Edward H. Frieden

Effect of Purified Relaxin on Uterine Glycogen and Protein in the Rat

Summary Purified, electrophoretically homogeneous porcine relaxin (1750 GPU/mg) increased uterine glycogen in both unprimed and estrogen-primed ovariectomized rats. In estrogen-primed animals the glycogenic response was linear with the logarithm of the dose from 3 to 30 μg/animal; in unprimed animals the effect of 10 μg was essentially the same as that achieved with 30 μg. Relaxin also increased wet and dry uterine weights and total nitrogen content, indicating true uterine growth. Uterotrophic, as well as glycogenic effects occurred without estrogen priming, although synergism of the two hormones was observed; this suggests that relaxin receptor concentrations in the uterus may be, to some degree, estrogen dependent. Relaxin did not affect the concentrations of diaphragm glycogen or plasma glucose; thus the direct effects of relaxin appear to be limited to control of metabolic events in the female reproductive system in a manner similar to insulin regulation of general body metabolism.

Stimulation of rat uterine collagen synthesis by relaxin.

Abstract Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.

Progesterone Inhibits the Uterotrophic Effect of Relaxin in Immature Rats

Abstract 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.

Effects of follicle stimulating hormone (FSH) upon steroid aromatization in vitro.

Summary Steroids labeled with 3H in positions 1 and 2 were incubated with rat ovary slices, and the formation of tritiated water determined. During three hr at 37°, approximately 3.5, 10.0, and 15% of the total tritium present in progesterone-1,2-3H, androstenedione-1,2-3H, and testosterone-1,2-3H respectively was converted to 3H2O by 100 mg of tissue. The addition of FSH had no effect upon the aromatization of any of the substrates tested, although one of the preparations tested contained a contaminant that significantly inhibited the aromatization of androstenedione.

Relaxin-insulin homology: Predictions of secondary structure and lack of competitive binding

Abstract 1. 1. Two methods for the prediction of protein secondary structure have been applied to the amino acid sequences of porcine relaxin and bovine insulin. A comparison has been carried out between the predicted secondary structure and the known structure of insulin. 2. 2. Circular dichroism spectra for relaxin in the UV (peptide bond) region have been analyzed and are consistent with 29% alpha helix and 19% beta structure in the hormone. 3. 3. No immunochemical homology was seen in radio immunoassay competition experiments using radioiodinated insulin and very large excesses of unlabeled relaxin. Three purified electrophoretic variants or isohormone species were tested. 4. 4. Homology at the level of receptor binding was evaluated in competition experiments using radioiodinated insulin, unlabelled relaxin and isolated peripheral mononuclear leukocytes bearing insulin receptors. 5. 5. Taken together these data are consistent with a significant homology in secondary structure between relaxin and insulin which parallels the striking correspondence in disulfide pattern. On the other hand, the data obtained in this study indicate no significant homology or overlap in immuno-chemical determinants nor any competition for binding to a human insulin receptor system.

Evidence for a "pro-relaxin" in porcine relaxin concentrates.

Summary From 5 to 10% of the biological activity of a porcine relaxin concentrate is contained in a fraction with a molecular weight which is considerably higher (∼42,000 daltons) than that of relaxin itself. Exposure of this material to a low concentration of trypsin results in the appearance of native relaxin, as judged by an increase in biological activity and changes in its chromatographic and electrophoretic properties. Presented at the 67th Annual Meeting of the American Society of Biological Chemists. San Francisco, California, June 6-10, 1976. From a thesis submitted by Li-An Yeh in partial fulfillment of the requirements for the M.S. degree. Supported in part by a research grant from Hynson, Westcott, and Dunning, Inc., Baltimore, Maryland.

Acute effects of testosterone propionate upon RNA synthesis in mouse kidney.

Summary In A. Cloudman female mice a single injection of 1 mg T.P. induces a significant increase in the incorporation of labeled orotic acid into kidney RNA. This increase is detectable within 6 hr and has disappeared after 68 hr. In all circumstances, specific activities of uridine nucleotides isolated from labeled RNA are greater than those of cytidine nucleotides; after T.P., however, CMP labeling increases more than does UMP labeling. Actinomycin D, in quantities sufficient to inhibit the β-glucuronidase response to T.P., does not diminish (in fact slightly increases) nucleic acid labeling, while reducing the T.P. effect by about 50%.

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice.

Abstract Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.

Stimulation by Pituitary Gonadotropin of the Synthesis of Estrogen by Rat Ovary Slices in vitro.

Summary Slices of ovaries from rats treated with chorionic gonadotropin produced biologically detectable amounts of estrogen when incubated under the proper conditions in vitro. FSH, TPN and Δ4-androstene, 3,17-dione, in various combinations, enhanced estrogen production. In the presence of maximally effective concentrations of androstenedione and FSH, one gram of ovarian slices formed the biological equivalent of about 1.2 μg of estradiol-17-β in 3 hours.

Structure of a porcine relaxin inactive on the mouse pubic ligament

In addition to B31 (CM-a) and B28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B. Sequence analysis indicates that the two major components of relaxin C, like the other porcine relaxins, consist of two polypeptide chains linked by disulfide bonds. The shorter (A) chains are identical to the A chains of the other porcine relaxins, except for the absence of the N-terminal arginine residue. The B chains display microheterogeneity; the B sequences of the two predominant species are the same as those of the other porcine relaxins through B25, but terminate at valine residue B25 or serine residue B26, respectively.