Source Of Relaxin Research Articles

Immunofluorescence studies on the localization of relaxin in the corpus luteum of the pregnant rat.

We have used 17 day pregnant Long-Evans rats to demonstrate, utilizing indirect fluorescent antibody methods, that relaxin is present in luteal cells of the ovary. Furthermore, this technique revealed that the highest concentration of relaxin is localized in the juxtanuclear area of the cell, presumably over the Golgi region. The role of granular cells of the uterine metrial gland as a source of relaxin in the pregnant rat is still not clarified. Using the indirect fluorescent antibody technique only one experiment out of six demonstrated the presence of relaxin in the granular cells of the metrial gland. However, other investigators claim to have localized relaxin in these granular cells by the fluorescent antibody technique. Failure to obtain such consistent results in our studies may have been a result of low concentrations of relaxin in the granular cells and/or the presence of an immunochemically different relaxin molecule in these cells.

The detection of relaxin in porcine, ovine and human plasma by radioimmunoassay.

A radioimmunoassay for porcine relaxin is described based on the inhibition of the reaction between I-labeled porcine relaxin and rabbit antiserum to porcine relaxin. The time course of the binding of labeled porcine relaxin to antiserum was determined by chromatoelectrophoresis. The sensitivity of the assay is greater than 4.0 ng of relaxin/ml of plasma. Immunoactivity in plasma from a late pregnant sheep and pig was identical to the standard porcine relaxin. Inhibitions obtained with plasmas from an early pregnant sheep, a ram, boar and normal man differed slightly from that of the standard porcine relaxin. On the other hand, definite partial crossreactions were shown by both pig and sheep plasma after ovariectomy and no inhibition was observed by plasma from a sheep and human after both ovariectomy and hysterectomy. {Endocrinology 91: 1113, 1972) T N 1926, Hisaw (1) described a peptide hor•*mone isolated from the corpora lutea of sow ovaries which produced a marked relaxation of the pelvic ligaments in guinea pigs. Bioassays were subsequently developed based upon the biological activities of relaxin, Hall (2). Current bioassay methods are impractical for measurements of relaxin in multiple samples taken in a wide variety of physiological conditions. Hemagglutination-inhibition has been used for relaxin measurements in saline extracts of ovaries from several species and for cross-reactivity studies by Cohen (3) and McClintock and Zarrow (4). The application of this technique to the estimation of plasma relaxin activity has not been reported. The relaxin preparation used for this study (NIH-R-P1) was extracted from the ovaries of pregnant sows and therefore represents ovarian relaxin as produced during pregnancy. The Uterine Relaxing Factor (URF) content of this preparation is not known. URF has not yet been identified as a separate moiety from relaxin, Griss, Keck, Englehorn and Tuppy (5), isolated from pig ovaries. However, in addition to the possible heterogeneity of ovarian relaxin, plasma may also contain relaxins produced by the decidua, endometrium and placenta, according to Dallenbach and Dallenbach-Hellweg (6). Received January 4, 1972. Address for correspondence: Dr. Gillian D. Bryant, Department of Anatomy & Reproductive Biology, 1960 East-West Road, Honolulu, Hawaii 96822. A sensitive and precise radioimmunoassay has been developed for ovarian relaxin and preliminary results have been obtained in plasma from pig, sheep and man. Evidence of a uterine source of relaxin is shown and discussed. Materials and Methods Buffers. Sodium salts were used in the preparation of buffers. Diluent buffer refers to a solution 0.5 mg/ml of bovine gamma globulin, Fraction II (Nutritional Biochemicals Corporation) in 0.05M-barbitone buffer pH 8.6 with 0.5 mg/ml iodoacetamide (Nutritional Biochemicals Corporation). Horse serum was obtained in liquid form from Grand Island Biological Company. Porcine relaxin. A preparation of porcine relaxin (NIH-R-Pl) was used as a standard and for the preparation of I-labeled porcine relaxin. Polyacrylamide gel electrophoresis at pH 7.6 and 9.6 showed a single band but further studies on the physicochemical homogeneity are in progress. i'-labeled porcine relaxin. This was prepared weekly by the method of Hunter and Greenwood (7) except that 0.5 mCi obtained from Cambridge Nuclear, Mass., U.S.A. was reacted with the hormone (10 Mg in 0.02 ml) and 30 M-g of chloramine-T. The 1 3 Ilabeled porcine relaxin was separated from the unreacted iodide on Sephadex G-25 (1.5 g) previously equilibrated with 1 ml of 0.05M-barbitone buffer, pH 8.6, containing 20 mg of bovine gamma globulin. Eluates (15 X 0.75 ml) in the 0.05M-barbitone buffer were collected into tubes containing 0.1 ml of diluent. The fraction containing the highest number of counts in the protein peak was repurified by passage through Sephadex G-50 (2.5 g) previously

The response of the pubic symphysis of the mouse to extracts of pregnant rabbit serum and pregnant sow ovaries and its application as an assay method.

GARDNER reported in 1935 that the pubic symphysis of the mouse separates during pregnancy and that a small amount of separation could be obtained by prolonged treatment of castracted mice with estrogens (Gardner, 1936). Hall and Newton (1946, 1947), however, showed that it was necessary to inject castrated mice with pregnant rabbit serum (PRS) in addition to a preliminary treatment with estrone in order to duplicate the rapid rate of separation observed during pregnancy. Further work by Hall (1948) indicated the necessity of pretreating the mice with estrogen for a period of five to eight days in order to obtain the maximum response to PRS. Because PRS is an excellent source of relaxin, and since the same type of response, i.e. pelvic relaxation, is obtained in both the mouse and guinea pig, Hall (1948) suggested that the measurement of pubic relaxation in the mouse could be used as an assay for relaxin.