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Purpose: BRAF V600E , a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin., Methods: Immunohistochemical expressions of BRAF V600E , TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004., Results: BRAF V600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAF V600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAF V600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors., Conclusions: Our study provides the first evidence of the potential interaction of BRAF V600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAF V600E in thyroid carcinogenesis. BRAF V600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

Unlabelled: Intrauterine infection frequently leads to preterm birth (PTB), with the pathophysiology involving activation of the innate immune system and its associated inflammatory response. The choriodecidua produces relaxin (RLN) and elevated levels are associated with preterm premature rupture of the fetal membranes. However, it is not increased in bacterially-mediated PTB, but may act as an endogenous sterile inflammatory mediator. Elevated systemic RLN levels from the corpus luteum are also associated with PTB, but the mechanism is unknown. In clinical obstetrics, intrauterine inflammation or infection can coexist with elevated RLN. Therefore, in this study, we further characterized the effects of RLN alone or together with an inflammatory mediator on the production of IL1B, CSF2 (GM-CSF), IL6, IL8 and TNF, from chorionic cytotrophoblasts (CyT), decidual fibroblasts (DF) and stromal cells (DSC), using interleukin-1 beta (IL1B) to mimic sterile inflammation or lipopolysaccharide (LPS) for bacterial infection. Endogenous differences between the cells showed that the CyT expressed more RLN, its receptor RXFP1 and the RXFP1 splice variant D. CyT also showed the most robust cAMP response to RLN with increased IL6 secreted after 4 h, preceded by increased transcription at 1 h, likely due to activation of RXFP1 and cAMP. When all cell types were treated with IL1B and RLN, RLN augmented secretion of IL6 and IL8 from CyT and DF, but not DSC. Similarly, RLN augmented LPS-induced IL6 secretion from CyT and DF. Despite the structural similarity between TLR4 and RXFP1, blocking TLR4 in CyT had no effect on RLN-induced IL6 secretion, suggesting specific activation of RXFP1. Thus, we have shown that in the presence of a low level of intrauterine inflammation/infection, elevated RLN could act on the CyT and DF to augment the inflammatory response, contributing to the pathophysiology of PTB., Summary: RLN augments the inflammatory responses induced by IL1B or LPS in chorionic cytotrophoblasts and decidual fibroblasts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.

In the absence of infection, decidual relaxin (RLN) expression is increased in patients with preterm premature rupture of the membranes (PPROM) resulting in preterm birth, but it is not known whether inflammation stimulates RLN expression or vice versa. This study examined the effect of lipopolysaccharide (LPS) on the expression of RLN mRNA and secreted protein and whether RLN treatment influences secretion of proinflammatory cytokines from the fetal membranes. Explants of human fetal membranes in vitro and rhesus monkey fetal membranes in vivo were treated with LPS, which increased expression of IL-6 but had no effect on RLN. RLN treatment stimulated IL-6 and IL-8 secretion from choriodecidual explants in a subset of patients, as well as from isolated chorionic cytotrophoblast cells but not decidual cells. In vivo results obtained in rhesus monkeys after intra-amniotic infusion of RLN demonstrated increased IL-6 and IL-8 concentrations in amniotic fluid. Our results indicate that increased decidual RLN expression is independent of LPS but may induce a local sterile inflammatory process which potentially contributes to extracellular matrix degradation and weakening of the fetal membranes.

The human has two relaxins, termed H1 and H2, both of which are biologically active and co-expressed in the decidua, placenta and prostate; in the corpus luteum, the main source of circulating relaxin, only the H2 form is expressed. The reasons for this differential expression of the relaxin genes are unknown. The possibility that their 3'-untranslated regions (UTRs) contribute to this differential expression by affecting their mRNA stabilities was investigated. Thus the 3'-UTRs of both relaxin genes were isolated through a combined 3'-rapid amplification of cDNA ends-PCR (RACE-PCR) using poly (A)(+)RNA from human decidua, placenta, prostate and corpus luteum. The sequences obtained for each 3'-UTR were identical in the tissues examined, were AT-rich (72%) and showed 91% homology between relaxin H1 and H2 when maximally aligned to include several gaps, the significance of which is unknown. Relaxin H1 has two, and relaxin H2 has one, poly (A)(+) signal, in addition to one cytoplasmic polyadenylation element 30 nucleotides upstream of this. The mRNA levels of relaxin H1 and H2 in the prostate adenocarcinoma LNCaP.FGC cell line were determined by quantitative competitive RT-PCR. Relaxin H1 had a 10-fold greater number of molecules (approximately 2.5x10(7)) per microgram of total RNA than relaxin H2 (approximately 2.5x10(6)). The stability of relaxin H1 and H2 mRNAs were compared in LNCaP cells treated with the transcription inhibitor actinomycin D (10 mM) for 0, 1, 2, 4, 8, 10, 14, or 24 h. Half-lives of 3.17 days for relaxin H1 mRNA and 11. 4 h for relaxin H2 mRNA were obtained from semi-logarithmic plots. Thus both mRNAs are relatively stable; however, relaxin H1 mRNA is considerably more stable than relaxin H2, at least in LNCaP cells. This difference in their mRNA stability may partly explain the greater level of expression of relaxin H1 in these cells.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

Objective: This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis., Study Design: Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis., Results: In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted., Conclusion: A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.

The decidua and placenta synthesize the human relaxins, termed H1 and H2, believed to be involved in collagen remodeling in the amnion and chorion in an autocrine/paracrine manner. The developmental regulation of the relaxin genes was quantitated in normal pregnancy by in situ hybridization histochemistry with six 48-mer oligonucleotide probes that detect both relaxin genes. A significant increase in relaxin expression occurred in both decidua (p < 0.01) and placenta (p < 0.05) at 12.5-14.4 wk gestation, with the mean peak value in the placenta more than double that of the decidua, suggesting a coordinate regulation of the relaxin genes. At term after spontaneous labor and delivery, a marginal increase in both decidual and placental relaxin gene expression occurred. Given these normal data, three abnormal preterm situations were investigated: 1) premature uterine contractions without prior rupture of the membranes, 2) premature rupture of the fetal membranes (PPROM), 3) cesarean section for medical reasons with intact membranes and no uterine contractions. Tissues showing intrauterine infection were eliminated. Significantly more relaxin was expressed in the preterm decidua from patients with PPROM when compared to patients in group 1 (p < 0.02) or group 3 (p < 0.008). These data were confirmed by Northern analysis with a relaxin cRNA probe. The placental tissues after PPROM also had a significantly higher and a uniform overexpression of relaxin in the placental syncytiotrophoblast. Tissues collected at term, in comparison, showed no such increases in decidua or placenta.

Interstitial collagen types I and III are the predominant collagens in the amniotic and chorionic connective tissues. However, this matrix also contains proteoglycans, fibronectin, laminin, and elastin, which together with the collagens may undergo partial degradation prior to fetal membrane rupture at term. In this study, stromelysin (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were immunolocalized in fetal membranes obtained at term prior to labor. MMP-3 stained the cells of the amniotic epithelium, fibroblasts and macrophages of the amniotic and chorionic matrix, and those of the chorionic cytotrophoblast; there was no staining in the maternal decidua. TIMP-1 showed a similar staining pattern, except that the staining was darker in some amniotic epithelial cells and was present in the maternal decidua. The maternal decidua produces the two human relaxins H1 and H2; the latter, when incubated with explants of human fetal membranes, caused a dose-dependent and significant increase in expression of the MMP-3 gene and its secreted protein into the media. A significant effect of relaxin H2 on 92-kDa gelatinase (MMP-9) gene expression was also shown--an effect requiring poly(A)+ RNA rather than total RNA. Both relaxin H1 and H2 caused a significant increase in secretion of MMP-9 protein and its enzyme activity in the media. The magnitude of the effects of the two relaxins was similar, in contrast to findings from other biological studies in which relaxin H2 was shown to be more active. Neither of the relaxins had any effect on 72-kDa gelatinase (MMP-2) activity or on the TIMP-1 protein or its activity. This study suggests that local relaxins may be involved in the degradation of the complex fetal membrane extracellular matrix and may cause activation of an enzyme cascade resulting in fully activated MMP-9. Such effects could be important in the degradative pathways occurring in the amnion and chorion in the peripartal period.

Decidual and placental relaxins have been proposed as autocrine/ paracrine hormones in the remodeling of collagen in the amnion and chorion in the last weeks of pregnancy. The matrix metalloproteinase-1 (MMP-1) is a key enzyme in the degradation of the interstitial collagens which predominate in the fetal membranes. Distribution of the MMP-1 gene and of the MMP-1 protein was shown by in situ hybridization and immunolocalization, respectively, in amnion, chorion, and decidua collected from patients before the onset of spontaneous labor. The distribution of MMP-1 in the chorionic cytotrophoblast and decidua coincided with that of the human relaxin receptor, detected by tissue section autoradiography in tissues collected at the same stage of pregnancy. Fetal membrane explants were used to study the effect of exogenous human relaxin H2. These responded by a dose-dependent increase in expression of the MMP-1 gene, in its secreted protein, and in its enzyme activity in the medium. A similar dose-dependent increase in the tissue plasminogen activator (tPA) gene and protein upon exposure of the explants to relaxin H2 suggested a coordinated cascade system, resulting in increases in secreted activities of MMP-1, MMP-3 (stromelysin), and MMP-9 (gelatinase B). There was no effect on the genes or proteins for MMP-2 (gelatinase A) or tissue inhibitor of metalloproteinase-1 (TIMP-1), showing the specificity of the response. This coordinated regulation by relaxin H2 of tPA, MMP-1, MMP-3, and MMP-9 would result in more complete degradation of the fetal membrane extracellular matrix components.

A Northern blot containing poly(A)+ RNA isolated from pregnant rabbit placenta, uterus, ovary, mammary gland, psoas muscle, and intestine was hybridized with an oligonucleotide probe to porcine preprorelaxin. Only the placenta and uterus exhibited high levels of a 1-kb mRNA that hybridized to the probe. Porcine preprorelaxin primers were used to generate and amplify cDNAs from placental and uterine poly(A)+ RNA. Southern blot analysis of the placental and uterine cDNAs each revealed a single PCR product that hybridized with the porcine relaxin probe. The sequence of the cDNAs exhibited 100% identity with SQ10, a preprorelaxin-like gene identified in rabbit tracheobronchial epithelial cells. Identical results were obtained with primers specific for SQ10. SQ10 is expressed in tracheobronchial epithelial cells during abnormal conditions, such as mechanical or toxic injury. The protein is believed to have a role in tracheal epithelial cell differentiation and transformation that occurs under the above conditions. This work shows, for the first time, the presence of high levels of message for SQ10 in normal reproductive tissues. The expression of SQ10 in the uterus and placenta may suggest an important role in cell differentiation and transformation that occurs in these tissues during pregnancy.

Objective: To study the effect of synthetic h RLX-1 peptide on human and porcine myometrium in vitro in comparison to the effect of h RLX-2 peptide., Study Design: Myometrial strips from humans and pigs in late pregnancy were allowed to spontaneously contract in an organ bath. Muscle activity was recorded isometrically in response to varying doses of h RLX-1., Results: Synthetic h RLX-1 peptide at 0.13 micrograms/mL caused sustained and complete inhibition of pregnant pig myometrial contractility, comparable to that produced by the h RLX-2 peptide, showing, for the first time, its biologic activity in this system. However, a range of concentrations of h RLX-1 peptide (0.07-7.32 micrograms/mL) had no effect on human myometrial contractions in vitro, whereas h RLX-2 peptide had some, albeit minimal, activity in this system., Conclusion: This result shows that h RLX-1 peptide does not appear to be an inhibitor of human myometrial activity and that although the pig myometrial relaxin receptor does not appear to distinguish between the two human relaxins, the human relaxin receptor might. Thus, the control of spontaneous myometrial activity during pregnancy in the human appears to be different from that in other species.

The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 micrograms, when incubated at 27 degrees C for 60 min, at pH 7.5 with Mn2+ and Mg2+ ion concentrations of 2.0 mM. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (Ka) of 2.15 nM. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC50 of 28 pM, while an IC50 of 60 pM and 280 pM was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2. Relaxins H2 and H1 can readily displace the binding of either 32P-labelled human relaxins H1 or H2 and gave very similar displacement curves.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of the two human relaxin genes termed H1 and H2 in human reproductive tissues ranges from high to very low copy number depending upon the tissue and reproductive state. The aim of this study was to use two approaches to identify total relaxin transcripts (HI and H2) at the cellular level by using a human relaxin H2 riboprobe and a series of six 48-mer synthetic oligoprobes. The results obtained with both methods were similar in all tissues studied; however, a lower background was achieved with the riboprobe. This was especially noticeable after long exposure times, and a better resolution was generally achieved without clustering of the signals. Treatment of the tissues with proteinase-K failed to increase the sensitivity in any tissue with either probe. The relative levels of expression of the total relaxin gene transcripts was estimated from the different exposure times needed to obtain a good hybridization signal. Thus, the order of expression was: corpus luteum of pregnancy > corpus luteum of the cycle > placenta and prostate > decidua parietalis. The results agree well with immunolocalization of the peptide hormone previously performed with both heterologous and homologous relaxin antibodies; the exception was the lack of hybridization signal over the cells of the chorionic cytotrophoblast of the chorion laeve. This suggests that the levels of relaxin gene expression was below the level of detectability with the in situ hybridization technique or that these cells sequester, but do not synthesize, relaxin. Expression in the term placenta varied greatly from tissue to tissue and within any one tissue. A similar variability has been noted for relaxin in this tissue by immunocytochemistry. Methodology for the detection of total relaxin transcripts at the cellular level when expressed in a wide range of copy number will allow the developmental regulation of relaxin gene expression in reproductive and nonreproductive tissues to be visualized.

Immunoreactive relaxin is present in human breast cyst fluid and postpartum milk without concurrent detectable serum levels, suggesting that the breast is a site of relaxin synthesis. Monoclonal and polyclonal antibodies to human relaxin H2 have been used to immunolocalize relaxins in normal, benign and neoplastic breast tissues with the avidin-biotin immunostaining technique. In view of the similarities in amino acid sequence between H1 and H2 relaxins, these antibodies to H2 relaxin are likely to detect either or both relaxins present in tissue sections. Staining patterns with these antibodies were identical and showed positive diffuse cytoplasmic staining in normal, lobular and ductal epithelium and in myoepithelial cells in breast tissues from normal prepubertal, cyclic, gestational, lactational and postmenopausal females. Relaxin staining was also present in epithelial and myoepithelial cells of ducts and lobules in benign breast disease as well as in metaplastic epithelium of apocrine microcysts. All breast carcinomas (infiltrating ductal, tubular, medullary, intraductal and infiltrating lobular carcinomas) had strong uniform cytoplasmic staining within the neoplastic epithelial cells. All staining was abolished in normal and neoplastic tissues when the polyclonal antibody was preabsorbed with relaxin. It was necessary to distinguish between the possibilities of relaxins being sequestered by breast tissue and local synthesis. Therefore, the expression of the H1, H2 or both human relaxin genes in normal and neoplastic breast tissues was studied by the isolation of RNA, synthesis of first strand cDNA and amplification by PCR using primer sets which amplified either both H1 and H2, or specifically only H1 or H2 relaxin. The coamplification of both relaxin genes was verified by Southern analysis, diagnostic restriction enzyme digestion and sequencing. The primer set for H1 relaxin detected H1 gene expression in 1 out of 8 normal and 9 out of 12 neoplastic breast RNA samples. The H2 relaxin gene was found to be expressed in 3 out of 8 of the normal samples but in all 12 of the neoplastic samples, suggesting that this gene is expressed at higher copy number in the neoplastic tissues. This is the first demonstration of the cellular immunolocalization of relaxin and relaxin gene expression in normal and neoplastic breast. This should allow further exploration of relaxin's role(s) in normal breast physiology and in its tumorigenesis.

Immunostaining with antibodies to human relaxin (H2) suggests the presence of a relaxin-like peptide in the gastrointestinal tract and its tumours. The peptide is present in the cytoplasm of non-neoplastic cells at sites of cell exfoliation: the surface cells of the stomach, the small intestinal villi and the surface cells of the colon. It also occurs in the cytoplasm of gastric parietal cells and carcinomas of both the stomach and the colon. Relaxin has been shown to stimulate collagenase gene expression and to down-modulate collagen synthesis and secretion in human skin fibroblasts. The distribution of relaxin shown here suggests that it could play a role in the detachment of non-neoplastic cells from their basement membranes at the end of the cell cycle and in the penetration of carcinoma cells through the basement membrane.

Relaxin (RLX) is a product of the human corpus luteum, pregnancy decidua and placenta, prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) are products of the cyclic endometrium and of the pregnancy decidua. All three proteins are thought to function interdependently in endometrium/decidua as local factors within the uterus without reaching the systemic circulation. In this study, the avidin-biotin immunoperoxidase method for immunolocalization with monoclonal or polyclonal antibodies has been applied to serial sections of endometria obtained from patients at different stages of the menstrual cycle and in early and late gestation. This allowed the cellular localization of the three proteins to be followed simultaneously through the reproductive stages from cyclic endometrium to term gestational decidua. The production, as opposed to sequestration of RLX from an ovarian source was demonstrated by the application in parallel of an antibody to the processed hormone and its connecting peptide. RLX was shown localized to the glandular and luminal epithelia in the proliferative and secretory phases. The decidualized stromal cells also immunostained for RLX in the late secretory phase and in early and late pregnancy. PRL was localized first to the glandular epithelium and then stroma, appearing after RLX, IGFBP-1 appeared later in the secretory phase and predominantly in the decidualized stromal cells confirming previous studies. In contrast, all three proteins were immunostained in early pregnancy and increased to term gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of the relaxin gene transcript in the endometrium of the late pregnant guinea pig has been determined. The strategy used was a combination of polymerase chain reaction (PCR) with primers designed from the mRNA sequence of porcine preprorelaxin, rapid amplification of cDNA ends-PCR, and blunt end cloning in M13 mp18. With heterologous primers, a 226-basepair (bp) segment of the guinea pig relaxin gene sequence was obtained and was used to design a guinea pig-specific primer for use with the rapid amplification of cDNA ends-PCR method. The latter allowed completion of the sequence of 336 bp, with a 96-bp overlap. The sequence obtained shows greater homology at both the nucleotide and amino acid levels with porcine and human relaxins H1 and H2 than with rat relaxin, supporting the thesis that the guinea pig is not a rodent. The transcription of the guinea pig endometrial relaxin gene during pregnancy was confirmed by Northern analysis of guinea pig endometrial tissues with a species-specific cDNA probe. The endometrial relaxin gene is transcribed during pregnancy, but not in lactation, consistent with the observed immunostaining for relaxin.

Mastectomy in the guinea pig increases the incidence of still births and neonatal deaths compared with intact animals. The guinea pig has the endometrial gland cells (EGC) as the major source of relaxin, and this hormone has possible roles in uterine quiescence, cervical dilatation, and lengthening of the interpubic ligament in pregnancy. An effect of mastectomy on uterine relaxin has been sought by immunocytochemical localization during the estrous cycle, mid and late pregnancy and on endometrial relaxin gene expression in the late pregnant mastectomized animal by Northern analysis. Endometrium from midpregnant (day 35) and late pregnant (day 63) and from lactating (days 5, 21, and 28) guinea pigs immunostained with antiserum to porcine relaxin by the avidinbiotin technique. By increasing the sensitivity of the latter, relaxin immunostaining was also detected for the first time in EGC from cyclic animals (days 9 and 14). A pattern and intensity of relaxin immunostaining could be readily assigned to each of the stages examined: estrous cycle, midpregnancy, late pregnancy, lactation, and post weaning. The dark uniform staining of the EGC in late pregnancy was followed by sporadic staining of the EGC in lactation, returning to the cyclic picture after weaning. Endometrium from mastectomized cyclic and late pregnant guinea pigs showed a reduction in the amount of immunostaining compared with the relevant control animals. The reduction was most pronounced in the mastectomized late pregnant guinea pig. This result was reflected in an apparently lower level of relaxin mRNA in the endometrium of these animals compared to intact controls. These data indicate a novel linkage between the mammary gland, either directly or indirectly, to the nonpregnant or pregnant uterus. The loss of this signal appears to be associated with subsequent problems at parturition which may be linked to the reduction in endometrial gland relaxin production. The nature of this signal from the mammary gland, normally considered to be an exocrine rather than an endocrine gland, is unknown.

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Relaxin is a peptide hormone whose A- and B-chains are derived by posttranslational cleavage from a single 185-amino acid preprorelaxin. Two genes in the human genome (H1 and H2) code for two polypeptides significantly different in amino acid sequence. The full spectrum of biological activities of these two polypeptides has not been examined, but transcription appears to be limited to the H2 relaxin gene in the human corpus luteum. Relaxin is also synthesized by the human decidua, placental trophoblast, and prostate gland; therefore, the expression of the human relaxin genes in these tissues has been examined using the reverse transcription polymerase chain reaction. The mRNA from decidua, placental trophoblast, and prostate was reverse transcribed and then amplified by polymerase chain reaction, using a series of oligonucleotide primers that were specific for but would not distinguish between human H1 and H2 relaxins. Using mRNA from these tissues, two amplified cDNA species were detected, whose identities were confirmed by Southern blots, HpaI and HpaII restriction enzyme analysis, and dideoxy sequencing. We have confirmed that the corpus luteum does not contain detectable H1 relaxin mRNA. However, we demonstrated for the first time relaxin H1 gene expression in the decidua, placental trophoblast, and prostate, and we have also shown that there are marked tissue differences in the relative amounts of expression of the H1 and H2 relaxin mRNA forms. The functional significance is unknown, but if both mRNAs are translated, differential expression of the two genes may result in tissue-specific differences in the production of these relaxins as well as in their binding and actions.

The human placenta and decidua are intrauterine production sites for a range of polypeptide hormones. Relaxin is one of these hormones, its production having been demonstrated by immunocytochemistry and Northern analysis. There are two relaxin genes in the human genome, termed H1 and H2; only the latter is expressed in cyclic and pregnant corpus luteum. However, it has recently been shown that both genes are expressed in the decidua and placenta. It is not known whether both are translated. These hormone(s) may act in a paracrine fashion and be partly responsible for the control of enzymes and inhibitors involved in collagen remodelling in the fetal membranes in the last weeks of pregnancy. An autocrine role of decidual relaxin and a possible decidual-cell/macrophage/extracellular-matrix interaction is described; this may act as a unit in the elaboration of a range of hormones.

The ovaries of the ascidian Herdmania momus were extracted for relaxin. Relaxin immunoactivity was eluted from Sephadex G-50 in the position of authentic porcine ovarian relaxin and these fractions were parallel to porcine relaxin in an homologous porcine relaxin radioimmunoassay. Poly(A)+ RNA from ascidian ovaries hybridized to a 32P-labeled porcine relaxin B-chain 48 mer oligonucleotide probe. There was no hybridization with 32P-labeled probes of equal length but was rather directed to the B-chain of human relaxin or to two different regions of the C-peptide of porcine relaxin. We conclude that the ascidian ovary produces a relaxin-like molecule, possibly with greater homology in the B-chain to mammalian relaxins than in the C-peptide region.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.

Granulosa cells isolated from the ovaries of pregnant mare serum gonadotropin (PMSG)-treated immature rats were cultured with relaxin or FSH. Both hormones increased the secretion of plasminogen activator into the culture medium. Relaxin caused a dose-related rise in plasminogen activator but did not increase cAMP or progesterone levels in the medium of freshly harvested granulosa cells, although they were responsive to FSH. This ability of relaxin to stimulate plasminogen activator synthesis without progesterone or cAMP rises indicates that the pathways of post-receptor events leading to stimulation of plasminogen activator differ markedly from those of the gonadotropins. Relaxin is thus a fully characterized peptide hormone produced by the ovary with a well-defined action upon the granulosa cell and may have an intraovarian role in the events leading to ovulation.

The effect of relaxin on prostaglandin E (PGE) production by human amnion in vitro was investigated. When amniotic discs were incubated in the presence of increasing concentrations of relaxin, two distinct effects were observed. Discs prepared from women delivered by caesarean section before the onset of labour showed a significant decrease in PGE output at relaxin concentrations of 0.5-2 micrograms/ml; the effect was abolished at higher relaxin concentrations. Discs obtained from women delivered after labour of spontaneous onset responded to the addition of relaxin (4-8 micrograms/ml) with a significant increase in PGE output, although this increase was only evident in patients in whom labour had started with intact membranes. These results suggest that relaxin, which is present in decidua and chorion laeve at term, may have a paracrine effect on the amnion, inhibiting PGE production during continuing pregnancy but favouring its production during spontaneous labour.

A crude relaxin preparation has been obtained from the basal plate region of electric cesarean section human placentae, as well as normal term placentae. The relaxin isolated has different charge properties by ion exchange chromatography from porcine relaxin, although it is of approximately the same molecular size. The most potent fraction obtained has approximately 0.7% of the immunoactivity of purified porcine relaxin when compared with porcine relaxin in a RIA based on porcine material, suggesting significant amino acid sequence differences between the putative human relaxin and its porcine counterpart.

125I-labeled porcine relaxin was injected into 27-day-old rats treated with pregnant mare's serum gonadotropin (PMSG) and known target tissues for relaxin, the myometrium, endometrium and cervix, and putative control tissues, heart, thigh muscle and duodenum, examined for binding by autoradiography. Specific binding in the target tissues was demonstrated by simultaneous injection of excess unlabeled relaxin. Radioactivity was located and quantified by grain counts predominantly over the inner, circular muscle layer of the myometrium and the cervix and to a lesser extent over the outer longitudinal muscle layer of the myometrium and the endometrium. The route of injection, the circulation time, or counting grains in transverse or longitudinal sections of myometrium made little difference in these results. Ovariectomy decreased, but not significantly, the grain count in all of the target tissues studied and estrogen treatment of ovariectomized animals restored the numbers of grains to approximately that of intact PMSG-treated rats. The degree of binding of the cervix was approximately that of the circular myometrial muscle. This work confirms the presence of specific receptors for relaxin in the rat uterus and cervix of primed rats and it also suggests that the inhibitory action of relaxin upon the myometrium is primarily on the inner circular muscle layer.

Dispersed cells from human amnion and chorion were cultured with and without relaxin. The addition of this hormone caused an increased secretion of both collagenase and plasminogen activator into the culture medium over a 32 h period, but had no effect on proteoglycanase or beta-glucuronidase secretion. The increase in plasminogen activator was dose-related to the amount of relaxin added in vitro. The results show that the fetal membranes are a novel target tissue for relaxin in the human, and suggest that relaxin in vivo may cause a similar release of collagenolytic enzymes, leading to the weakening and eventual rupture of the fetal membranes.

Relaxin (Rlx) is shown in vitro to increase the release of plasminogen activator (PA) activity from granulosa cells obtained from 28-day-old rats after priming 48 h before with PMSG. Priming with PMSG was essential for the subsequent marked increase in PA by the addition of Rlx to these cells in vitro. Under the same conditions Rlx also increased the release of both total collagenase and total proteoglycanase activities but not of beta-glucuronidase activity. The total collagenase and proteoglycanase activities of control cells are made up of essentially equal amounts of their respective active and latent enzymes. Rlx stimulation increases the amounts of the respective active enzymes while the latent collagenase and proteoglycanase activities are unchanged or decreased, respectively. The enzyme beta-glucuronidase was not stimulated by Rlx and appears not to be involved in follicular proteoglycan degradation. Granulosa cells harvested from preantral follicles responded most to FSH by PA production whereas cells from antral follicles responded more to LH, reflecting the known changes in concentration of FSH and LH receptors on these cells. The release of PA is maximal by all four hormones studied (FSH, LH, prostaglandin E1, and Rlx) on granulosa cells harvested from rats 48 h after PMSG treatment and this suggests that the follicles at this time are a mixture of both preantral and antral stages. The PA response to FSH is lost by 60 h after PMSG at the same time that the response to prostaglandin E1 is maintained at the same level, whereas that to Rlx and LH, although still significantly higher than controls, were decreased. By 70 h after PMSG, postovulatory, the responses to all hormones studied were lost. Thus, the involvement of PA in ovarian connective tissue alterations appears to be greatest in the period of follicular antrum formation rather than just before ovulation. Rlx is one of a number of hormones involved in the sequence of events culminating in follicle connective tissue remodeling as shown by its action on the release of three intrafollicular enzymes.

Relaxin is an ovarian peptide that is released just prior to parturition to effect changes in the tissues of the birth canal that aid in the delivery of the fetus. Structural studies established that the two constituent polypeptide chains, composed of 22 and 30 amino acids, are joined by two interchain disulfide bonds with an additional third intrachain bridge. As well as the identical pattern of disulfide bonds, relaxin shows an overall 25% identity with insulin. Furthermore, the sequence of relaxin can be incorporated into the known three-dimensional structure of insulin without significant distortion of the main polypeptide chain backbone. The discovery of the insulin-relatedness of relaxin brings to three the number of growth factors that share a common structural gene precursor with insulin. Nerve growth factor and insulin-like growth factor have already been so identified. Inclusion in this hormone family suggests that the mechanism of action may involve internalization as well as complexation with cell surface receptors of target cells.

A biologically-active succinylated porcine relaxin was iodinated by a modification of the Bolton-Hunter method. Fibroblasts cultured from the mouse pubic symphysis and human skin were used to investigate relaxin binding sites. 125I-labelled relaxin binding to both cell types was time- and temperature-dependent. An accelerated rate of labelled hormone degradation (90%) was observed when both cell types were incubated at 37 degrees C. Specific relaxin binding sites on the mouse and human cells were observed as other peptides, such as insulin, epidermal growth factor, glucagon, hFSH and human prolactin, failed to inhibit relaxin binding. Further results indicate that porcine relaxin is mitogenic to these specific fibroblasts because increasing concentrations (10(-9) to 10(-6) M) of this hormone stimulated cell growth in vitro. These data suggest that the effect of relaxin at the target tissue level is mitotic in nature.

The production of PRL by the human decidua is generally accepted, but the production of relaxin by this tissue is not. The two hormones were localized in decidual tissue using the avidin-biotin immunoperoxidase procedure with antisera to human PRL and to a synthetic 14-amino acid sequence of the connecting peptide of human relaxin (hCp14). The object of using the hCp14 antiserum was to verify relaxin production by the detection of C-peptide and/or prorelaxin. Cells of the parietal decidua adherent to the fetal membranes stained with both antisera, and immunostaining for both hormones in the same cell was seen. Also, the decidua-like cells of the placental basal plate stained with both antisera. The chorionic cytotrophoblast stained with the antiserum to hCp14, but not the antiserum to human PRL, whereas the placental syncytiotrophoblast stained for PRL and/or human placental lactogen (hPL), but not hCp14. The PRL staining in all tissues was lost when anti-PRL serum absorbed with human placental lactogen (hPL) was used. This finding suggests that the antiserum to PRL could not distinguish between PRL and hPL. It appears, therefore, that the parietal decidua cells and the decidua-like cells of the placental basal plate may be capable of producing both relaxin and PRL, while the syncytiotrophoblast produces hPL and possibly PRL.

Plasma relaxin levels were measured in animals at different stages of lactation and related to the amount of nuzzling and suckling behavior exhibited by the piglets. Only in some acute suckling episodes was relaxin secreted rapidly and episodically in spite of normal piglet and sow behavior and interaction. However, when the piglets were removed from the dams 6 h before suckling, the sows were very restless and the relaxin response to suckling was delayed. Oxytocin injection in lactating but nonsuckled sows caused an episodic secretion of relaxin similar to suckling itself. The source of relaxin in the lactating sow may be the old corpus luteum, since progesterone levels increased acutely, somewhat reflecting the profile of relaxin increase over the suckling episode.

The pre-partum plasma concentrations of progesterone, relaxin, PGF-2 alpha and 13,14-dihydro-15-keto PGF-2 alpha (PGFM) were determined in 6 sows treated with meclofenamic acid (5.0 mg/kg body wt daily, from Day 109 to Day 113 of pregnancy), and 2 sows which farrowed prematurely. The inhibition of PG synthesis with meclofenamic acid did not prevent either the decline in plasma progesterone or the release of relaxin. Three distinct stages in the progressive changes of the above hormones were apparent before parturition: (1) the gradual decline in the concentrations of progesterone from about 7 days pre partum to reach less than 4.5 ng/ml at farrowing; (2) a surge in relaxin levels (greater than 100 ng/ml) by 2 days pre partum; and (3) sharp increases in the concentrations of PGF-2 alpha and PGFM within 24 h of parturition. Maximal concentrations of PGF-2 alpha (greater than 0.5 ng/ml) and PGFM (greater than 20.0 ng/ml) occurred during farrowing. It is suggested that PG from an extra-ovarian source may not be responsible for initiating functional regression of the corpora lutea in the sow.

Relaxin (Rlx) classically causes uterine quiescence during pregnancy and cervical dilatation prior to parturition. Its actions involve major changes in the components of the extracellular matrix of these tissues. The activities of three enzymes, collagenase, proteoglycanase and beta-glucuronidase, major determinants of the integrity of the extracellular matrix have been measured in the rat uterus and cervix in different reproductive states. The results show that there are marked differences in the changes of these enzymes occurring in the uterus and cervix during the course of pregnancy and the puerperium. It was not possible to directly relate these changes to a single hormonal event over this period of major endocrine fluctuations. Two models have therefore been used in an attempt to delineate the effects of oestrogen and Rlx on the tissue enzyme levels or their secretion into culture medium. In the first model cyclic animals were treated with oestrogen alone or oestrogen followed by Rlx and tissue enzyme levels measured. The addition of Rlx treatment reversed an inhibiting effect of oestrogen alone on both uterine and cervical collagenase and proteoglycanase activities, at the same time as completely obliterating the stimulating effect of oestrogen on uterine and cervical beta-glucuronidase activity. A second model used in vivo oestrogen priming of cyclic rats followed by in vitro Rlx treatment and measurement of the enzymes secreted into the culture medium over 7 days. The results showed as in the first model that Rlx treatment could in particular overcome the inhibiting effect of oestrogen on uterine proteoglycanase secretion without affecting beta-glucuronidase levels. In contrast, the effect of Rlx on the cervix was to decrease collagenase and proteoglycanase secretion whilst not affecting the beta-glucuronidase levels.

The endometrium of late pregnant guinea-pigs was found to contain mRNA for relaxin. Poly(A)+RNA hybridized to 32P-labelled porcine relaxin-specific oligonucleotide probes. These probes corresponded to the C-peptide region of the prohormone. There was no hybridization to a 32P-labelled probe to the B-chain of porcine relaxin even under conditions of low stringency. The size of the relaxin mRNA was approximately 1.0 kb and similar to that found for relaxin mRNA from the pregnant sow ovary. This is the first study on relaxin mRNA from a uterine source.

Relaxin is a product of the endometrial gland cells in the guinea pig. Mammary tissue was collected from intact cyclic animals, on days 35 and 63 of pregnancy, days 5 and 21 of lactation, and day 28 postpartum. Tissue was collected from six cyclic hysterectomized animals. Sections of mammary gland were immunostained with the avidin-biotin immunoperoxidase method, and antisera to porcine relaxin which were raised to different preparations in different laboratories. Light uniform staining was evident in the cytoplasm of all of the cuboidal epithelial cells forming the mammary duct system in cyclic animals; mammary gland from hysterectomized animals showed similar staining. At midpregnancy light staining was seen in some epithelial cells, and by late pregnancy there was only faint staining. On day 5 of lactation there was intense and uniform staining throughout the epithelial cells of the alveoli. By day 21 of lactation the cells still immunostained for relaxin, but on day 28 postpartum there was a return to the cyclic staining pattern. Endometrium from animals at 55-60 days of pregnancy and mammary gland from day 6 of lactation were used for poly(A)+ RNA isolation and Northern analysis. Three 48-mer oligonucleotide probes were used. Poly(A)+ RNA from endometrium of late pregnant guinea pigs and from the mammary gland in lactation hybridized with the same two probes and failed to hybridize with a third under moderate stringency conditions. The results suggest that the major source of relaxin in the guinea pig is sequential, the pregnant uterus and the lactating mammary gland. The local and/or systemic significance is not known.