Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Pregnant mouse corpora lutea: immunocytochemical localization of relaxin and ultrastructure

Relaxin was localized in corpora lutea of pregnant mouse ovaries by using the unlabeled antibody peroxidase-antiperoxidase technique and a highly specific rabbit antirat relaxin serum. Relaxin immunostaining was first observed in luteal cells located at the periphery of corpora lutea on Day 10 of gestation. The number of relaxin immunostained cells and the intensity of the stain gradually increased to reach a maximum between Days 16 and 18 of gestation. While a few luteal cells were specifically stained for relaxin on Day 1 postpartum, no luteal cells were stained on Day 2 postpartum. Ultrastructural studies of luteal cells from pregnant mouse ovaries revealed the presence of a distinct electron-dense, membrane-bound granule population, which was first observed on Day 12 of gestation. The granules increased in number to reach a maximum between Days 16 and 18 of gestation, and were absent by Day 2 postpartum. The appearance and disappearance of this granule population closely paralleled the relaxin immunostaining in the luteal cells. We suggest that the granules may be the subcellular sites of relaxin storage in the pregnant mouse ovary.     

Immunohistochemical co-localization of placental lactogen II and relaxin in the golden hamster (Mesocricetus auratus)

Two hormones with lactogenic activity are produced by the hamster placenta during the second half of pregnancy. One of these hormones, hamster placental lactogen II (haPL-II), has been well characterized; however, its cellular source is not known. In the present study, haPL-II was localized in placental tissues using a specific antibody and the avidin-biotin-peroxidase immunohistochemical technique. Because relaxin has been localized in the hamster placenta, it was of interest to determine if haPL-II and relaxin are localized in the same cells. haPL-II immunoactivity was observed in primary and secondary giant trophoblast cells of the placenta on Days 12, 14, and 15 of pregnancy. On Day 15 positive staining was also observed in large cells located within mesometrial arteries and in eosinophilic bodies associated with degenerating sheathed arteries of the decidua basalis. haPL-II-positive staining was not observed in placentae from Days 8 or 10 of pregnancy. On Day 14, haPL-II was colocalized with relaxin in 75% of the giant trophoblast cells observed. Therefore, it is probable that these hormones are synthesized and secreted by the same cell.     

Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta

The purpose of this investigation was to identify the cellular origin of placental lactogen-I (PL-I) expression in the mouse placenta and to cytologically define the transition from PL-I to PL-II expression during gestation. PL-I mRNA expression was assessed by in situ hybridization, and expression of PL-I and PL-II protein was determined by immunocytochemical analysis. PL-I mRNA and protein were localized to trophoblast giant cells. Trophoblast giant cells ceased producing PL-I at midgestation and began expressing PL-II. PL-I immunoreactivity was present in trophoblast giant cells on Days 9 and 10 of gestation but was not detectable in trophoblast giant cells on Day 11 of gestation. Immunoreactive PL-II-producing giant cells were detected first on Day 10 of gestation, continuing on Day 11 of gestation. Expression of PL-I and PL-II signals a significant functional transition in trophoblast giant cells of the developing mouse placenta.     

Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.     

Determination of the source of immunoreactive relaxin in the cat

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.     

Rabbit endometrial relaxin: immunohistochemical localization during preimplantation, pregnancy, and lactation

Relaxin was localized in rabbit endometrium (but not ovary) on Days 4-30 of pregnancy and Days 2-5 of lactation. The hormone was not observed on Days 2 and 3 post coitus. Relaxin was found in endometrial glands throughout the length of the uterus on Days 4-9 post coitus. Later, on Days 11-23, relaxin was localized in both uterine endometrial gland cells and luminal epithelial cells. At this time, staining was observed only in the endometrium directly associated with implantation sites. Areas between implantation sites were devoid of staining. On Days 25-30 of pregnancy, relaxin was found mainly in uterine luminal epithelial cells. Few glands were observed with relaxin. During the first week of lactation, the staining profile was the same as that observed on Days 25-30. Relaxin was not found in the endometrium of pseudopregnant rabbits (Days 1, 4, 8, 12, and 16). The early appearance of uterine relaxin at the time the blastocyst migrates into the uterine cavity coupled with the hormone's later confinement to implantation sites suggests that the blastocyst initiates and the conceptus maintains uterine relaxin.     

Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.     

Ultrastructural morphology and relaxin immunolocalization in giant trophoblast cells of the golden hamster placenta

Relaxin immunoreactivity was previously demonstrated in three cell types within the hamster placenta; fetal primary and secondary giant trophoblast cells (GTCs) and maternal endometrial granulocytes. The objectives of the present research were to examine the ultrastructure of the GTCs and identify the intracellular relaxin storage site. Primary GTCs, first present on day 8 of gestation, were characterized by numerous polyribosomes and large heterogeneous cytoplasmic inclusions suggesting phagocytic activity. Primary and secondary GTCs from days 10, 14, and 15 of gestation contained numerous polyribosomes, mitochondria with tubular cristae, and extensive Golgi complex, and abundant rough endoplasmic reticulum, all characteristics of a cell actively involved in protein synthesis. Membrane-bound secretory granules were not present. Relaxin was immunolocalized within the Golgi complex of primary and secondary GTCs using the avidin-biotin-peroxidase method. Following differential centrifugation of hamster placental homogenates and radioimmunoassay (RIA) of subcellular fractions, the majority of relaxin immunoactivity was detected in the postmicrosomal fraction; however, the majority of relaxin immunoactivity from similarly treated pig corpora lutea was present in the mitochondrial/granule fraction. These data indicate that hamster placental relaxin is not stored in membrane-bound secretory granules but is contained within the extensive Golgi complex of the GTC.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig

Relaxin was localized in luteal cells of ovaries from nonpregnant, pseudopregnant, and pregnant pigs using porcine relaxin antiserum and peroxidase-antiperoxidase light microscopy immunohistochemistry. The number of immunoreactive cells seemed to increase from Days 17 to 106 of gestation. Luteal cells from pseudopregnant (Day 110) and nonpregnant (Day 14 of the estrous cycle) pigs were also positive for relaxin. However, less than 3% of the luteal cells in the nonpregnant animals were immunoreactive. Electron microscopy immunocytochemistry using porcine relaxin antiserum and goat antirabbit immunoglobulin G-colloidal gold demonstrated that relaxin was packaged in the small membrane-bound granules in luteal cells of pregnant as well as pseudopregnant and nonpregnant pigs. The intensity of labeling (number of gold particles) of the granules increased with pregnancy. There was a 10-fold increase in labeling of granules with the 10-nm versus 25-nm diameter gold. The goat antirabbit labeled with the smaller 10-nm gold particles was necessary to demonstrate the apparent low levels of relaxin in the luteal cells of the nonpregnant pigs. These data further indicate that pregnancy is not required for relaxin synthesis. However, physiologic significance of relaxin in corpora lutea of nonpregnant pigs has not been determined.     

Effect of oestradiol-17 beta on ovarian and serum concentrations of relaxin during the second half of pregnancy in the rat

Immunoactivity concentrations of ovarian relaxin, serum relaxin and serum progesterone were determined from Day 12 through Day 18 of pregnancy in rats treated with oil or oestradiol-17 beta after hysterectomy or hypophysectomy and hysterectomy on Day 12. Relaxin and progesterone concentrations increased between Days 12 and 18 in sham-operated rats but failed to increase or declined in oil-treated hysterectomized or hypophysectomized-hysterectomized animals. Oestradiol treatment increased serum concentrations of relaxin and progesterone in hypophysectomized-hysterectomized rats on Day 15 and increased the concentrations of ovarian and serum relaxin and serum progesterone in hysterectomized rats on Day 18. These data are consistent with the concept that placental support for the promotion and maintenance of relaxin and progesterone concentrations from Day 12 through Day 18 may be mediated, at least in part, through a common mechanism(s) which involves oestradiol.     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile.

A porcine relaxin radioimmunoassay was developed to evaluate the profile of immunoreactive relaxin in rabbit plasma. Relaxin was nondetectable in pseudopregnant (Days 1, 4, 5-8, 12, and 16), nonpregnant, and male rabbits. However, in pregnant rabbits, relaxin was detected during the peri-implantation period (Days 4-9). Peak concentrations were reached on Day 15 and were maintained until parturition (Day 32). Relaxin concentrations abruptly decreased on Day 1 postpartum to low but detectable concentrations that were unchanged during the first week postpartum. In contrast, progesterone concentrations peaked earlier (Day 13), decreased after Day 25, and were not detectable on Day 1 postpartum. The effect of ovariectomy on the profile of plasma relaxin was evaluated. Four pregnant rabbits were ovariectomized (Day 13) and treated with medroxyprogesterone acetate to maintain pregnancy. As in normal pregnant rabbits, relaxin was observed initially during the peri-implantation period (Days 4-9) and increased to peak concentrations by Day 16. These concentrations were maintained until parturition and abruptly decreased on Day 1 postpartum to low yet detectable concentrations during the first week postpartum. The concentrations of relaxin in the plasma of ovariectomized medroxyprogesterone-treated rabbits were not different from those in three sham controls. These results indicate that the ovary is not a significant source of relaxin in pregnant rabbits. The unique observation of the presence of relaxin during the peri-implantation period suggests that this hormone has a role in preparing the rabbit uterus for implantation. The continued presence of relaxin during the first week postpartum may represent residual hormone, or it may suggest a physiological role during the early postpartum period.     

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.     

Relaxin activity in the pregnant cat

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.     

Ergocryptine and pregnancy maintenance in hamsters.

Ergocryptine (ECR) terminated pregnancy in hamsters when administered on Day 5; when ECR was given on Day 6 the response was diminished, and pregnancy continued after ECR treatment on Day 7. The abortifacient action of ECR in Day 5 pregnant hamsters was overcome by exogenous prolactin but not FSH and LH. When sera collected from hamsters on different days of gestation were examined for their ability to neutralize the effect of ECR in Day 5 pregnant hamsters, a peak of luteotrophic activity was observed in sera collected on Days 10 and 11. The results of these studies suggest that in hamsters the role of hypophyseal prolactin in luteal support is diminished by Day 7 of pregnancy, and the appearance of luteotrophic activity in sera collected on Days 10 and 11 may be indicative of a placental luteotrophin.