Effect of estradiol and relaxin on growth of fibroblastic cells isolated from guinea pig mammary glands

Fibroblasts were isolated from the mammary glands of guinea pigs and grown in 96-well culture plates. They were treated with a factorial arrangement of porcine relaxin (0.0, 0.5, 1.0 or 1.5 micrograms/ml) and estradiol-17 beta (0, 200, 400 or 600 pg/ml). Tritiated thymidine or uridine was added to a final activity of 25 nCi per well and the cells incubated at 37 degrees C for 48 h. Cells were then harvested onto filter paper and counted for tritium. Controls (0.0 micrograms/ml relaxin and 0 pg/ml estradiol) incorporated 3.7 nCi of tritiated thymidine and 4.8 nCi tritiated uridine. Both relaxin and estradiol altered the incorporation of thymidine and uridine. There was also an interaction between the two hormones. Thymidine incorporation with no estradiol and 1.5 micrograms/ml relaxin was 129% of controls. The optimum incorporation of thymidine occurred with 0.5 micrograms/ml relaxin and 400 pg/ml estradiol. This combination of hormones gave a response of 145% of controls. Uridine incorporation followed a different pattern. Relaxin alone at a concentration of 1.5 micrograms/ml gave a near-optimum response of 141% of control. The optimum combination of relaxin and estradiol for uridine incorporation was 1.5 micrograms/ml relaxin and 400 pg/ml estradiol, which gave a response of 156% of controls. These data indicated that relaxin and estradiol alter DNA and RNA synthesis in mammary fibroblasts and thus may be important in controlling the growth of the mammary gland stroma.     

Effect of estradiol and relaxin on collagen and non-collagen protein synthesis by mammary fibroblasts

In order to determine the effect of relaxin and estradiol on collagen and noncollagen synthesis by mammary gland fibroblasts, fibroblasts isolated from guinea pig mammary glands were grown on plastic or Cytodex-3 collagen coated microcarriers. On plastic, estradiol (600 pg/ml) increased the incorporation of tritiated glycine into collagen. Non-collagenous protein synthesis was increased at all concentrations of estradiol, but it was greatest with 200 pg/ml estradiol. On Cytodex-3, 400 pg/ml estradiol increased the synthesis of collagen and non-collagenous protein. Relaxin (1 microgram/ml) did not affect collagen synthesis but decreased the synthesis of non-collagenous protein.     

Inhibition of postpartum uterine involution in the rat by relaxin

The possible contribution of relaxin to the support of uterine accommodation during late gestation by retarding tissue lysis was examined using the involuting postpartum uteri of unilaterally pregnant rats. In otherwise intact animals, twice-daily administration of 0.1 mg of relaxin (porcine fraction B) significantly retarded the regression of both gravid and, to a greater extent, nongravid tissue during the first 4 days postpartum, and collagenolysis was similarly delayed. Immediate postpartum ovariectomy had little effect on the uterus, although 5 micrograms estradiol benzoate daily suppressed uterine involution in the gravid tissue to about 50% and was even more effective in the nongravid uterus. Relaxin alone had little effect on the gravid uterus following ovariectomy, but augmented estrogen to the extent that less than half of the tissue and its collagen were lost during 4 days. The effect on nongravid tissue was even more striking in that the combination of estrogen and relaxin prevented any degradation of tissue in general or of collagen. Although we have reported that relaxin can stimulate uterine collagen synthesis as well as uterine growth, the magnitude of its postpartum effect in the presence of estrogen suggests a stabilizing or anticatabolic effect upon the uterus.     

Relaxin and beta-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by beta-estradiol. We postulated that relaxin and beta-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, beta-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered beta-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 microg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. Beta-estradiol, relaxin, or beta-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or beta-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by beta-estradiol, relaxin, or beta-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

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.     

Influence of submandibular salivary glands on hormone responsiveness of mouse mammary glands

Surgical removal of the submandibular salivary glands (sialoadenectomy) of female Balb/c mice significantly (P less than 0.05) reduced mammary development as judged by development scores and mammae DNA levels. Reduction in mammae development score by sialoadenectomy was observed in both mice saline injected and mice treated with estradiol and progesterone. Autografts of submandibular salivary tissue or daily administration of EGF to sialoadenectomized mice partly alleviated the atrophy of the mammary gland induced by sialoadenectomy (P less than 0.05). The results of our studies are consistent with a model of mammary gland developmental regulation that includes the submandibular salivary gland as a mediator of mammogenesis via secretion of EGF.     

Progesterone inhibits the uterotrophic effect of relaxin in immature rats

Injection of progesterone for 3 days before treatment with relaxin inhibited the trophic effect of the peptide in both estrogen-primed and unprimed uteri. The depression in collagen concentration and increase in apparent rate of proline incorporation into collagen induced by relaxin alone were also eliminated, indicating a fundamental blockade of the effect of relaxin in this experimental design as well as a close association of changes in collagen concentration with tissue hypertrophy. The effect of relaxin on incorporation of proline into soluble protein was not blocked by progesterone, however, suggesting a separate mechanism for this anabolic effect of relaxin.     

Synergism between prolactin and ovarian hormones of DNA synthesis in rats mammary gland.

Autoradiography with [30H] thymedine has been used to measure the rate of DNA synthesis in the mammary epithelium of hypophysectomized-ovariectomized rats under the influence of estradiol, progesterone, and prolactin. Controls and animals treated with estradiol did not increase [3-H] thymidine incorporation, while progesterone alone had a slight stimulatory effect. Prolactin alone stimulated some [3-H] thymidine uptake in ductal and alveolar epithelium, but when combined with either estradiol or progesterone synergistic effects were observed. Estradiol with prolactin stimulated incorporation primarily in the ductal epithelium, whereas progesterone with prolactin stimulated both ductal and alveolar epithelium.     

Cyclic adenosine 3'5'-monophosphate and the relaxant action of relaxin in the rat uterus in vivo.

The relationship between relaxant actions of relaxin in the uterus and changes in uterine cAMP concentrations was assessed in anaesthetized bilaterally ovariectomized nonpregnant rats. Relaxin i.v. bolus (5 micrograms kg-1) did not change cAMP concentrations but inhibited uterine contractions with rapid onset. Uterine contractions were significantly reduced by 30-70% for 60 min. Relaxin (50 micrograms kg-1) produced a short-lived (up to 5 min) and small (up to 3.2-fold) increase in cAMP concentrations plus a marked (90%) and prolonged inhibition of uterine contractions (70-90% over 60 min). Salbutamol (an agonist at beta 2-adrenoceptors, 100 and 500 micrograms kg-1) produced a similar degree and time course of inhibition of uterine contractions to that of relaxin but a more marked (19-fold) increase in cAMP concentrations. Glibenclamide, a blocker of ATP-sensitive potassium channels, which has been shown to antagonize relaxin as a uterine relaxant, did not prevent the relaxin-induced rise in cAMP concentrations. It is suggested that the uterine relaxant action of relaxin may not result from an increase in uterine cAMP concentrations.     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

Effects of estradiol and progesterone on accumulation of relaxin- and carbohydrate-containing granules in endometrial gland cells of the guinea pig

Guinea pigs were spayed and given various regimens of injections of estradiol and progesterone. The following were monitored in each animal: pubic separation (relaxin stimulation), resorption of the vaginal membrane (estrogen priming), and the presence of PAS-positive granules and/or relaxin in endometrial gland cells (EGC). Injections of estradiol alone resulted in resorption of the vaginal membrane, accumulation of PAS-positive granules in EGC of all animals, and accumulation of relaxin in EGC of two of four animals; but they did not cause pubic separation. Progesterone injections did not result in resorption of the vaginal membrane, separation of pubes, or accumulation of PAS-positive granules; however, one of three animals demonstrated a few EGC that contained relaxin. Animals that received both estradiol and progesterone exhibited PAS-positive granules and relaxin in EGC as well as separated pubes, but did not have resorbed vaginal membranes. Upon examination with the electron microscope, EGC from animals that received estradiol alone exhibited remarkable numbers of secretory granules that contained a carbohydrate-rich material. Secretory granules were not prominent in EGC from animals that received progesterone alone. Estradiol and progesterone injections resulted in accumulation of secretory granules in EGC that contained relaxin and a carbohydrate-rich material. The observations that estradiol and progesterone induce relaxin production in EGC support the hypothesis that uterine relaxin plays an important role in pregnancy and/or parturition in the guinea pig.     

A physiological dose of estradiol with progesterone affects striatum biogenic amines

The acute effect of estradiol and progesterone on dopamine and serotonin metabolism in rat striatum was studied. One subcutaneous injection of 17 beta-estradiol (300 ng) and progesterone (150 micrograms) into intact male rats increased plasma levels of these steroids, while testosterone, corticosterone, and estrone remained unchanged. Dehydroepiandrosterone, androstane-3 beta, 17 beta-diol and dihydrotestosterone remained undetectably low. Prolactin decreased and androstane-3 alpha, 17 beta-diol, and 17-OH progesterone increased, but less than estradiol and progesterone. Peak levels of striatal dopamine, dihydroxyphenylacetic acid, and homovanillic acid were observed 15-45 min after steroid injection with a return to control values after 45-60 min, while serotonin and 5-hydroxyindoleacetic acid levels were slightly decreased. An injection of estradiol (70 ng) with progesterone (70 micrograms) to ovariectomized female rats left plasma prolactin levels unchanged, while striatum dopamine and serotonin as well as their metabolite concentrations peaked 15-60 min after steroid injection and returned to control values after 45-75 min. To allow for a better comparison of the action of these steroids, the effect of estradiol or progesterone alone and in combination on the brain of ovariectomized rats was compared in the same experiment. A similar increase in metabolites of dopamine levels was observed after these steroids alone or in combination, while dopamine levels were increased only after progesterone alone or in combination with estradiol. An injection of estradiol or progesterone to ovariectomized rats led to peak steroid concentrations at approximately the same time in the brain and plasma. In addition, plasma and brain steroid levels were significantly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts

Relaxin is believed to play a role in connective tissue remodeling during pregnancy (Bell, R.J., Eddie, L. W., Lester, A. R., Wood, E. C., Johnston, P.D., and Niall, H. D. (1987) Obstet. Gynecol. 69, 585-589; MacLennan, A. H. (1983) Clin. Reprod. Fertil. 2, 77-95). In the present study, normal human fibroblasts exposed to concentrations of a synthetic bioactive relaxin peptide from 0.1 to 10 ng/ml synthesized and secreted the metalloproteinase procollagenase, which was immunoprecipitable as a doublet of 52 and 57 kDa by a monoclonal antibody to human collagenase. The stimulation in procollagenase protein expression was reflected in an elevation in procollagenase mRNA levels. Media conditioned for 48 h by relaxin-treated fibroblasts (0.1 ng/ml) contained 1.7 units/ml activatable collagenase compared with 0.2 units/ml by untreated fibroblasts. In addition, relaxin caused a modest decrease in the levels of tissue inhibitor of metalloproteinases, as detected by reverse zymography and Northern analysis. Relaxin was also a potent modulator of the collagen secretory phenotype of these fibroblasts. Relaxin at 100 ng/ml down-regulated collagen secretion by 40%. When fibroblasts were treated simultaneously with cytokines such as transforming growth factor beta or interleukin 1 beta, which stimulated collagen synthesis to at least 9-fold of basal levels, relaxin at 100 ng/ml was able to down-regulate collagen expression by up to 88%. This decrease was reflected by changes at the mRNA level. These results indicate that relaxin can cause significant collagen turnover both by stimulating collagenase expression and by down-modulating collagen synthesis and secretion.     

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

Cytochemical detection of carbohydrate and immunocytochemical detection of relaxin in the same secretory granule

We administered estradiol and progesterone to spayed guinea pigs, with resultant accumulation of secretory granules in endometrial gland cells. By initially employing protein A-colloidal gold immunolocalization of relaxin, followed by cytochemical staining of carbohydrate with the thiocarbohydrazide-silver proteinate method on the same section, we showed clearly that the secretory granules were composed of a central core containing relaxin and a cortex of carbohydrate-rich material. Use of normal rabbit serum rather than relaxin antiserum, and omission of periodic acid, demonstrated the specificity of the technique.