Pregnant Sow Ovaries Research Articles

Does a “500 million‐year‐old hormone” disprove Darwin?

In a paper published by the FASEB Journal in 1999, Danielle Georges and Christian Schwabe described gene sequences from the tunicate Ciona intestinalis that were indistinguishable from porcine relaxin (1). Newer data contradict that finding: recently published analyses of the C. intestinalis genome do not confirm the presence of relaxin sequences (2). What might seem like a narrow issue of scientific fact has broader implications. To the delight of creationists and fans of intelligent design, the presence of similar relaxin sequences in pigs and sea squirts–species separated by 500 million years–has been used to cast genomic doubt on Darwinian evolution. Indeed, Schwabe has repeatedly cited the tunicate relaxin data, and the nearly identical relaxin sequences he has identified in whales, (3) not only to support his own “Genomic Potential Hypothesis” but also to refute modern evolutionary biology (4). Schwabe’s hypothesis would replace Darwin’s theory of common descent with the view of the independent origin of all species, thereby making the origin of life and the origin of species one and the same problem. Moreover, the “Genomic Potential Hypothesis” rejects natural selection of random variations as the mechanism that underlies speciation and adaptation. Thus, albeit Schwabe sees chemistry as the sole force generating genomes and species the independent origin of all species asserted by his “Genomic Potential Hypothesis” coincides with the creationist view of the origin of species. Since Schwabe therefore is regularly cited by creationists as an “atheistic scientist” who rejects neoDarwinism, it seems worthwhile to critique his experimental work in the context of his “Genomic Potential Hypothesis.” It’s an hypothesis that wants to have it both ways. On the one hand, Schwabe argues that the high degree of diversity found in the relaxin sequences of different species would date speciation events much earlier than phylogenies based on other data. On the other hand, he argues that the identity of tunicate, cetacean, and porcine relaxin sequences cannot be explained by Darwinian selection of random mutants. This concomitant use of sequence diversity and sequence identity to deny the theory of evolution appears to us at least curious or at worst tautologous. The 1999 The FASEB Journal paper immediately evoked critical responses summarized by an editor’s comment (5) that the conclusions rested entirely on PCR and microsequencing data, two methods quite prone to contamination. The authors rejected those concerns and replied that the experiments were carried out in two separate labs and that Georges had never worked with porcine material before (6). Not so: Georges had indeed used polyA -RNA from pregnant sow ovaries (the organ in which relaxin is produced) in work published well before The FASEB Journal paper (7). At any rate, since the genomic sequence was reported to lack introns, the cDNA could have served as a good probe for Southern blot analysis of genomic DNA, a far more robust technique (a description of Southern blot analysis appears in the Methods section of The FASEB Journal paper but such data are not presented in the Results section). Indeed, this approach would have enabled the authors to decide whether the sequence variation they observed in one tenth of the PCR products was introduced during amplification, whether it resulted from a polymorphism of the gene or if two different relaxin genes reside within the Ciona genome as the authors speculated. In point of fact, polymorphisms would contradict the “Genomic Potential Hypothesis,” because, as rigidly interpreted, the Schwabe hypothesis would predict the independent origins of individuals carrying different alleles (see below). Still, the authors adduce the fact that Ciona relaxin is more similar to an infrequent allele of porcine relaxin. They suggest an “occasional exchange” in the respective position of the porcine gene: but that of course implies a mutation. Curiously, Schwabe willingly accepts that “chance mutations” may cause inherited human diseases (8) but denies that mutations can drive evolution. In addition it should be

Endogenous heterogeneity of relaxin and sequence of the major form in pregnant sow ovaries.

Using an extraction procedure that minimized proteolysis, followed by gel filtration, cation-exchange FPLC, and reverse-phase HPLC, the present study unambiguously showed the presence of multiple isoforms of relaxin in the ovaries of pregnant sows. Four relaxin isoforms were isolated (designated as R-I1, R-I2, R-II1 and R-III1). They had a similar migration pattern on SDS/urea PAGE, and showed no significant difference in biological activity. HPLC separation of the reduced and S-pyridylethylated relaxin variants indicated that R-I1, R-I2 and R-III1 each contained multiple relaxin molecules which showed variability of the B-chain, whereas R-II1 contained mostly a single relaxin molecule with only slight B-chain variability. R-II1 was thus considered likely to be the major form of relaxin stored in the ovary. Sequence analysis revealed that R-II1 contained 22 amino-acid residues in the A-chain and 29 residues in the B-chain, with a total molecular mass of 5814.8 Da. It was thus equivalent to CM-a' relaxin designated by Sherwood and O'Byrne (1974).

Characterization of the circulating form of porcine relaxin: biological activity and terminal amino acids.

Relaxin is structurally related to insulin, and it induces pregnancy-related changes in the reproductive tract of several mammalian species. Relaxin isolated from the ovaries of pregnant sows has been used for primary structure determination, for much of the biological characterization of the hormone, and for the development of RIAs. Immunoreactive (IR) relaxin is found in peripheral blood during pregnancy in pigs and other species, but it has not been established that the substance identified by RIA is structurally or biologically equivalent to the native ovarian hormone. IR relaxin was, therefore, isolated from peripheral plasma of late pregnant gilts (days 112-114) for bioassay and determination of terminal amino acid residues. IR relaxin was monitored by a specific homologous RIA during fractionation of plasma by gel filtration, cation exchange, and hydrophobic binding to octadecysilica. IR relaxin circulates unbound and is equipotent with ovarian relaxin in the mouse pubic ligament bioassay. Amino acids released from IR relaxin by pyroglutamic aminopeptidase and carboxypeptidase-Y were converted to their 4-dimethylamino-azo-4'-sulfonyl derivatives for identification by HPLC. The B-chain of IR relaxin had an amino-terminal pyroglutamic acid. Amino acids sequentially released from the carboxy-terminal indicated a chain length of 28-30 amino acids, suggesting a heterogeneity reminiscent of that of ovarian relaxin isolated by other methods. Arginine was released from the free amino-terminal by dimethylaminozaobenzene-isothiocyanate degradation, indicating an intact A-chain of 22 amino acids. Blood immunoreactive relaxin in pigs is, therefore, a secreted biologically active form of relaxin with an amino acid composition similar to that of the form stored in the corpus luteum.

Structure of a porcine relaxin inactive on the mouse pubic ligament

In addition to B 31 (CM-a) and B 28 (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 B 25, but terminate at valine residue B 25 or serine residue B 26, respectively.

Isolation and characterization of high molecular weight forms of relaxin.

The striking structural homology with insulin suggests that relaxin may also be derived from a 9,000 dalton single peptide chain precursor. Therefore, in the past few years, we have been searching for a precursor of relaxin using two different approaches, namely, a) isolation of the precursor from pregnant sow ovaries and b) demonstration of the precursor in in vitro biosynthetic studies.

Relaxin inhibition of KCl-induced uterine contractions in vitro: an alternative bioassay.

The effect on the rat uterus of the ovarian peptide hormone, relaxin, has been studied in vitro. Partial (20%) replacement of the NaCl in physiological saline by KCl resulted in a strong contraction of the uterus which was reversed by relaxin. The preparation contracted by this level of KCl substitution was shown to be very sensitive to the action of extracts of pregnant sow ovaries containing relaxin in comparison with the ones contracted by higher and lower degrees of KCl substitution. The ovarian extracts induced a concentration-dependent reversal of the contraction of the uterus; this activity of relaxin increased with the degree of purification of the hormone. Relaxin induced a slowly developing relaxation whereas epinephrine relaxed the same preparation very rapidly until a plateau relaxation was reached. The concentration-response curves to relaxin at different states of purification were parallel and were also parallel and comparable in potency to the ones measured for similar preparations from other laboratories. These results suggest that rat uterus contracted in vitro by KCl-substituted saline should be a valuable and reproducible bioassay for relaxin.

Relaxin Production by the Porcine Ovarian Graafian Follicle in vitro

Immunoreactive rela.xin was shown to be present in pools of porcine follicular fluid collected from individual ovaries from cyclic and pregnant sows. In the cyclic animals, the highest relaxin level was obtained from ovaries containing active corpora lutes, but at no stage during the cycle did relaxin content of follicular fluid approach that found in ovaries of pregnant sows. Fractionation of follicular fluid on Sephadex C-SO revealed at least three immunoreactive relaxin peaks of differing molecular weight: >40,000, 20,000, and one corresponding to authentic relaxin (6300). Serum of pregnant sows when fractionated in a similar fashion contained more of the highest molecular weight immunoreactive relaxin while only ‘\‘20% of the immunoreactivity was associated with 6300 relaxin. Isolated ovarian follicles and follicular tissue released relaxin throughout prolonged periods in culture. Relaxin output in vitro was significantly higher for tissue obtained from pregnant than from nonpregnant sows. Examination of the relationship between steroid hormone and relaxin release revealed that follicle wall segments, washed free of follicular fluid, released both more relaxin and steroids than did intact follicles. Of the steroids examined, the pattern of relaxin most resembled that found for progesterone, which showed an increase after 48 h of culture as the tissue underwent spontaneous luteinization. The relationship between steroid hormone and relaxin biosynthesis was further studied in these cultures by the addition of inhibitors of steroid biosynthesis. Aminoglutethimide effectively reduced production of all steroids studied without significantly reducing the total production of relaxin over the culture period. Tniostane and androstriene 3, 17-dione were only partially effective in reducing steroidogenesis but caused a significant reduction in total relaxin production. Follicular tissue therefore synthesizes and secretes relaxin in vitro, but the cellular origin, the relationship with steroidogenesis, and the routes of release remain to be established.

Evidence for proteolysis during purification of relaxin from pregnant sow ovaries.

The proteolytic degradation of relaxin during its isolation from pregnant sow ovaries has been examined. Ovaries from pregnant sows were selected and divided into three groups according to the stages of pregnancy. Each group was extracted with and without protease inhibitors. It was found that protease(s) were present in all groups of ovaries and that a 2-4 fold increase in yield of total relaxin was obtained when isolation and purification was carried out in the presence of protease inhibitors. However the ratio of the three forms of relaxin remain unchanged.

High molecular weight forms of relaxin in pregnant sow ovaries

The existence of a prohormone for relaxin has been investigated by purification from extracts of pregnant sow ovaries. Radioimmunoassay of column fractions from large scale extraction of frozen pregnant sow ovaries detected two species of high molecular weight relaxin immunoactivity besides the 6,300 dalton relaxin. On further purification, these two species were resolved into three forms with apparent molecular weights of 19,000, 13,000 and 10,000 daltons respectively. Each was biologically active and could be converted by trypsin to the 6,300 dalton relaxin. They may represent intermediates of relaxin.

Separation of relaxin activities in extracts of ovaries of pregnant sows by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis has been employed to separate three fractions that have relaxin activity from crude extracts of ovaries of pregnant sows. Bioassays indicated that all fractions had the ability to inhibit spontaneous uterine contractions in vitro and to induce formation of interpubic ligaments in oestrogen-primed mice. The presence of the various fractions in crude extracts of sow ovaries was monitored with the polyacrylamide gel system on days 40, 70 and 100 of pregnancy. Both the total amount and the relative proportions of the fractions were found to change as pregnancy progressed.

Relaxin, a male hormone? Immunocytological localization of a related antigen in the boar testis.

Convincingly demonstrated by immunocytological methods in females of several mammalian species, relaxin has not yet been localized in the male. Immunocytologically, a related antigen was identified in adult normal boar testes using and anti- [NIH P-relaxin/HSA] antiserum free of anti HSA Abs. A strong reaction was observed in interstitial cells, a weaker but very clear one in Sertoli cells. NIH P-relaxin and HC1-acetone extracts of either corpora lutea from pregnant sows or boar testes inhibited the immunofluorescence of the reactive structures in the boar testes as well as in ovaries of pregnant sows. Ethanol-acetone precipitates from boar rete testis or caudal epididymal fluids inhibited the reaction of interstitial and Sertoli cells, but this inhibition in the sow was limited only to degenerative ovarian structures, probably due to an insufficient level of inhibiting antigen in these two seminal fluids, in contrast with the very high concentration of relaxin in luteal cells of pregnant sows. Specific immunofluorescence was observed neither in ectopic testes of adult monocryptorchid boars (contrary to scrotal testes in these same animals) nor in testes of prepuberal pigs. The specificity and meaning of these results are discussed.

Primary structure of the B-chain of porcine relaxin

The sequence of the B-chain of relaxin, and ovarian peptide hormone isolated from ovaries of pregnant sows, has been shown to have the following primary structure: PCA-Ser-Thr-Asn-Asp-Phe-Ile-Lys-Ala-Cys-Gly-Arg-Glu-Leu-Val-Arg-Leu-Trp-Val-Glu-Ile-Cys-Gly-Val-Trp-Ser (2820 daltons). The heterogeneity of relaxin observed during purification procedures is likely to be due to variations in the C-terminal region of the B-chain, in particular the substitution of Gln for Glu 20, and the possible addition of arginine or serylarginine at the C terminus. The B-chain exhibited a distribution of sulfhydryl residues relative to one another that is identical to that found in the B-chain of insulin. A similar analogy has already been demonstrated for the A-chains of relaxin and insulin.

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 isolation and purification of an ovarian polypeptide with uterin-relaxing activity

1. 1.|A strongly basic polypeptide with uterine-relaxing activity was extracted from the ovaries of pregnant sows using aqueous acetone containing hydrochloric acid. Purification was achieved by an isoelectric precipitation and by heat denaturation of inactive contaminants followed by fractionation with trichloroacetic acid, gel filtration on Sephadex, and chromatography on DEAE-Sephadex. The material obtained proved to be chromatographically and electrophoretically pure. 2. 2.|In addition to reducing (in nanogram amounts) the motility of the isolated rat uterus, the purified hormone preparation displayed a strong activity on the pubic symphysis characteristic of relaxin. It was concluded that the ovarian uterine-relaxing factor and relaxin are identical or very closely similar polypeptides.

Isolation of a highly purified polypeptide with uterus relaxing activity

A highly purified polypeptide with a powerful uterus relaxing activity has been isolated from pregnant sow ovaries. The homogenity has been checked, using various electrophoretic and chromatographic techniques.

Reversal of the decidual-inhibiting effect of relaxin by an inactive derivative.

ABSTRACT Relaxin preparations, isolated from the ovaries of pregnant sows, have previously been shown to inhibit the formation of decidua in the uteri of ovariectomized, pseudopregnant rats treated with progesterone. This effect has been employed to study the interaction of an inactive derivative and the native hormone. When an excess of thioglycollate- or mercaptoethanol-inactivated relaxin is administered simultaneously with native relaxin, the inhibitory effect of the latter is nearly completely abolished. Preparations from the ovaries of non-pregnant sows, of low relaxin content, have a similar effect. It is suggested that the ability to inhibit the effects of a protein hormone may be shared by naturally-occurring proteins structurally related to the native hormone.

The effect of relaxin extracts, progesterone and oestradiol on maintenance of pregnancy, parturition and rearing of young after ovariectomy in mice.

SUMMARY 1. 216 mice, spayed on the 14th day of their first pregnancies, were injected daily thereafter with progesterone, either alone, with oestradiol or relaxin extracts or both; no injections were given after parturition. Extracts of relaxin obtained from pregnant rabbit serum and pregnant sow ovaries were used. 2. When given alone, 1 mg/day progesterone maintained pregnancy to full term in 83%, and 0·5 mg/day in only 30% of mice. Parturition was often delayed, prolonged and difficult, with consequent death of the foetus in utero, even when injections were stopped on the 18th day. A high proportion of young were stillborn, and none were reared. The symphysis pubis remained closed and cartilaginous. 3. 1·5 μg/day oestradiol did not synergize the action of progesterone in maintaining pregnancy, but there were fewer delayed deliveries and a higher incidence of live births; a small number of mice reared some of their young. The innominate bones did not separate, but the symphysis was less rigid than when progesterone was given alone. 4. Relaxin extracts had a synergizing action when all three hormones were given together, 0·5 mg/day (but not 0·25 mg) of progesterone then being enough for maintenance of pregnancy in over 80% of mice. The experiments have not established whether oestradiol was necessary for this synergism. There was a positive relation between punctual and normal delivery of live young and potency of the relaxin extracts in producing full pregnancy changes in the pelvis. More mice reared some of their young. None of the hormone combinations used completely compensated for the absence of the ovaries.

Purification of relaxin by ion exchange resins

The preparation of relaxin extracts from pregnant sow ovaries is described. Studies with ion exchange resins show that relaxin is a neutral substance. By the concomitant use of strong cationic and strong anionic ion exchange resins, a shortened extraction procedure was devised which allows the removal of most of the extraneous nitrogenous content of the relaxin extracts.

A comparison of the response of the pubic symphysis of the mouse and guinea pig to extracts of pregnant sow ovaries.

THE response of the pubic symphysis of the estrogen-primed mouse to relaxin-containing extracts of pregnant rabbit serum (PRS) and of pregnant sow ovaries (PSO) has been reported previously (Kliman, Salhanick and Zarrow, 1953). Significant symphyseal separations were obtained with both types of extracts, although some differences were noted in the slopes of the dose-response curves of preparations from each of these two sources. However, roentgenographic measurement of the average increments in pubic separation produced by single doses of such extracts gave reliable quantitative determinations of activity. The effectiveness of crude extracts was confirmed by their ability to produce significant relaxation in all treated mice, and to reproduce the rate of separation which normally occurs during pregnancy. Subsequently, studies were made of the action, on the pubic symphysis of the mouse, of relaxin preparations of different specific activities. It has been found that some preparations which are very potent...

Effect of relaxin upon decidual reactions in the rat.

SummaryPreparations of relaxin from the ovaries of pregnant sows and from the blood of pregnant rabbits inhibit the development of a decidual reaction in the uterus of the castrate-pseudopregnant rat following traumatization and treatment with progesterone. Partially purified relaxin samples also contain a heat-labile factor which synergizes with progesterone in the production of deciduomata. On the basis of experiments with relaxin preparations covering a wide range of specific activities, and the effects of a variety of chemical reagents, it is concluded that the inhibitory property of such preparations is a specific response to relaxin.