RE) 54 . Our SRE-luciferase assay and qPCR analysis of SRF signaling genes showed that GRP64 regulates SRE activation in hESC during  in vitro  decidualization. ATF-6 is membrane-bound transcription factor that activated by endoplasmic reticulum stress response 55 . ATF-6 was interacted with SRF for target gene expression by SRE activation 55 . GNA12 is part of G alpha subunit and regulates a variety of cellular responses including activation of Jun N-terminal kinase 56  and SRE 57 . GNA12 mediated signaling is related to the Rho-family of small GTPase (Rho, Tan, and Cdc42) which regulates cellular activities such as target gene expression and controlling actin cytoskeleton 57 . SRC is a critical factor of decidualization in the SRC deficient mice study 58 . SRC also bind to SRF for co-activate the SRE-mediated transactivation of CBP 59 . CBP activates transcription that interacted with transcription factor managed by cAMP response element binding protein (CREB) domain, and had a SRE in promoter region regulated by SRC-SRF 59 . Therefore, our results suggest that GPR64 regulates SRE activation through SRF-related transcription factors.\nIn summary, we first addressed that the  Gpr64  is a target gene of P4-PGR signaling in the uterus. Inhibition of  GPR64  by siRNA-mediated knockdown suppresses decidualization of hESCs. Attenuation of  GPR64  decreased the expression of  PGR ,  FOXO1 ,  CRISPLD2 ,  PTCH1 , and  COUP-TFII  in hESCs during  in vitro  decidualization. These results suggest that  Gpr64  plays an important role for successful decidualization and is a target of P4-PGR signaling in mice as well as humans.\n\nMice were cared for and used in the designated animal care facility according to Michigan State University’s institutional guidelines. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. For the early pregnancy study, wild-type C57BL/6 female mice at 8 weeks of age were mated with wild-type C57BL/6 male mice and uterine samples from pregnant mice were obtained at different days of pregnancy. The morning of vaginal plug observation was designated as 0.5 days post coitum (dpc) (n = 3). For the study of steroid hormone regulation, wild-type and PRKO mice 5  at 6 weeks of age were ovariectomized. Two weeks post-surgery, ovariectomized mice were injected with vehicle (sesame oil; Veh), P4 (1 mg/mouse), and estradiol (0.1 μg/mouse). Mice were euthanized at 6 hours after injection (n = 3 per genotype per treatment). Uterine tissues were immediately frozen at the time of dissection and stored at −80 °C for RNA extraction or fixed with 4% (v/v) paraformaldehyde for immunohistochemistry.\nTotal RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was produced from 1 μg of total RNA using random hexamers and MMLV Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA). Real-time PCR was performed using the real-time PCR SYBR Green detection system (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). mRNA quantities were normalized against the housekeeping gene, 18 S RNA. The sequences of the primers used for mouse  Gpr64  were 5′-GCCCTTCCTCACCAGAAGAG-3′ and 5′-ATAAGGGCATGATCAAGGGG-3′, human  GPR64  were 5′-CTGCAGGATCCCATTGTCTG-3′ and 5′-TGAAAGGGGTTGAATCTCCC-3′, for  IGFBP1  were 5′-CTATGATGGCTCGAAGGCTC-3′ and 5′-TTC TTGTTGCAGTTTGGCAG-3′, for  PRL  were 5′-CATCAACAGCTGCCACACTT-3′ and 5′-C GTTTGGTTTGCTCCTCAAT-3′, for  PTCH1  were 5′-TGTGCGCTGTCTTCCTTCTG-3′ and 5′-ACGGCACTGAGCTTGATTC-3′, for  CRISPLD2  were 5′-CGGACGAGATGAATGAGGTG-3′ and 5′-TGACCGCAGAGGTTTTCTTG-3′, for  ATF-6  were 5′-GCCTTTATTGCTTCCAGCAG-3′ and 5′-TGAGACAGCAAAACCGTCTG-3′, for  GNA12  were 5′-ATGGTCTCCTCCAGCGAGTA-3′ and 5′-CTTGATGCTCACGGTCTTCA-3′, for  SRC  were 5′-AGGGGAGTTTGCTGGACTTT-3′ and 5′-AGGTTCTCTCCCACCAGGAT-3′, for  CBP  were 5′-GAATGCCGTACCCTACTCCA-3′ and 5′-GGCTGTCCAAATGGACTTGT-3′, and for 18S were 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.\nImmunohistochemistry analysis was performed as previously described 60 . Uterine cross sections from paraffin-embedded tissue were cut into 6 μm sections, mounted on silane-coated slides (12-550-15, Fisher Scientific, Pittsburgh, PA), deparaffinized and rehydrated in a graded alcohol series. Sections were pre-incubated with 10% normal rabbit serum in phosphate-buffered saline (PBS; pH 7.5) and then incubated with anti-GPR64 (1:500 dilution, sc-69492, Santa Cruz, Santa Cruz, CA) antibody in PBS supplemented with 10% normal goat serum overnight at 4 °C. The next day, sections were washed with PBS and incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB-Vector Laboratories, Burlingame, CA) then counterstained with hematoxylin and coverslipped with permount. Imuunostaining was analyzed using microscopy software from NIS Elements, Inc. (Nikon, Melville, NY).\nChIP was performed as previously described 61 . Briefly, ovariectomized mice were injected with vehicle (sesame oil) or P4 (1 mg/mouse) after two weeks post-surgery. Uteri were removed from euthanized mice at 2 hours after injection (n = 10 per treatment). For each ChIP reaction, 100 μg of chromatin was immunoprecipitated by 4 μg of antibodies against PGR (sc7208; Santa Cruz Biotechnology, Santa Cruz, CA). Eluted DNA was amplified with specific primers using SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA). Primers used in PCR were as follows: PRE (forward: 5′-GGGGACTCCTTTTTGGTGGA-3′; reverse: 5′-TCAGAAGCCACCAGACCGTG-3′) and negative control (NC) (forward: 5′-GCCCACCGTTATCGTCTTCC-3′; reverse: 5′-CAGGGGGATCGTAGGCTGAG-3′). The resulting signals were normalized to input activity.\nWe used previously isolated Human endometrial stromal cells (hESCs) for this study 34 ,  62 . hESCs were then maintained in phenol red–free RPMI-1640 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) depleted of steroids by pre-treatment with dextran-coated charcoal (Sigma Aldrich, St. Louis, MO) (Charcoal-stripped FBS; CS-FBS), and 1% penicillin streptomycin (P/S; Gibco, Grand Island, NY). Cells were cultured in monolayer at 37 °C in 5% CO 2 . The induction of  in vitro  decidualization has been previously described 46 ,  62 . To induce  in vitro  decidualization, cells were washed with PBS and incubated to OPTI-MEM medium (Gibco, Grand Island, NY) containing 2% CS-FBS, 10 nM estradiol (E2; Sigma-Aldrich, St. Louis, MO), 1 mM medroxyprogesterone acetate (MPA; Sigma-Aldrich, St. Louis, MO), 50 μM cAMP (Sigma-Aldrich, St. Louis, MO), and 1% P/S. Differentiation medium was changed every 48 hours for a total of 6 days. For  GPR64  knockdown, small interfering RNA (siRNA) was obtained from Dharmacon (Lafayette, CO). Human  GPR64  siRNA was transfected using Lipofectamine 2000 reagent (Invitrogen Crop., Carlsbad, CA) prior to  in vitro  decidualization.\nhESCs were were transiently co-transfected with 1 ug/well of the cis-reporter plasmids pSRE-luc (PathDetect, La Jolla, CA) and 100 ng/well pRL-TK (Promega, Madidon, WI) with or without  GPR64  siRNA for 24 hours in 24 well culture dish. After 24 hour, transfection medium was replaced with low serum growth medium (0.5% FBS). The assay was terminated 30 hour post transfection medium change. Luminescence were determined using Dual-Luciferase reporter assay system reagent kit (Promega, Madidon, WI) according to the manufacturer’s instructions and measured using victor 3 multiabel plate reader (PerkinElmer, Groningen, The Netherlands).\nStatistical analyses were performed with the Student’s t-test for data with two groups. For data containing more than two groups, we performed analysis of variance (ANOVA) test and analyzed by Tukey or Bonferroni test for pairwise t-test. All data are presented as means ± SEM. p < 0.05 was considered statistically significant. All statistical analyses were performed using the Instat package from GraphPad (San Diego, CA, USA).\n\nFig. S1. Histological analysis confirmed the specificity of GPR64 antibody in in mouse epididymis tissue.\nFig. S1. Histological analysis confirmed the specificity of GPR64 antibody in in mouse epididymis tissue.","source_license":"CC-BY-4.0","license_restricted":false}