Relaxin 2/RXFP1 Signaling Induces Cell Invasion via the ß-Catenin Pathway in Endometrial Cancer.

Relaxin is known to play an important role in animal pregnancies, including those of humans. It is suggested that relaxin induces aggressive cell growth and invasiveness in several types of cancer, including endometrial cancer. However, the mechanisms of relaxin remain largely unclear. In this study, we examined the effects of relaxin 2 (RLN2), the major circulating relaxin in humans, on human endometrial carcinoma cell lines. RLN2 treatment induced invasion in HEC-1B and Ishikawa cells. RLN2-induced cell invasion was significantly decreased by transfection of relaxin receptor 1 (RXFP1) siRNAs. The ß-catenin inhibitor, XAV939, also significantly inhibited the RLN2-induced cell invasions. Both a decrease of cadherin expression and an increase of ß-catenin phosphorylation were observed in response to the RLN2 treatment in HEC-1B and Ishikawa cells. We then examined RLN2 and RXFP1 expression in 80 human endometrioid endometrial carcinoma tissues. RLN2 immunoreactivity was detected in the human endometrial carcinoma cells and had a correlative tendency with histological grade and RXFP1. These results suggest that adherens junctions in cancer cells are weakened by the breakdown of the cadherin/catenin complex, which is induced by ß-catenin phosphorylation via RLN2/RXFP1 signaling.

Significance of relaxin-2 expression in hepatocellular carcinoma: relation with clinicopathological parameters.

BACKGROUND: A number of putative roles, including the modulation of tumor growth, neovascularization, metastasis and oncogenic progression, have been correlated to relaxin-2 overexpression. However, the clinical significance of relaxin-2 expression in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the expression of relaxin-2 in HCC and determine its correlation with tumor progression and prognosis. PATIENTS AND METHODS: 180 HCC patients who had undergone curative liver resection were selected and immunohistochemistry was performed to analyze relaxin-2 expression in the respective tumors. RESULTS: Immunohistochemistry confirmed relaxin-2 overexpression in HCC tissues compared with their adjacent nonneoplastic tissues (p < 0.01). Additionally, immunostaining showed more relaxin-2 positive cells in the higher tumor grade (III) than in the lower tumor stage (I, II; p = 0.026). Moreover, HCC patients with high relaxin-2 expression were significantly associated with lower 5-year overall survival (p < 0.01) and lower 5-year disease-free survival (p < 0.01), respectively. Furthermore, immunostaining showed more relaxin-2 positive cells in the tumor recurrence (ETR) patients than non-ETR patients (p = 0.001). The Cox proportional hazards model further showed that relaxin-2 was an independent poor prognostic factor for both 5-year disease-free survival (hazards ratio [HR] = 1.872, 95% confidence interval [CI] = 1.18-5.146, p = 0.023) and 5-year overall survival (HR = 3.637, CI = 1.443-7.15, p = 0.001) in HCC. CONCLUSIONS: Our data suggest for the first time that the overexpression of relaxin-2 protein in HCC tissues is of predictive value on tumor progression and poor prognosis.

Progesterone, pregnanediol-3-glucuronide, relaxin and oestrone sulphate concentrations in saliva, milk and urine of female alpacas (Vicugna pacos) and their application in pregnancy diagnosis.

The pregnancy-associated hormones, progesterone (P4), pregnanediol-3-glucuronide (PdG), relaxin (RLN) and oestrone sulphate (E1S) in plasma, saliva, milk and urine of alpacas were measured in order to assess their potential use for pregnancy diagnosis. Samples were obtained from 36 female alpacas before mating and at different stages throughout pregnancy (confirmed by ultrasonography). The hormone concentrations were determined using enzyme immunoassays. Milk samples were also tested using a commercial on-farm P4 kit, designed for dairy cattle. Although the concentration of P4 in plasma, milk and urine, and the concentration of PdG in urine were significantly higher in pregnant than in non-pregnant alpacas, there was no difference in the concentrations of P4 or PdG in saliva. The on-farm milk P4 kit showed a sensitivity of 90 per cent for diagnosis of pregnancy and a specificity of 69 per cent for non-pregnancy. The concentration of RLN in plasma increased significantly after the second month, and concentration of E1S in plasma and urine during the last month of pregnancy, whereas, there were no significant differences in RLN or E1S concentrations in saliva and milk between pregnant and non-pregnant alpacas. Values of P4, RLN and E1S in plasma, and PdG and E1S in urine are comparable with the previous reports in alpacas and, therefore, can be confirmed as an indicator for pregnancy. This is the first study to include determination of pregnancy-associated hormones in the saliva and milk of alpacas. However, saliva seems to be unsuitable for pregnancy diagnosis in alpacas, whereas, P4 in milk, as well as PdG and E1S in urine, seem to be adequate tools for diagnosis.

Characterization and biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.

Expression and functional role of hepatocyte growth factor and its receptor (c-met) during fetal mouse testis development.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine able to regulate different cellular functions. HGF action is mediated by its receptor, c-met, a glycoprotein with tyrosine kinase activity. We previously demonstrated that c-met is expressed in the newly formed seminiferous cords of the mice embryonic testes and that HGF acts as a morphogenetic factor. In this paper, we report that at 15.5 days post-coitum (dpc) c-met is expressed in the testicular cords, whereas at 18.5 dpc c-met expression is almost exclusively localized in the interstitial tissue of the testis in particular in the fetal Leydig cells. In addition, we demonstrate that HGF gene is expressed during the fetal period of testis development, heavily detectable in the interstitial compartment of 18.5 dpc testes. Interestingly, HGF is not expressed in the Leydig cells that, as above reported, express the HGF receptor. Looking for the functional role of HGF on Leydig cells, we evaluated the amount of testosterone secreted by testes isolated from 18.5 dpc embryos and cultured in the presence of HGF. The results of the in vitro organ culture show that, at this age, HGF increases the amount of testosterone secreted in the culture medium. On the contrary, HGF does not modulate the amount of testosterone secreted by testes isolated from 15.5 dpc embryos. In conclusion, we report that HGF is produced in the interstitial compartment of the developing testis but not by the Leydig cells. Conversely, the HGF receptor c-met is expressed in the Leydig cells and HGF modulates Leydig cell function during the late period of prenatal development.

Quantitation of estrogen receptors and relaxin binding in human anterior cruciate ligament fibroblasts.

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

The role of relaxin in endometrial cancer.

Relaxin (RLN) is a naturally occurring hormone that is known to modulate connective tissue remodeling in the uterus and cervix. Our goal was to investigate the role of RLN in endometrial cancer. RLN expression was evaluated using immunohistochemistry in 57 samples of invasive endometrial carcinoma (EC) and ten benign endometrial tissues. 67% of high-stage (III/IV) tumors demonstrated strong RLN expression compared to 37% of low-stage (I/II) cases. Strong RLN expression associated significantly with high-grade and depth of myometrial invasion. Notably, strong RLN expression was associated with a significantly shorter overall survival (p < 0.005) compared to weak or moderate expression. Using RT-PCR, the expression of RLN and its receptor (LGR7) was detected in EC cell lines (HEC-1B and KLE); in addition, LGR7 was expressed in 86% of 15 primary EC tissue samples. Exogenous RLN stimulation caused a significant increase in migration and invasion in both cell lines, but did not stimulate proliferation in vitro. Addition of the MMP inhibitor, FN439 abolished the stimulatory effect of RLN on invasion in both HEC-1B and KLE cells. RLN stimulation caused a significant increase in levels of activated MMP-2 in KLE cells and activated MMP-9 in HEC-1B cells compared to unstimulated cells. Inhibition of endogenous RLN signaling via siRNA targeted to LGR7 caused a significant reduction of EC cell invasiveness. Our results indicate that RLN overexpression is significantly asso- ciated with aggressive features such as high-grade and deep myometrial invasion. We provide the first evidence that overexpression of RLN is associated with poor clinical outcome in women with EC. RLN stimulation enhances the invasive potential of endometrial cancer cells by upregulating MMPs. In turn, downregulation of endogenous RLN signaling decreases invasiveness of endometrial cancer cells. These novel findings may have therapeutic implications in the management of patients with endometrial carcinoma.

Early human preantral follicles have relaxin and relaxin receptor (LGR7), and relaxin promotes their development.

The regulatory mechanisms of early follicle development are not clearly understood. Although relaxin is a peptide that controls cell proliferation and differentiation in many tissues, its role in human follicular development is unclear. In this study we cultured slices of human ovarian cortical tissue in the presence and absence of recombinant human relaxin. Ovarian tissue was obtained by biopsy during gynecological laparotomy or laparoscopy (14 women; mean age +/- sem, 29.0 +/- 6.1 yr; range, 17-37 yr). A significantly higher proportion of secondary follicles (14.5% vs. 5.0% in the control group; P < 0.01) and a significantly decreased proportion of primordial follicles (30.1% vs. 47.4% in the control group; P < 0.05) were found in tissues cultured with relaxin for 7 d. Immunocytochemical studies with the anti-C-peptide of prorelaxin and antirelaxin antibodies revealed the localization of relaxin in the oocyte and in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles. The presence of the relaxin receptor LGR7 was observed in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles by immunocytochemical and in situ hybridization analyses. These results suggest that relaxin plays a role through its receptor during the early stage of follicle development.

Interleukin-6, but not relaxin, predicts outcome of rescue cerclage in women with cervical incompetence.

OBJECTIVE: We investigated the potential roles of relaxin and subclinical intra-amniotic inflammation by quantitating amniotic fluid relaxin and interleukin-6 concentrations for the prediction of outcome of rescue cerclage in women with cervical incompetence. STUDY DESIGN: Cervical incompetence was diagnosed when cervical dilatation exceeded 2 cm with intact but bulging membranes and no detectable uterine activity. Each woman underwent amniocentesis to facilitate the performance of a rescue cerclage between 15 and 27 weeks of gestation (n=40 women). Forty-five additional women who underwent amniocentesis for chromosomal testing between 16 and 27 weeks of gestation served as a control group. All control patients were delivered of chromosomally normal infants at>37 weeks of gestation. All cases and control patients were singleton gestations. Interleukin-6 and relaxin were determined in all amniotic fluid samples by enzyme-linked immunosorbent assay. RESULTS: Amniotic fluid interleukin-6 levels were significantly higher in women with cervical incompetence than in control patients (control patients, 50.4 pg/mL [range, 19.4-97.4 pg/mL] vs cervical incompetence patients, 5459.1 pg/mL [range, 1131.4-14425.7 pg/mL] ; P < .001). In contrast to interleukin-6, relaxin levels did not differ between the 2 groups (control patients, 67.5 pg/mL [range, 35.1-153.5 pg/mL] vs cervical incompetence patients, 45.6 pg/mL [range, 30.1-75.5 pg/mL]; P=.061). There was a significant difference in interleukin-6 levels in women with shorter latencies (P < .01 for all latency intervals that were examined: delivery within 24 hours, 3 days, 7 days, before 33 and 37 completed weeks of gestation). Linear regression analysis with the use of the latency interval from cerclage to delivery as the dependent and with interleukin-6 as the independent variable revealed a significant inverse relationship (r=-0.62; P < .001 after log transformation of interleukin-6). There was no relationship on regression analysis between relaxin and the latency interval. CONCLUSION: Amniotic fluid interleukin-6 is increased in patients with cervical incompetence, which suggests that subclinical inflammation may contribute to cervical incompetence. Further, an elevated interleukin-6 level predicts a cerclage short-latency interval between cerclage and delivery. In contrast with interleukin-6, amniotic fluid relaxin does not appear to contribute to cervical incompetence-induced cervical dilation.

Adenovirus-mediated expression of human prorelaxin promotes the invasive potential of canine mammary cancer cells.

This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.

Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells.

Extensive evidence demonstrates pronounced effects of relaxin on the differentiation of human endometrial cells in vitro. In vivo data in rhesus monkeys suggest a role for relaxin in the development of endometrial vascular architecture. In women, pregnancy can be established and maintained in the absence of circulating relaxin. Thus, local synthesis by the endometrium is necessary if relaxin plays a physiological role in human endometrial function. Although relaxin protein and the prorelaxin C peptide have been localized to human endometrium, no data for relaxin synthesis have been provided to date. We therefore assessed relaxin mRNA and protein levels in cultured, defined human endometrial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of relaxin mRNA in human stromal and glandular epithelial cells. Secretion of the protein into the media of cultured cells of both types was also detected. Relaxin stimulated the expression of vascular endothelial growth factor in glandular epithelial and stromal cells that were isolated from tissue that had been taken during the secretory phase of the cycle. Relaxin inhibited the expression of procollagenase from both glandular epithelial cells, with a more marked inhibition demonstrated from cells that were isolated from tissue that had been taken during the secretory phase, and from stromal cells. These data demonstrate that human endometrial cells synthesize relaxin, and they support the concept that relaxin fosters endometrial conditions that are required for implantation in women.

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

Concentrations of tissue-type plasminogen activator and relaxin in normal and induced-cystic follicles of gilts.

Manipulation of one ovary in prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) results in cysts on the manipulated ovary and corpora lutea (CL) on the non-manipulated (control) ovary. Because tissue-type plasminogen activator (tPA) might play a role in follicular rupture and because relaxin might increase tPA production, concentrations of tPA and relaxin in manipulated and control follicles were measured at different stages of development. Prepubertal gilts were treated with 1000 IU PMSG followed by 750 IU hCG at 72 hr later. Follicles on one ovary in each gilt were manipulated at laparotomy 48 hr after PMSG administration. Gilts were ovariectomized at 72, 90, 108, 114, 144, and 216 hr after PMSG. Concentrations of tPA and relaxin were determined for follicular fluid from follicles dissected free of ovarian stroma and snap frozen in liquid nitrogen and media from follicles cultured for 48 hr. Relaxin did not differ between treatment groups (manipulated and control) at any time (P > 0.05); whereas, tPA was greater in control follicles at 114 hr after PMSG than in manipulated follicles (P < 0.01). The effect of pyrilamine, a histamine-1 receptor antagonist, on tPA concentrations was determined in manipulated and control follicles collected at 3, 12, 24, 42, and 66 hr after manipulation. Concentrations of tPA were similar in control and manipulated follicles for gilts treated with pyrilamine, but again control follicles had greater (P < 0.05) tPA concentrations at 114 hr after PMSG. Thus, tPA seems to be involved in ovulation, and blockage of ovulation and subsequent cyst formation results from inadequate tPA activity in manipulated follicles.

Role of high-performance liquid chromatographic protein analysis in developing fermentation processes for recombinant human growth hormone, relaxin, antibody fragments and lymphotoxin.

Development of efficient and reliable fermentation processes for protein pharmaceuticals is aided by the availability of accurate quantitative and qualitative product analyses. We have developed a variety of single and dual column chromatographic separations that meet the needs of process development and examples will be provided of how the resulting data has been used to optimize the culture process. For single column methods, reversed-phase chromatography has been the most versatile, permitting the reliable quantitation of many yeast, Chinese hamster ovary (CHO) cell and Escherichia coli-expressed products in the matrix of culture broth or cell extract. Analysis of secreted human growth hormone synthesized in E. coli, along with clipped and unprocessed forms, will be discussed. Another reversed-phase assay for direct analysis of a peptide product (B-chain relaxin) and its degradation products secreted into E. coli fermentation medium has allowed the purification of the responsible protease. Cation-exchange has proven extremely useful for the direct analysis of antibody fragment synthesized in E. coli, allowing the separation and quantitation of the desired Fab' and Fab'2, as well as the unwanted products of glutathione addition and translational read-through. Assay development is often complicated by the presence of host proteins with chromatographic behavior that is similar to that of the product. Commercial instrumentation now permits the facile development of multidimensional chromatographic assays. We show examples of coupled receptor affinity-reversed-phase assays for a mistranslation product and for covalent multimers of E. coli-synthesized lymphotoxin.

Pathogenetic relevance of the pregnancy hormone relaxin to inborn hip instability.

The etiology of inborn hip dysplasia is unknown. In general, a multifactorial genesis is assumed. The influence of hormones on the development of the fetal hip joint and its stability is discussed as well as mechanical influences. This study was carried out with the intention to examine the correlation between the concentration of the pregnancy hormone relaxin and the stability of the hip joint in newborns. Both hips of 90 newborn children were examined clinically and sonographically. In 25 hips (13.9%), pathological sonograms according to the classification of Graf were found. The relaxin concentration was measured in cord blood using a heterologous radioimmunoassay. Statistical evaluation revealed an insignificant decrease of relaxin concentration with increasing sonographic hip instability. The results indicate that hip instability frequently occurs with decreasing relaxin concentration. These facts contradict the earlier assumption that hip instability coincides with increased relaxin concentrations in newborns. We assume that there is a worse preparation of the pelvis and the birth canal during pregnancy due to the lower relaxin concentration and thus that there could be a higher pressure on the fetus in the perinatal phase. A decreased relaxin concentration seems to have no direct effect on the hip joint tissue, but indirectly there is consequent rigidity of the tissue in mother and child, which can further promote the development of hip instability.

In vitro transformation of rabbit cytotrophoblast cells into syncytiotrophoblast: stimulation of hormone secretion by progesterone and dibutyryl cyclic 3',5'-adenosine monophosphate.

Rabbit placenta syncytiotrophoblast cells exhibit immunostaining for the hormone relaxin. The objectives of this study were to evaluate the ability of cultured rabbit trophoblast cells to secrete immunoreactive (IR) relaxin and then to study the effects of progesterone, which is essential for maintenance of the placenta and of pregnancy in the rabbit, on that secretion. On Day 1, both treated and untreated trophoblast cell cultures consisted of 90% relaxin-negative mononuclear cells (cytotrophoblast and fibroblast) and 10% relaxin-positive multinuclear cells (syncytiotrophoblast). Media from untreated cultures, collected throughout 9 days of culture, contained low but constant levels of relaxin. Electron microscopy studies indicated that relaxin was localized in dense granules of the multinuclear cells and that these cells formed by fusion of mononuclear cytotrophoblast. Progesterone treatment (40 and 80 ng/ml) increased (p < 0.0001) media concentrations of relaxin, increased the number of desmosomes between cytotrophoblast cells (12 vs. 4 for controls on Day 5), and increased the percentage of multinuclear cells (73% of the cell population vs. 20% for controls on Day 7). Specificity of the progesterone effect was evaluated by treatment of cultures with dibutyryl cAMP (dbcAMP), insulin, hCG, estradiol-17 beta, prostaglandin E2, and prostaglandin F2 alpha. Only dbcAMP (2 mM and 4 mM) produced an increase (p < 0.0001) in media concentrations of relaxin. These results indicate that, like the intact placenta, cultured cytotrophoblast cells fuse to form syncytiotrophoblast and that the latter contain IR relaxin.

Transmission of relaxin from lactating bitches to their offspring via suckling.

The 6-kDa polypeptide hormone relaxin (Rlx) has been identified in human and bovine milk, and we recently reported its presence in canine milk. We postulated that Rlx might be transferred via suckling to the newborn pups, where, by virtue of its known effects to increase the distensibility of the pelvic connective tissues, it could play a role in causing the excessive laxity of the capsule and ligaments of the coxofemoral joint that precedes the development of hip dysplasia in genetically predisposed animals. Rlx was found in the serum of dysplastic (HD+) bitches for up to 6 wk of lactation, whereas it was detected in the serum of nondysplastic (HD-) bitches for only 1-2 wk of lactation. Rlx concentrations in milk were up to 60-fold greater than in serum. Milk Rlx levels varied markedly, but were highest during the first week of lactation and decreased thereafter. There were no significant differences in milk Rlx concentrations between HD+ and HD- bitches. Although the source of Rlx in milk is unknown, it cannot be the ovary or uterus, since hystero-ovariectomy performed at the time of cesarean section did not eliminate Rlx from milk during subsequent lactation. In serum samples taken from newborn pups before suckling, there were significant quantities of Rlx, demonstrating that the hormone enters the fetus in utero. However, Rlx rapidly disappears from serum of pups prevented from suckling for five hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical identification of a relaxin-like protein in gastrointestinal epithelium and carcinoma: a preliminary report.

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.

Measurement of periimplantational relaxin concentrations in the macaque using a homologous assay.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry.

Light-microscope immunocytochemistry using the peroxidase-antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo-urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows. Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin. These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.