Abstract
Although GPR64 has an important role for male fertility, its physiological roles in the female reproductive system are still unknown. In the present study, immunohistochemical analysis reveals a spatiotemporal expression of GPR64 in the uterus during early pregnancy. Observation of remarkable induction of GPR64 expression in uterine decidual cells points to its potential physiological significance on decidualization. The decidualization of uterine stromal cells is a key event in implantation. Progesterone (P4) signaling is crucial for the decidualization of the endometrial stromal cells for successful pregnancy. Therefore, we examined ovarian steroid hormone regulation of GPR64 expression in the murine uterus. P4 induced GPR64 expression in the epithelial and stromal cells of the uterus in ovariectomized wild-type mice, but not in PRKO mice. ChIP analysis confirmed that PGR proteins were recruited on progesterone response element of Gpr64 gene in the uteri of wild-type mice treated with P4. Furthermore, the expression of GPR64 was increased in human endometrial stromal cells (hESCs) during in vitro decidualization. Interestingly, small interfering RNA (siRNA)-mediated knockdown of GPR64 in hESCs remarkably reduced decidualization. These results suggest that Gpr64 has a crucial role in the decidualization of endometrial stromal cells.
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Introduction
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 effects2, 3.
P4-PGR signaling is essential in the uterus for successful implantation, decidualization, and glandular development4, 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 responses5, 6, and have led to the identification of several P4-PGR signaling pathways within the uterus7.
During early pregnancy, the uterine stromal cells undergo a process called decidualization. P4-PGR signaling is critical in the process of decidualization8. 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 embryo9. 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 profiles11,12,13. Multiple transgenic mouse models demonstrate that the decidualization process is important for the maintenance of pregnancy5, 14,15,16,17,18.
Decidualization 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 pregnancy10. 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 process19,20,21. 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.
G-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 protein22, 23. GPCRs have a pivotal role in cancer development and progression24, 25. The levels of GPR64 are significantly overexpressed in the Wnt signaling-dependent subgroup of medulloblastoma26 and higher in Ewing sarcomas27. GPR64 promotes tumor invasion and metastasis through induction of the placental growth factor (PGF) and metalloproteinase (MMP1) expression27. 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 microarrays28.
Furthermore, GPR64 is crucial for male fertility29. 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 littermates29. 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.
Results
The expression of Gpr64 in the mouse uterus during early pregnancy
To 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 control30, 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).
Regulation of Gpr64 expression by P4 and E2 in the mouse uterus
During early pregnancy, P4 and temporal E2 induction are important for embryo implantation32. 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.
Gpr64 as a target gene of progesterone receptor in the mouse uterus
To 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.
To 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 data33. 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.
Role of Gpr64 in human endometrial stromal cells (hESCs) during in vitro decidualization
To examine the role of GPR64 in decidualization, we used a well-characterized in vitro decidualization model in hESCs34. 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 (IGFBP1and 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.
To 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).
PGR 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.
Activation of serum response element (SRE) by GPR64 in hESCs
GPR64 activates serum response element (SRE) for cell adhesion and migration in HEK293 cells35. 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.
Discussion
In 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 pregnancy36,37,38,39. 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 analysis7 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.
P4 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 decidualization5,6,7. 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 uterus40, 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 dpc42,43,44. 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 PGR44,45,46. 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 levels42, 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.
According 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 mice7, 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 system48. 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.
We 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 infertility29. 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 ducts29. 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.
P4 is closely associated with endometrial stromal cell decidualization49. hESC from patients with endometriosis, with P4-resistance50 and PRKO mice demonstrate a decidualization defect, supporting a serious role for P4-PGR signaling in decidualization in both humans and mice5. 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 uteri51, 52. COUP-TFII, has been shown to promote decidualization of hESCs via induction of bone morphogenetic protein 2 (BMP2) and inhibition of ESR1 activation51. 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 decidualization53. Additionally, CRISPLD2 is a target gene regulated by P4-PGR response and it has critical role during in vitro decidualization of hESCs46. Our study shows that expression of PGR, FOXO1, CRISPLD2, PTCH1, and COUP-TFII were significantly decreased in GPR64-deficient hESCs.
GPR64 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation29. It is co-localized apical and subapical F-actin in male excurrent duct epithelia31. 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.
GPRs 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 G1254. 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 response55. ATF-6 was interacted with SRF for target gene expression by SRE activation55. GNA12 is part of G alpha subunit and regulates a variety of cellular responses including activation of Jun N-terminal kinase56 and SRE57. 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 cytoskeleton57. SRC is a critical factor of decidualization in the SRC deficient mice study58. SRC also bind to SRF for co-activate the SRE-mediated transactivation of CBP59. 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-SRF59. Therefore, our results suggest that GPR64 regulates SRE activation through SRF-related transcription factors.
In 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.
Methods
Animals and tissue collection
Mice 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 mice5 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.
RNA isolation and quantitative real-time PCR
Total 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′.
Immunohistochemistry
Immunohistochemistry analysis was performed as previously described60. 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).
Chromatinimmunoprecipitation (ChIP)
ChIP was performed as previously described61. 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.
Human endometrial stromal cell culture and in vitro decidualization
We used previously isolated Human endometrial stromal cells (hESCs) for this study34, 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% CO2. The induction of in vitro decidualization has been previously described46, 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.
SRE-luciferase assay
hESCs 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).
Statistical analysis
Statistical 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).
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Acknowledgements
We would also like to thank Paige Wells for manuscript preparation. We would also like to thank John P. Lydon (Baylor College of Medicine, Houston, TX) for PRKO mice and Amanda Sterling for manuscript preparation. This work was supported by the Bio-industry Technology Development Program (IPET312060-5), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea to J.M.L., NIH R01HD084478 and American Cancer Society Research Grant RSG-12-084-01-TBG to J.W.J.
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J.Y.Y., J.I.A., and T.H.K. conceived and designed the experimental approach, performed experiments and prepared the manuscript. S.Y. and J.Y.A. designed the experimental approach and analyzed the results. J.M.L. and J.W.J. conceived and designed the experimental approach, performed data analysis and prepared the manuscript.
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Yoo, JY., Ahn, J.I., Kim, T.H. et al. G-protein coupled receptor 64 is required for decidualization of endometrial stromal cells. Sci Rep 7, 5021 (2017). https://doi.org/10.1038/s41598-017-05165-8
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DOI: https://doi.org/10.1038/s41598-017-05165-8
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