The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells

Abstract

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart– and neural crest derivatives–expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs. Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart– and neural crest derivatives–expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs. The human endometrium undergoes growth and regeneration during each menstrual cycle. In the absence of an implantation, the endometrium sheds, is repaired, proliferates, and terminally differentiates to prepare for subsequent embryo implantation (1Cha J. Sun X. Dey S.K. Mechanisms of implantation: strategies for successful pregnancy.Nat. Med. 2012; 18 (23223073): 1754-176710.1038/nm.3012Crossref PubMed Scopus (634) Google Scholar, 2Evans J. Salamonsen L.A. Winship A. Menkhorst E. Nie G. Gargett C.E. Dimitriadis E. Fertile ground: human endometrial programming and lessons in health and disease.Nat. Rev. Endocrinol. 2016; 12 (27448058): 654-66710.1038/nrendo.2016.116Crossref PubMed Scopus (108) Google Scholar). These sequential events are controlled by the interplay of ovarian steroid hormones, estradiol (E2) and progesterone (P4) (3Fox C. Morin S. Jeong J.W. Scott Jr., R.T. Lessey B.A. Local and systemic factors and implantation: what is the evidence?.Fertil. Steril. 2016; 105 (26945096): 873-88410.1016/j.fertnstert.2016.02.018Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). During the proliferative phase, human endometrial epithelial cells and stromal cells (ESCs) increase in response to E2, leading to a thickening of the stratum functionalis of the endometrium. After ovulation, under P4 dominance, the endometrium, which is regulated by autocrine, paracrine, and transcription factors, transitions from the proliferative to the secretory phase (4Patel B. Elguero S. Thakore S. Dahoud W. Bedaiwy M. Mesiano S. Role of nuclear progesterone receptor isoforms in uterine pathophysiology.Hum. Reprod. Update. 2015; 21 (25406186): 155-17310.1093/humupd/dmu056Crossref PubMed Scopus (150) Google Scholar). The endometrial transition leads to spontaneous decidualization of human ESCs. Decidualization co-occurs with functional and morphological changes in human ESCs (5Gellersen B. Brosens J.J. Cyclic decidualization of the human endometrium in reproductive health and failure.Endocr. Rev. 2014; 35 (25141152): 851-90510.1210/er.2014-1045Crossref PubMed Scopus (385) Google Scholar). Uterine natural killer (uNK) cells are the predominant leukocyte in normal human endometrium (6Nagler A. Lanier L.L. Cwirla S. Phillips J.H. Comparative studies of human FcRIII-positive and negative natural killer cells.J. Immunol. 1989; 143 (2530273): 3183-3191PubMed Google Scholar). Approximately 70–80% of uNK cells are characterized as CD56brightCD16− (7King A. Loke Y.W. On the nature and function of human uterine granular lymphocytes.Immunol. Today. 1991; 12: 432-43510.1016/0167-5699(91)90014-KAbstract Full Text PDF PubMed Scopus (265) Google Scholar). Activated uNK cells can produce angiogenic factors, which promote spiral artery remodeling, and secrete cytokines that direct the migration and invasion of the trophoblast by interaction with trophoblast surface antigens (8Jokhi P.P. King A. Sharkey A.M. Smith S.K. Loke Y.W. Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction.J. Immunol. 1994; 153 (7525703): 4427-4435PubMed Google Scholar, 9Dosiou C. Giudice L.C. Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives.Endocr. Rev. 2005; 26 (15689572): 44-6210.1210/er.2003-0021Crossref PubMed Scopus (289) Google Scholar, 10Kalkunte S.S. Mselle T.F. Norris W.E. Wira C.R. Sentman C.L. Sharma S. Vascular endothelial growth factor C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetal interface.J. Immunol. 2009; 182 (19299706): 4085-409210.4049/jimmunol.0803769Crossref PubMed Scopus (148) Google Scholar). Importantly, the uNK cells and other leukocytes in the endometrium do not express P4 receptors (11Henderson T.A. Saunders P.T. Moffett-King A. Groome N.P. Critchley H.O. Steroid receptor expression in uterine natural killer cells.J. Clin. Endocrinol. Metab. 2003; 88 (12519888): 440-44910.1210/jc.2002-021174Crossref PubMed Scopus (217) Google Scholar). The activation and survival of uNK cells have been associated with the decidual-specific factor, interleukin-15 (IL15), in the human endometrium (12Kitaya K. Yamaguchi T. Honjo H. Central role of interleukin-15 in postovulatory recruitment of peripheral blood CD16(−) natural killer cells into human endometrium.J. Clin. Endocrinol. Metab. 2005; 90 (15713701): 2932-294010.1210/jc.2004-2447Crossref PubMed Scopus (83) Google Scholar). Previously, we showed that one transcription factor, heart and neural crest derivatives-expressed transcript 2 (HAND2), was up-regulated by E2 and a representative progestin, medroxyprogesterone (MPA), in a time- and dose-dependent manner for ESC decidualization (13Cho H. Okada H. Tsuzuki T. Nishigaki A. Yasuda K. Kanzaki H. Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells.Fertil. Steril. 2013; 99 (23036802): 248-25510.1016/j.fertnstert.2012.08.056Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Originally, HAND2 was identified as a basic helix-loop-helix transcription factor for embryonic right ventricles (14Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.Nat. Genet. 1997; 16 (9171826): 154-16010.1038/ng0697-154Crossref PubMed Scopus (541) Google Scholar, 15Yamagishi H. Olson E.N. Srivastava D. The basic helix-loop-helix transcription factor, dHAND, is required for vascular development.J. Clin. Invest. 2000; 105 (10675351): 261-27010.1172/JCI8856Crossref PubMed Scopus (116) Google Scholar). In human ESCs, silencing of HAND2 reduced both the morphological differentiation and the decidual-specific factors, including prolactin, forkhead box O1A (FOXO1A), interleukin-15 (IL15), fibulin-1, and tissue inhibitor of metalloproteinase-3 (16Shindoh H. Okada H. Tsuzuki T. Nishigaki A. Kanzaki H. Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.Fertil. Steril. 2014; 101 (24745730): 1781-1790.e1-510.1016/j.fertnstert.2014.03.013Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Therefore, it is conceivable that IL15 transcription is directly or indirectly regulated by HAND2. To the best of our knowledge, no studies have evaluated transcription factors directly affecting IL15 in human ESCs. We have applied in silico analysis here to identify HAND2 motifs (CHTCTGG) (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) and found candidate sequences in the upstream and intronic region of the human IL15 locus. The present study aimed to examine whether HAND2 participates in IL15 transcriptional regulation in human ESCs. First, we examined whether HAND2 and IL15 up-regulated in the human endometrium as as ESCs. During the secretory phase from HAND2 and IL15 with the proliferative phase from is a HAND2 and IL15 in the human endometrium In the endometrium of the in the was as the proliferative phase In the secretory phase, to a secretory in the as ESCs in the endometrium from in the proliferative phase to with and of cells in the secretory phase as to PCR histological analysis that HAND2 and IL15 in the secretory phase in the endometrium with the proliferative phase both are to expressed in ESCs, HAND2 and IL15 in not ESCs in the endometrium during the secretory phase analysis that during the secretory phase, are with IL15 exhibited with HAND2 in the secretory cells with HAND2 did not with IL15 the that of cells with the a for IL15 transcription by HAND2. 2 upstream from transcription human IL15 and or IL15 gene C. M. A. of 2000; 16 PubMed Scopus Google Scholar, A. for PubMed Scopus Google Scholar) IL15 upstream and or and are not and are the from to in the human IL15 upstream region are not a and was We found the putative for HAND2 (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) in the upstream region and intronic of human IL15 in silico analysis. HAND2 motifs are as at to the transcription in the upstream region of the human IL15 gene and putative HAND2 motif was also found in the of the human IL15 and is as at sequences have been to gene expression by a of and as D. of gene expression in 15 PubMed Scopus Google Scholar). of the transcription to an the of in the human IL15 gene is 153 it has been suggested that the putative HAND2 motif at is in Therefore, analysis was to the human IL15 upstream region the upstream putative motif, the putative motif in negative and and and and purified analysis the and revealed a for the human IL15 upstream region the putative HAND2 motif was found in the the and PCR the putative HAND2 motif in with the and or the and showed no was from purified with normal or which as negative or negative with the and and the and We also the of HAND2 to the HAND2 motifs at with ESC from in HAND2 the at of the IL15 upstream region the at and the negative region to the of the upstream including the at of the IL15 gene IL15 transcription in ESCs, we a luciferase reporter with under with E2 and E2 and an absence of ESCs transcriptional in the are no and E2 and luciferase activity of with no in ESCs, from a with the secretory phase, E2 and luciferase activity with no It is suggested that the upstream region of the IL15 gene can regulate IL15 transcription in ESCs under E2 and in intronic in ESCs, showed in luciferase activities E2 and with E2 and the of an transcription factor for IL15 to E2 and in ESCs. we examined whether HAND2 regulates IL15 transcription the upstream region of including the at in ESCs. of the with the or exhibited luciferase expression in ESCs activity from with the exhibited no increase with the and in ESCs in the with the with in ESCs the luciferase activity of deletion and substitution of the putative HAND2 motif The luciferase activity from with was up-regulated in secretory phase ESCs from with These that is no in IL15 in each ESC the or with showed activity with with are no in the and To examine whether HAND2 IL15 transcription is in ESCs, we a reporter in cells the showed luciferase activities in cells in ESCs, no was in the IL15 upstream region HAND2 in cells that HAND2 IL15 transcription in ESCs, or the IL15 upstream region and by HAND2 in ESCs. In our HAND2 (16Shindoh H. Okada H. Tsuzuki T. Nishigaki A. Kanzaki H. Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.Fertil. Steril. 2014; 101 (24745730): 1781-1790.e1-510.1016/j.fertnstert.2014.03.013Abstract Full Text Full Text PDF PubMed Scopus (18) Google expression was with HAND2 that transcription is regulated by HAND2 and that regulates IL15 transcription in ESCs. We have also identified putative motifs T. G. in are in that gene PubMed Scopus Google Scholar, M. D. M. M. C. X. A. J. HAND2 a of transcriptional that the 2014; Full Text Full Text PDF PubMed Scopus Google Scholar, E. D. L.A. A. A. transcription factors with the cytokines and to cell 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Lin S. X. W. L.A. 2013; PubMed Scopus Google Scholar) in the upstream region of human IL15 gene at and Therefore, we examined whether IL15 was regulated by in ESCs. analysis was to the putative motif at and at and purified analysis the and to and to showed no the with normal and and no recruitment of to the IL15 upstream region in our ESCs as in analysis with and ESCs X. E. W.E. is required for of transcriptional of endometrial stromal Endocrinol. 2015; PubMed Scopus Google Scholar). it has been that binds to human region in ESCs with decidualization K. S. M. T. H. H. The upstream region of growth expression in endometrial stromal cells during Full Text Full Text PDF PubMed Scopus Google as a we with a for the region the human including in our ESCs the of was expressed in study and was to upstream region under our E2 and To confirm the of the IL15 upstream we a reporter HAND2 the luciferase activities in the IL15 upstream region in ESCs from a is no found We also examined the of as a in the IL15 upstream region found no of as the variants in the motif no is to in the transcriptional of the upstream region of IL15 E2 and is the to that HAND2 is a transcription factor that directly regulates IL15 expression in human ESCs. We have identified a in the region of the human IL15 gene as a putative HAND2 motif analysis and a luciferase reporter assay, a human IL15 upstream region and in ESCs. a functional HAND2 and putative HAND2 motif in human IL15 We did not functional and IL15 expression as X. E. W.E. is required for of transcriptional of endometrial stromal Endocrinol. 2015; PubMed Scopus Google Scholar). analysis showed no region and with a of the human endometrium, endometrium not induce thickening of the stratum functionalis embryo In decidualization of the endometrium is under in a of which and and the (5Gellersen B. Brosens J.J. Cyclic decidualization of the human endometrium in reproductive health and failure.Endocr. Rev. 2014; 35 (25141152): 851-90510.1210/er.2014-1045Crossref PubMed Scopus (385) Google Scholar). Therefore, the upstream are not and and transcriptional regulation of IL15 that the HAND2 motif in the upstream region of the IL15 gene is the HAND2 and IL15 transcription in ESCs in motif is to have been by and sequences the putative HAND2 motif in the deletion the deletion of and motifs in the upstream region of the IL15 we found that nuclear receptor a factor, expression through an nuclear receptor response in the upstream region of the gene S. T. T. H. M. M. M. E. regulation of the gene by PubMed Scopus Google Scholar). We a or of into the upstream region of the box in reporter to whether the transcriptional activity or In the with the expression a was by a each to other motifs or the with of to the Therefore, we have examined the activities of not a deletion also a substitution to confirm that the putative HAND2 motif in the human IL15 transcription in ESCs. to at to or to the from the transcription and of the other putative motifs not in substitution both showed no response HAND2. Therefore, HAND2 regulate IL15 transcription the putative HAND2 motif at in the upstream region of the IL15 gene in ESCs. analysis that a of ESCs express IL15 expression by ESCs M. M. E. K. A. S. J. A. Sharkey A.M. et of the maternal-fetal in PubMed Scopus Google Scholar). Therefore, HAND2 transcription is in each or HAND2 is expressed at a in ESCs, the such as of the IL15 HAND2 is for each that HAND2 is not expressed in HAND2 regulated by in each of expression of IL15 no of uNK that a of uNK cells IL15 as a factor associated with immune the cell interaction M. M. E. K. A. S. J. A. Sharkey A.M. et of the maternal-fetal in PubMed Scopus Google Scholar). which is expressed the surface of uNK ESCs through with and receptors the surface of ESCs as in activation by cells by S. V. C. to Med. PubMed Scopus Google Scholar). We that interaction IL15 transcription directly or indirectly HAND2 in ESCs. Therefore, in it is to in which the is to or a to the response of and IL15 E2 and We found the putative HAND2 motif, the motif, at in of the IL15 locus. from the transcription have been to gene expression as D. of gene expression in 15 PubMed Scopus Google Scholar, regulation of gene Immunol. Google Scholar). The showed that gene expression the of a gene T. G. in are in that gene PubMed Scopus Google Scholar, The of of gene expression in J. PubMed Scopus Google Scholar, J. M. V. increase gene expression in PubMed Scopus Google Scholar, J.H. of the the expression of gene PubMed Scopus Google Scholar, expression from the in PubMed Scopus Google Scholar). our showed no of HAND2 at the putative HAND2 motif at found in an of the IL15 not in in ESCs. it is that HAND2 up-regulates the of transcription factors, such as and in the M. D. M. M. C. X. A. J. HAND2 a of transcriptional that the 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). the IL15 receptor is to regulated by transcription factors E. D. L.A. A. A. transcription factors with the cytokines and to cell 2015; Full Text Full Text PDF PubMed Scopus Google we that HAND2 regulate IL15 responsiveness from both and receptor a of study, human ESCs in cell the ESC Therefore, D. Lin S. X. W. L.A. 2013; PubMed Scopus Google Scholar) of human ESCs has been for as it is to cell with ESC and whether identified are ESCs or other cell human ESCs have been J. S. Decidualization and of a functional in human endometrial cell with Reprod. 12 PubMed Scopus Google Scholar) or of cell by human of human in of endometrial stromal cells not Reprod. 2005; PubMed Scopus Google Scholar, G. G. A. S. E. M. M. human endometrial stromal cell with normal PubMed Scopus Google Scholar, A. K. S. Brosens J.J. A.M. B. of a human endometrial stromal cell Endocrinol. 2009; PubMed Scopus Google Scholar, K. M. T. K. H. of in and differentiation of human endometrial stromal cells.J. Reprod. PubMed Scopus Google Scholar). These cell are the decidualization and response to have not been a human ESC has been cell is to such as decidualization to human ESCs M. T. T. T. of an human endometrial stromal cell with functional to ovarian Endocrinol. PubMed Scopus (18) Google Scholar). Therefore, the cell a in In we found that human IL15 transcription is directly regulated by HAND2 a putative HAND2 motif in the upstream region of the human IL15 in HAND2 is that with E2 and HAND2 regulate IL15 transcription in with other study was by the of and was from each study also by the of endometrial from of of with regular menstrual cycles. of the showed such as ESCs purified from endometrial as was from each human endometrium from phase, 12 from secretory the The was with a with The expression levels a and the in gene expression levels evaluated by the of gene expression PCR and the PubMed Scopus Google Scholar) as an (13Cho H. Okada H. Tsuzuki T. Nishigaki A. Yasuda K. Kanzaki H. Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells.Fertil. Steril. 2013; 99 (23036802): 248-25510.1016/j.fertnstert.2012.08.056Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The into The and for in After for at the with for to After with the with (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) in at and with for to the After with for the with with an in for IL15 and HAND2 was an or and After with the in the for 15 and with with and sequential was with or After the or was with with an The was to human ESCs. ESCs in and 2 to the of steroid hormones, at under a of in was ESCs for each the cells cells in ESCs at a of by as H. T. T. Yasuda K. Kanzaki H. The of a the growth of human endometrial stromal cells in Reprod. PubMed Scopus Google Scholar). The reporter and from The which was as an for transfection The upstream region of the human IL15 transcription was with the is and the is The PCR into The deletion which an deletion at the was and and The deletion which an deletion at the was and and substitution reporter at was with and and at was with and are as in The expression was from HAND2 was into at and was into to have no no and no deletion by analysis for with a and an with was to a S. T. T. H. M. M. M. E. regulation of the gene by PubMed Scopus Google Scholar, S. S. T. S. T. K. T. H. of in J. Med. Google Scholar). ESCs with E2 and for 12 ESCs with for at a of was to with and a nuclear was To and the nuclear was with for at The of the was by with for at and for 2 at The was at at and and the was in at a with 2 of (17Boeva V. Louis-Brennetot C. Peltier A. Durand S. Pierre-Eugène C. Raynal V. Etchevers H.C. Thomas S. Lermine A. Daudigeos-Dubus E. Geoerger B. Orth M.F. Grünewald T.G.P. Diaz E. Ducos B. et al.Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.Nat. Genet. 2017; 49 (28740262): 1408-141310.1038/ng.3921Crossref PubMed Scopus (125) Google Scholar) to the HAND2 to the as a or 2 of normal as a negative with for 2 at with and and for PCR purified from the analysis was polymerase and a with the one at for by 35 of at for at for and at for was to the levels of transcription factor and the of to the was ESCs into cell at a of of luciferase with transfection with E2 and the or of luciferase and one expression to the The of and in each of reporter or of and of expression or Approximately ESCs with and of cell was to an luciferase from the and luciferase from the in a and the to the luciferase activity was by luciferase by luciferase luciferase activity was the which was with or with a and a or levels expressed as the of at and HAND2 and in ESCs analysis transfection. from cell in and inhibitor for in to and with for and with or in at or as The After we also the by and to the was the HAND2 and IL15 expression in the proliferative and secretory phase HAND2 and IL15 expression was in the luciferase reporter not Therefore, we an with for normal or a for with at analyses The and during the study are from the We are to our in the of and for with estradiol progesterone endometrial stromal cell uterine natural killer interleukin 15 and neural crest derivatives–expressed transcript 2 medroxyprogesterone PCR nuclear receptor response analysis of

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