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A simple biological assay for relaxin measurement
Camp. Biochem. Physiol. Vol. 99C, No. l/2, pp. 35-39, 1991 Printed in Great Britain
0
0306492/91 $3.00 + 0.00 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 (Received 21 June 1990) 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 electrophysiological 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.
Although many biochemical studies have been carried out on the role of R, the biological assays to determine its activity were developed on mouse interpubic 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, despite 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 pregnant rat uterus in in vivo as well as in in vitro preparations.
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 nonidentical 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 reproductive tract; however, there is evidence for a nonluteal 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, approximately, 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 progesteron level falls (Schwabe and McDonald, 1977).
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
A. R. DEL ANGEL MEZA et al.
36
Table 2. Protein concentration from the ovarian extracts (280 nm)
ice after being minced and finely ground at 4°C in a 15-ml tissue grinder, and the 2.5 g samples were worked according to the method of Sherwood et al. 1979). Gel jltration on Sephadex G -50 Gel filtration of 1 ml of bovine insulin (1 ml = 80 U) and the rat relaxin extract (15 mg protein) was conducted on a 2 x 90 cm column at 4°C which was equilibrated and run with 0.01 M ammonium acetate-acetic acid buffer (pH 5.0). The flow rate was maintained at 8 ml/hr with fractions collected at 30 min intervals, protein determinations of fractions obtained were made in a Waters HPLC system with u.v.-visible detector Model 480. Insulin and protein fractions derived from relaxin extract obtained by Sephadex were processed by ion exchange chromatography (IEC) conducted on a 1.2 x 5.5 cm column of Dowex AG 50 WX4 (20&400 mesh) which was equilibrated and run with the same buffer and conditions used in gel filtration, only the flow rate was maintained at 6 ml/hr. Protein was also determined in all fractions obtained. The bioassay method used is based upon the ability of R to cause inhibition of spontaneous smooth muscle contractions. It was done in vivo and in vitro as follows; in vitro assay: fresh uterine and ileum segments (2.5cm) from multiparous rats were suspended in a 40-ml double-wall glass chamber in order to isolate tissue containing Hartmann’s solution (pH 7.2). The chamber was aereated and the temperature controlled at 37°C. One of the muscle segments was set and the other attached to a force transducer FT03C. Contractions were recorded in a polygraph (Grass 7D), and after regular contractions were established (l&15 min), protein fractions from IEC were injected into the chamber in a volume of 200 ~1 (47 fig protein). It was allowed 5 min to act on the ileum and uterine muscles, and after this period the chamber was rinsed with Hartmann’s solution @H 7.2). Insulin (200~1) was assayed on ileum to determine whether that contraction was specifically due to the presence of R and not to a compound similar in structure and molecular weight. The in uivo method animals were divided into two groups. Ether was employed for anesthesia, and laparatomies were performed. The uterus was exposed in the first group and the ileum in the second. One segment of each muscle was attached to a transducer of the polygraph, and after regular contractions were established (l&15 min), 200 ~1 of relaxin extract from IEC was dropped over the two kind of muscles. It was allowed to act for 5 min, and after this period, the tissues were rinsed with Hartmann’s solution (pH 7.2).
Protein field (q/IO
Ovarian extract 1 2 3 4
was 0.085 & 0.016 g in each rat at 20th day of pregnancy. Four groups of ovarian tissue were collected, dialized and the proteins measured. A different protein concentration was observed in each one of them (Table ‘2). However, the ovarian extract run on a Sephadex G-50 was adjusted to a total amount of 15.0mg of protein. The higher protein separation Sephadex fractions were observed around 54-55 hr (12-13 fractions) after the ovarian extract was applied on the column. Insulin, however, was separated on fraction approx. 57 hr (17-19 fractions) approximately after its application (Fig. 1A). On the other hand, the insulin was seen to evaluate with maximum absorbance around 5-6 fractions after being run on a Dowex column. Those protein fractions obtained from dialysis and further run on an ion exchange column were eluted at 3.5 hr after application (Fig. 1B). The percentages of protein recovered from the Sephadex and Dowex columns at the end of the process were 22% and 0.41% respectively (Table 3). With the purpose of determining whether the protein fraction obtained from ion exchange chromatography had some physiological activity, the relaxin extract activity was assayed on uterus and ileum segments of rats in vivo, as well as in vitro. Two hundred microliters of protein fraction produced a significant reduction of 60-63% on contraction strength and only 13-21% on muscular shade (Fig. 2A), but its frequency was increased in 43%, whereas the muscle shade increased only 13-2 1% and its frequency was reduced in 43% (Fig. 2B).
Table 3. Protein recoveries throughout Fraction
RESULTS
of fresh ovarian tissue was adjusted to 2.5 g from approx. fifteen rats since the mean weight The amount
Ovarian extract Sephadex G-50 Dowex AG 50 WX4
the procedure
Protein yield (mg/g fresh tissue)
% Recovery
6.712 + 2.0 1.514 * 0.03 0.028 f 0.01
100 22.56 0.41
Table 1. Methods used to uurifv relaxin from different snecies Species Sow ovaries
Rat ovaries Sow and rat ovaries Human placental Tissue Tiger shark ovaries Sow and cow ovaries Rabbit placental tissue Spiny dogfish ovaries Eauine olacental tissue
)rl)
49.70 11.187 17.485 39.646
Bioassay Mouse interpubic ligament Mouse interpubic ligament Mouse interpubic ligament Estrogen administration on mouse Uterus in vitro Mouse and guinea-pig interpubis ligament Mouse uterus Estrogen administration on mouse uterus in vitro Mouse and guinea-pig interpubic ligament Mouse intemubic ligament
Reference (Sherwood ef al., 1984) (Sherwood et al., 1979) (Walsh and Niall, 1980) (Yamamoto et al., 1981) (Reining ef a/., 1981) (Reining et al., 1981) (Field er al., 1982) (Eldridge and Field 1985) (Bullesback et al., 1986) (Stewart and Paukoff 1986)
A simple biological assay for relaxin measurement RELAXIN
B
INSULIN
RELAXIN
37
EXTRACT
EXfRlCt
Fig. 1. Protein concentration (280 nm) in both insulin and relaxin extract derived from ovary tissue. (A) Gel filtration (Sephadex G-SO),(B) ion exchange chromatography.
However, the response of ileum segments to the protein fractions produced an important reduction (100%) in contraction strength, and its frequency was only 50% in both in vivo and in vitro experiments, whereas the muscular shade decreased in 50%, in in vivo studies changes were not observed in the in vitro experiments (Figs 3A and B). Finally, using insulin under the same conditions there was no change in the physiological activity registered in the tissues previously studied. DISCUSSION
The insulin and R had a similar chemical structure (Schwabe and McDonald, 1977; Bagnell et al., 1977) although insulin is normally not used as standard reference to obtain R. However, in the present work it was used for the purpose of obtaining the elution time of the relaxin extract in the chromatographic procedures. Thus Figs 1 and 2 showed that the eluted
peaks of relaxin extract and insulin had little differences in the time run probably due to the fact that R is heavier than insulin (5950-6015 and 5800 daltons respectively; Sherwood and O’Byrne, 1974; Sherwood, 1979). The results obtained in this work showed a final yield of 0.41% using only 15 mg of protein applied on Sephadex G-50 (Table 2) while other authors using 70 mg of protein charge have reported a 0.49% final yield. In this manner, it is important to note that it is possible to use less fresh tissue in order to obtain a similar yield of the relaxant peptide. The evaluation of biological activity of R has been tested mainly in preparations of the murine interpubit ligaments (Sherwood and O’Byrne, 1974; Sherwood, 1979; Reinging et al., 1981; Bullesbach, 1986; Stewart and Papkoff, 1986). Later, since it had been proposed that this tissue is the most sensitive method of evaluating the relaxant effects of the R, some authors have been using the rodent uterus (Stewart
A. R. DEL ANGELMEZA et al.
38
N
I
=x
SWOJ6 j NOISN31
m
(swol6 ) NOISN31
I
A simple biological assay for relaxin measurement and Stanbenfelt, 1981; Fields et al., 1982; Eldridge and Fields, 1985). However, in the present work, two different kinds of smooth muscle tissue which exhibit similar features were used; in the ileum and the uterus, in in viva as in in vitro studies, this preparation exhibited an important decrease in their mechanical activities when exposed to the fraction obtained from IEC and used as R, and returned them to their control values of activity when washed. This is the first evidence presented where the R hormone has an effect on a different tissue other than its possible blank organ, for instance, on the ileum of the rat. The effect of R on these tissues has two fundamental components; it decreases the contraction strength (maximum recorded) as well as the basal tension of the smooth muscle (declivity of the basal activity). With the purpose of determining whether that effect was due to R and not to insulin, we probed the last one on the same bioassays. However, it did not have any relaxant recording effect on that preparation. On the other hand, R is classically considered to be a hormone of pregnancy, transported in the bloodstream to target smooth muscle and connective tissue of the reproductive tract. Thus, it induces inhibition of myometrical activity and softening of the uterine cervix, as well as pubic symphysis and pelvic joints (Downing and Sherwood, 1985). However, there is evidence for a non-luteal source of R and its role in non-pregnant animals responsible for permitted follicle rupture (Bagnell et al., 1988), inhibiting milk ejection (Summerlee et ai., 1984; Jones and Summerlee, 1986), as well as spermatozoa fertilization capacity (Bagnell et al., 1988; Essig et al., 1982; Jong et al., 1988). In this way, it is possible that a modifi-
cation in that hormone level may be influenced by a different type of diet, since undernourished rats have shown an important lengthening of gestation time (Zamenhof and Van Marthens, 1978; Del Angel and Feria-Velasco, 1982). On this basis, in our laboratory, using a maize-base diet as the only protein source, important changes in the estrus cycle, and longer gestation and parturition time have been observed (unpublished data). Furthermore, it is important to show an economic, easy and practical extraction method for R under the above conditions. Finally, it is also necessary to perform further studies to evaluate the role that R may play on delivery development of animals submitted to different diet conditions. REFERENCES
Bagnell C., Ainsworth L., Bryant-Greenwood G. D. and Greenwood F. C. (1988) Follicular relaxin: a role in the paracrine control of ovarian function. Serono Symposia Publication, 35. The Control of Follicle Development Ovulation and Luteal Function: Lessons from in vitro Ferzit~zu~jon(Edited by Naftolin Frederick and de Cherney H.1, pp. 35-44. Raven Press, New York. Bull&bach k: E.., Gowan L. K., Schwabe, C., Steinetz C. C. and O’Bvrne E. M. (1986) Isolation. ourification and the sequence . of relaxin from spin;’ dogfish (Squalus acanthias). Eur. J. Biochem. 161, 335-341. Del Angel A. R. and Feria-Velasco A. (1982) Effects of
protein restriction on growth of adult and developing rats
39
(first and second generations). Arch Invest. Med., Mex. 13, 43-49. Downing S. J. and Sherwood 0. D. (1985) The physiological role of relaxin in the pregnant rat. I. The influence of relaxin on parturition. -~n~oc~j~olog~ 116, lZw1204. Eldridee R. K. and Fields P. A. 11985) Rabbit ulacental rela&: purification and immu;ohisiochemicai localization. Endocrinology 117, 2512-2519. Essig M., Schoenfeld C., D’Eletto R. A., Dubin L., Steinetz B. G., O’Byrne M. E. and Weiss G. (1982) Relaxin in human seminal plasma. Ann. N. Y. Acad. Sci. 380, 224-230. Fields M. J., Roberts R. and Fields P. A. (1982a) Octadecylsilica and carboxymethyl cellullose isolation of bovine and porcine relaxin. In Relaxin: Srructure, Function and Evolution (Edited by Steinetz B. G., Schwabe C. and Weiss G.), pp. 3746. The New York Academy of Sciences, New York. Fields P. A., Larkin L. H. and Pardo R. J. (1982b) Purification of retaxin from the placenta of rabbit. In Relaxin: Structure, Function and- Evolution (Edited by Steinetz B. G., Schwabe C. and Weiss G.), pp. 75-86. The New York Academy of Sciences, New York. Jones S. A. and Summerlee A. J. S. (1986) Relaxin acts centrally to inhibit oxytocin release during parturition: an efffect that is reversed bv naloxane. J. Endocr. 111, 99-102. Jong M., Park M. D., Ewing K., Milfer F., Friedman Ch. I. and Kim M. H. 119881 Effects of relaxin on the fertilization capacity bf h;man spermatozoa. Am. J. Obstet. Gynec. 158, 974-979. Kwok S. and Bryant-Greenwood G. (1977) Primary structure of porcine relaxin: homology with insulin and related growth factors. Nature 267, 544-546. Re&ging J. W., Daniel L. N., Schwabe C., Gowan L. K. and O’Byrne E. M. (1981) Isolation and ~haracte~zation of relaxin from the sand tiger shark (Odontaspis taurus). Endocrinology 109, 537-543. Schwabe C. and McDonald J. K. 11977) Relaxin: a disulfide homolog of insulin. Science 19?, 914-915. Sherwood 0. D. (1979) Purification and characterization of rat relaxin. Endocrinology 104, 886-892. Sherwood 0. D., Crenekovic V. E., Gordon W. L. and Rutherford J. (1980) Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 107, 691697. Sherwood 0. D., Key R. H., Tarbell M. K. and Downing S. J. (1984) Dynamic changes of multiple forms of serum immunoactive relaxin during pregnancy in the rat. Endocrinology 114, 806-8 I 3. Sherwood 0. D. and O’Byrne E. M. (1974) Puri~~ation and characterization of porcine relaxin. Archs Biochem. Biophys. 160, 185-196. Stewart D. R. and Stanbenfelt G. (1981) Relaxin activity in the pregnant mare. Biol. Reprod. 25, 281-289. Stewart D. R. and Paokoff H. (1986) Purification and characterization of equine relaxin. kdocrinology 219, 1093s1099. Summerlee A. J. S., O’Byrne K. T., Paisley A. C., Brezee M. F. and Porter D. G. (1984) Relaxin effects on the central control of oxytocin release. Nature 309, 371-374. Walsh J. R. and Niall H. D. (1980) Use of an octadecylsilica purification method minimizes proteolysis during isolation of porcine and rat relaxin. Endocrinology 107, 1258-1260. Yamamoto S., Kwok S. C. M., Greenwood F. C. and Brvant-Greenwood G. D. (1981) Relaxin purification from human placental basal plates: J. clin. en&r. Metab. 52, 601604. Zamenhof S. and Van Marthens E. (1978) The effects of chronic undernutrition on generation of rat development. 3. Nutr. 108, 1719-1722.
0
0306492/91 $3.00 + 0.00 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 (Received 21 June 1990) 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 electrophysiological 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.
Although many biochemical studies have been carried out on the role of R, the biological assays to determine its activity were developed on mouse interpubic 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, despite 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 pregnant rat uterus in in vivo as well as in in vitro preparations.
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 nonidentical 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 reproductive tract; however, there is evidence for a nonluteal 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, approximately, 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 progesteron level falls (Schwabe and McDonald, 1977).
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
A. R. DEL ANGEL MEZA et al.
36
Table 2. Protein concentration from the ovarian extracts (280 nm)
ice after being minced and finely ground at 4°C in a 15-ml tissue grinder, and the 2.5 g samples were worked according to the method of Sherwood et al. 1979). Gel jltration on Sephadex G -50 Gel filtration of 1 ml of bovine insulin (1 ml = 80 U) and the rat relaxin extract (15 mg protein) was conducted on a 2 x 90 cm column at 4°C which was equilibrated and run with 0.01 M ammonium acetate-acetic acid buffer (pH 5.0). The flow rate was maintained at 8 ml/hr with fractions collected at 30 min intervals, protein determinations of fractions obtained were made in a Waters HPLC system with u.v.-visible detector Model 480. Insulin and protein fractions derived from relaxin extract obtained by Sephadex were processed by ion exchange chromatography (IEC) conducted on a 1.2 x 5.5 cm column of Dowex AG 50 WX4 (20&400 mesh) which was equilibrated and run with the same buffer and conditions used in gel filtration, only the flow rate was maintained at 6 ml/hr. Protein was also determined in all fractions obtained. The bioassay method used is based upon the ability of R to cause inhibition of spontaneous smooth muscle contractions. It was done in vivo and in vitro as follows; in vitro assay: fresh uterine and ileum segments (2.5cm) from multiparous rats were suspended in a 40-ml double-wall glass chamber in order to isolate tissue containing Hartmann’s solution (pH 7.2). The chamber was aereated and the temperature controlled at 37°C. One of the muscle segments was set and the other attached to a force transducer FT03C. Contractions were recorded in a polygraph (Grass 7D), and after regular contractions were established (l&15 min), protein fractions from IEC were injected into the chamber in a volume of 200 ~1 (47 fig protein). It was allowed 5 min to act on the ileum and uterine muscles, and after this period the chamber was rinsed with Hartmann’s solution @H 7.2). Insulin (200~1) was assayed on ileum to determine whether that contraction was specifically due to the presence of R and not to a compound similar in structure and molecular weight. The in uivo method animals were divided into two groups. Ether was employed for anesthesia, and laparatomies were performed. The uterus was exposed in the first group and the ileum in the second. One segment of each muscle was attached to a transducer of the polygraph, and after regular contractions were established (l&15 min), 200 ~1 of relaxin extract from IEC was dropped over the two kind of muscles. It was allowed to act for 5 min, and after this period, the tissues were rinsed with Hartmann’s solution (pH 7.2).
Protein field (q/IO
Ovarian extract 1 2 3 4
was 0.085 & 0.016 g in each rat at 20th day of pregnancy. Four groups of ovarian tissue were collected, dialized and the proteins measured. A different protein concentration was observed in each one of them (Table ‘2). However, the ovarian extract run on a Sephadex G-50 was adjusted to a total amount of 15.0mg of protein. The higher protein separation Sephadex fractions were observed around 54-55 hr (12-13 fractions) after the ovarian extract was applied on the column. Insulin, however, was separated on fraction approx. 57 hr (17-19 fractions) approximately after its application (Fig. 1A). On the other hand, the insulin was seen to evaluate with maximum absorbance around 5-6 fractions after being run on a Dowex column. Those protein fractions obtained from dialysis and further run on an ion exchange column were eluted at 3.5 hr after application (Fig. 1B). The percentages of protein recovered from the Sephadex and Dowex columns at the end of the process were 22% and 0.41% respectively (Table 3). With the purpose of determining whether the protein fraction obtained from ion exchange chromatography had some physiological activity, the relaxin extract activity was assayed on uterus and ileum segments of rats in vivo, as well as in vitro. Two hundred microliters of protein fraction produced a significant reduction of 60-63% on contraction strength and only 13-21% on muscular shade (Fig. 2A), but its frequency was increased in 43%, whereas the muscle shade increased only 13-2 1% and its frequency was reduced in 43% (Fig. 2B).
Table 3. Protein recoveries throughout Fraction
RESULTS
of fresh ovarian tissue was adjusted to 2.5 g from approx. fifteen rats since the mean weight The amount
Ovarian extract Sephadex G-50 Dowex AG 50 WX4
the procedure
Protein yield (mg/g fresh tissue)
% Recovery
6.712 + 2.0 1.514 * 0.03 0.028 f 0.01
100 22.56 0.41
Table 1. Methods used to uurifv relaxin from different snecies Species Sow ovaries
Rat ovaries Sow and rat ovaries Human placental Tissue Tiger shark ovaries Sow and cow ovaries Rabbit placental tissue Spiny dogfish ovaries Eauine olacental tissue
)rl)
49.70 11.187 17.485 39.646
Bioassay Mouse interpubic ligament Mouse interpubic ligament Mouse interpubic ligament Estrogen administration on mouse Uterus in vitro Mouse and guinea-pig interpubis ligament Mouse uterus Estrogen administration on mouse uterus in vitro Mouse and guinea-pig interpubic ligament Mouse intemubic ligament
Reference (Sherwood ef al., 1984) (Sherwood et al., 1979) (Walsh and Niall, 1980) (Yamamoto et al., 1981) (Reining ef a/., 1981) (Reining et al., 1981) (Field er al., 1982) (Eldridge and Field 1985) (Bullesback et al., 1986) (Stewart and Paukoff 1986)
A simple biological assay for relaxin measurement RELAXIN
B
INSULIN
RELAXIN
37
EXTRACT
EXfRlCt
Fig. 1. Protein concentration (280 nm) in both insulin and relaxin extract derived from ovary tissue. (A) Gel filtration (Sephadex G-SO),(B) ion exchange chromatography.
However, the response of ileum segments to the protein fractions produced an important reduction (100%) in contraction strength, and its frequency was only 50% in both in vivo and in vitro experiments, whereas the muscular shade decreased in 50%, in in vivo studies changes were not observed in the in vitro experiments (Figs 3A and B). Finally, using insulin under the same conditions there was no change in the physiological activity registered in the tissues previously studied. DISCUSSION
The insulin and R had a similar chemical structure (Schwabe and McDonald, 1977; Bagnell et al., 1977) although insulin is normally not used as standard reference to obtain R. However, in the present work it was used for the purpose of obtaining the elution time of the relaxin extract in the chromatographic procedures. Thus Figs 1 and 2 showed that the eluted
peaks of relaxin extract and insulin had little differences in the time run probably due to the fact that R is heavier than insulin (5950-6015 and 5800 daltons respectively; Sherwood and O’Byrne, 1974; Sherwood, 1979). The results obtained in this work showed a final yield of 0.41% using only 15 mg of protein applied on Sephadex G-50 (Table 2) while other authors using 70 mg of protein charge have reported a 0.49% final yield. In this manner, it is important to note that it is possible to use less fresh tissue in order to obtain a similar yield of the relaxant peptide. The evaluation of biological activity of R has been tested mainly in preparations of the murine interpubit ligaments (Sherwood and O’Byrne, 1974; Sherwood, 1979; Reinging et al., 1981; Bullesbach, 1986; Stewart and Papkoff, 1986). Later, since it had been proposed that this tissue is the most sensitive method of evaluating the relaxant effects of the R, some authors have been using the rodent uterus (Stewart
A. R. DEL ANGELMEZA et al.
38
N
I
=x
SWOJ6 j NOISN31
m
(swol6 ) NOISN31
I
A simple biological assay for relaxin measurement and Stanbenfelt, 1981; Fields et al., 1982; Eldridge and Fields, 1985). However, in the present work, two different kinds of smooth muscle tissue which exhibit similar features were used; in the ileum and the uterus, in in viva as in in vitro studies, this preparation exhibited an important decrease in their mechanical activities when exposed to the fraction obtained from IEC and used as R, and returned them to their control values of activity when washed. This is the first evidence presented where the R hormone has an effect on a different tissue other than its possible blank organ, for instance, on the ileum of the rat. The effect of R on these tissues has two fundamental components; it decreases the contraction strength (maximum recorded) as well as the basal tension of the smooth muscle (declivity of the basal activity). With the purpose of determining whether that effect was due to R and not to insulin, we probed the last one on the same bioassays. However, it did not have any relaxant recording effect on that preparation. On the other hand, R is classically considered to be a hormone of pregnancy, transported in the bloodstream to target smooth muscle and connective tissue of the reproductive tract. Thus, it induces inhibition of myometrical activity and softening of the uterine cervix, as well as pubic symphysis and pelvic joints (Downing and Sherwood, 1985). However, there is evidence for a non-luteal source of R and its role in non-pregnant animals responsible for permitted follicle rupture (Bagnell et al., 1988), inhibiting milk ejection (Summerlee et ai., 1984; Jones and Summerlee, 1986), as well as spermatozoa fertilization capacity (Bagnell et al., 1988; Essig et al., 1982; Jong et al., 1988). In this way, it is possible that a modifi-
cation in that hormone level may be influenced by a different type of diet, since undernourished rats have shown an important lengthening of gestation time (Zamenhof and Van Marthens, 1978; Del Angel and Feria-Velasco, 1982). On this basis, in our laboratory, using a maize-base diet as the only protein source, important changes in the estrus cycle, and longer gestation and parturition time have been observed (unpublished data). Furthermore, it is important to show an economic, easy and practical extraction method for R under the above conditions. Finally, it is also necessary to perform further studies to evaluate the role that R may play on delivery development of animals submitted to different diet conditions. REFERENCES
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protein restriction on growth of adult and developing rats
39
(first and second generations). Arch Invest. Med., Mex. 13, 43-49. Downing S. J. and Sherwood 0. D. (1985) The physiological role of relaxin in the pregnant rat. I. The influence of relaxin on parturition. -~n~oc~j~olog~ 116, lZw1204. Eldridee R. K. and Fields P. A. 11985) Rabbit ulacental rela&: purification and immu;ohisiochemicai localization. Endocrinology 117, 2512-2519. Essig M., Schoenfeld C., D’Eletto R. A., Dubin L., Steinetz B. G., O’Byrne M. E. and Weiss G. (1982) Relaxin in human seminal plasma. Ann. N. Y. Acad. Sci. 380, 224-230. Fields M. J., Roberts R. and Fields P. A. (1982a) Octadecylsilica and carboxymethyl cellullose isolation of bovine and porcine relaxin. In Relaxin: Srructure, Function and Evolution (Edited by Steinetz B. G., Schwabe C. and Weiss G.), pp. 3746. The New York Academy of Sciences, New York. Fields P. A., Larkin L. H. and Pardo R. J. (1982b) Purification of retaxin from the placenta of rabbit. In Relaxin: Structure, Function and- Evolution (Edited by Steinetz B. G., Schwabe C. and Weiss G.), pp. 75-86. The New York Academy of Sciences, New York. Jones S. A. and Summerlee A. J. S. (1986) Relaxin acts centrally to inhibit oxytocin release during parturition: an efffect that is reversed bv naloxane. J. Endocr. 111, 99-102. Jong M., Park M. D., Ewing K., Milfer F., Friedman Ch. I. and Kim M. H. 119881 Effects of relaxin on the fertilization capacity bf h;man spermatozoa. Am. J. Obstet. Gynec. 158, 974-979. Kwok S. and Bryant-Greenwood G. (1977) Primary structure of porcine relaxin: homology with insulin and related growth factors. Nature 267, 544-546. Re&ging J. W., Daniel L. N., Schwabe C., Gowan L. K. and O’Byrne E. M. (1981) Isolation and ~haracte~zation of relaxin from the sand tiger shark (Odontaspis taurus). Endocrinology 109, 537-543. Schwabe C. and McDonald J. K. 11977) Relaxin: a disulfide homolog of insulin. Science 19?, 914-915. Sherwood 0. D. (1979) Purification and characterization of rat relaxin. Endocrinology 104, 886-892. Sherwood 0. D., Crenekovic V. E., Gordon W. L. and Rutherford J. (1980) Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 107, 691697. Sherwood 0. D., Key R. H., Tarbell M. K. and Downing S. J. (1984) Dynamic changes of multiple forms of serum immunoactive relaxin during pregnancy in the rat. Endocrinology 114, 806-8 I 3. Sherwood 0. D. and O’Byrne E. M. (1974) Puri~~ation and characterization of porcine relaxin. Archs Biochem. Biophys. 160, 185-196. Stewart D. R. and Stanbenfelt G. (1981) Relaxin activity in the pregnant mare. Biol. Reprod. 25, 281-289. Stewart D. R. and Paokoff H. (1986) Purification and characterization of equine relaxin. kdocrinology 219, 1093s1099. Summerlee A. J. S., O’Byrne K. T., Paisley A. C., Brezee M. F. and Porter D. G. (1984) Relaxin effects on the central control of oxytocin release. Nature 309, 371-374. Walsh J. R. and Niall H. D. (1980) Use of an octadecylsilica purification method minimizes proteolysis during isolation of porcine and rat relaxin. Endocrinology 107, 1258-1260. Yamamoto S., Kwok S. C. M., Greenwood F. C. and Brvant-Greenwood G. D. (1981) Relaxin purification from human placental basal plates: J. clin. en&r. Metab. 52, 601604. Zamenhof S. and Van Marthens E. (1978) The effects of chronic undernutrition on generation of rat development. 3. Nutr. 108, 1719-1722.