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Expression of the endothelin-1 and oxytocin genes in the hypothalamus of the pregnant rat
BRAIN RESEARCH ELSEVIER
Brain Research 648 (1994) 59-64
Research Report
Expression of the endothelin-1 and oxytocin genes in the hypothalamus of the pregnant rat Mara J. Horwitz a, K e n n e t h D. Bloch b, N o r m a B. Kim a, Janet A. Amico a,, "Department of Medicine, E-1140 Biomedical Science Tower, Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA b Cardiac Unit, Medical Services Massachusetts General Hospital, Boston, MA, USA
Accepted 22 February 1994
Abstract
Endothelin (ET)-I, a neuropeptide and possible neuromodulator, has been found in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) of the rat in the distribution of oxytocin (OT) neurons. Within the hypothalamus of the pregnant rat, we investigated the developmental expression of the ET-1 gene and the possibility of coordinate expression of the ET-1 and OT genes. Blots containing hypothalamic mRNAs from 4-, 14-, 18-, and 21-day-old pregnant rats were hybridized to a 32p-labeled probe specific to the rat ET-1 gene. Hypothalamic tissue contained an ET-1 transcript of = 2.3 kb size. ET-1 mRNA abundance increased significantly in the SON and PVN from early to late gestation (P = 0.005 and 0.05, respectively). Blots containing hypothalamic mRNA were rehybridized to a 32p-labeled probe specific to exon C of the rat OT gene. OT gene expression increased significantly within both the hypothalamic SON (P = 0.0009) and PVN (P = 0.003) as gestation advanced. The sizes of the hypothalamic ET-1 and OT transcript sizes remained unchanged throughout gestation. Hypothalamic ET-1 and OT transcripts display stage-specific increases during gestation. ET-1 may be a neuroendocrine regulator of pregnancy-related events in the rat, and may act alone or in concert with OT. Key words: Endothelin; Gestation; Hypothalamus; Neurohypophysis; Oxytocin; Pregnancy
1. Introduction
The endothelins (ET) are a family of three structurally-related peptides, ET-1, ET-2, and ET-3, which are encoded by three distinct ET genes [5]. ET-1, which was originally isolated from porcine endothelial ceils [24], has been found in several non-vascular tissues [16], including those of the central nervous system, CNS. The presence of ET-1 immunoreactive peptide within the CNS suggests a neurotransmitter or neuromodulatory role for this peptide. Within the CNS of the rat, ET-1 immunoreactivity has been found in the hypothalamoneurohypophysial system [25], which is the primary site of synthesis and storage of the hormones oxytocin (OT) and vasopressin (AVP). O T and AVP
* Corresponding author. Fax: (l) (412) 648-7047, Janet A. Amico, M.D. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00266-F
are synthesized in the paired hypothalamic supraoptic and paraventricular nuclei (SON and PVN) and transported axonally to their storage site in the posterior lobe of the pituitary gland. Within the SON and PVN of the rat hypothalamus, ET-1 immunoreactivity has been reported to concentrate in the region of O T and AVP neurons [25]. ET-1 immunoreactivity has also been colocalized to the same neurosecretory granules as O T and AVP in the rat posterior pituitary gland [13]. Yoshizawa and coworkers have shown that water deprivation, a potent stimulus for depletion of posterior pituitary stores of O T and AVP, decreased posterior pituitary stores of ET-1 immunoreactivity in the rat [25]. Although in situ hybridization histochemistry has identified ET-1 m R N A in the porcine PVN [25], similar studies of ET-1 m R N A in the PVN or SON of the rat have not yet been reported. Since ET-1 immunoreactive peptide colocalizes to O T and A V P neurons of the rat hypothalamus, conditions that are known to enhance the abundance of hypothalamic O T a n d / o r A V P mRNAs, such as preg-
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nancy [20,26,27], lactation [20,26,27], and hyperosmolar states [1,9,10,12,17,21,23,28], may be accompanied by enhanced expression of ET-1 mRNA. OT is best known for its role in pregnancy and parturition [4] and milk ejection during lactation [22]. OT is released from the posterior pituitary gland with high circulating concentrations in late gestation [4] and during suckling [4,22]. The abundance of OT mRNA has been reported to increase within the SON and PVN of the rat in late gestation [20,26,27]. Additionally, the hypothalamic OT transcript has been reported by one [26,27], but not another [20], laboratory to undergo lengthening of the polyadenylate tail as gestation advances in the rat. In the present study, we examined the developmental expression of the ET-1 gene in the rat hypothalamus during gestation to determine whether the ET-1 transcript would also undergo changes in abundance and size.
2. Materials and methods 2.1. Animals and tissue preparation Timed primiparous Sprague-Dawley rats (250-550 g body weight) were obtained from Zivic-Miller laboratories (Allison Park, PA) and were housed individually in plastic maternity cages in a temperature(22°C) and humidity-controlled room with an 08:00-20:00 h photoperiod. Animals were fed rat chow pellets (Wayne Lab-Blox, Chicago, IL) and had access to tap water ad libitum. Animals were sacrificed in groups of four by rapid guillotine decapitation between 09:00 and 11:00 h on days 4, 14, 18, and 21 of gestation. At the time of sacrifice, the entire brain was removed from the pregnant animals and placed ventral surface upward in a Jacobowitz slicer (Zivic-Miller). Vertical razor blade cuts were made 1 mm rostral to the optic chiasm and 1 m m rostral to the mammillary bodies as previously described [2,3,14]. This coronal section of hypothalamus, which contained SON and PVN, was placed on a Petri plate for free-hand dissection of individual SON and PVN. Dissection technique guaranteed that the complete SON and PVN were contained within the sample [2,3,14]. Animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.2. Preparation of RNA Bilaterally pooled SON and PVN from each animal were placed in separate tubes containing RNAzol, 0.8 ml (Cinna-Biotecx, Houston, TX) and immediately homogenized by repeated trituration with a 21-gauge needle and syringe. Chloroform, 0.1 vol., was added and the samples were vortexed and maintained on ice for 15 min prior to centrifugation for 15 min at 12,000x g at 4°C. The aqueous layer was transferred to a clean polypropylene tube and the chloroform extraction was repeated. R N A was precipitated by the addition of one vol isopropanol and overnight storage at - 20°C, and then centrifuged at 12,000 x g for 30 min. The R N A pellet was washed twice with 800/zl 70% e t h a n o l / 3 0 % diethylpyrocarbonate (DEPC)-water. The pellet
was dissolved in DEPC-water and stored in one-nuclei aliquots from each animal at - 7 0 ° C .
2.3. Blot hybridization Samples of total R N A (one nucleus) were denatured for electrophoresis in 10 m M boric acid, 0.1 m M E D T A , 10% glycerol, 6% formaldehyde, and 50% deionized formamide. Samples were incubated at 65°C for 5 min and cooled on ice. O n e ~1 of 5% ethidium bromide was added to each sample. Nucleic acid electrophoresis was carried out on a horizontal 1% a g a r o s e / 3 % formaldehyde gel, unless otherwise specified. The positions, integrity, and abundance of 28S and 18S ribosomal R N A s were visualized and photographed under ultraviolet (UV) light. A 2:1 ratio of 28S to 18S ribosomal R N A s was found in the R N A samples for this study, thus verifying good integrity of RNA. Equal quantities of ribosomal R N A s among samples verified equal loading. Nucleic acids were transferred to a nylon m e m b r a n e (Gene Screen, New England Nuclear, Boston, MA) overnight by capillary action and immobilized by U V cross-linking. Prehybridization was done at 50°C in a sealed plastic bag in 10 ml of buffer comprised of 50% deionized formamide, 0.25 M sodium phosphate, NaHPO4, pH 7.2; 0.25 M NaCI; 1 m M EDTA; 100 txg/ml denatured salmon sperm DNA; and 7% SDS. Hybridization was performed at 50°C for 16 h in the same buffer with the addition of a 32p-labeled c D N A probe. Following hybridization, the filter was washed to a final stringency of 0.1% S D S - 2 × S S C (1 x S S C was 0.15 M NaC1; 0.015 M Na citrate, pH 7.0) at 50°C. The blots were exposed to Kodak X-omat A R x-ray film with 2 intensifying screens at - 70 ° for 9 days for ET-1 and 4 h for OT. Blots were hybridized to the ET-1 probe prior to hybridization to the O T probe. Autoradiograms of hybridized blots were scanned (Model 620 densitometer, Bio-Rad Laboratories, Richmond, CA) and the arbitrary densitometry unit measure of each band minus background for the film was used in calculating the relative abundance of cytoplasmic m R N A s . Autoradiograms were selected for densitometry with exposure times within the linear range of the film. To control for loading and transfer of RNA, m e m b r a n e s were rehybridized with a 32p-labeled D N A probe to the /3-subunit of the mouse actin gene. Actin abundance in the PVN and SON showed uniform signed intensity among samples, verifying uniform loading and transfer.
2.4. Preparation of radiolabeled probes The probe used to detect ET-1 m R N A was prepared from a rat ET-1 genomic clone and does not cross hybridize with ET-2 or ET-3. The rat ET-1 genomic fragment was cloned into the SacI and AccI sites of p G E M 4 (Promega, Madison, WI) and hybridizes to nucleotides 263 through 409 of the rat ET-1 c D N A described by Sakurai et al. [65]. The O T probe was prepared from a genomic clone for rat O T provided by Dr. T h o m a s Sherman, Department of Behavioral Neuroscience, University of Pittsburgh. The rat O T probe was prepared by digesting the p G E M 4 - O T 3c clone with the enzymes EcoRI and SaclI to yield a 169-bp c D N A for exon C of the O T gene [2,3,18]. For probe labeling, each plasmid D N A was digested with the appropriate restriction endonuclease known to cleave the insert D N A from its plasmid DNA. Probes were labeled using R a n d o m Primed D N A Labeling Kit (Boehringer M a n n h e i m Biochemical) according to the directions of the manufacturer. Sufficient 32p (1 / x C i / n g D N A [a-32p]dCTP, 3000 C i / m m o l aqueous solution, New England Nuclear) was added to the labeling reactions to yield specific activities estimated at 1 × 109 C P M / ~ g . The labeled probe was separated from unlabeled nucleotide on a Sephadex G-50 spin column, equilibrated and eluted with 1 x STE (10 m M Tris-H C1, pH 8.0; 1 m M E D T A , pH 8.0; and 100 m M NaCI).
M.J. Horwitz et al. / Brain Research 648 (1994) 59-64 2.5. Statistical analysis
61
Day 4 - SON r
Results are expressed as mean_+S.D. O T and ET-1 m R N A levels. Statistical comparisons for hypothalamic O T and ET-1 m R N A concentrations among groups were assessed using analysis of variance ( A N O V A ) or Kruskal-Wallis test, as appropriate, followed by post-hoc two sample t test or Wilcoxon rank sum test, respectively, between pairs of m e a n values. All statistical assumptions were met. Significance was P < 0.05.
3. R e s u l t s
The size and abundance of ET-1 mRNAs were assessed in the hypothalamic SON and PVN of day 4, 14, 18, and 21 pregnant rats. Accumulation of ET-1 m R N A increased significantly in the SON ( P = 0.005, Kruskal-Wallis test), and PVN ( P = 0 . 0 5 , ANOVA) with advancing gestation, Fig. 1. Significant differences
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Fig. 2. Northern blot hybridization of ET-1 m R N A in the supraoptlc nucleus (SON) of pregnant rats. The blot contains the SON from rats on pregnancy days 4 and 18. The Size of the ET-1 transcript was --- 2.3 kb. The 28S and 18S ribosomal R N A s identify m R N A species of = 5 kb and 2 kb, respectively.
in SON ET-1 m R N A abundance were found for gestational days 4 versus 14, 18, and 21 (each P = 0.02), and day 14 vs. 21 ( P = 0.05), with peak levels of ET-1 on day 18 gestation in the SON. However, by day 21 of gestation, ET-1 mRNA in the SON declined signifi-
SON
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Fig. 1. ET-1 m R N A in the SON (top panel) and PVN (bottom panel) of day 4, 14, 18, and 21 pregnant rats (n = 4 per group). The quantity of ET-1 m R N A , which was measured by densitometry of the bands on the autoradiogram, increased significantly in the SON and the PVN from early to late gestation ( P = 0.005 and 0.05, respectively). Significant differences in S O N ET-1 m R N A were found for gestational days 4 vs. 14", 18", and 21" (each, P = 0.02, indicated by asterisks), day 14 vs. 21 ( P = 0.05) and day 18 vs. 21 ( P = 0.03), whereas significant differences in PVN ET-1 m R N A were found for days 4 vs. 21" ( P = 0.05) and 14 vs. 21" ( P = 0.02) gestation.
4
14 18 Days of Gestation
21
Fig. 3. Quantitative assessment of OT m R N A in the SON (top panel) and PVN (bottom panel) of day 4, 14, 18, and 21 pregnant animals (n 4 per group). The quantity of O T m R N A was measured by densitometry of the bands on the autoradiogram. As gestation advanced, O T m R N A abundance increased significantly in the SON ( P = 0.0009) and PVN ( P = 0.003). Significant increases were found on day 4 vs. 18" and 21" (each, P = 0.0001), day 4 vs. 14" ( P = 0.009) and day 14 vs. 21 ( P = 0 . 0 5 ) in the SON and on day 21" vs. 4 ( P = 0 . 0 4 ) , 14 ( P = 0.02), and 18 ( P = 0.009) in the PVN of pregnant animals•
m.J. Horwitz et al. /Brain Research 648 (1994) 59-64
62
cantly compared to day 18 ( P = 0.03, Wilcoxon rank sum test). Significant differences in PVN ET-1 m R N A abundance were found for pregnancy days 4 vs. 21 and 14 vs. 21 ( P = 0.05, and P = 0.02, respectively, t-test). The size of the ET-1 transcript in the hypothalamus of pregnant rats was approximately 2.3 kb, identical to that reported in vascular tissues [5,16,24], Fig. 2. No size change was found in hypothalamic ET-1 transcripts during gestation, Fig. 2. Identification of ET-1 as the primary ET species in the pregnant rat SON and PVN was done by rehybridization of the blots to a 32p_labeled probe to ET-3. A very faint to non-detectable transcript was found after exposure of the autoradiogram for 21 days. The low or absent ET-3 signal precluded quantitation by densitometry. The blots containing m R N A from the SON and PVN of the pregnant rats were rehybridized to a .~2p_ labeled probe specific for the rat O T gene, and m R N A abundance was assessed by densitometry. Accumulation of O T m R N A was found to increase significantly in the SON and PVN as gestation advanced, Fig. 3, ( P = 0.0009 and 0.003, respectively, ANOVA). Significant differences in SON O T m R N A accumulation were found for gestational days 4 vs. 18 and 21 (each P = 0.0001), day 4 vs. 14 ( P = 0.009), and day 14 vs. 21 ( P = 0 . 0 5 , t-test), whereas significant differences in PVN O T m R N A were found for pregnancy days 4 vs. 21 ( P = 0.04), 14 vs. 21 ( P = 0.02), and 18 vs. 21 ( P = 0.009, t-test). The increase in abundance of the O T gene in the SON as pregnancy advanced was not accompanied by an increase in the size of the O T transcript. To further
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Fig. 4. Autoradiogram of SON m R N A from gestational days 4 (D4) and 21 (D21) animals and a mixture of the two (mix). The blot was hybridized to a probe specific for exon C of the rat OT gene. The size of the OT transcript was the same in day 4 and 21 gestational hypothalami. A mixture of R N A from gestational day 4 and 21 SON revealed a single transcript for the O T gene. The larger width of the middle band (mix) is due to the larger amount of R N A in that sample ( 1 / 2 nucleus) compared to day 4 and 21 samples which each contained 1 / 4 nucleus. The 28S and 18S ribosomal R N A s identify species of = 5 kb and 2 kb, respectively.
Day 18 - Placenta
28S ET-1
18S Fig. 5. Northern blot hybridization of ET-1 m R N A in the placenta of pregnant rats. Each lane contains 80 /~g of total R N A from the placentas of day 18 pregnant rats hybridized to a 32p-labeled probe to ET-1. The size of the placental ET-1 transcript was 2.3 kb. The 28S and 18S ribosomal R N A s are 5 kb and 2 kb, respectively.
resolve possible size differences in early vs. late gestational hypothalami, pooled R N A from the SON of day 4 and day 21 gestational rats ( 1 / 4 nucleus/lane) and a mixture of the two R N A pools ( 1 / 2 nucleus/lane) were electrophoresed on a 1.5% agarose gel. The size of the O T transcript was similar in the SON of day 4 and 21 gestational rats, and the mixture of these RNAs showed a single transcript, Fig. 4. A comparison of hypothalamic O T transcript size with that of extra hypothalamic O T transcripts was done by electrophoresing on a 1.5% agarose gel pools of R N A from the placenta and hypothalamus of day 21 pregnant animals and measuring the distance that each sample migrated from the well. Migration was less for the hypothalamic than the placental O T transcript, indicating a larger size for the hypothalamic O T transcript (data not shown for OT). In contrast, placental and hypothalamic ET-1 transcripts were both = 2.3 kb, Figs. 2 and 5.
4. Discussion
In the present study we used blot hybridization to demonstrate that ET-1 m R N A is present in the hypothalamic PVN and SON of the pregnant rat. Since ET-1 immunoreactivity was reported to localize in the distribution of O T neurons of the PVN and SON [25], we hypothesized that hypothalamic ET-1 gene expression may be increased during pregnancy, a condition characterized by enhanced accumulation of O T m R N A within these nucei [20,26,27]. We found ET-1 m R N A accumulation to be enhanced 1.5 fold in the PVN and 2-fold in the SON as gestation advanced. O T m R N A increased 2-fold in the PVN and 3-4-fold in the SON of the same animals as gestation advanced. Although both ET-1 and O T m R N A s increased in the SON, the changes did not exactly parallel one another. Increases in both genes were found on days 14 and 18 gestation compared to day 4. However, by day 21 gestation, O T levels in the SON increased further, while ET-1 de-
M.J. Horwitz et al. / Brain Research 648 (1994) 59-64
creased significantly. In the PVN, however, ET-1 and OT were more closely correlated with one another with significant increases in both genes on day 21 vs. day 4 of pregnancy. This study is the first to document stage specific increases in the expression of ET-1 mRNA in the hypothalamus of the pregnant rat. ET-1 appears to be the predominant ET gene isoform (ET-1, 2, or 3) in the pregnant rat SON and PVN. ET-2 mRNA is = 1500 nucleotides in length [15] and the transcript we identified in the PVN and SON of pregnant rats was = 2.3 kb. Moveover, hybridization of the blots in this study to a probe to rat ET-3 found a very weak or absent signal. The OT system has been a useful model for the study of neuropeptide gene expression. OT gene expression is enhanced in a variety of conditions in which peripheral OT secretion is enhanced, including advancing gestation [20,26,27], lactation [20,26,27], and chronic osmotic stimulation [1,10,12,20,21,23,28]. Moreover, the length of the OT transcript has been reported to increase in each of these conditions [12,26,27] due to elongation of the polyadenylate tail. Zingg and Lefebvre have found that the degree of polyadenylation of the OT transcript increased as pregnancy advanced [27,65], yet other investigators [20] have not reported OT transcript size changes with advancing gestation. Since mRNA transcripts of varying size can be distinguished by their migration rates through agarose, we did experiments to determine potential size differences in the hypothalamic OT transcripts of early and late pregnant rats. We observed no increase in OT transcript size as gestation advanced, but similar to other laboratories [20,26,27] found enhanced accumulation of OT mRNA in late gestation. The differences between the findings of Zingg and Lefbvre's studies [26,27] and our study are not explained by differences in the strain of rat (Sprague-Dawley rats were used in both studies) stage of pregnancy (day 21 pregnant animals were examined in both studies) or environmental conditions (animals were maintained in a normal state of hydration in both studies). Although Zingg and Lefebvre [26,27] have convincingly shown a size change in the hypothalamic OT transcript of late pregnant rats, the finding was not observed in the animals in our study. Thus although the change in OT transcript size may occur in pregnant rats, this change appears to be found less consistently than the enhanced accumulation of OT mRNA in pregnant rats. The reasons for these observations are not yet understood, primarily because factors regulating the length of the polyadenylate tail of the OT transcript have yet to be defined. Nevertheless, this study and other studies [2,12,25,26] support the concept that the factors governing accumulation and length of the OT transcript are independent. The size of the OT transcript depends upon the tissue source, and the size differences are also due to
63
variation in the length of the polyadenylate tail [6,8]. Other investigators have found size heterogeneity between hypothalamic and placental OT transcripts [8] and corpus luteal and hypothalamic OT transcripts [6]. In the present study, we confirmed the finding of Lefebvre and coworkers [8] that the hypothalamic OT transcript size was larger than placental OT. In contrast, the ET-1 hypothalamic and placental transcripts were both 2.3 kb in size. The abundance of ET-1 mRNA in the rat hypothalamus was far less than that of OT. The low abundance of ET-1 in the hypothalamus may favor a paracrine or autocrine role for this peptide rather than an endocrine role. Indeed, ET-I has been shown to exert local actions within the neurohypophysial axis [11,19] and has been colocalized within OT granules [13]. ET-1 may act as a modulator of OT expression or may have independent effects upon other hormonal systems that are important in gestation, such as the adrenocorticotropin-adrenal or gonadotropin-gonadal axes [7]. In summary, a study of the developmental expression of the OT and ET-1 systems in the hypothalamus of the pregnant rat showed that there are gestation-dependent increases in these two genes. The physiological function of hypothalamic ET-1 in gestation is not known, but ET-1 may be important as a neuroendocrine regulator of pregnancy a n d / o r parturition in the rat.
Acknowledgements The authors are grateful to Thomas G. Sherman, Ph.D., Department of Behavioral Neuroscience, University of Pittsburgh, for supplying the genomic clone for rat OT; Janine Janosky, Ph.D., Department of Clinical Epidemiology and Family Medicine, University of Pittsburgh School of Medicine, for statistical analysis of data; and to Christine Milcarek, Ph.D. and Stephen Phillips, Ph.D., Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, for consultation regarding molecular biology techniques. We thank Anna Halloran for expert technical assistance with animal studies and Michele Dobransky for excellent secretarial assistance. Supported in part by funds from the Department of Veterans Affairs (J.A.A.) and NHLBI Grant HL45895 (K.D.B.). J.A.A. is recipient of a Career Development Award from the Department of Veterans Affairs. Mara J. Horwitz is supported by NIH Grant No. 5T32DK0752-18 for training in Endocrinology and Metabolism. Presented in part at the Society for Neuroscience, 23rd Annual Meeting, Washington, DC, November 7-12, 1993.
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References [1] Burbach, J.P.H., Van Tol, H.H.M., Bakkus, M.H.C., Schmale, H. and Ivell, R., Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by Solution hybridization assays, J. Neurochem., 47 (1986) 1814-1821. [2] Crowley, R.S. and Amico, J.A., Gonadal steroid modulation of oxytocin and vasopressin gene expression in the hypothalamus of the osmotically stimulated rat, Endocrinology, 133 (1993) 2711-2718. [3] Crowley, R.S., Insel, T.R., O'Keefe, J.A. and Amico, J.A., Cytoplasmic oxytocin and vasopressin gene transcripts decline postpartum in the the hypothalamus of the lactating rat, Endocrinology, 133 (1993) 2704-2710. [4] Higuchi, T., Honda, K., Fukoka, T., Negoro, H. and Wakabayashi, T., Release of oxtyocin during suckling and parturition in the rat, J Endocrinol., 105 (1985) 339-346. [5] Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Masaki, T., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three seperate genes, Proc. Natl. Acad. Sci. USA, 86 (1989) 2863-2867. [6] Ivell, R. and Richter, D., The gene for the hypothalamic peptide hormone oxytocin is highly expressed in the bovine corpus luteum: biosynthesis, structure, and sequence analysis, EMBO J., 3 (1984) 2351-2354.. [7] Krsmanovic, L., Stojilkovic, J., Balla, T., Al-Damluji S., Weiner, R. and Cart, K., Receptors and neurosecretory actions of endothelin in hypothalamic neurons, Proc. Natl. Acad. Sci., USA, 88 (1991) 11124-11128. [8] Lefebvre, D., Giaid, A. and Zingg, H., Expression of the oxytocin gene in rat placenta, Endocrinology, 130 (1992) 1185-1192. [9] Lightman, S.L. and Young, W.S. IIl, Vasopressin, oxytocin, dynorphin, enkephalin, and corticotrophin-releasing factor mRNA stimulation in the rat, J. Physiol., 394 (1987) 23-39. [10] Majzoub, J.A., Rich, A., Van Boom, J. and Habener, J.F., Vasopressin and oxytocin mRNA regulation in the rat assessed by hybridization with synthetic oligonucleotides, J. Biol. Chem., 258 (1983) 14061-14064. [11] Matsumura, K., Abe, I., Tsuchihashi, T., Tominaga, M., Kobayashi, K. and Fujishima, M., Central effect of endothelin on neurohormonal responses in conscious rabbits, Hypertension, 17 (1991) 119-1196. [12] Murphy, D. and Carter, D., Vasopressin gene expression in the rodent hypothalamus: transcriptional and posttranscriptional responses to physiological stimulation, Mol. Endocrinol, 4 (1990) 1051-1059. [13] Nakamura, S., Naruse, M., Naruse, K., Shioda, S., Nakai, Y. and Uemura, H., Colocalizaiton of immunoreactive endothelin-1 and neurohypophyseal hormones in the axons of the neural lobe of the rat pituitary, Endocrinology, 132 (1993) 530-533. [14] Robinson, A.G., Roberts, M.M., Evron, W.A., Verbalis, J.G. and Sherman, T.G., Hyponatremia in rats induces down regulation of vasopressin synthesis, J. Clin. Invest., 86 (1990) 10231029.
[15] Saida, K. and Mitsui, Y. cDNA cloning, sequence analysis and tissue distribution of a precursor for vasoactive intestinal contraction. Bioehim. Biophys. Acta, 1089 (1991) 404-406. [16] Sakurai, T., Yanagisawa, M., Inoue, A., Ryan, U,, Kimura, S., Mitsoi, Y., Goto, K. and Masaki, T., cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA, Biochem. Biophys. Res. Commun., 175 (1991) 44-47. [17] Sherman, T.G., Civelli, O., Douglas, J., Herbert, E. and Watson, S.J., Coordinate expression of hypothalamic pro-dynorphin and pro-vasopressin mRNAS with osmotic stimulation, Neuroendocrinology, 44 (1986) 222-228. [18] Sherman, T.G., Day, R., Civelli, O., Douglas, J., Herbert, E., Akil, H., and Watson, S.H., Regulation of hypothalamic magnocellular neuropeptides and their mRNAs in the Brattleboro rat: Coordinate responses to further osmotic challenge, .L Neurosci., 8 (1988) 3785-3796. [19] Shichiri, M., Hirata, Y., Kanno, K., Ohta, K., Emori, T. and Marumo, F., Effect of endothelin-1 on release of argininevasopressin from perifused rat hypothalamus, Biochem. Biophys. Res. Commun., 163 (1989) 1332-13337. [20] VanTol, H.M., Bolwerk, E.L., Liu, B. and Burbach, J.P., oxytocin and vasopressin gene expression in the hypothalamo-neurohypophyseal system of the rat during the estrous cycle, pregnancy, and lactation, Endocrinology, 22 (1988) 945-951. [21] VanTol, H.H.M., Voorhuis, D.T.A.M. and Burbach, J.P.H., Oxytocin gene expression in discrete hypothalamic magnocellur cell groups is stimulated by prolonged salt loading, Endocrinology, 120 (1987) 71-76. [22] Wakerly, J.B., Clarke, G. and Summerlee, A.J.S., Milk ejection and its control. In E. Knobil and J.D. Neill (Eds.), The Physiology of Reproduction, Raven Press, New York, 1988, Vol. 2, pp. 2283-2321. [23] Yagil, C. and Sladek, C.D., Effect of extended exposure to hypertonicity on vasopressin messenger ribonucleic acid content in hypothalamo-neurohyphyseal explants, Endocrinology, 127 (1990) 1428-1435. [24] Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y, Goto, K. and Masaki, T., A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 332 (1988) 411-415. [25] Yoshizawa, T., Shinmi, O., Giaid, A., Yanagisawa, M., Gibson, S., Kimura, S., Uchiyama, U., Polak, J., Masaki, T. and Kanazawa, 1., Endothelin: A novel peptide in the posterior pituitary system, Science, 247 (1992) 462-464. [26] Zingg, H. and Lefebvre, D.L., Oxytocin and vasopressin gene expression during gestation and lactation, Mol. Brain Res., 4 (1988) 1-6. [27] Zingg, H.H. and Lefebvre, D.L., Oxytocin mRNA: increase of polyadenylate tail size during pregnancy and lactation, Mol. Cell. Endocrinol., 65 (1989) 59-62. [28] Zingg, H.H., Lefebvre, D.L. and Almazan, G., Regulation of vasopressin gene expression in rat hypothalamic neurons, J. Biol. Chem., 261 (1986) 12956-125959.
Brain Research 648 (1994) 59-64
Research Report
Expression of the endothelin-1 and oxytocin genes in the hypothalamus of the pregnant rat Mara J. Horwitz a, K e n n e t h D. Bloch b, N o r m a B. Kim a, Janet A. Amico a,, "Department of Medicine, E-1140 Biomedical Science Tower, Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA b Cardiac Unit, Medical Services Massachusetts General Hospital, Boston, MA, USA
Accepted 22 February 1994
Abstract
Endothelin (ET)-I, a neuropeptide and possible neuromodulator, has been found in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) of the rat in the distribution of oxytocin (OT) neurons. Within the hypothalamus of the pregnant rat, we investigated the developmental expression of the ET-1 gene and the possibility of coordinate expression of the ET-1 and OT genes. Blots containing hypothalamic mRNAs from 4-, 14-, 18-, and 21-day-old pregnant rats were hybridized to a 32p-labeled probe specific to the rat ET-1 gene. Hypothalamic tissue contained an ET-1 transcript of = 2.3 kb size. ET-1 mRNA abundance increased significantly in the SON and PVN from early to late gestation (P = 0.005 and 0.05, respectively). Blots containing hypothalamic mRNA were rehybridized to a 32p-labeled probe specific to exon C of the rat OT gene. OT gene expression increased significantly within both the hypothalamic SON (P = 0.0009) and PVN (P = 0.003) as gestation advanced. The sizes of the hypothalamic ET-1 and OT transcript sizes remained unchanged throughout gestation. Hypothalamic ET-1 and OT transcripts display stage-specific increases during gestation. ET-1 may be a neuroendocrine regulator of pregnancy-related events in the rat, and may act alone or in concert with OT. Key words: Endothelin; Gestation; Hypothalamus; Neurohypophysis; Oxytocin; Pregnancy
1. Introduction
The endothelins (ET) are a family of three structurally-related peptides, ET-1, ET-2, and ET-3, which are encoded by three distinct ET genes [5]. ET-1, which was originally isolated from porcine endothelial ceils [24], has been found in several non-vascular tissues [16], including those of the central nervous system, CNS. The presence of ET-1 immunoreactive peptide within the CNS suggests a neurotransmitter or neuromodulatory role for this peptide. Within the CNS of the rat, ET-1 immunoreactivity has been found in the hypothalamoneurohypophysial system [25], which is the primary site of synthesis and storage of the hormones oxytocin (OT) and vasopressin (AVP). O T and AVP
* Corresponding author. Fax: (l) (412) 648-7047, Janet A. Amico, M.D. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00266-F
are synthesized in the paired hypothalamic supraoptic and paraventricular nuclei (SON and PVN) and transported axonally to their storage site in the posterior lobe of the pituitary gland. Within the SON and PVN of the rat hypothalamus, ET-1 immunoreactivity has been reported to concentrate in the region of O T and AVP neurons [25]. ET-1 immunoreactivity has also been colocalized to the same neurosecretory granules as O T and AVP in the rat posterior pituitary gland [13]. Yoshizawa and coworkers have shown that water deprivation, a potent stimulus for depletion of posterior pituitary stores of O T and AVP, decreased posterior pituitary stores of ET-1 immunoreactivity in the rat [25]. Although in situ hybridization histochemistry has identified ET-1 m R N A in the porcine PVN [25], similar studies of ET-1 m R N A in the PVN or SON of the rat have not yet been reported. Since ET-1 immunoreactive peptide colocalizes to O T and A V P neurons of the rat hypothalamus, conditions that are known to enhance the abundance of hypothalamic O T a n d / o r A V P mRNAs, such as preg-
60
M.J. Horwitz et al. / Brain Research 648 (1994) 59-64
nancy [20,26,27], lactation [20,26,27], and hyperosmolar states [1,9,10,12,17,21,23,28], may be accompanied by enhanced expression of ET-1 mRNA. OT is best known for its role in pregnancy and parturition [4] and milk ejection during lactation [22]. OT is released from the posterior pituitary gland with high circulating concentrations in late gestation [4] and during suckling [4,22]. The abundance of OT mRNA has been reported to increase within the SON and PVN of the rat in late gestation [20,26,27]. Additionally, the hypothalamic OT transcript has been reported by one [26,27], but not another [20], laboratory to undergo lengthening of the polyadenylate tail as gestation advances in the rat. In the present study, we examined the developmental expression of the ET-1 gene in the rat hypothalamus during gestation to determine whether the ET-1 transcript would also undergo changes in abundance and size.
2. Materials and methods 2.1. Animals and tissue preparation Timed primiparous Sprague-Dawley rats (250-550 g body weight) were obtained from Zivic-Miller laboratories (Allison Park, PA) and were housed individually in plastic maternity cages in a temperature(22°C) and humidity-controlled room with an 08:00-20:00 h photoperiod. Animals were fed rat chow pellets (Wayne Lab-Blox, Chicago, IL) and had access to tap water ad libitum. Animals were sacrificed in groups of four by rapid guillotine decapitation between 09:00 and 11:00 h on days 4, 14, 18, and 21 of gestation. At the time of sacrifice, the entire brain was removed from the pregnant animals and placed ventral surface upward in a Jacobowitz slicer (Zivic-Miller). Vertical razor blade cuts were made 1 mm rostral to the optic chiasm and 1 m m rostral to the mammillary bodies as previously described [2,3,14]. This coronal section of hypothalamus, which contained SON and PVN, was placed on a Petri plate for free-hand dissection of individual SON and PVN. Dissection technique guaranteed that the complete SON and PVN were contained within the sample [2,3,14]. Animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.2. Preparation of RNA Bilaterally pooled SON and PVN from each animal were placed in separate tubes containing RNAzol, 0.8 ml (Cinna-Biotecx, Houston, TX) and immediately homogenized by repeated trituration with a 21-gauge needle and syringe. Chloroform, 0.1 vol., was added and the samples were vortexed and maintained on ice for 15 min prior to centrifugation for 15 min at 12,000x g at 4°C. The aqueous layer was transferred to a clean polypropylene tube and the chloroform extraction was repeated. R N A was precipitated by the addition of one vol isopropanol and overnight storage at - 20°C, and then centrifuged at 12,000 x g for 30 min. The R N A pellet was washed twice with 800/zl 70% e t h a n o l / 3 0 % diethylpyrocarbonate (DEPC)-water. The pellet
was dissolved in DEPC-water and stored in one-nuclei aliquots from each animal at - 7 0 ° C .
2.3. Blot hybridization Samples of total R N A (one nucleus) were denatured for electrophoresis in 10 m M boric acid, 0.1 m M E D T A , 10% glycerol, 6% formaldehyde, and 50% deionized formamide. Samples were incubated at 65°C for 5 min and cooled on ice. O n e ~1 of 5% ethidium bromide was added to each sample. Nucleic acid electrophoresis was carried out on a horizontal 1% a g a r o s e / 3 % formaldehyde gel, unless otherwise specified. The positions, integrity, and abundance of 28S and 18S ribosomal R N A s were visualized and photographed under ultraviolet (UV) light. A 2:1 ratio of 28S to 18S ribosomal R N A s was found in the R N A samples for this study, thus verifying good integrity of RNA. Equal quantities of ribosomal R N A s among samples verified equal loading. Nucleic acids were transferred to a nylon m e m b r a n e (Gene Screen, New England Nuclear, Boston, MA) overnight by capillary action and immobilized by U V cross-linking. Prehybridization was done at 50°C in a sealed plastic bag in 10 ml of buffer comprised of 50% deionized formamide, 0.25 M sodium phosphate, NaHPO4, pH 7.2; 0.25 M NaCI; 1 m M EDTA; 100 txg/ml denatured salmon sperm DNA; and 7% SDS. Hybridization was performed at 50°C for 16 h in the same buffer with the addition of a 32p-labeled c D N A probe. Following hybridization, the filter was washed to a final stringency of 0.1% S D S - 2 × S S C (1 x S S C was 0.15 M NaC1; 0.015 M Na citrate, pH 7.0) at 50°C. The blots were exposed to Kodak X-omat A R x-ray film with 2 intensifying screens at - 70 ° for 9 days for ET-1 and 4 h for OT. Blots were hybridized to the ET-1 probe prior to hybridization to the O T probe. Autoradiograms of hybridized blots were scanned (Model 620 densitometer, Bio-Rad Laboratories, Richmond, CA) and the arbitrary densitometry unit measure of each band minus background for the film was used in calculating the relative abundance of cytoplasmic m R N A s . Autoradiograms were selected for densitometry with exposure times within the linear range of the film. To control for loading and transfer of RNA, m e m b r a n e s were rehybridized with a 32p-labeled D N A probe to the /3-subunit of the mouse actin gene. Actin abundance in the PVN and SON showed uniform signed intensity among samples, verifying uniform loading and transfer.
2.4. Preparation of radiolabeled probes The probe used to detect ET-1 m R N A was prepared from a rat ET-1 genomic clone and does not cross hybridize with ET-2 or ET-3. The rat ET-1 genomic fragment was cloned into the SacI and AccI sites of p G E M 4 (Promega, Madison, WI) and hybridizes to nucleotides 263 through 409 of the rat ET-1 c D N A described by Sakurai et al. [65]. The O T probe was prepared from a genomic clone for rat O T provided by Dr. T h o m a s Sherman, Department of Behavioral Neuroscience, University of Pittsburgh. The rat O T probe was prepared by digesting the p G E M 4 - O T 3c clone with the enzymes EcoRI and SaclI to yield a 169-bp c D N A for exon C of the O T gene [2,3,18]. For probe labeling, each plasmid D N A was digested with the appropriate restriction endonuclease known to cleave the insert D N A from its plasmid DNA. Probes were labeled using R a n d o m Primed D N A Labeling Kit (Boehringer M a n n h e i m Biochemical) according to the directions of the manufacturer. Sufficient 32p (1 / x C i / n g D N A [a-32p]dCTP, 3000 C i / m m o l aqueous solution, New England Nuclear) was added to the labeling reactions to yield specific activities estimated at 1 × 109 C P M / ~ g . The labeled probe was separated from unlabeled nucleotide on a Sephadex G-50 spin column, equilibrated and eluted with 1 x STE (10 m M Tris-H C1, pH 8.0; 1 m M E D T A , pH 8.0; and 100 m M NaCI).
M.J. Horwitz et al. / Brain Research 648 (1994) 59-64 2.5. Statistical analysis
61
Day 4 - SON r
Results are expressed as mean_+S.D. O T and ET-1 m R N A levels. Statistical comparisons for hypothalamic O T and ET-1 m R N A concentrations among groups were assessed using analysis of variance ( A N O V A ) or Kruskal-Wallis test, as appropriate, followed by post-hoc two sample t test or Wilcoxon rank sum test, respectively, between pairs of m e a n values. All statistical assumptions were met. Significance was P < 0.05.
3. R e s u l t s
The size and abundance of ET-1 mRNAs were assessed in the hypothalamic SON and PVN of day 4, 14, 18, and 21 pregnant rats. Accumulation of ET-1 m R N A increased significantly in the SON ( P = 0.005, Kruskal-Wallis test), and PVN ( P = 0 . 0 5 , ANOVA) with advancing gestation, Fig. 1. Significant differences
12-
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Fig. 2. Northern blot hybridization of ET-1 m R N A in the supraoptlc nucleus (SON) of pregnant rats. The blot contains the SON from rats on pregnancy days 4 and 18. The Size of the ET-1 transcript was --- 2.3 kb. The 28S and 18S ribosomal R N A s identify m R N A species of = 5 kb and 2 kb, respectively.
in SON ET-1 m R N A abundance were found for gestational days 4 versus 14, 18, and 21 (each P = 0.02), and day 14 vs. 21 ( P = 0.05), with peak levels of ET-1 on day 18 gestation in the SON. However, by day 21 of gestation, ET-1 mRNA in the SON declined signifi-
SON
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Fig. 1. ET-1 m R N A in the SON (top panel) and PVN (bottom panel) of day 4, 14, 18, and 21 pregnant rats (n = 4 per group). The quantity of ET-1 m R N A , which was measured by densitometry of the bands on the autoradiogram, increased significantly in the SON and the PVN from early to late gestation ( P = 0.005 and 0.05, respectively). Significant differences in S O N ET-1 m R N A were found for gestational days 4 vs. 14", 18", and 21" (each, P = 0.02, indicated by asterisks), day 14 vs. 21 ( P = 0.05) and day 18 vs. 21 ( P = 0.03), whereas significant differences in PVN ET-1 m R N A were found for days 4 vs. 21" ( P = 0.05) and 14 vs. 21" ( P = 0.02) gestation.
4
14 18 Days of Gestation
21
Fig. 3. Quantitative assessment of OT m R N A in the SON (top panel) and PVN (bottom panel) of day 4, 14, 18, and 21 pregnant animals (n 4 per group). The quantity of O T m R N A was measured by densitometry of the bands on the autoradiogram. As gestation advanced, O T m R N A abundance increased significantly in the SON ( P = 0.0009) and PVN ( P = 0.003). Significant increases were found on day 4 vs. 18" and 21" (each, P = 0.0001), day 4 vs. 14" ( P = 0.009) and day 14 vs. 21 ( P = 0 . 0 5 ) in the SON and on day 21" vs. 4 ( P = 0 . 0 4 ) , 14 ( P = 0.02), and 18 ( P = 0.009) in the PVN of pregnant animals•
m.J. Horwitz et al. /Brain Research 648 (1994) 59-64
62
cantly compared to day 18 ( P = 0.03, Wilcoxon rank sum test). Significant differences in PVN ET-1 m R N A abundance were found for pregnancy days 4 vs. 21 and 14 vs. 21 ( P = 0.05, and P = 0.02, respectively, t-test). The size of the ET-1 transcript in the hypothalamus of pregnant rats was approximately 2.3 kb, identical to that reported in vascular tissues [5,16,24], Fig. 2. No size change was found in hypothalamic ET-1 transcripts during gestation, Fig. 2. Identification of ET-1 as the primary ET species in the pregnant rat SON and PVN was done by rehybridization of the blots to a 32p_labeled probe to ET-3. A very faint to non-detectable transcript was found after exposure of the autoradiogram for 21 days. The low or absent ET-3 signal precluded quantitation by densitometry. The blots containing m R N A from the SON and PVN of the pregnant rats were rehybridized to a .~2p_ labeled probe specific for the rat O T gene, and m R N A abundance was assessed by densitometry. Accumulation of O T m R N A was found to increase significantly in the SON and PVN as gestation advanced, Fig. 3, ( P = 0.0009 and 0.003, respectively, ANOVA). Significant differences in SON O T m R N A accumulation were found for gestational days 4 vs. 18 and 21 (each P = 0.0001), day 4 vs. 14 ( P = 0.009), and day 14 vs. 21 ( P = 0 . 0 5 , t-test), whereas significant differences in PVN O T m R N A were found for pregnancy days 4 vs. 21 ( P = 0.04), 14 vs. 21 ( P = 0.02), and 18 vs. 21 ( P = 0.009, t-test). The increase in abundance of the O T gene in the SON as pregnancy advanced was not accompanied by an increase in the size of the O T transcript. To further
D4 Mix D21 28 18s--
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Fig. 4. Autoradiogram of SON m R N A from gestational days 4 (D4) and 21 (D21) animals and a mixture of the two (mix). The blot was hybridized to a probe specific for exon C of the rat OT gene. The size of the OT transcript was the same in day 4 and 21 gestational hypothalami. A mixture of R N A from gestational day 4 and 21 SON revealed a single transcript for the O T gene. The larger width of the middle band (mix) is due to the larger amount of R N A in that sample ( 1 / 2 nucleus) compared to day 4 and 21 samples which each contained 1 / 4 nucleus. The 28S and 18S ribosomal R N A s identify species of = 5 kb and 2 kb, respectively.
Day 18 - Placenta
28S ET-1
18S Fig. 5. Northern blot hybridization of ET-1 m R N A in the placenta of pregnant rats. Each lane contains 80 /~g of total R N A from the placentas of day 18 pregnant rats hybridized to a 32p-labeled probe to ET-1. The size of the placental ET-1 transcript was 2.3 kb. The 28S and 18S ribosomal R N A s are 5 kb and 2 kb, respectively.
resolve possible size differences in early vs. late gestational hypothalami, pooled R N A from the SON of day 4 and day 21 gestational rats ( 1 / 4 nucleus/lane) and a mixture of the two R N A pools ( 1 / 2 nucleus/lane) were electrophoresed on a 1.5% agarose gel. The size of the O T transcript was similar in the SON of day 4 and 21 gestational rats, and the mixture of these RNAs showed a single transcript, Fig. 4. A comparison of hypothalamic O T transcript size with that of extra hypothalamic O T transcripts was done by electrophoresing on a 1.5% agarose gel pools of R N A from the placenta and hypothalamus of day 21 pregnant animals and measuring the distance that each sample migrated from the well. Migration was less for the hypothalamic than the placental O T transcript, indicating a larger size for the hypothalamic O T transcript (data not shown for OT). In contrast, placental and hypothalamic ET-1 transcripts were both = 2.3 kb, Figs. 2 and 5.
4. Discussion
In the present study we used blot hybridization to demonstrate that ET-1 m R N A is present in the hypothalamic PVN and SON of the pregnant rat. Since ET-1 immunoreactivity was reported to localize in the distribution of O T neurons of the PVN and SON [25], we hypothesized that hypothalamic ET-1 gene expression may be increased during pregnancy, a condition characterized by enhanced accumulation of O T m R N A within these nucei [20,26,27]. We found ET-1 m R N A accumulation to be enhanced 1.5 fold in the PVN and 2-fold in the SON as gestation advanced. O T m R N A increased 2-fold in the PVN and 3-4-fold in the SON of the same animals as gestation advanced. Although both ET-1 and O T m R N A s increased in the SON, the changes did not exactly parallel one another. Increases in both genes were found on days 14 and 18 gestation compared to day 4. However, by day 21 gestation, O T levels in the SON increased further, while ET-1 de-
M.J. Horwitz et al. / Brain Research 648 (1994) 59-64
creased significantly. In the PVN, however, ET-1 and OT were more closely correlated with one another with significant increases in both genes on day 21 vs. day 4 of pregnancy. This study is the first to document stage specific increases in the expression of ET-1 mRNA in the hypothalamus of the pregnant rat. ET-1 appears to be the predominant ET gene isoform (ET-1, 2, or 3) in the pregnant rat SON and PVN. ET-2 mRNA is = 1500 nucleotides in length [15] and the transcript we identified in the PVN and SON of pregnant rats was = 2.3 kb. Moveover, hybridization of the blots in this study to a probe to rat ET-3 found a very weak or absent signal. The OT system has been a useful model for the study of neuropeptide gene expression. OT gene expression is enhanced in a variety of conditions in which peripheral OT secretion is enhanced, including advancing gestation [20,26,27], lactation [20,26,27], and chronic osmotic stimulation [1,10,12,20,21,23,28]. Moreover, the length of the OT transcript has been reported to increase in each of these conditions [12,26,27] due to elongation of the polyadenylate tail. Zingg and Lefebvre have found that the degree of polyadenylation of the OT transcript increased as pregnancy advanced [27,65], yet other investigators [20] have not reported OT transcript size changes with advancing gestation. Since mRNA transcripts of varying size can be distinguished by their migration rates through agarose, we did experiments to determine potential size differences in the hypothalamic OT transcripts of early and late pregnant rats. We observed no increase in OT transcript size as gestation advanced, but similar to other laboratories [20,26,27] found enhanced accumulation of OT mRNA in late gestation. The differences between the findings of Zingg and Lefbvre's studies [26,27] and our study are not explained by differences in the strain of rat (Sprague-Dawley rats were used in both studies) stage of pregnancy (day 21 pregnant animals were examined in both studies) or environmental conditions (animals were maintained in a normal state of hydration in both studies). Although Zingg and Lefebvre [26,27] have convincingly shown a size change in the hypothalamic OT transcript of late pregnant rats, the finding was not observed in the animals in our study. Thus although the change in OT transcript size may occur in pregnant rats, this change appears to be found less consistently than the enhanced accumulation of OT mRNA in pregnant rats. The reasons for these observations are not yet understood, primarily because factors regulating the length of the polyadenylate tail of the OT transcript have yet to be defined. Nevertheless, this study and other studies [2,12,25,26] support the concept that the factors governing accumulation and length of the OT transcript are independent. The size of the OT transcript depends upon the tissue source, and the size differences are also due to
63
variation in the length of the polyadenylate tail [6,8]. Other investigators have found size heterogeneity between hypothalamic and placental OT transcripts [8] and corpus luteal and hypothalamic OT transcripts [6]. In the present study, we confirmed the finding of Lefebvre and coworkers [8] that the hypothalamic OT transcript size was larger than placental OT. In contrast, the ET-1 hypothalamic and placental transcripts were both 2.3 kb in size. The abundance of ET-1 mRNA in the rat hypothalamus was far less than that of OT. The low abundance of ET-1 in the hypothalamus may favor a paracrine or autocrine role for this peptide rather than an endocrine role. Indeed, ET-I has been shown to exert local actions within the neurohypophysial axis [11,19] and has been colocalized within OT granules [13]. ET-1 may act as a modulator of OT expression or may have independent effects upon other hormonal systems that are important in gestation, such as the adrenocorticotropin-adrenal or gonadotropin-gonadal axes [7]. In summary, a study of the developmental expression of the OT and ET-1 systems in the hypothalamus of the pregnant rat showed that there are gestation-dependent increases in these two genes. The physiological function of hypothalamic ET-1 in gestation is not known, but ET-1 may be important as a neuroendocrine regulator of pregnancy a n d / o r parturition in the rat.
Acknowledgements The authors are grateful to Thomas G. Sherman, Ph.D., Department of Behavioral Neuroscience, University of Pittsburgh, for supplying the genomic clone for rat OT; Janine Janosky, Ph.D., Department of Clinical Epidemiology and Family Medicine, University of Pittsburgh School of Medicine, for statistical analysis of data; and to Christine Milcarek, Ph.D. and Stephen Phillips, Ph.D., Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, for consultation regarding molecular biology techniques. We thank Anna Halloran for expert technical assistance with animal studies and Michele Dobransky for excellent secretarial assistance. Supported in part by funds from the Department of Veterans Affairs (J.A.A.) and NHLBI Grant HL45895 (K.D.B.). J.A.A. is recipient of a Career Development Award from the Department of Veterans Affairs. Mara J. Horwitz is supported by NIH Grant No. 5T32DK0752-18 for training in Endocrinology and Metabolism. Presented in part at the Society for Neuroscience, 23rd Annual Meeting, Washington, DC, November 7-12, 1993.
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