Camp. Biochem. Physiol. Vol. 99C, No. l/2, pp. 35-39, 1991 Printed in Great Britain 0306492/91 $3.00 + 0.00 0 1991 Pergamon Press plc A SIMPLE BIOLOGICAL ASSAY FOR RELAXIN MEASUREMENT A. R. DEL ANGEL MEZA,* C. BEAS-ZARATE,~F. L. ALFARO and A. MORALES-VILLAGRAN *Unidad de Investigacibn Biomedica de Occidente, I.M.S.S., Facultad de Ciencias, Universidad de Guadalajara and tFacultad de Medicina, Universidad de Guadalajara, Apdo. Postal 4-160, Guadalajara, Jalisco, MBxico zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON (Received 21 June 1990) zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Abstract-l. Relaxin (R) is considered a gestation hormone with an insulin-like molecular structure. Its physiological importance is significant in the reproduction process. 2. Different methods of biologically assaying R have been published but electrophysiology techniques on uterus and ileum of rat have never been used. 3. A protein fraction was obtained from ovarian tissue of the rat and used to measure electrophysio- logical activity in viva and in vitro. 4. Protein recovered with R activity was similar to that in previous reports. 5. Reduction of 100% in contraction strength and SO% in its frequency was observed in ileum and in uterus respectively; it was only of 60%, but its frequency increased 43%. 6. Methodological considerations and some physiological aspects are discussed. INTRODUCI’ION Relaxin (R) is considered as a classical gestation hormone, having a chemical structure like insulin, since in both hormones the molecular weight is approximately 6000 daltons and they have two non- identical chains (A) and (B) joined by similar disulfide cross-links distribution. This and other homologies provided a basis for categorising R as a member of a family of insulin-like growth factors with both structural and functional similarities in modulating cell growth and activity (Schwabe and McDonald, 1977; Kwok and Bryant-Greenwood, 1977). Relaxin is produced in high levels by corpora lutea of pregnancy, transported in the bloodstream target smooth muscle and connective tissue for the repro- ductive tract; however, there is evidence for a non- luteal source of R and its roles in the non-pregnant animals (Bagnell et al., 1988). Furthermore, during late pregnancy, R inhibits myometrical activity and induces marked softening of the uterine cervix as well as pubic symphysis and pelvic joints (Sherwood et al., 1980; Sherwood and O’Byrne, 1974; Fields et al., 1982). In this way, R may ensure successful pregnancy parturition and fetal survival (Downing and Sherwood, 1985). In rats this hormone is detectable in the serum by the 10th day of gestation, and increases to 40-80 ng/ml, approxi- mately, by the 20th day, while in antepartum (21st to 22nd or gestation day) the levels appear as 150-180 ng/ml and remain at that level for 18-24 hr preceding birth, when they decline to low levels around the first day of lactation (Sherwood et al., 1984). Consequently, R is important in controlling myometrial activity and delivery when the proges- teron level falls (Schwabe and McDonald, 1977). Although many biochemical studies have been carried out on the role of R, the biological assays to determine its activity were developed on mouse inter- pubic ligament, and only one of them on mouse uterus (Table 1) (Sherwood and O’Byme, 1974; Sherwood, 1979; Steward and Stanbenfelt, 1981; Reinging et al., 1981; Fields et al., 1982; Eldridge and Fields, 1985; Bullesbach et al., 1986; Stewart and Papkoff, 1986), which is one of the principal targets of this hormone during gestation. Nevertheless, de- spite the importance of R in the reproduction process, it is still difficult to obtain a standard which could be used as a reference to study R in other circumstances. Thus, the aim of this work was to obtain an R fraction, under our methodological conditions, which could be used as a reference standard to perform biological assays by electrophysiology studies in preg- nant rat uterus in in vivo as well as in in vitro preparations. MATERIALS AND METHODS Female Wistar rats (Rattus norvegicus) between 2.5 and 3 months of age and 200-250 g body weight were used in all experiments. The animals were maintained in individual cages, fed a stock laboratory diet (Purina Chow) and water ad libitum under controlled conditions of light (12 hr light; 12 hr darkness), temperature (22°C) and environmental humidity (45550%). Rats were mated with healthy adult males, and the dates in which sperm was found in the vagina were designated as first day of pregnancy. Ovaries were collected on the 20th day of gestation from ether-anesthetized rats and those were cleaned of connective tissue fat, weighed and arranged in phosphate-buffered saline (PBS) (0.14 M sodium chloride and 0.01 M sodium phosphate), pH 7.0 at PBS to a tissue weight ratio of 1 ml: 100 mg of fresh tissue. The ovaries were frozen in dry 35