Molecular Human Reproduction, Vol. 7, No. 10, 963-970,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Characterization of relaxin binding in the uterus of the marmoset monkey
1 Deutsches Primatenzentrum, Kellnerweg 4, 37077 Göttingen and 2 Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany
Abstract
The ovarian peptide hormone relaxin (RLX) plays an important role in the regulation of the endometrium both during the cycle and in early pregnancy. RLX interacts with specific receptors on endometrial stromal cells causing these to decidualize. In order to characterize the molecules with which RLX interacts in the primate uterus, a methodology based on a fully bioactive preparation of biotinylated porcine RLX was applied to cryosections of the uterus of female marmoset monkeys. Specific RLX binding was weakly detected in the proliferative phase in isolated endometrial stromal cells. In the secretory phase, the positively reacting cells increased in staining intensity and in number and also included some epithelial cells. Further increases occurred in pregnancy, but RLX binding in the endometrium decreased at the end of the cycle if pregnancy did not occur. The myometrium showed weak staining which did not vary through the cycle, but increased in pregnancy. Electrophoretic analysis of the RLX-binding moieties in these tissue sections indicated that a protein of ~40 kDa was the principal RLX-binding molecule, while minor specific bands were detectable at ~100 and ~200 kDa. The binding of biotinylated RLX could be specifically suppressed by co-incubation with unlabelled RLX, but not by insulin, IGF-I or biotin. This technique therefore allows the detection and molecular characterization of specific RLX binding in the primate uterus. In the marmoset monkey, the pattern of specific binding closely reflects the RLX-dependent physiology during implantation and early pregnancy, implying the probable involvement of a specific RLX receptor.
endometrium/marmoset monkey/pregnancy/relaxin/relaxin receptor
Introduction
The peptide hormone relaxin (RLX) is responsible for a wide range of functions, with responsive tissues in the brain, heart, kidney and skin, besides the more classical organs of the female reproductive system (Sherwood, 1994
). Structurally RLX is closely related to insulin, with the same heterodimeric subunit composition, resulting from post-translational removal of a signal peptide and excision of a C (connecting)-domain from a common precursor molecule. Despite the length of time since RLX was first described and its structural similarity to insulin, very little is known about the RLX receptor. Scatchard analyses in different tissues have shown homogeneous high affinity binding sites with a Kd in the range of 0.2–1.5 nmol/l (Büllesbach et al., 1995; Osheroff, 1995; Palejwala et al., 1998). Attempts to assess the molecular size by cross-linking to iodinated relaxin have resulted in diverse estimates varying between 36 and 220 kDa (Büllesbach et al., 1995; Osheroff, 1995; Palejwala et al., 1998). Whereas from its structural similarity to insulin and IGF-I, one might expect the receptor for RLX also to belong to the family of membrane-linked tyrosine kinases, paradoxically the major second messenger described in the intracellular response to specific RLX stimulation is cAMP (Fei et al., 1990; Parsell et al., 1996; Zarreh-Hoshyari-Khah et al., 2001). A dose-dependent increase in cAMP has been observed in myometrial smooth muscle cells (Hsu et al., 1985), in endometrial stromal and epithelial cells (Chen et al., 1988; Fei et al., 1990), and in the monocyte cell line, THP-1 (Parsell et al., 1996; Zarreh-Hoshyari-Khah et al., 2001). The most plausible current explanation is that the RLX receptor is a novel type of tyrosine kinase linked by a kinase cascade to inhibition of a specific phosphodiesterase, and consequent up-regulation of cAMP (Bartsch et al., 2001).
Because the molecular cloning of the RLX receptor has so far resisted all attempts using conventional methods, it seems likely that we still have too little information upon which to design a reliable cloning strategy. To relieve this situation, we have therefore set about a more detailed characterization of the receptor, by developing specific molecular tools with which to define the molecule at the protein level. Here we describe a novel approach to assess relaxin binding in the uterus of the marmoset monkey. This not only allows the specific binding to be characterized morphologically, but also allows the molecular characteristics of the specific binding entity to be determined. The marmoset monkey is a New World primate which, in its pattern of relaxin expression and its responses to the hormone, appears to be very similar to the human, and thus an ideal model system in which to investigate relaxin physiology (Steinetz et al., 1995; Einspanier et al., 1997, 1999).
Materials and methods
Animals and tissues
Tissues were collected at defined stages of the cycle and early pregnancy from female marmoset monkeys aged 3–6 years and weighing 350–500 g (n = 12 animals). Cycle staging was carried out by regular progesterone measurements in peripheral blood (0.1 ml, twice per week) and the application, at day 12 of the luteal phase, of a synthetic prostaglandin F2
(PF2; 0.8 µg/per animal; estrumate, Mallinckrodt Vet, Burgwedel, Germany) which induces luteal regression and the onset of new waves of follicle development. Another set of animals (n = 4), which was part of an ovarian stimulation protocol, was also used in this study. Daily FSH injections (1.5 and 3 IU per day human recombinant FSH; Serono, Freiburg, Germany) started on the day of PGF2-induced luteolysis, and uteri were collected 3 days later. Tissues from the other adult female marmoset monkeys were then collected at different stages of the cycle and early pregnancy, as indicated. Tissue samples were divided with part being frozen at –80°C for RLX-binding studies and part being fixed in 5% paraformaldehyde for later histological examination. Institutional approval was obtained for all experiments.
Bioactivity of biotinylated porcine relaxin
Highly pure porcine RLX, obtained by extraction of the native hormone from pig corpora lutea and estimated to be >85% pure, was a generous gift of Dr O.D.Sherwood, and indicated only a single stainable band on denaturing polyacrylamide gel electrophoresis (PAGE) (not shown). The RLX was biotinylated as described in detail elsewhere (Einspanier et al., 1999). Others have previously shown that RLX can be labelled with biotin without impinging on its receptor-binding properties (Büllesbach and Schwabe, 1990; Min and Sherwood, 1996). In order to check whether the biotinylation influenced the binding properties of the RLX to its specific receptor, biotinylated RLX (bio-RLX) was first tested in the THP-1 human monocyte and primary endometrial stromal cell (ESC) bioassay systems, exactly as described previously (Zarreh-Hoshyari-Khah et al., 2001), by comparing the effect of the bio-RLX on the cAMP concentration in the cells with that induced by unbiotinylated porcine RLX as a control.
Specific ligand binding to uterine tissue sections
The procedure employed to assay for specific ligand binding was originally validated with rat testis sections for the detection and quantification of insulin and atrionatriuretic peptide receptors (D.Müller and R.Middendorf, unpublished results). For the RLX-binding assays, several parameters were initially varied until optimal sensitivity and specificity were obtained. In the final protocol, 8 µm cryosections were lyophilized (–4°C) for 2 h, allowed to adapt to room temperature for 1 h, then rinsed briefly in HEPES buffer (50 mmol/l HEPES, 150 mmol/l NaCl, 5 mmol/l MgCl, 0.1% BSA, pH 7.5) allowed to adapt to room temperature again for 1 h and washed for a second time in HEPES buffer. The biotinylated porcine relaxin was diluted to 0.5 µmol/l in HEPES buffer, added to the sections in a 50 µl total volume, and the sections were incubated for 15 h at 4°C in a humidified chamber. The slides were then exposed to UV irradiation (30 W, wavelength 254 nm, 10 cm distance) for 10 min and then rinsed in HEPES buffer for 5 min. This irradiation procedure was repeated, and then the remaining bound bio-RLX was visualized by incubation with avidin–biotin–peroxidase (ABC; Vector Laboratories, Burlingame, USA) for 30 min, followed by two washes in HEPES buffer each for 5 min; the chromogen substrate AEC (Vector Laboratories) was then added for 20 min, before the slides were rinsed sequentially in tap and distilled water and embedded in mounting medium (Immuno-Mount; Shandon, Frankfurt, Germany). For some sections (e.g. myometrium), 3,3'-diaminobenzidine (DAB) staining (Vector Laboratories) was used instead of AEC. For both protocols, as controls, porcine RLX, human insulin (Serono), human IGF-I (Peprotech, New York, NY, USA), or unconjugated biotinyl-e-aminocaproic acid-N-hydroxysuccinimide ester (Sigma, Deisenhofen, Germany), all at 1 mmol/l concentrations (i.e. 2000-fold molar excess) in HEPES buffer, were applied to the slides after the application of the biotinylated porcine RLX. As a further control, some experiments were repeated using a sample of biotinylated porcine RLX kindly provided by Dr O.D.Sherwood, and shown to be bioactive in the in-vivo mouse interpubic ligament assay (Min and Sherwood, 1996).
Electrophoretic analysis of tissue sections incubated with bio-RLX
Individual tissue sections were incubated with bio-RLX as above, and processed up to the UV cross-linking step. These were then scraped and dissolved into 60 µl standard denaturing PAGE loading buffer, containing only 5% sodium dodecyl sulphate, then heated to 100°C for 5 min, loaded onto 9% polyacrylamide gels, and electrophoresed at ~100 mA. Electrophoretically separated proteins were then electrotransferred overnight onto 0.45 µm pore nitrocellulose membranes, as for standard Western blotting. Biotinylated proteins were then detected by incubating the membranes for 30 min at room temperature in horseradish peroxidase–streptavidin conjugate (Vector Laboratories; stock solution 1 mg/ml) diluted 5000:1 in TBST buffer (20 mmol/l Tris–HCl, pH 7.6, 137 mmol/l NaCl, 0.05% Tween-20) additionally containing 10% Western Blocking Reagent (Roche Diagnostics, Mannheim, Germany; prepared according to the manufacturer's instructions) and 0.005% thimerosal. Peroxidase activity was visualized using the ECL chemiluminescence system (Amersham–Pharmacia Biotech, Freiburg, Germany).
Results
Porcine RLX was biotinylated in such a way that not more than three biotin groups were attached to each RLX molecule. In order to check that the biotinylation did not interfere with RLX binding to a putative receptor, a dose–response analysis was carried out to compare pure porcine RLX with the biotinylated hormone (bio-RLX), using the primary ESC-cell bioassay (Fei et al., 1990; Zarreh-Hoshyari-Khah et al., 2001). The results (Figure 1A) clearly show that there is no difference in the specific activity of the bio-RLX compared to the pure standard, both indicating EC50 values of ~1.5 nmol/l. Similar results were also obtained using the THP-1 human monocyte bioassay system (Parsell et al., 1996; Zarreh-Hoshyari-Khah et al., 2001) (Figure 1B). Thus we can expect that binding of the bio-RLX to a receptor molecule will be equivalent to binding of the unmodified hormone.
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Specific RLX binding to cryosections of the non-pregnant marmoset uterus collected in the proliferative phase of the endometrial cycle elicited only very weak signals restricted to the endometrial stroma (Figure 2A
). These signals increased in intensity through the proliferative phase (Figure 2C and D). When animals were additionally treated with FSH during the early proliferative phase as part of an ovarian stimulation paradigm, there was also a slight increase in bio-RLX staining intensity (Figure 2B). After ovulation, when the endometrium had entered the secretory phase under the influence of increasing ovarian progesterone, there was a definite increase in bio-RLX binding to the cells of the endometrial stroma (Figure 2E), and this extended into the mid-secretory phase (Figure 2F). Additionally, weak staining could then be observed in some epithelial cells of the endometrial glands (not shown). Later in the cycle, however, in the non-gravid uterus bio-RLX binding to individual cells was reduced (Figure 2G). In animals which had entered a persistent secretory phase due to polycystic ovarian syndrome (PCOS), though not pregnant, staining intensity for bio-RLX binding was maintained or even increased (Figure 2H) to give an appearance similar to that seen in early pregnancy (Figure 2I, see below). Finally, during pregnancy, staining intensity for bio-RLX binding was markedly increased in the endometrial stroma, particularly in the luminal regions and in the decidua (Figure 2I and J). Control sections (Figure 2K), which had been additionally co-incubated with an excess of unlabelled RLX, showed no specific staining.
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In the early pregnant uterus (day 30–40), a strong signal was present in the decidual cells as well as in the area of maternal and fetal contact (Figure 3A and B
), in contrast with control sections incubated with excess unlabelled RLX (Figure 3C and D). Whereas the fetal membranes were negative (Figure 3A and E), the fetal tissue itself gave a positive signal (Figure 3A and E).
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During the cycle, very weak specific RLX binding was also evident in the myometrium, mostly associated with blood vessels (Figure 4A–C
), but this did not appear to vary greatly in staining intensity from one phase to another. In pregnancy, however, RLX binding in the myometrium was increased (Figure 4D and E; compared with control, Figure 4F).
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As controls to check for the specificity of the bio-RLX binding, sections of uterus from day 3 of the secretory phase were additionally treated with an excess of unlabelled porcine RLX, which completely inhibited all staining (compare Figure 5A and B
). Similar applications of IGF-I (Figure 5C), insulin (Figure 5D) or biotin-ester (not shown) were all without effect on the signal intensity. Neither kidney tissue nor liver showed evidence of any specific RLX binding (Figure 5E and G) by comparison with appropriate controls (Figure 5F and H).
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The advantage of the methodology we have developed is that we can analyse the properties of those molecular entities which are directly adjacent to the bio-RLX ligand, and hence likely to be part of the RLX receptor or associated with it. By using UV light as a cross-linking agent, only molecules directly adjacent to the ligand are detectable. Therefore, the RLX-binding moiety giving rise to the specific signals seen in the tissue sections could be subsequently solubilized and analysed by PAGE (Figure 6
). By comparing the proteins solubilized from sections where bio-RLX has bound with those where ligand binding has been competitively inhibited by unlabelled RLX, the specific RLX-binding molecules could be identified (arrows). The major RLX-binding moiety was a protein of ~40–42 kDa. Minor specific bands were also evident at higher molecular weights (100 and 200 kDa). Such specific and competable bands were only evident in the uterine samples. Liver sections used as negative controls showed only non-specific staining unrelated to the presence or not of bio-RLX.
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Discussion
In the marmoset monkey, RLX circulates in peripheral blood with a maximum concentration in the first half of pregnancy (Steinetz et al., 1995; Einspanier et al., 1999). This is very similar to the pattern in women in whom maximum concentrations of serum RLX occur in the first trimester, but unlike the pattern observed in rodents and pigs, where highest RLX concentrations are found in the immediate prepartum period (Sherwood, 1994). Not only is RLX found during pregnancy, but it is also detected at lower levels in most mammals, including the marmoset, during the cycle, particularly in the late secretory phase (Einspanier et al., 1999). Most of the circulating RLX in primates is synthesized within the corpus luteum of the ovary; however, immunoreactive RLX and its mRNA have also been detected within the uterus (Bryant-Greenwood et al., 1993; Einspanier et al., 1997), suggesting a local synthesis. While the classic role for RLX in pregnancy is the softening of the cervix and widening of the pubic symphysis, recently attention has been drawn to RLX functions in the cycle and early pregnancy, particularly in the context of implantation (Stewart et al., 1990; Gosh et al., 1997; Einspanier et al., 1999). RLX has been identified as probably the most important factor responsible for the induction of endometrial stromal cell differentiation in the human (Telgmann and Gellersen, 1998). This differentiation, known as decidualization, and associated with specific patterns of new gene expression (Beier and Beier-Hellwig, 1998), occurs in the second half of the cycle as an essential prerequisite for successful implantation should a blastocyst be formed. RLX, acting through the second messenger cAMP, induces both a morphological as well as a biochemical differentiation of the endometrial stromal cells, such that these begin to express factors important for implantation and angiogenesis, e.g. vascular endothelial growth factor (Unemori et al., 1999).
In this study, we have shown specific RLX binding in the endometrium during the cycle and in early pregnancy. At present we have no further information on the phenotypic identity of those stromal cells indicating specific RLX binding. It will be particularly interesting to use co-localization studies using known endometrial markers to characterize these cells further. Comparing our findings with those of another group (Kohsaka et al., 1998) for the human uterus show that similar cell types are being labelled, though in the latter study substantial RLX binding was also observed in the luminal and glandular epithelium. Such staining was not seen in the marmoset. A direct comparison is, however, not possible since no information was available either on the stage of the cycle when the human tissues were collected nor on their endocrine status.
In the present study, RLX binding was also evident in the myometrium, though it appeared to be of a lower intensity than in the stromal cells; staining intensity did not vary through the cycle, but did increase in pregnancy. This expression would accord with a role for RLX in modulating uterine smooth muscle contractility, as observed in the rat (Hughes et al., 1997). Whether RLX influences myometrial function in the human is still controversial (e.g. Petersen et al., 1991; Sherwood, 1994), though to date there are no studies reporting on possible RLX effects in early pregnancy when the circulating RLX concentrations in women are maximal.
To date there is very little information available on the presence and distribution of RLX receptors, particularly in uterine tissue. Osheroff and colleagues working in the rat, using 32P-labelled ligand-binding autoradiography (Osheroff et al., 1990), have shown RLX binding both in the cervix and in the uterus, though the poor resolution of this analytical system did not allow for any cellular localization. RLX binding was shown to increase markedly upon oestradiol treatment of ovariectomized animals (Osheroff et al., 1990). Gaining resolution by using a 33P-labelled RLX, Tan and colleagues have identified RLX binding in rat atria, cerebral cortex and myometrium (Tan et al., 1999). In the rat, pig and human, Sherwood and colleagues (Kuenzi and Sherwood, 1995; Min and Sherwood, 1996; Kohsaka et al., 1998), using a biotinylated RLX preparation, have indicated RLX binding predominantly in the epithelial cells of the cervix and breast. By measuring iodinated RLX binding to membrane preparations, binding sites for RLX have also been localized to stromal fibroblasts of the human lower uterine segment (Palejwala et al., 1998). This study, taken together with the physiological studies applying RLX to cultures of human endometrial stromal cells (Fei et al., 1990; Telgmann and Gellersen, 1998), implies that in primates, including the human, within the female reproductive tract there are RLX-binding sites in stromal cells, and possibly in epithelial cells (Chen et al., 1988). In rodents, RLX receptors are definitely present on myometrial smooth muscle cells (Hsu et al., 1985; Grazi et al., 1988), and possibly also on epithelial cells. There is thus a discrepancy in the cellular localization of RLX binding between rodents and primates, and this may be associated with species specificity in the way the uterus is regulated.
In the present study, we have devised tools and techniques which allow a rigorous validation of the results. The biotinylated RLX preparation used is of high purity and has full bioactivity in primate (human) bioassay systems by comparison with a non-biotinylated control RLX preparation. Within the uterus of the marmoset monkey, RLX binding is detected mostly in the endometrial stromal cells, but with some weak epithelial signals. The temporal pattern of RLX binding in the marmoset uterus reflects closely what we would expect based on our knowledge of RLX physiology in primates. Within the cycle, the principal uterine function of RLX is during the secretory phase, where, as in the human, RLX is involved in the decidualization of the stromal cells in anticipation of fertilization occurring with subsequent blastocyst implantation. Logically, this decidualization process recedes if no pregnancy occurs, but continues to increase in the first trimester concomitant with implantation and trophoblast invasion. Thus the results obtained here strongly support the view that the RLX binding observed reflects the endogenous RLX physiology, and hence probably the localization of RLX receptors. Sections of uterus from a marmoset suffering PCOS also showed strong RLX binding similar to the situation in the early pregnant uterus. This up-regulation could be due to high peripheral steroid levels, which are present in this clinical picture as well as during early pregnancy.
An advantage of the methodology applied in this study is that the tissue sections used for RLX binding can be subsequently solubilized and submitted to denaturing PAGE analysis, with the ligand cross-linked to its intimate binding partner. In the present case, this revealed that bio-RLX bound mostly to form a ~42 kDa protein (of which 6 kDa will be due to the bio-RLX itself), with lower levels of binding to larger molecular weight moieties. This is in good agreement with the results of one group (Osheroff and King, 1995) who, using [32P]RLX in cell cultures, identified binding proteins of 36 and 220 kDa. It has been shown that there was tyrosine phosphorylation of a 220 kDa protein upon RLX stimulation of primary uterine cells in culture, and this was presumed to represent autophosphorylation of a RLX receptor (Palejwala et al., 1998). These results differ from those of others (Büllesbach et al., 1995), who, also using cross-linking and solubilization, reported a RLX-binding moiety of between 60–120 kDa. A direct comparison of such results is difficult because UV cross-linking, as used in the present study, is likely only to covalently link those molecules which are directly adjacent to one another, whereas the chemical crosslinking used in other studies can join molecules which are slightly further apart.
One interpretation of the present results would be that these binding proteins indeed represent the RLX receptor, which, like the receptors for insulin and IGF-I, comprises two subunits, the smaller extracellular subunit being responsible for ligand binding, and the larger transmembrane and intracellular moiety containing the tyrosine kinase activity and site(s) for autophosphorylation. This larger subunit would be linked to the ligand-binding subunit by the cross-linking procedure, and may itself be part of a homodimeric complex. Another plausible interpretation might be that the smaller, more prominent RLX-binding protein at ~35–40 kDa is similar to those IGF-I-binding proteins that are of similar size and are unrelated to the IGF receptor, but which play a key role in the regulation of IGF function (Hossenlopp et al., 1986; Op De Beeck et al., 1997; Weber et al., 1999). The aim of future research will be to clarify the molecular identity of these RLX-binding proteins and the role they play in RLX physiology. The marmoset, by exhibiting such a close correlation between RLX binding and RLX physiology in this important peri-implantation phase, is again confirmed as an ideal primate model in which to investigate the actions of this important reproductive hormone.
Acknowledgements
We are very grateful to Professor O.D.Sherwood (Urbana-Champaign, IL, USA) for the generous gift of pure porcine relaxin and the control sample of its biotinylated derivative, to Professor Keith Hodges (DPZ) for providing research facilities and to Professor Freimut Leidenberger (IHF) for his continued support of this project. We should also like to acknowledge the advice of Dr Ralph Middendorf during the development of the ligand-binding technique. This study was funded by the Deutsche Forschungsgemeinschaft (Ei333/8-1 and in part Iv7/9-1).
Notes
3 To whom correspondence should be addressed. E-mail: aeinspa{at}gwdg.deorivell{at}ihf.de 
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Submitted on April 24, 2001; accepted on July 27, 2001.
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 R. A. Bathgate, R. Ivell, B. M. Sanborn, O. D. Sherwood, and R. J. Summers International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides. Pharmacol. Rev., March 1, 2006; 58(1): 7 - 31. [Abstract] [Full Text] [PDF]
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 C. P. Bond, L. J. Parry, C. S. Samuel, H. M. Gehring, F. L. Lederman, P. A. W. Rogers, and R. J. Summers Increased Expression of the Relaxin Receptor (LGR7) in Human Endometrium during the Secretory Phase of the Menstrual Cycle J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3477 - 3485. [Abstract] [Full Text] [PDF]
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 O. D. Sherwood Relaxin's Physiological Roles and Other Diverse Actions Endocr. Rev., April 1, 2004; 25(2): 205 - 234. [Abstract] [Full Text] [PDF]
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 J. J. Luna, A. Riesewijk, J. A. Horcajadas, R. d. van Os, F. Dominguez, S. Mosselman, A. Pellicer, and C. Simon Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle Mol. Hum. Reprod., February 1, 2004; 10(2): 85 - 90. [Abstract] [Full Text] [PDF]
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A. L. Siebel, H. M. Gehring, I. G. T. Reytomas, and L. J. Parry Inhibition of Oxytocin Receptor and Estrogen Receptor-{alpha} Expression, But Not Relaxin Receptors (LGR7), in the Myometrium of Late Pregnant Relaxin Gene Knockout Mice Endocrinology, October 1, 2003; 144(10): 4272 - 4275. [Abstract] [Full Text] [PDF]
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