Progesterone receptor-mediated up-regulation of transthyretin in preimplantation mouse uterus

Transthyretin (TTR), a carrier for thyroxine and retinol, has its mRNA expressed in the glandular endometrial epithelium and its protein detected in the glandular endometrial epithelium and the uterine lumen. TTR mRNA is dramatically upregulated in the pre-implantation mouse uterus as well as progesterone-treated ovariectomized mouse uterus, and in both situations the upregulation of TTR is blocked by treatment with progesterone receptor antagonist RU486. Transthyretin (TTR) is a secreted protein that functions as a carrier for thyroid hormone thyroxine (T4) and retinol (1). Numerous investigations have suggested that TTR is associated with female reproduction under both physiological and pathological conditions. The specific localization of TTR protein in the subapical compartment of villous trophoblasts suggests that placental TTR may involve maternal-fetal thyroid hormone and retinol transport (2). The importance of TTR in placenta is supported by the observation that TTR is down-regulated in the placental villous trophoblasts of early pregnancy loss in human (3). In addition, monomeric TTR, an indication of oxidative stress, is significant increased in the amniotic fluids of patients with preeclampsia (4). Although TTR-deficient mice were reportedly to have normal fertility (5), the following observations suggest that TTR may play a role in the early pregnancy. TTR was identified as a protein marker in the receptive stage of endometrium in human (6). TTR is one of the major proteins in uterine flushing whose protein content increases dramatically at implantation in mouse (7). TTR is dysregulated in the post-implantation day 5.5 uterus deficient of cytosolic phospholipase A2α (Pla2g4a(−/−)) (8), which has delayed uterine receptivity (9). Interestingly, our microarray data indicate dysregulation of TTR in the pre-implantation day 3.5 mouse uterus deficient of the third receptor for lysophosphatidic acid (LPA), Lpar3, which also has delayed uterine receptivity (10). Dysregulation of LPAR3 has also been implicated in the defective uterine receptivity in women with endometriosis (11). In this study we used mouse as a model to determine the localization and regulation of TTR in the early pregnancy uterus. Wild type (WT) and Lpar3-deficient (Lpar3(−/−)) mice (C57Bl6/129svj mixed background) were derived from Dr. Jerold Chun's mouse colony at The Scripps Research Institute (10). All methods used in this study were approved by the Animal Subjects Programs of the University of Georgia and conform to National Institutes of Health guidelines and public law. Previously reported procedures were followed: mating (mating night as day 0), uterine tissue collection, and total RNA isolation (10, 12); microarray analysis (13); in situ hybridization (14); 17β-estradiol (E2) and progesterone (P) treatment (15); and estrogen receptor antagonist and progesterone receptor antagonist treatment (16–20). Realtime PCR reactions were performed in 384-well plates using Sybr-Green I intercalating dye on ABI 7900 (Applied Biosystems). Immunohistochemistry was performed using goat-anti-TTR antibody (1:50, Santa Cruz) and ABComplex/HRP method. Statistical analyses were done using two-tail, unequal variance Student's t test. The significant level was set at p<0.05. Lpar3 expression peaks in the preimplantation day 3.5 mouse uterus and deletion of Lpar3 leads to delayed uterine receptivity [10]. To understand the molecular mechanism of LPAR3 in uterine receptivity, day 3.5 WT and Lpar3(−/−) uteri were screened for differentially expressed genes by microarray analysis, which indicated that TTR was down-regulated in day 3.5 Lpar3(−/−) uterus (data not shown). To learn more about TTR in the early pregnancy, we examined the temporal expression of TTR in the whole uterus of both WT and Lpar3(−/−) mice during early pregnancy using realtime PCR. TTR expression was very low in day 0.5 uterus. It increased a few hundred folds in the uterus from day 0.5 to day 3.5. However, it seemed that from day 3.5 to day 4.5, TTR expression dropped slightly but significantly in the WT uterus (Fig. 1A). No significant difference in the TTR mRNA levels was detected between WT and Lpar3(−/−) uterus at day 0.5 and day 2.5. At day 3.5, TTR expression levels were lower in the Lpar3(−/−) uterus, which confirmed the microarray results. Interestingly, TTR expression levels were higher in the day 4.5 Lpar3(−/−) uterus compared to that in the day 4.5 WT uterus and comparable to that of day 3.5 WT uterus (Fig. 1A). In situ hybridization localized TTR mRNA expression specifically in the glandular endometrial epithelium (GE) of day 4.5 WT uterus (Fig. 1B). Immunohistochemistry localized TTR protein mainly in the GE and the uterine lumen of day 4.5 WT uterus (Fig. 1C). It is unknown why TTR mRNA levels were decreased in the day 3.5 Lpar3(−/−) uterus. Since Lpar3 is mainly expressed in the luminal endometrial epithelium (LE) while TTR is mainly expressed in the GE, the reduced TTR mRNA levels in the day 3.5 Lpar3(−/−) uterus may not be a direct effect of the lack of Lpar3 but could be an indirect effect caused by the delayed implantation in Lpar3(−/−) uterus [10]. Since reduced or lack of GE are observed in primary decidual zone (Fig. 1B, 1C), the higher TTR mRNA levels in the day 4.5 Lpar3(−/−) uterus compared to that of day 4.5 WT uterus, which was lower than that in the day 3.5 WT uterus, may reflect reduced GE content in the day 4.5 WT uterus, in which implantation had occurred but not that in day 4.5 Lpar3(−/−) uterus [10] or day 3.5 WT uterus. Interestingly, higher TTR expression was also observed in the day 5.5 Pla2g4a(−/−) implantation site with deferred implantation (8). These observations raise the possibility that increased TTR expression in the post-implantation uterus may be an indication of delayed uterine receptivity. The differential temporal expression of TTR in the peri-implantation uterus (Fig. 1A) and the previous report that TTR levels increase in the uterine flushing upon embryo implantation (7) suggest hormonal regulation of uterine TTR. In ovariectomized mice, E2 treatment for one hour did not affect TTR expression levels, for six hours showed marginal suppression of TTR expression, for 24 hours significantly reduced TTR levels about 6 folds, and for 74 hours reduced TTR levels about 300 folds (data not shown). A positive control Lif (Leukemia inhibitory factor), an E2 target gene (21), was dramatically induced by E2 (data not shown), indicating the suppression of TTR by E2 was specific. P treatment from 4 to 54 hours significantly induced TTR levels (Fig. 1D). In situ hybridization revealed that P-induced TTR expression was mainly shown in the GE (data not shown). However, this induction was abolished upon co-administration of E2 for 6 and 24 hours but not for 1 hour (Fig. 1E). These results demonstrate that TTR is coordinately regulated by both E2 and P in the uterus of ovariectomized WT mice. Fig. 1A showed upregulation of TTR in the pre-implantation uterus. To determine the molecular mechanism of TTR upregulation in the preimplantation uterus, day 2.5 pregnant WT females were treated with estrogen receptor (ER) antagonist ICI 182 780 or progesterone receptor (PR) antagonist RU486. The uterine tissues were dissected on preimplantation day 3.5. Realtime PCR indicated that ER antagonist ICI 182 780 did not have an effect on TTR expression but PR antagonist RU486 reduced TTR expression levels about 100 fold in the pre-implantation uterus (Fig. 1F). The dramatic upregulation of TTR in the pre-implantation WT uterus (Fig. 1A) is in accordance with the increased secretion of P during the same period of time (22), and this upregulation is PR-dependent but ER-independent (Fig. 1F). Data from another set of ovariectomized mice demonstrated that P-induced uterine TTR expression was completely abolished upon pre-treatment with RU486 (Fig. 1G), which further confirmed the critical role of PR in P-induced upregulation of uterine TTR expression. The regulation of TTR by P signaling in the uterus has also been indicated in another study, which suggests the role of P-SRC-2 (p160 steroid receptor coactivator 2) in TTR regulation. Microarray analysis reveals that TTR is among the genes that are upregulated by P treatment in ovariectomized WT mouse uterus (23). P-induced upregulation of TTR in uterus is attenuated upon deletion of SRC-2 (23), suggesting that TTR is under the control of SRC-2. This suggestion is supported by the upregulation of SRC-2 by P and main localization of SRC-2 in the GE, although SRC-2 is also detected in other uterine compartments, such as LE (23). Our results also demonstrate that co-treatment of E2 plus P for 6 and 24 hours in ovariectomized mice prevents P-induced uterine TTR expression (Fig. 1E). These data suggest that E2 counteracts with P-PR signaling to regulate TTR expression in the ovariectomized uterus. It is unknown whether this interaction is ER-dependent or ER-independent. We analyzed the 3kb upstream sequence of TTR gene using MatInspector (24). There are three putative progesterone response elements and three putative estrogen response elements in this region. The mechanism of transcriptional regulation of TTR by PR and ER has not been established. The detection of TTR protein in both GE and uterine lumen is in line with TTR as a secreted protein, which is one of the major proteins identified in the uterine flushings (6, 7, 25, 26). The secreted TTR protein functions as a carrier for T4 and retinol. Numerous studies have indicated the importance of T4 and retinol in early pregnancy. Uterus is a site with active thyroid hormone metabolism (27), a process regulated by P and E2 during pregnancy (28). Lack of T4 in thyroparathyroidectomized rats significantly reduces the number of implantation sites (~50%) (29). Retinol is required for female reproduction. The enzymes for retinol synthesis and metabolism are differentially expressed in the uterus during estrous cycle and early pregnancy (30). Microarray analysis has demonstrated that the enzymes involved in retinoic acid synthesis and metabolism, e.g., ADH5, Aldh1a1, and Cyp26a1, are induced by P-PR axis (31). Interestingly, retinol-binding protein synthesis is confined to the GE and under P control in baboon (32), similar as that of TTR in mice (Fig. 1B–G). Since TTR is a carrier for T4 and retinol, both of which are associated with embryo implantation (27, 29–31), understanding the localization and regulation of TTR in the early pregnancy uterus will help understand the roles of T4 and retinol in the early pregnancy. Acknowledgments The authors thank Dr. James N. Moore and Dr. Zhen Fu in the College of Veterinary Medicine, University of Georgia for giving free access to the ABI 7900 Realtime PCR machine and the imaging system, respectively. The authors also thank Mr. Eric Miller at Genomics and Proteomics Core Facility Medical College of Georgia for the microarray analysis. This work was supported by the startup funding from the Office of the Vice President for Research at University of Georgia, Athens, Georgia, USA. Footnotes All authors, H.D., S.X., J.C., Y.X., and X.Y., have nothing to disclose.

- 1.Raz A, Shiratori T, Goodman DS. Studies on the protein-protein and protein-ligand interactions involved in retinol transport in plasma. J Biol Chem. 1970;245:1903–12. [PubMed] [Google Scholar] - 2.McKinnon B, Li H, Richard K, Mortimer R. Synthesis of thyroid hormone binding proteins transthyretin and albumin by human trophoblast. J Clin Endocrinol Metab. 2005;90:6714–20. doi: 10.1210/jc.2005-0696. [DOI] [PubMed] [Google Scholar] - 3.Liu AX, Jin F, Zhang WW, Zhou TH, Zhou CY, Yao WM, et al. Proteomic analysis on the alteration of protein expression in the placental villous tissue of early pregnancy loss. Biol Reprod. 2006;75:414–20. doi: 10.1095/biolreprod.105.049379. [DOI] [PubMed] [Google Scholar] - 4.Vascotto C, Salzano AM, D'Ambrosio C, Fruscalzo A, Marchesoni D, di Loreto C, et al. Oxidized transthyretin in amniotic fluid as an early marker of preeclampsia. J Proteome Res. 2007;6:160–70. doi: 10.1021/pr060315z. [DOI] [PubMed] [Google Scholar] - 5.Episkopou V, Maeda S, Nishiguchi S, Shimada K, Gaitanaris GA, Gottesman ME, et al. Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone. Proc Natl Acad Sci U S A. 1993;90:2375–9. doi: 10.1073/pnas.90.6.2375. [DOI] [PMC free article] [PubMed] [Google Scholar] - 6.Beier HM, Beier-Hellwig K. Molecular and cellular aspects of endometrial receptivity. Hum Reprod Update. 1998;4:448–58. doi: 10.1093/humupd/4.5.448. [DOI] [PubMed] [Google Scholar] - 7.Aitken RJ. Changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration. J Reprod Fertil. 1977;50:29–36. doi: 10.1530/jrf.0.0500029. [DOI] [PubMed] [Google Scholar] - 8.Burnum KE, Tranguch S, Mi D, Daikoku T, Dey SK, Caprioli RM. Imaging mass spectrometry reveals unique protein profiles during embryo implantation. Endocrinology. 2008;149:3274–8. doi: 10.1210/en.2008-0309. [DOI] [PMC free article] [PubMed] [Google Scholar] - 9.Song H, Lim H, Paria BC, Matsumoto H, Swift LL, Morrow J, et al. Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for `on-time' embryo implantation that directs subsequent development. Development. 2002;129:2879–89. doi: 10.1242/dev.129.12.2879. [DOI] [PubMed] [Google Scholar] - 10.Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, et al. LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature. 2005;435:104–8. doi: 10.1038/nature03505. [DOI] [PMC free article] [PubMed] [Google Scholar] - 11.Wei Q, St Clair JB, Fu T, Stratton P, Nieman LK. Reduced expression of biomarkers associated with the implantation window in women with endometriosis. Fertil Steril. 2009;91:1686–91. doi: 10.1016/j.fertnstert.2008.02.121. [DOI] [PMC free article] [PubMed] [Google Scholar] - 12.Ye X, Skinner MK, Kennedy G, Chun J. Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling. Biol Reprod. 2008;79:328–36. doi: 10.1095/biolreprod.108.068783. [DOI] [PMC free article] [PubMed] [Google Scholar] - 13.Pelch KE, Schroder AL, Kimball PA, Sharpe-Timms KL, Wade Davis J, Nagel SC. Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women. Fertil Steril. 2009 doi: 10.1016/j.fertnstert.2009.03.086. [DOI] [PMC free article] [PubMed] [Google Scholar] - 14.Diao HL, Su RW, Tan HN, Li SJ, Lei W, Deng WB, et al. Effects of androgen on embryo implantation in the mouse delayed-implantation model. Fertil Steril. 2008;90:1376–83. doi: 10.1016/j.fertnstert.2007.07.1341. [DOI] [PubMed] [Google Scholar] - 15.Brown N, Deb K, Paria BC, Das SK, Reese J. Embryo-uterine interactions via the neuregulin family of growth factors during implantation in the mouse. Biol Reprod. 2004;71:2003–11. doi: 10.1095/biolreprod.104.031864. [DOI] [PubMed] [Google Scholar] - 16.Surveyor GA, Gendler SJ, Pemberton L, Das SK, Chakraborty I, Julian J, et al. Expression and steroid hormonal control of Muc-1 in the mouse uterus. Endocrinology. 1995;136:3639–47. doi: 10.1210/endo.136.8.7628404. [DOI] [PubMed] [Google Scholar] - 17.Ni H, Yu XJ, Liu HJ, Lei W, Rengaraj D, Li XJ, et al. Progesterone regulation of glutathione S-transferase Mu2 expression in mouse uterine luminal epithelium during preimplantation period. Fertil Steril. 2009;91:2123–30. doi: 10.1016/j.fertnstert.2008.04.053. [DOI] [PubMed] [Google Scholar] - 18.Das SK, Tan J, Raja S, Halder J, Paria BC, Dey SK. Estrogen targets genes involved in protein processing, calcium homeostasis, and Wnt signaling in the mouse uterus independent of estrogen receptor-alpha and -beta. J Biol Chem. 2000;275:28834–42. doi: 10.1074/jbc.M003827200. [DOI] [PubMed] [Google Scholar] - 19.Mohamed OA, Dufort D, Clarke HJ. Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol Reprod. 2004;71:417–24. doi: 10.1095/biolreprod.103.025692. [DOI] [PubMed] [Google Scholar] - 20.Cheon YP, Li Q, Xu X, DeMayo FJ, Bagchi IC, Bagchi MK. A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation. Mol Endocrinol. 2002;16:2853–71. doi: 10.1210/me.2002-0270. [DOI] [PubMed] [Google Scholar] - 21.Yang ZM, Chen DB, Le SP, Harper MJ. Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus. Mol Reprod Dev. 1996;43:470–6. doi: 10.1002/(SICI)1098-2795(199604)43:43.0.CO;2-M. [DOI] [PubMed] [Google Scholar] - 22.Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet. 2006;7:185–99. doi: 10.1038/nrg1808. [DOI] [PubMed] [Google Scholar] - 23.Jeong JW, Lee KY, Han SJ, Aronow BJ, Lydon JP, O'Malley BW, et al. The p160 steroid receptor coactivator 2, SRC-2, regulates murine endometrial function and regulates progesterone-independent and -dependent gene expression. Endocrinology. 2007;148:4238–50. doi: 10.1210/en.2007-0122. [DOI] [PubMed] [Google Scholar] - 24.Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, et al. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics. 2005;21:2933–42. doi: 10.1093/bioinformatics/bti473. [DOI] [PubMed] [Google Scholar] - 25.Hearn JP, Renfree MB. Prealbumins in the vaginal flushings of the marmoset, Callithrix jacchus. J Reprod Fertil. 1975;43:159–61. doi: 10.1530/jrf.0.0430159. [DOI] [PubMed] [Google Scholar] - 26.Guise MB, Gwazdauskas FC. Profiles of uterine protein in flushings and progesterone in plasma of normal and repeat-breeding dairy cattle. J Dairy Sci. 1987;70:2635–41. doi: 10.3168/jds.S0022-0302(87)80333-8. [DOI] [PubMed] [Google Scholar] - 27.Galton VA, Martinez E, Hernandez A, St Germain EA, Bates JM, St Germain DL. The type 2 iodothyronine deiodinase is expressed in the rat uterus and induced during pregnancy. Endocrinology. 2001;142:2123–8. doi: 10.1210/endo.142.5.8169. [DOI] [PubMed] [Google Scholar] - 28.Wasco EC, Martinez E, Grant KS, St Germain EA, St Germain DL, Galton VA. Determinants of iodothyronine deiodinase activities in rodent uterus. Endocrinology. 2003;144:4253–61. doi: 10.1210/en.2003-0490. [DOI] [PubMed] [Google Scholar] - 29.Baksi SN. Effects of thyroparathyroidectomy on implantation and early gestation in rats. J Reprod Fertil. 1973;32:513–6. doi: 10.1530/jrf.0.0320513. [DOI] [PubMed] [Google Scholar] - 30.Vermot J, Fraulob V, Dolle P, Niederreither K. Expression of enzymes synthesizing (aldehyde dehydrogenase 1 and reinaldehyde dehydrogenase 2) and metabolizaing (Cyp26) retinoic acid in the mouse female reproductive system. Endocrinology. 2000;141:3638–45. doi: 10.1210/endo.141.10.7696. [DOI] [PubMed] [Google Scholar] - 31.Jeong JW, Lee KY, Kwak I, White LD, Hilsenbeck SG, Lydon JP, et al. Identification of murine uterine genes regulated in a ligand-dependent manner by the progesterone receptor. Endocrinology. 2005;146:3490–505. doi: 10.1210/en.2005-0016. [DOI] [PubMed] [Google Scholar] - 32.Fazleabas AT, Donnelly KM, Mavrogianis PA, Verhage HG. Retinol-binding protein in the baboon (Papio anubis) uterus: immunohistochemical characterization and gene expression. Biol Reprod. 1994;50:1207–15. doi: 10.1095/biolreprod50.6.1207. [DOI] [PubMed] [Google Scholar] - 33.Jeong JW, Lee HS, Lee KY, White LD, Broaddus RR, Zhang YW, et al. Mig-6 modulates uterine steroid hormone responsiveness and exhibits altered expression in endometrial disease. Proc Natl Acad Sci U S A. 2009;106:8677–82. doi: 10.1073/pnas.0903632106. [DOI] [PMC free article] [PubMed] [Google Scholar]