BRAINRESEARCH

Brain Research 738 (1996) 48-52

Research report

Differential regulation of oxytocin and vasopressin messenger ribonucleicacid levels by gonadal steroids in postpartum rats

Abraham Thomas, Norma B. Kim, Janet A. Amico *Department of Medicine, University of Pittsburgh School of Medicine and Department of Veterans Affairs, Medical Center, Pittsburgh, PA 15261, USA

Accepted 18 June 1996

Abstract

We previously reported that sequential estradiol and progesterone exposure followed by progesterone withdrawal increases oxytocin(OT),but not arginine vasopressin (AVP), messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus (PVN) of therat. Substitution of testosterone for progesterone and subsequent testosterone withdrawal in the estrogen-primed rat increases PVN AVPmRNA levels. At the end of pregnancy (day 21), rats are exposed to high estrogen and declining progesterone and testosteroneconcentrations. Coincident with these changes in circulating gonadal steroid hormones are increases in OT and AVP mRNAs. Ifprogesterone levels are sustained at term, OT levels are attenuated and if testosterone is sustained, AVP mRNA levels are attenuated.Immediately postpartum, however, OT and AVP mRNA levels decline compared to term levels. To further determine the role of estrogenin the regulation of OT and AVP mRNAs, we performed two experiments. In the first experiment, we administered estrogen during theperipartum period to determine if estrogen supplementation prevents the relative attenuation of OT and AVP mRNAs that is seen afterparturition. Day 18 pregnant rats were given estradiol-filled or empty capsules and sacrificed on day 2 of lactation. By Northern analysis,significant differences in PVN AVP, but not OT, mRNA were found between the estrogen- and sham-treated lactational animals,P <0.02. In the second experiment, we determined if sustaining estrogen after progesterone is removed in steroid-treated ovariectomizedrats is essential for the increase in OT mRNA. Ovariectomized rats were given either empty capsules or sequential estradiol- andprogesterone-filled capsules and both were sustained for 12 days. When progesterone-filled capsules were removed, estradiol-filledcapsules were either removed or left in place, and the animals were sacrificed 48 h later. PVN OT mRNA was analyzed by Northern blothybridization. OT mRNA was increased in both of the steroid-treated groups to the same degree, compared to sham-treated animals,P = 0.04. In summary, estrogen supplementation during early lactation prevents the attenuation of PVN AVP, but not OT, mRNA afterparturition. In the estrogen-primed ovariectomized rat, it is not necessary to sustain estrogen to see the effects of progesterone withdrawalupon PVN OT mRNA.

Keywords: Arginine vasopressin; Estrogen; Lactation; Oxytocin; Paraventricular nucleus; Progesterone

1. Introduction

During the 48 h preceding and following parturition,dramatic changes in oxytocin (OT) and arginine vaso-Pressin (AVP) mRNA levels occur in the hypothalamicparaventricular and supraoptic nuclei (PVN and SON) ofthe rat [3]. OT and AVP mRNA levels are at their peak atterm (day 21 of gestation) [7,11,12] and precipitouslydecline after delivery [3]. We recently reported that thechanging pattern of gonadal steroid hormones that occursduring pregnancy and lactation accounts for these changesin OT and AVP mRNA levels [4,9]. Specifically, rising

“ Corresponding author. Fax: + 1 (412) 648-7047; e-mail: jamico +@pitt.edu

estrogen levels during pregnancy combined with decliningprogesterone and testosterone levels at term account forthe increases in OT and AVP mRNAs, respectively [4,9].If progesterone levels are sustained on day 21 of preg-nancy, OT, but not AVP, mRNA levels are attenuated [4].If testosterone levels are sustained at term, AVP mRNAlevels are attenuated [9]. Thus, progesterone and testos-terone are inhibitory to OT and AVP expression, respec-tively. In the setting of prior estrogen priming, the declinesin progesterone and testosterone are stimulator to OT andAVP mRNAs, respectively.

The peak levels of OT and AVP mRNA levels at termare not sustained following delivery [3], possibly due to thechanging gonadal steroid milieu postpartum. During weekone postpartum, estrogen levels are low [6], except for the

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A. Thomas et al. /Brain Research 738 (1996) 48-52 49

brief postpartum surge of estradiol that occurs with thepostpartum estrus [5], progesterone levels rise [6] and bothOT and AVP mRNA levels are attenuated compared toterm levels [3]. Thus, even if estrogen alone were sustainedpostpartum, OT mRNA levels might not increase becauseprogesterone, which is inhibitory to OT expression [4],rises postpartum. On the other hand, the increase in AVPmRNA at term pregnancy might persist postpartum ifestrogen levels were sustained because there is no postpar-tum rise in testosterone [8], which is inhibitory to AVPmRNA [9]. To test this hypothesis, we determined theeffects of sustaining estrogen during early lactation uponPVN OT and AVP rnRNA levels. We report that estrogensupplementation sustains AVP, but not OT, mRNA levelsin early lactation.

We also used a second experimental paradigm in theovariectomized rat to determine whether the ‘upregulatory’effects upon OT of progesterone withdrawal in the estro-gen-primed rat is dependent upon sustaining estrogen afterprogesterone levels decline. In a prior report [4], sequentialestrogen and progesterone administration to, and subse-quent progesterone removal from, ovariectomized rats in-creased OT rnRNA. In that study, estrogen was maintainedat the time of progesterone withdrawal. In the presentstudy, we maintained estrogen at the time of progesteronewithdrawal and compared this effect to simultaneouslywithdrawing both. We now report that progesterone with-drawal is critical to the increase in OT mRNA, irrespectiveof whether estrogen is maintained or simultaneously with-drawn.

2. Materials and methods

2.1. Animals and tissue removal

First-time pregnant Sprague–Dawley rats weighing250–300 g (experiment 1) or 225–250 g ovariectomizedSprague–Dawley rats (experiment 2) were obtained fromZivic-Miller laboratories (Allison Park, PA) and werehoused in plastic maternity cages or mesh cages, respec-tively, under controlled conditions (lights on, 0600-1800h; room temperature, 22”C). Laboratory rat chow (WayneLab-Blox, Chicago, IL) and water were available ad libi-tum. For the lactation studies, animals delivered on siteand litters were culled to 10 pups on day one of lactation.Day O was the day of delivery.

At the end of an experiment, animals were killed byrapid guillotine decapitation between 0900 and 1100 h.Trunk blood was collected into chilled borosilicate tubes,centrifuged at 4°C, and the serum was taken for estradioland progesterone measurements. For Northern blot hy-bridization, brains were removed and placed in a modifiedJacobovitz brain slicer (Zivic-Miller). A thick section ofhypothalamus (4 mm) containing the bilateral PVN wasremoved. The PVNS were microdissected from this section

and were processed in RNAzo1 (Cinna-Biotecx, Houston,TX) for extraction of RNA [3,4].

For experiments requiring steroid implant, SILASTICbrand tubing (Dow-Corning, Midland, MI, o.d. = 0.125”;id. = 0.078”) was filled with crystalline estradiol or pro-gesterone, (Sigma Chemicals, St. Louis, MO) or left emptyand placed S.C.under methoxyflurane anesthesia. Implantswere removed under methoxyflurane anesthesia.

Animal studies were approved by the committee onanimal care and use of the University of Pittsburgh Schoolof Medicine and were conducted in accordance with theNIH Guide for the Care and Use of Laboratory Animals.

2.1.1. Experiment 1We used Northern analysis to study the effect of sus-

taining estradiol during early lactation on PVN OT andAVP mRNAs. Day 18 pregnant Sprague–Dawley ratswere implanted with a 5 mm estradiol-filled (4 animals) orempty capsule (4 animals). Dams were sacrificed by rapidguillotine decapitation on day 2 of lactation. The bilateralPVNS were microdissected from a thick section of hypo-thalamus (4 mm) and both were placed in a tube contain-ing RNAzo1 for extraction of RNA. Trunk blood wascollected at sacrifice for steroid hormone levels.

2.1.2. Experiment 2In this experiment we compared the effect of simultane-

ous estrogen and progesterone withdrawal (Ez – /P – ) tothat of progesterone withdrawal and estrogen continuation(E2/P – ) in the ovariectomized rat. On day 1, animals ingroups of 5–6 received a 2 mm estradiol-filled capsule oran empty capsule. On day 3 the sham treatment groupreceived empty capsules while the estradiol-treateci ani-mals received 3 X 30 mm progesterone-filled capsules. Onday 14, the sham group had the empty capsules removedwhile the steroid-treated animals had the estradiol capsuleseither removed (Ez – /P – ) or left in (Ez/P – ) whenprogesterone capsules were removed. While the animalswere under methoxyflurane anesthesia on day 14 with theimplants in place, tail blood was collected for steroidhormone levels. The animals were sacrificed by rapidguillotine decapitation 48 h later on day 16 of the regimen.The bilateral PVNS were microdissected from a thicksection of hypothalamus (4 mm) and placed in a tubecontaining RNAzo1 for extraction of RNA. Trunk bloodwas collected at sacrifice for steroid hormone levels.

2.2. Northern blot hybridization

Total RNA was extracted and prepared for Northernblot hybridization as previously described [3,4]. A samplevolume equal to one half nucleus of bilaterally pooledPVN was fractionated on a 1% agarose/3% formaldehydegel. Only samples that were free of RNA degradation (asdetermined by photographs of the ethidium bromide stainedgels) were included in the analysis. DNA probes were

50 A. Thomas etaL/Brain Research 738 (1996) 48-52

prepared by random primer labeling cDNAs to exon C ofthe rat OT and AVP genes (provided by Dr. ThomasSherman, Department of Neuroscience, University of Pitts-burgh) with a32P-deoxy-CTP to specific activities of 1 X

IOx-g cpm/Lg. Hybridizations were done at 50”C for 16h. Specific hybridization of the OT and AVP probes hasbeen previously documented [3,4]. Blots were washed to afinal stringency of 0.1% SDS-2 X SSC (1 X SSC = 0.15 MNaCl and 0.015 M sodium citrate, pH 7.0). Blots wereexposed to Kodak X-Omat AR-5 film at –70°C (1 day forOT, 7 days for AVP, and 2.5 h for actin). Blots werehybridized first to the OT probe, then stripped, and ex-posed to film to verify absence of residual signal prior tohybridization to the AVP probe. Autoradiograms of hy-bridized blots were scanned (Model 620 densitometer,Bio-Rad Laboratories, Richmond, CA) and the arbitrarydensitometry measure of each band was used in calculatingthe relative abundance of mRNAs. Autoradiograms wereselected for densitometry with exposure times within thelinear range of the film. PVN RNA among the groups ineach experiment were hybridized and exposed simultane-ously.

To ensure equal loading and uniform transfer of RNAamong samples, membranes were rehybridized to anCX32P-labeled DNA probe to the (3-subunit of the mouseactin gene (provided by Dr. Stephen Phillips, Departmentof Molecular Genetics and Biochemistry, University ofPittsburgh School of Medicine). Results are expressed asOT mRNA per half nucleus and are presented as a percent-age of sham-treated animals.

2.3. Assays

Estradiol and progesterone were assayed with doubleantibody radioimmunoassay kits purchased from Diagnos-tic Products Corporation (Los Angeles, CA), and Diagnos-tic Systems Laboratory (Webster, TX) respectively, usingduplicate measurements. The minimum detectable concen-tration of estradiol and progesterone in their respectiveassays are 5 pg/ml and 1 rig/ml.

2.4. Statistics

In experiment 1, Northern blot hybridization data andestradiol and progesterone levels were analyzed by un-paired t-tests. In experiment 2, the Northern blot hy-bridization data and estradiol and progesterone levels wereanalyzed by analysis of variance (ANOVA) with post-hocFishers protected least significant difference (PLSD) test.Significance was at a level of P <0.05.

~ 350

-300

E2250cl)z~ 200&.<z 150KE 100z>n 50

0-,Sham Eatradiol

Treatment

Fig. 1. A: quantitative analyses of Northern blot hybridizations of PVNOT, AVP, and actin mRNAs of sham-treated (4/group) vs. estradiol-treated (4/group) day 2 lactating rats. Data are presented as a percent ofthe sham-treated group and are the mean ~ S.E.M. Significant differenceswere found for AVP, but not OT and actin, mRNAs. B: the autoradio-gram of the Northern blot analysis of PVN AVP mRNA of sham-treatedvs. estradiol-treated (E2) day 2 lactating rats.

vs. 5 ~ 0.4 pg/ml, respectively, P < 0.002), but proges-terone levels were not significantly different (32 + 5 vs.27 + 2 rig/ml, respectively). Despite the differences inestradiol levels, PVN OT mRNA levels were not signifi-cantly different between the two groups, Fig. 1A. Incontrast, PVN AVP mRNA levels were significantly higherin animals receiving estradiol-filled implants, P <0.02,Fig. 1A. Actin was not significantly different between thetwo groups, indicating that differences in AVP mRNAwere not likely due to variation in sample loading ortransfer, Fig. 1A. The autoradiogram of the Northern blothybridized to the AVP probe illustrates these increases inAVP, Fig. IB.

Table ISerum estradiol (E2) and progesterone (P) levels in ovariectomized ratswith silastic implants

Treatment n Estradiol (pg/ml) Progesterone (rig/ml)

Day 14 Sacrifice Day 14 Sacrifice

3. Results

3.1. Experiment 1

At sacrifice on day 2 of lactation, estradiol levels werehigher in the estradiol- vs. sham-implanted group (25 + 4

Sham 5 7*2 4*1 6*1 4*1E2 /P– 6 60f 8 30* 2 25+ 2 8+2Ez –/P– 6 66* 17 6+0.4 24 ~ 2 8*2

Ez /P– = only progesterone capsules were removed on day 14.Ej – /P– = both estradiol (E2) and progesterone (F’) capsules wereremoved on day 14; tail blood was obtained on day 14 and trunk blood atsacrifice for steroid assays

A. Thomas et al. /Brain Research 738 (1996) 48-52 51

A m-

$

-200azaEz 100

>&

o

B 1-

I ●

ShamTre%ment

E2-/P-

Sham EzIP- Ez-/P-~~~ 1

Fig. 2. A: quantitative analysis of PVN OT and actin mRNAs ofsham-treated (5/group) and steroid-treated (6/group) rats. In bothsteroid-treated groups the animals received sequential estradiol and pro-gesterone and subsequent removal of progesterone. In the group desig-nated Ez /P –, the estrogen was not removed with the progesterone,whereas in the group labeled, E2 –/P –, the estrogen was removed withthe progesterone. Data are presented as a percent of the sham-treatedgroup and represent mean+ S.E.M. Significant differences were presentbetween the hormone-treated and the sham-treated groups but not be-tween the two hormone-treated groups. B: the autoradiogram of theNorthern blot of PVN mRNA hybridized to a 32P-labeled cDNA probe toOT. The groups consist of ovariectomized animsds receiving sham im-plants or two steroid regimens in which sequential estrogen and proges-terone are administered. In one group (E2 /P– ), progesterone, but notestrogen, implants are removed whereas in the other group(Ez – /P–)progesterone and estrogen implants are removed simultaneously.

3.2. Experiment 2

Estradiol and progesterone levels verified the release ofthe appropriate steroid in the steroid implanted-ovariecto-rrtized animals, Table 1. On day 14, prior to the removal ofcapsules, estradiol levels were significantly higher in theestradiol- vs. sham-implanted animals, P <0.008, Table1, and progesterone levels were significantly higher in theprogesterone- vs. sham-treated animals, P <0.0001, Table1. At sacrifice on day 16 of the protocol, estradiol levels inthe group in which the estradiol was not removed (E2/P – )were higher than in the animals receiving empty capsules(sham) or in which estradiol-filled capsules had beenremoved (Ez – /P – ), P <0.0001, Table 1. The differ-ences in the absolute level of estradiol on day 14 and atsacrifice in the Ez/P – group may be due to the differentmethods of sample collection (e.g. tail blood on day 14 vs.trunk blood at sacrifice). At sacrifice, progesterone levelswere not significantly different among the groups, Table 1.PVN OT mRNA was higher in the steroid-treated groupscompared to the sham-treated groups, P = 0.04, but notsignificantly different between the two steroid-treatedgroups, Fig. 2A. The equal abundance of actin mRNA

among the groups indicated that differences in OT mRNAwere not likely due to variation in sample loading ortransfer, Fig. 2A. The autoradiogram of the Northern blothybridized to the OT probe is shown in Fig. 2B.

4. Discussion

The 48 h prior to and after delivery in the rat areaccompanied by rapid fluxes in circulating levels of estro-gen, progesterone and testosterone. Progesterone and tes-tosterone levels, which rise during gestation, precipitouslydecline prepartum [1,2]. Twenty-four hours after deliveryprogesterone, but not testosterone, levels begin to rise[5,6,8]. Estrogen levels are high during gestation but re-main low ( <10 pg/ml) during the first week of lactation[6], except for the brief postpartum surge of estradiol thatoccurs with the postpartum estrus [5]. PVN OT and AVPmRNAs rise dramatically in the 24 h prior to parturitionthen are attenuated, relative to day 21 of pregnancy, for thefirst 10 days of lactation [3]. While it has been shown thatthe decline in progesterone and testosterone, respectively,prepartum are essential to the respective increases in PVNOT and AVP mRNAs at term, it is not known why theprepartum increases in OT and AVP mRNAs are notsustained during early lactation. We theorized that if hy-poestrogenernia contributed to the relative attenuation inthese RNA species, then sustaining estrogen during earlylactation may increase one or both of these mRNAs post-partum. In this study, we implanted late pregnant rats withestrogen capsules to sustain estrogen levels postpartum.We found that PVN AVP, but not OT, mRNA levels werehigher in lactating animals that received estrogen supple-mentation. Thus, factors other than hypoestrogenetrtia alonemust account for the attenuation of OT mRNA postpartum.Since progesterone appears to be inhibitory to OT mRNA,the rise in progesterone postpartum is likely an importantfactor contributing to the attenuation in OT mRNA in earlylactation. Thus despite estrogen supplementation postpar-tum, the rising progesterone levels appear to suppress OTmRNA.

In contrast to OT mRNA, AVP mRNA is regulated byestrogen and testosterone [9]. The decline in testosterone atterm pregnancy is essential to the increase in AVP mRNAprepartum. If testosterone levels are sustained prepartum,term pregnancy levels of AVP mRNA are attenuated [9].While high levels of progesterone may regulate postpartumOT mRNA levels, high levels of testosterone postpartumcannot be invoked as a possible regulator of AVP mRNAlevels because testosterone levels do not increase postpar-tum [8]. In contrast to OT mRNA, low estrogen levelsappear to regulate AVP rnRNA postpartum since supple-mentation of animals with estrogen significantly increasedAVP mRNA postpartum compared to matched cohortsreceiving sham implants.

52 A. Thomas et al. /Brain Research 738 (1996) 48–52

While it is clear that estrogen priming and progesteronewithdrawal are both necessary for increased Ievek of PVNOT mRNA [4], the role of sustained estrogen levels afterprogesterone levels have fallen has not been previouslystudied. We now report that in the steroid-implantedovariectomized rat, it is not necessary to sustain estrogenafter progesterone withdrawal to increase OT mRNA.Ovariectomized animals receiving sequential estrogen andprogesterone implants and subsequent removal of proges-terone implants had comparable increases in PVN OTmRNA irrespective of whether estrogen was removed si-multaneously with progesterone or continued after proges-terone was withdrawn. Thus increased OT mRNA levelsoccur even if estrogen levels are low after progesterone iswithdrawn. Thus, during physiological events in whichboth estrogen and progesterone decline simultaneously, thelevel of PVN OT mRNA should still increase. Althoughthe simultaneous decline in both of these ovarian hor-mones does not occur during parturition in the rat, both fallsimultaneously at parturition in primate species, includinghumans [10]. Although levels of OT mRNA in the PVN ofprimate species receiving this specific steroid supplementa-tion or at parturition have not yet been investigated, it willbe of interest to determine if estrogen priming and proges-terone withdrawal increases OT mRNA in the PVN ofprimates as well.

In summary, in the estrogen-primed progesterone with-drawn rat, sustaining estradiol is not necessary to increasePVN OT mRNA. Furthermore, estrogen supplementationduring early lactation can sustain AVP, but not OT, mRNAlevels during early lactation. This latter observation sug-gests factors other than hypoestrogenemia alone, perhapsthe rising progesterone level, cause the attenuation of OTmRNA during early lactation. The mechanisms underlyingthese steroid-induced changes in OT and AVP mRNAsremain to be elucidated.

We thank Dr. Thomas Sherman for the rat OT and AVPcDNAs, and Dr. Stephen Phillips for the mouse (3 actincDNA. We are grateful for the expert technical assistanceof Ms. Jean Chiang and Ms. Debby Hollingshead as wellas the superb secretarial skills of Ms. Michele Dobranskyand Ms. Tammy Molinaro.

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[5] McNeilly, A.S., Suckfing and the control of gonadotropin secretion.In E. Knobil and J,D. Neill (Eds.), The Physiology of Reproduction,Raven Press, New York, 1994, pp. 1179-1212.

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[7] Spinolo, L.H., Raghow, R, and Crowley, W.R., Oxytocin messengerRNA levels in hypothalamic, paraventriculw, and supraoptic nucleiduring pregnancy and lactation in rats: evidence for regulation byafferent stimuli from the offspring, Ann. NY Acad. Sci., 652 (1992)425-428.

[8] Taya, K., Komura, H., Watanabe G. and Sasamoto, S., Peripheral

This project was supported by funds from the Depart-ment of Veterans Affairs (J.A.A.). A.T. was supported byNIH grant 5T32-DK-0752 for training in Endocrinologyand Metabolism. J.A.A. was a recipient of a Career Devel-opment Award from the Department of Veterans Affairs.Presented as an abstract at the annual meeting of theSociety for Neuroscience, November 13–17, 1995, SanDiego, CA.

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