Chronic estrogen exposure maintains elevated levels of progesterone receptor mRNA in guinea pig hypothalamus

Molecular Brain Research, 10 (1991) 167-172 © 1991 Elsevier Science Publishers B.V. 0169-328X/91/$03.50 ADONIS 0169328X91800824

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Chronic estrogen exposure maintains elevated levels of progesterone receptor mRNA in guinea pig hypothalamus Douglas A. Bayliss 1 and David E. Millhorn 1'2 1Department of Physiology and 2Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27599-7545 (U.S.A.)

(Accepted 8 January 1990) Key words: In situ hybridization; Progesterone receptor; Estrogen; Guinea pig; Hypothalamus; Steroid

We performed in situ hybridization on hypothalamic sections from ovariectomized guinea pig using a cocktail of three ass-labeled oligonucleotides complementary to mammalian progesterone receptor (PR) cDNA. PR mRNA was readily detected in hypothalamic neurons from guinea pigs pretreated with 17fl-estradiol benzoate (E2B), but not from animals which did not receive supplemental E2B. The distribution of PR mRNA-containing cells corresponded well with previous Iocalizationsof PR in guinea pig. In contrast to earlier reports of E2B regulation of PR mRNA in rat hypothalamus, however, we found that PR mRNA remained elevated during chronic exposure to E2B (up to 10 days) in guinea pig. Progesterone has a number of central nervous system effects, some of which are estrogen (E2) dependent. Thus, pretreatment with E 2 is critical for the facilitation by progesterone of sexual behaviors 5'8, luteinizing hormone-releasing hormone ( L H R H ) release 16 and respiration 2 in ovariectomized animals. Because these effects of progesterone are mediated by progesterone receptor (PR)-containing cells located in the hypothalamus 2'7'15, their E 2 dependence is thought to be a result of the ability of E 2 to induce progestin binding sites (i.e. PR) in hypothalamus 18. The increase in PR caused by E 2 could be mediated, in part, by altered levels of PR mRNA. Indeed, it was recently reported that E 2 increases PR m R N A levels in cells of the rat hypothalamus 21'23. That effect was maximal by 24 h after E 2 injection, and appeared to be decreasing toward control by 48 h, even after a second E 2 injection 23. These results prompted the interpretation that the increase in PR m R N A cannot be sustained even with a continuous E 2 challenge 21'23. The reported short-lived induction of PR m R N A by E223 appears to correlate well with the timing of the estrogen/progesterone interaction in facilitating sexual behavior and LH release in rodents 1~. Although progesterone stimulates sexual receptivity and respiration through a similar mechanism 2, its effects on respiration are much longer-lasting. For example, in human females, the stimulation of respiration attributed to progesterone lasts for days during the luteal phase of the menstrual

cycle and throughout the entire gestation period 9. In addition, chronic administration of progesterone causes prolonged respiratory stimulation in humans 26 and guinea pigs 12'14. Since this response is mediated by estrogendependent PR 2'3, it would seem that a continued elevated level of PR would be necessary to maintain the longlasting stimulation of respiration. In fact, high levels of PR have been detected immunohistochemically in guinea pig brain after periods of E 2 pretreatment lasting as long as 4 days 6'27. Furthermore, given the relatively short half-life of PR (7-10 h) which is apparently unaffected by E22°, one might expect chronic E 2 treatment to cause prolonged increases in the rate of PR synthesis, perhaps mediated by a sustained elevation in the level of PR mRNA. In the present experiments, in situ hybridization was used to determine [1] if E 2 increases levels of PR m R N A in guinea pig hypothalamus, and [2] to investigate whether PR m R N A remains elevated during chronic E 2 exposure. Hartley strain female guinea pigs (200-400 g) were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), ovariectomized and after a period of at least 2 weeks the animals were either pretreated with vehicle (i.e. sesame oil) or with 100/~g (1.0 mg/ml in sesame oil) of 17fl-estradiol benzoate (E2B) for periods of between 1 and 10 days. The animals were then deeply anesthetized (pentobarbitol i.p., 100 mg/kg), and decapitated. The brain was removed, the hypothalamus

Correspondence: D.E. Miilhorn, 78 Medical Sciences Research Wing, Department of Physiology, University of North Carolina, Chapel Hill, NC 27599-7545, U.S.A.

168 blocked and frozen quickly over dry ice. Sections (10/~m) were cut in a cryostat at 100-300/~m intervals throughout the rostrocaudal extent of the hypothalamus and the preoptic area and thaw-mounted onto twice-gelatin coated slides and either stored at -80 °C or immediately processed for in situ hybridization as previously described 4. Briefly, sections were warmed to room temperature, fixed in 4% paraformaldehyde, rinsed extensively in 0.1 M phosphate-buffered saline (PBS) and once in 0.1 M triethanolamine/0.9% saline with 0.25% acetic anhydride before being dehydrated and delipidated through graded ethanols and chloroform. Sections were then hybridized to 0.3-1 x 10 6 cpm of probe (see below) in hybridization buffer [50% deionized formamide; 4 x SSC (1 x SSC is 0.15 M NaCI/0.015 M sodium citrate, pH = 7.2); 10% dextran sulfate; 0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin; 500/~g/ml sonicated, denatured salmon sperm DNA; 250/~g/ml yeast tRNA; and 100 mM dithiothreitol] overnight at 37 °C. The sections were washed in 1 x SSC for 1 h at 55 °C (4 × 15 min rinses) and for 1 h at room temperature, dipped briefly in water and ethanol (95%), air-dried, and apposed to X-ray film (Hyperfilm-flmax, Amersham, Arlington Hts., IL) for 3-10 days before being dipped in nuclear track emulsion (Kodak NTB2, 2:1 with 0.6 M ammonium acetate) and exposed for 4-6 weeks. Film and emulsion were developed and fixed and the sections counterstained with toluidine blue (0.25%) before being analyzed under bright-field and dark-field microscopy. Synthetic oligodeoxyribonucleotides (oligonucleotides) were used as probes for in situ hybridization. Probe sequences, determined from published progesterone receptor cDNA sequences, were chosen to be complementary to highly conserved regions of the mammalian PR

coding sequence 17'19 which are not shared among other members of the steroid receptor superfamily ~°. The sequences used are complementary to nucleotides 15851614 (PRAS-1), 1619-1648 (PRAS-2) and 2395-2430 (PRAS-3) of human PR c D N A ~9. Oligonucleotides were labeled using a-thio-[35S]dATP (New England Nuclear, Boston, MA; >1000 Ci/mmol) and terminal deoxynucleotidyl transferase (BRL) to specific activities of approximately 8-15 x 10 6 cpm/pmol. Labeled probes were purified from unincorporated deoxynucleotides by gel filtration (Sephadex G50; Pharmacia, Piscataway, N J), dried and resuspended in hybridization buffer (3-10 x 106 cpm/ml). Initial hybridizations carried out with two of the oligonucleotide probes alone (PRAS-1 and -2) yielded essentially identical results, but the intensity of the signal was weak. We found, however, that a more intense cellular labeling could be achieved by combining the probes (PRAS-1, -2, and -3), which were directed against different regions of PR m R N A in a hybridization cocktail. Therefore, we used this strategy in all but the initial experiments. We found m R N A for PR in hypothalamus from E2B-treated guinea pig but PR m R N A was undetectable in hypothalamus from animals which were not pretreated with estrogen. This is readily apparent in the arcuate nucleus, which was completely devoid of labeled cells in animals treated with vehicle (i.e. sesame oil) alone (Fig. 1A), but had a high density of PR mRNA-containing cells in E2B-treated animals (Fig. 1B). This was the case throughout all regions of the hypothalamus where PR mRNA-containing neurons were found. Thus, estrogen pretreatment was necessary for detection of PR m R N A in ovariectomized guinea pig. The disposition of peri-

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Fig. 1. Induction by estrogen of progesterone receptor mRNA in guinea pig hypothalamus. A cocktail of 3 35S-labeled PR oligonucleotide probes (PRAS-I, -2, and -3) was applied to coronal sections (10/tm) of hypothalamus from guinea pigs which were ovariectomized. Dark-field photomicrographs of hypothalamus at the level of the mammillary recess (MR) are shown. There was no evidence of expression of PR mRNA in the arcuate nucleus of the oil-treated animal (A) whereas we found a strong hybridization signal indicative of PR mRNA expression in arcuate nucleus of E2B-treated animals (B).

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Fig. 2. Photomicrographs of PR mRNA-containing cells in hypothalamus of guinea pig. Coronal sections of hypothalamus from OVX, E2B-treated (4 day) guinea pig were hybridized to a cocktail of three asS-labeled probes (PRAS-1, -2, and -3). Bright-field photomicrographs show labeled cells (dark arrows) found in the arcuate (A,B), medial preoptic (C) and ventromedial (D) nuclei of the hypothalamus. PR mRNA-containing neurons in the arcuate nucleus tended to be more strongly labeled than those in the other regions. Unlabeled cells are indicated by open arrows.

170 karya containing PR m R N A corresponded well with the reported localization of the receptor by immunohistochemistry 6"27 and autoradiography 24. Photomicrographs of cells which express PR m R N A from select regions of the hypothalamus are shown in Fig. 2. The arcuate nucleus contained the highest density and most strongly labeled cells (Fig. 2A, B). A large number of perikarya containing PR m R N A were found in the anterior hypothalamic and medial preoptic nucleus (Fig. 2C). We also found neurons containing PR m R N A in the ventromedial hypothalamic nucleus (Fig. 2D) and distributed throughout the periventricular nucleus, especially in its ventral aspects. When compared to the cells of the arcuate nucleus, however, PR mRNA-containing cells in other regions of the hypothalamus were generally less numerous and expressed lower levels of PR mRNA. To investigate the time course of the induction of PR m R N A , we studied animals after various periods of exposure to E2B. This effect of different periods of E 2 pretreatment is best illustrated by examining film autoradiographs of the densely packed PR mRNA-containing

neurons of the arcuate nucleus (Fig. 3). There was again no evidence of PR m R N A in the oil treated animal (Fig. 3A). A modest increase in PR m R N A was found in the arcuate nucleus after only 1 day of EzB treatment (not shown). A substantial increase in PR m R N A was found in the arcuate nucleus after 2 days of E2B treatment (Fig. 3B). PR m R N A was still elevated after 4 days of E2B treatment (Fig. 3B, C) and remained at high levels for as long as we administered estrogen (up to 10 days). Qualitatively similar results were observed by microscopic evaluation of other hypothalamic nuclei (e.g. ventromedial nucleus, anterior hypothalamic nucleus). This prolonged effect does not appear to be a function of the dose administered because PR m R N A was expressed at elevated levels after 4 days of E2B treatment with a range of doses (0.1, 1.0, 10 and 100/~g/kg) (not shown). We have demonstrated for the first time the presence of PR m R N A in cells of guinea pig hypothalamus. Furthermore, we have found that estrogen increases PR m R N A in those cells and that PR m R N A remains elevated during continued exposure to E2B. The speci-

Fig. 3. Time course for induction of progesterone receptor mRNA by estrogen. Coronal sections of hypothalamus from ovariectomized guinea pig were hybridized to a cocktail of three 35S-labeled oligonucleotide probes (PRAS-1, -2, and -3). Film autoradiographs at the level of the caudal arcuate nucleus are shown from animals pretreated with oil for 4 days (A) or E2B for 2 (B), 4 (C), and 10 (D) days. Thus, PR mRNA was elevated by E2B pretreatment, even for periods as long as 10 days.

171 ficity of the hybridization signal for PR m R N A is indicated by a number of observations. Firstly, the distribution of PR mRNA-containing cells we report is essentially identical to previous immunohistochemical6'27 and autoradiographic 24 determinations of the location of PR-containing cells in guinea pig hypothalamus. The distribution of PR mRNA-containing cells was markedly different from that reported for cells expressing glucocorticoid receptor m R N A 1, which is the steroid receptor m R N A most closely related in sequence to PR m R N A 19. In addition, we found that probes directed against different regions of PR m R N A gave essentially identical results. Together, these results attest to the specificity of the probes used for detection of PR mRNA. Theoretically, the reported upregulation of PR by E 2 in hypothalamic cells could involve any number of steps in the pathway from synthesis to degradation of PR. Our results, and those of Romano et al. 23, indicate that a major point of control by E 2 is at the level of PR mRNA. In addition, similar results have been noted in MCF-7 cells, a hormone-dependent breast cancer cell line, in which E 2 does not alter PR turnover 2° despite causing increases in both PR m R N A and PR 2°'22. It remains to be clarified whether the increase in PR m R N A by E 2 represents an effect on rate of transcription or on m R N A stability, both of which have been implicated in other actions of estrogen 25. In their investigation of rat hypothalamus, Romano et al. 23 found that the expression of PR m R N A was maximal after 24 h, but began to decrease between 24 and 48 h of constant E 2 exposure. This would imply that an autoregulatory mechanism exists which would govern the duration of the E 2 effect on PR. For example, chronic treatment with E 2 may downregulate its own receptor 13, rendering the cells less responsive to E 2. However, in the present study of guinea pig hypothalamus, we found that the time to maximal levels of PR m R N A was longer and that PR m R N A remained elevated, even after prolonged (i.e. 10 days) E 2 exposure. The longer-lasting effect which we report is probably not due to the manner in

which E 2 was administered. In both studies E 2 was administered subcutaneously by injection. Furthermore, the differences do not appear to be a function of the higher dose (100 #g) used in our time-course experiments since we found that PR m R N A remains elevated for at least four days with a dose of E2B as small as 0.1/~g. It remains to be determined whether these disparate results from studies of E 2 regulation of PR m R N A represent an actual species difference in the mechanism by which rat and guinea pig respond to E 2 or simply a difference in the kinetics by which a similar mechanism operates. In addition, the longer-lasting effect of E 2 on PR m R N A is not peculiar to either guinea pig or to hypothalamus; in human breast cancer cells, treatment with E 2 continues to enhance PR m R N A and PR coordinately for at least 3-4 days2°,22.

1 Aronsson, M., Fuxe, K., Dong, Y., Agnati, L.E, Okret, S. and Gustafsson, J.-A., Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 9331-9335. 2 Bayliss, D.A., Cidlowski, J.A. and Millhorn, D.E., The stimulation of respiration in ovariectomized cat is mediated by an estrogen-dependent hypothalamicmechanism requiring gene expression, Endocrinology, 126 (1990) 519-527. 3 Bayliss, D.A., Miilhorn, D.E., Gailman, E.A. and Cidlowski, J.A., Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7788-7792. 4 Bayliss, D.A., Wang, Y.-M. Zahnow, C.A., Joseph, D.R. and Millhorn, D.E., Localization of histidine decarboxylase mRNA in rat brain, Mol. Cell. Neurosci., 1 (1990) 3-9.

5 Beach, EA., Importance of progesterone to induction of sexual receptivity in spayed female rats, Proc. Soc. Exp. Biol. Med., 51 (1942) 369-371. 6 Blaustein, J.D., King, J.C., Toft, D.O. and Turcotte, J., Immunocytochemical localization of progestin receptor-immunoreactivity in guinea pig hypothalamus and preoptic area, Brain Res., 424 (1988) 1-15. 7 Blaustein, J.D. and Olster, D.H., Gonadal steroid hormone receptors and social behaviors. In J. Balthazart (Ed.), Advances Comparative and Environmental Physiology, Vol. 3, Springer, Berlin, 1989, pp. 31-104. 8 Boling, J.L. and Blandau, R.J., The estrogen-progesterone induction of mating responses in the spayed female rat, Endrocrinology, 25 (1939) 359-364. 9 Dempsey, J.A., OIson, E.B. and Skatrud, J.B., Hormones and

In conclusion, the results of this study indicate that PR m R N A is increased by E 2 in neurons of guinea pig hypothalamus and remains elevated in those cells in response to prolonged treatment with E 2. This finding, therefore, suggests a cellular basis for chronic PRmediated effects, such as the long-lasting stimulation of respiration in humans and guinea pigs 9A2A4. In addition, these results demonstrate the utility of oligonucleotide probes for in situ hybridization studies of regulation of gene expression in the central nervous system, even for detecting extremely low abundance mRNAs such as that encoding PR. The novel approach of applying multiple oligonucleotide probes in a hybridization cocktail nJarkedly enhanced the sensitivity of the method, without complicating the protocols.

The authors express their appreciation to Luisa E. Klingler for her expert technical assistance and thank Dr. P.J. Brooks for providing probes used in earlier experiments. They also thank Dr. D.R. Joseph for his help throughout these studies. This work was supported by Grants HL33831 and AHA881108(D.E.M.). D.A.B. was supported in part by a Postgraduate Scholarship from NSERC (Canada) and a fellowship from Glaxo Pharmaceutical (Research Triangle Park, NC). D.E.M. is a Career Investigator of the American Lung Association.

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