Plasma relaxin concentrations in the pig during the periparturient period: Association with prolactin, estrogen and progesterone concentrations

THERIOGENOLOGY

PLASMA RELAXIN CONCENTRATIONS IN THE PIG DURING THE PERIPARTURIENT PERIOD: ASSOCIATION WITH PROLACTIN, ESTROGEN AND PROGESTERONE CONCENTRATIONS J.Z. Kendalll, G.E. Richards2, LI-N. Shihl, T S. Fart-is2 Department of Obstetrics and Department of Gynecology1 and Pediatrics2, University of Texas Health Science Center at Houston, 6431 Fannin, Houston, Texas 77030

Received

for

publication: Accepted:

January 21, 1982 March 25, 1982 ABSTRACT

Concentrations of relaxin, prolactin, unchromatographed estradiol 17s (E2s) and progesterone (P4) were measured in serial samples of inferior vena caval blood, in three pigs, during late pregnancy, Maximal relaxin concentrations occurred 60 to 24h and parturition. and ranged from 60 to 286ng/ml. before parturition, Prolactin increased from 12.5ng/ml, 48 to 36 hours before concentrations parturition, to between 79 to 184ng/ml. At the time of the relaxin concentrations surge, levels were high, and P4 were E2B A surge in prolactin decreasing, thus raising the E2s/P4 ratio. concentrations followed upon a decline of P4 to less than long/ml, coinciding with the increase in relaxin concentrations in 2 gilts, and following the surge in relaxin in the third. Udder development occurred near the time of increased relaxin concentrations. 'Milk let followed maximal relaxin and prolactin concentrations in two down' gilts, and the increase in prolactin, rather than the increase in relaxin concentration, in the third.

INTRODUCTION: Relaxin is a protein hormone synthesized in large amounts by the porcine Relaxin is corpora lutea of pregnancy.lp2 stored in secretory granules in the luteal ce113p4 until just before birth, when it is released into the circulation over one to two days.5 Possible functions durin the perinatal period include cervical L? a tocolytic ripening and dilatation effect on the myometrium7, and stimulation of mammiry gland development.8 The mechanism regulating relaxin release before parturition is not known. Progester ne delays parturition, 6 at term. Oxytocin and PGF2p The effects of changes in luteinlzing hormone, estrogen, and prolactin have not been reported. prolactin release in PGF2c, also stimulates Kendall, Ph.D., Address Correspondence to: J.Z. Obstetrics and Gynecology, University of Texas Health at Houston, 6431 Fannin, P.O. Box 20036, Houston, Texas

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THERIOGENOLOGY released at term after suggest a possible The interrelationship of prolactin and relaxin release before birth. purpose of our study was to measure the plasma concentrations of relaxin in the same sample in which prolactin, progesterone (Pq), an indicator of luteal function, and unchromatographed estradiol 17 were also estimated. The profile of all 4 hormones in serial B' y collected samples from individual pigs before parturition has not previously been reported. Although cause and effect cannot be concluded from our study, any apparent suggestion of a temporal relationship between these hormones, may provide direction for further investigation of the endocrine regulation of relaxin secretion before birth in the pig.

~,“~,,ltate~onPereg:rapnattm,P,‘tq’13andTheqr,O1aoCbt:'e"rvi,Zi ons

MATERIALS

AND METHODS

Animals and Blood Samples Theatternplasrna hormones was measured in 3 late preqnant cross bred gilts. The day of mating was designated Day 0 of pregnancy. At day 90, 92 or 95 of gestation, an indwelling polyvinyl catheter (i.d. 0.86mm, o.d. 1.27mm) was placed into the inferior vena cava by way of a small uterine vein exposed at laparotomy.14 In two of the animals, a balloon catheter was placed in an amniotic sac.15 The catheters were fed subcutaneously, and enclosed in a cloth pouch glued to the sacral area. The balloon catheter was connected to a Statham pressure transducer, continuous intra-uterine and pressure was recorded using a Narco Polygraph pen recorder. The onset and length of labor was monitored. The pigs were housed individually in metabolic cages. They were fed a full diet, 3kg per day, of a 16% protein ration. Blood samples were taken daily from the vena cava catheter. Samples were collected regularly at 1600 h. In 2 gilts, additional samples were collected at 0800 h and 2400 h. One animal was sampled at 2 h intervals for 10 h during labor. The gestation length of the gilts was 114 to 116 days, parturition was normal (~12 h), and the litter size 8 to 11 live piglets. Piglets were removed from the dam on the day of birth, and maternal blood samples collected 24 to 48 h postpartum. All samples were collected into heparinized syringes, transferred to chilled polystyrene tubes, and centrifuged immediately at 15009 for 15 min at 4oC. The plasma was divided into aliquots and stored at -200C until assayed. The gilts were observed frequently for signs of udder enlargement and the expression of milk ('milk let down'). Radioimmunoassays Relaxin: Relaxln immunoactivity measured according to the was procedure of Loumaye et al.16 Porcine relaxin, elq7uplwqausantuisv;sf;; CM-a and CM-a' and CM-B (2500 to 3000 U/mg) radio-iodination. Relaxin antisera (CM-a + CM-a' + CM-B antigen prepared in our laboratory) was used at a final dilution of 1:70,000. Porcine relaxin standards (NIH-PR, 2500-3000 U/mg) were assayed in triplicate. Unknown serum is assayed in triplicate at 3 to 4 dilutions ranging from 5~1 to 200~1. Antibody bound relaxin was precipitated by goat antirabbit C( globulin diluted l/32 in PBS (P4 Antibodies Incorp. Ca.). The mean slope of the regression line for 6

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THERIOGENOLOGY radioimmunoassays was -0.82 + 0.01 SE. The slope of the dose response curve obtained with multiple volumes of late pregnancy pig serum was similar to the dose response curve obtained with standard relaxin. FSH, LH, or prolactin did not react in Porcine insulin, proinsulin, The quantities of porcine relaxin the system even in up amounts. (NIH-PR) measured after the addition of 400pg to huffer, 10~1 or 200~1 of boar serum, were 442 + 33.8pg, 459 + 69.4pg, and 445 + 21.3pg, The intra-assay coefficiefi of variation for-a plasma respectively. sample of ZOng/ml obtained from a pig during late pregnancy was 6.0%. The interassay coefficient of variation was 17.9% (n = 91). Sensitivity of the assay was 16pg. Prolactin was measured by radioimmunoassay . Prolactin: uslng18a method previously described for measurement of canine prolactln. The only modification to the method was substitution of purified porcine prolactin (pPRL) for canine prolactin as the reference used for iodination and reference The same pPRL was standard. Antibody bound PRL is precipitated by sheep antirabbit y standard. Porcine plasma displaced iodinated diluted l/4 in PBS. globulin, a manner that was parallel to the purified porcine prolactin in Purified porcine growth hormone displaced porcine prolactin standard. Porcine relaxin did iodinated pPRL with a cross reactivity of 10%. Sensitivity of the assay was 0.1 not cross react in ug amounts. Intra-assay ng/tube or 1 ng/ml when 100~1 samples were assayed. coefficient of variation was lC% at a plasma concentration of 0.5 Interassay and 7% at a plasma concentration of 5 ng/ml. w/ml, coefficient of variation was 12% at a plasma concentration of 5 ng/ml. determined by Concentrations of P4 Progesterone: were as described by Gordon and Sherwoodl9, with some radioimmunoassay, antisera against generated modifications. The P4 -dione-11-hemisuccinate: cross reacts 4-pregnen-lla-ol-3,20 BSA with lla-hydroxysignificantly progesterone [;';J%j, 17a-hydroxyprogesterone . 5a-pregnan-3,20-dione (3.98%), Duplicati desoxycorticosterone (1.78%), and corticosterone (1.0%). 100~1 aliquots of plasma were extracted once with 3cc of petroleum ether. Sensitivity of the assay was 6pg.20 Serial dilutions of a plasma sample gave a P4 value of 10.1 + 0.34ng/ml (mean + SD, n = Recovery of 50 pg, 100 pg, 250 pg, 500 pg, 1000 pg at-2 2,500 pg 8). P4 (auadruplicate assays) added to a 100~1 plasma sample containing The 12Opp P4 was 102%, 103%, 99%, 96% 96%, and 98% respectively. intra-assay coefficient of variation for one plasma sample was 4.3% control sample, (mean = 7.0 + 0.3ng/ml SD, n = II), and for another 9.4 %(mean =x.4 + 0.61ng/ml SD, n = 11). Interassay CV for the same samples was 10.6%: estradiol concentrations of Plasma unchromatographed Estrogen: 1-1 were determined by RIA using an antibody raised against 1,?,5,?10) -Estratrien-3,17-dial-6-one-6(carboxymethyloxime) BSA, at This antibody, which cross reacts 1.8% with a dilution of 1:125,000. and 0.48 %with 17~~ estradiol was kindly estrone, 4.9 %with estriol, Estradiol -170 -2,4,6,7:3H (Sp. Act provided by Dr. O.D. Sherwood. 85 Ci/mmol Amersham, Ill.) was used as the radioactive ligand. Plasma (lml) was extracted in duplicate with 4cc of dichloromethane (DCM). The amount of radiolabelled E2s extracted, using this procedure, was 86.3 + 2.9%. The dried extract was redissolved in O.OlM PBS (O.l% overnight at 4oC with antibody gelatin W~V), pH 7.5, and incubated

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Bound and free hormone was separated by and 30,000 dpm of label. charcoal suspension (final concentration using 0.25~~ dextran .coated The supernatant was decanted into 1Occ of Econofluor (New 0.12%;). Ill.) and counted in a Packard Tricarb England Nuclear, Chicago, Two controls in duplicate were spectrophotometer (1% counting error). included in each assay- one consisting of follicular phase pig plasma, The intra-assay coefficient of the other, a pregnant pig plasma pool. variation for these standard samples was 5.9%; the interassay The sensitivity of the assay, as coefficient of variation 8.2%. Solvent blanks were <5pg. defined by Ekins and Newman 26 was 3pg. Recovery of 25pg, 5Opg, lOOpg, (quadruplicate ana'lysis) added to lml aliquots of plasma sample containing 8pg E2a/ml, was 118x, 88%, and 90%. Serial dilutions of a plasma sample ranging from 250~1 to 1000~1 of undiluted plasma gave E2s value of 309pg/ml, and a coefficient of variation of 18.4% Selected plasma samples were assayed for Epa with and without a Plasma was extracted in duplicate with chromatography procedure. DCM. Samples were redissolved in benzene methanol 85:15, applied to LH 20 columns (16.0cm x 0.9cm), and eluted with the same solvent ratio.21 of radiolabelled The amount recovered when this E20 procedure was included was 71.9 + 3.4%. Calculations: Calculations fir all hormone assays were made by computer using a logilogit transformed standard curve.22

RESULTS Relaxin concentrations increased markedly in individual gilts 60, and subsequently declined (Fig. 1). 24, and 40 h before parturition, The prepartum peaks of plasma prolactin reached maximum values of 79, 143, and 184 ng/ml in these three silts. Prior to the prepartum surges in plasma relaxin and prolactin, relaxin concentrations were and baseline concentrations of prolactin low, 4.0 + l.eng/irl S.D., were 12.57 7.0 ng/ml S.D. Relaxin concentrations declined to very Prolactin low values-also following delivery, 1.5 + l.Ong/ml S.D. concentrations were less than 25ng/ml, 20 to 48 h postpartum. concentrations declined gradually during late Although the P4 the mean concentration of P4 was 20.7ng/ml, until 2 to 3 pregnancy, fell sharply to an average days before birth, when P4 concentrations Progesterone concentrations prepartum value of 2.1 + 0.83ng/ml S.D. declined further postpartum (0.96 2 0.37ng/ml S.D.) (Fig. 1). The concentration of Epa was already high, greater than lOOpg/ml, 5 to 6 days before birth (Figs. 1 and 2). Peak concentrations were attained within the 24 hours preceding delivery of the first piglet. Eps concentrations continued to rise during the 8 h preceding birth in the gilt sampled at intervals of 2 h during labor. After delivery, E2 concentrations declined to less than 20pglml at 24 h. 9.lsible swelling of the mammary glands was observed 16 to 36 h before birth. Milk could be expressed within I2 to 24 h of delivery (Fig. 1). ratio ('milk let down') and prolactin EzB/P~ concentrations were rising at this time. In one gilt, the relaxin surge preceded 'milk-let down', in the other two, the peak relaxin concentration coincided with the onset of 'milk let down'.

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FIG.

1.

concentrations relaxin, prolactin, Plasma of unchromatographed estradiol 17s and progesterone in three individual pigs during late pregnancy. The arrow denotes the onset of udder edema; the bar denotes the prepartum period of milk ejection. Day 0 represents the time of delivery of the first piglet.

DISCUSSION There are only a few studies of prolactin in the circulation during sow,23~ISy24 The mean baseline pregnancy and parturition in the concentration of prolactin, the prepartum rise in prolactin secretion, and the maximum prola tin concentrations are similar in all studies of measurements.fSy24 Our findings after farrowing are in serial accord with other re orts of rolactin in sows during early lactation 2!,26,27,28 Prolactin levels remain elevated in The relationship, observed here, of udder enlargement, and 'milk let down', to relaxin release, has also been reported by others du;i;g normal, induced, and pro10;~;xiregna:,cy.ga~d2 In the rat, somatotropin, synerglse tk prolactin, P4 accelerate mammary gland differentiltion.8 We found a similar time course between prolactin secretion, mammary gland growth, and lactation in our pigs. Further investigations concerning the effect of induction of parturition, prolongation of pregnancy, and treatment with bromocriptine are needed to determine the relative importance of prolactin, or relaxin, in the onset of lactation in the pig.

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160160140lZDlooao6040zo-

Days framParturition

FIG.

2.

Changes in inferior vena caval plasma concentration (with and without --during late pregnancy in 1 pig. Each point mean of duplicate determinations L SD.

estradiol 17 chromatography B represents the

prolactin secretion before parturition is not The stimulus to obvious from our study. In contrast to the cow,29 prolactin concentrations in pigs do not show marked fluctuation during stress provoking situations.30,31 Thus, it is possible that prolactin secretion is not stimulated markedly by the stress of labor. In cycling pigs, a rise in prolactin concentration occurs about the day of estrus, and it has been suggested that the estrogen rise, occurring prior to the onset of estrus, stimulates both LH and prolactin release.32933 already late Estrogen levels elevated in are pregnancy in the pig and, within the limitations of our sampling regimen, no further rise was seen in E2e preceding rising prolactin concentrations. In all three pigs, however, during a rise in prolactin Concentration the P4 concentration and maximum was falling, prolactin release occurred when P4 levels were less than long/ml. Prolactin release was, therefore, associated with corpus luteum regressi n and a rise in the E26/P4 ratio. Van Landeghem and Van de We1 3 4 and Dusza and Krzymowska35 reported a second surge in prolactin during the early follicular phase of the cycle, also coincident with a decline in P4 levels. Nonetheless, when pregnancy is secreted at term, is prolonged by treatment with P4, prolactin and again upon withdrawal of plasma P4 concentrations, suggesting stimulate that stimuli, other than circulating P4, prolactin secretion.13 The pattern for plasma P4, and the concentration of EzB are in agreement with that previously reported.36*28*3;;38 The pattern of total that described for and for E2f3 resembles EZ% unconjugated estrogens,39 with the highest concentration of EZB In the present occurring some time during the two days before birth. The study, the RIA is more descriptive for total estrogens. discrepancy seen in our study between plasma E2e assayed with and

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THERIOGENOLOGY without chromatography (Fig. 21, likely results from cross reaction Estrone and estrone sulfate are the major estrogens in with estrone. dam and fetus during late pregnancy,40 and are 10 to 16 times in of plasma estradiol 17, concentrations.39 The secretory excess however, is essentially identical.39 pattern of the three estrogens, The pattern of relaxin secretion and the plasma concentration, observed in our study, are in general agreement with those described parturition in Sherwood and after the ’ by before and colleagues.S,41 The occurrence of maximum r!l'zxin secretion in our interval than the mean study preceded parturition by a longer It is possible, however, that because qenerally described by others. in concentrations of relaxin, occurring 2-3 days before ~:r:~J~:iX~~Sjs the true peaks were missed by the less frequent sampling in our experiment. Nonetheless, the time of the mean relaxin peak secretion still fel? within the previously reported range. There was no evidence of abnormal parturition in our study; labor was less than 12 h in the 2 gilts retaining intra-uterine catheters, and all gilts completed farrowing within a few hours of delivery of the first piglet. Evidence that the prepartum surge in relaxin may be associated with termination of luteal function, and declining P4 secretion, was provided previously in reports of relaxin and P4 concentrations in and in prostaglandin induced parturition.5y12 normal parturition, between the The striking temporal relationship, in our study, falling concentrations of P4 and relaxin, suggest that concentrations of plasma P4 may be the stimulus to increase relaxin In conflict with this view is the observation that relaxin secretion. concentrations rise at term in pregnancies prolonged by exogenous progesterone,' despite high levels of plasma P4 maintained by sources.42743 Another interpretation of and endogenous exogenous the relationship of relaxin secretion to corpus luteum regression is that luteotrophic or luteolytic factors regulate both relaxin and progesterone secretion. Prolactin and estrogen, considered by some investigators to be part of the luteotrophic complex necessary for maintenance of secretory function of the corpus luteum in the pregnant sow,44945 could be prime contenders. It is somewhat surprising, therefore, to find that prolactin and estrogen increase during the time of luteal regression when a fall in secretion of these hormones a rise in prostaglandin F201 might have been expected. Nonetheless, reTaxin46 and (P;;;:iin.$ t;;s tzmeof co;;; increase ,,p;;Jn and prolactin gilts, concentrations increased simultaneously. A similar close association relaxin and prolactin concentrations during between increases in suckling in sheep, led Bryant and Chamley47 to speculate, either a direct cause and effect between prolactin and relaxin secretion, or The separation control of both hormones by another, such as oxytocin. of the two peaks, in one gilt in our study, would tend to refute an essential causal relationship between prolactin and relaxin secretion during normal parturition; although more frequent sampling may be reauired to detect specific fluctuations. Nonetheless, a recent study41 has failed to confirm a previous observation that relaxin is significant secreted in concentrations in the during sow The relaxin suckling.48 close association of and prolactin secretion, in two of our gilts, may suggest a common regulatory

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indicates that Substantial evidence has accumulated which and stimulates release of relaxin is luteolytic in the pig, PGF2, production. with Treatment decline in P4 and a PGF2, significantly increases serum prolactin above control levels.13 If regulating the onset of PGF2, is indeed the final common denominator in P4 secretion, and the release of the relaxin surge, the decline then the hormonal profile seen in the gilts in our study prolactin, differential responses to PGF2, by may necessitate, on occasions, Recently Nara and colleagues46 stimulated the three hormones. independently of a fall in P4 concentration, with relaxin release, low doses of PGF2,. factor.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the gifts of porcine prolactin and porcine growth hormone from Dr. C.H. Li, of porcine relaxin used for radioiodination from Dr. O.D. Sherwood, of porcine relaxin used for cold ligand provided by the NIAMDD Hormone Distribution Program, and of progesterone and estrogen antisera from Dr. W. Gordon. We wish interest and encouragement. We are to thank Dr. B. Held for his for skilled technical grateful to Ms. M.E. Buoy and Ms. D. Bridges assistance. The study was supported hy a Biomedical Research Support Grant, Univ. TX. Hlth Sci. Cntr. at Houston. REFERENCES 1.

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