Putative estrogen receptor β and α mRNA expression in male and female rhesus macaques

Molecular and Cellular Endocrinology 146 (1998) 59 – 68

Putative estrogen receptor b and a mRNA expression in male and female rhesus macaques Candace Y. Pau a,c, K.-Y. Francis Pau a, Harold G. Spies a,b,* a

Di6ision of Reproducti6e Sciences, Oregon Regional Primate Research Center, 505 NW 185th A6enue, Bea6erton, OR 97006, USA b Department of Cell and De6elopmental Biology, Oregon Health Sciences Uni6ersity, Portland, OR 97201, USA c Aloha High School student, Aloha, OR 97007, USA Received 9 July 1998; accepted 3 September 1998

Abstract The profound effects of estrogen on different tissues may involve at least two estrogen receptor (ER) subtypes, ERa and the recently discovered ERb. Where and how the two ER subtypes differentially or cooperatively mediate estrogen actions, however, are still unknown. In this study, we report the cloning of a specific ERb cDNA fragment and the expression of ERa and ERb mRNAs in various endocrine and non-endocrine tissues of male and female rhesus macaques. Total RNA from monkey tissues was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using human-specific ERb primers. A 126 bp RT-PCR product was identified in ovarian tissue and subsequently transfected into pGEM-T vectors for DNA sequencing. The cloned rhesus monkey ERb fragment contained a sequence nearly identical to the corresponding sequence in the human (four mismatched nucleotides out of 126). Because complete monkey ERb and ERa DNA sequences have not been established, the expression of the ERb and ERa fragments in monkey tissues by RT-PCR reflects ‘putative’ ERb and ERa mRNA expression, respectively. Both ERb and ERa mRNAs were present in male and female reproductive organs, in several endocrine and non-endocrine tissues, and in various regions of the brain, whereas several tissues, including liver, frontal cortex, caudate nucleus, locus coeruleus and cerebellum, expressed only ERa message. In some brain regions, i.e. the putamen, internal capsule, hippocampus and paraventricular hypothalamus, the ERb fragment was expressed in the female but not in the male. These data suggest that ERa mRNA is widely distributed in both female and male tissues, while ERb mRNA is more widely distributed in female than in male brain. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estrogen receptor; DNA sequence; Ovary; Hypothalamus; Brain; Monkey; Primate

1. Introduction The profound effects of estrogen on the reproductive, nervous, cardiovascular and many other body systems are mediated by estrogen receptors (ER) which belong to a superfamily of ligand-activated transcription factors (Tsai and O’Malley, 1994; Katzenellenbogen, 1996). At least two genomic ER subtypes, ERa (Greene et al., 1986; Green et al., 1986) and ERb (Kuiper et al., 1996; Mosselman et al., 1996; Enmark et al., 1997), * Corresponding author. Tel.: +1-503-6905299; fax: + 1-5036905563; e-mail: [email protected].

have been cloned in humans and rodents, and new members of the ER family continue to be identified (Petersen et al., 1998). The discovery of ERb provokes many questions as to the nature, characteristics and distribution of the a and b subtypes and their interactions in mediating various estrogenic and anti-estrogenic actions (Katzenellenbogen and Korach, 1997). The expression of ERb in the prostate also intensifies the search for estrogenic functions in the male (Kuiper, et al., 1996; Lau, et al., 1998; Prins, et al., 1998). In this investigation, we report the cloning of a ERb cDNA fragment by human-specific primers and the distribution of ERb in selected tissues of the rhesus macaque.

0303-7207/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 8 ) 0 0 1 9 7 - X

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We present a comparative analysis of ERb vs. ERa distribution as well as gender differences in expression of ERb and ERa. Because complete ERb and ERa DNA sequences have not been published in the monkey, the expression of the ERb and ERa fragments may reflect ‘putative’ monkey ERb and ERa mRNAs, respectively.

ered by centrifugation and washed with 75% ethanol. The RNA pellet was air-dried and dissolved in diethyl pyrocarbonate (DEPC) treated water. The concentration of RNA was estimated based on absorbance at 260 nm, and an aliquot of total RNA was subjected to 1% gel electrophoresis to confirm its quality and integrity. Isolated total RNA samples were stored at −80°C immediately following these procedures.

2. Materials and methods

2.4. Cloning of a rhesus monkey-specific ERb cDNA fragment

2.1. Animals Adult rhesus monkeys (4.5 – 6.5 kg) were housed in individual cages in an environmentally controlled room (lights-on from 07:00 – 19:00 h; temperature between 21 – 25°C). Purina monkey chow (Ralston – Purina, St. Louis, MO) was fed twice daily, fresh fruit was supplied once daily, and tap water was provided ad libitum.

2.2. Tissue preparation One male and one female (during the follicular phase of the menstrual cycle) rhesus monkey were killed under deep anesthesia (30 mg pentobarbital/kg b.w., i.v.). Peripheral organs and tissues were quickly removed, dissected into blocks of approximately 100 mg each, placed in sterile, 2 ml vials, fresh-frozen on dry ice and kept at −80°C until processed for RNA isolation as described below. The brains and brainstems were removed from the skull and spinal cord, coronally sectioned into seven slices each, and placed horizontally on a RNase-free platform cooled with dry ice. Various brain and brainstem regions were micro-dissected, frozen in vials and stored at −80°C for RNA isolation. The brain of another female rhesus was perfused with 4% paraformaldehyde (pH 9.5) during the follicular phase of the menstrual cycle, grossly dissected into several blocks, post-fixed with 4% paraformaldehyde for 4 h, cryo-protected in 20% sucrose-4% paraformaldehyde for 3 days, fast-frozen in isopentane/dry iceethanol bath, and stored at −80°C for in situ hybridization as previously described (Pau et al., 1997, 1998).

2.3. RNA isolation Total RNA from each organ or brain tissue was isolated according to the procedures of Chomczynski and Sacchi (1987) as described previously (Yang et al., 1997). Briefly, each sample was homogenized in 10 vol. of TRI Reagent (Molecular Research Center) with a tissue homogenizer. Homogenate was then supplemented with 0.2 volume chloroform and centrifugated to isolate the aqueous phase. RNA was precipitated by addition of isopropanol (0.5-vol.). The pellet was recov-

The sense and antisense primers corresponding to nt 178198 (5%-TGGTGTGAAGCAAGATCGCTA-3%) and nt 283-303 (3%-GAAGTGAGCATCCCTCTTTGA5%), respectively, of the human ERb cDNA sequence were designed to generate a monkey-specific cDNA fragment of 126 bp. The procedures for cloning by reverse transcription-polymerase chain reaction (RTPCR) have been described previously (Pau et al., 1997). Briefly, the first-strand DNA was reverse-transcribed with total RNA templates (5 mg) isolated from a rhesus monkey ovary, a poly-T oligonucleotide primer and Superscript DNA polymerase (Promega, Madison, WI). The RT product and the ERb primers were used in PCR performed with Taq polymerase (Promega, Madison, WI) and a thermocycler (MJ Research, Watertown, MA) for 35 cycles at 92°C (1 min), 50°C (2 min) and 72°C (3 min), with the final extension lengthened to 15 min. A single PCR product corresponding to a 126-bp DNA fragment was produced, excised and purified from the gel and subcloned into the vector pGEM-T (Promega, Madison, WI). Sequencing of the 126-bp DNA fragment was performed by the Sequenase 2.0 kit (US Biochemicals, Cleveland, OH) with the vector primers T3 and SP6.

2.5. Expression of monkey ERa and ERb mRNA by RT-PCR The sense (5%-CTGTTTGCTCCTAACTTGCTCT-3%) and antisense (3%-GAGGTACGGAAACAATGAGTAC-5%) oligonucleotide primers for ERa RT-PCR were constructed according to the 363 nt rhesus monkey ERa fragment in the ligand binding domain (Chandrasekher et al., 1994). Primers for ERb RT-PCR were the same as described above. Several modifications of the RT-PCR procedures were included. Before RT, each total RNA sample (1 mg) was treated with DNAase I (Ambion, Austin, TX) to eliminate genomic and vector DNAs according to the procedures described elsewhere (Huang et al., 1996). The activity of DNAase I was deactivated by heating the samples at 75°C for 5 min. The final RT mix was split into 2 equal aliquots (10 ml each) for ERa and ERb PCR using the ‘Touchdown’ procedures provided by the MJ Research

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Fig. 1. Comparison of the amino acid and nucleotide sequences between the human and a cloned, rhesus monkey ERb cDNA fragments.

thermocyler that included a progressive, 0.5°C decrease in annealing temperature (from 70 to 50°C). The PCR products were separated by 2.5% gel electrophoresis (Metaphor agarose, FMC BioProducts, Rockland, ME) and visualized by ethidium bromide/UV illumination.

2.6. Southern blot analysis The identity of the ERa and ERb primer-directed PCR products was determined by Southern blot analysis using internal primers constructed between nt 55-76 (5%-AAAAGGAAGGTTAGTGGGAACC-3%) for ERb and between nt 233-252 (5%-ACAAGATCACAGACACTTTGAT-3%) for ERa. For Southern blotting, the gel with separated PCR products was transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) and hybridized at 50°C overnight with ERb and ERa internal primers radiolabelled with gamma [32P] ATP and T4 polynuclease kinase. Hybridized signals were visualized on X-ray films (Kodak XAR 5, Eastman Kodak, Rochester, NY).

2.7. In situ hybridization Brain sections 20 mm in thickness were mounted on poly-L-lysine-treated slides and processed for in situ hybridization (ISH) as described previously (Pau et al., 1997). Briefly, 35S-labeled antisense ERa and ERb cRNA probes were transcribed in vitro by using the appropriate polymerase (T7 for ERa and SP6 for ERb) and purified by the NICK column (Pharmacia, Biotech, Piscataway NJ). Only the fraction containing peak 35S radioactivity was diluted with hybridization buffer (65.8% formamide, 13% Dextran sulphate, 0.26 M NaCl, 1.3× Denhardt’s solution, 0.013 M Tris, 0.0013 M EDTA, pH 8.0). For hybridization, the probes were

denatured at 65°C for 5 min and pipetted onto the sections (80 ml/slide). The slides were covered with a glass coverslip and sealed with dextropropoxyphene before incubation at 58°C for 20 h. After hybridization, the slides were washed four times at 5 min each in 4× SSC before RNase digestion (20 mg/ml for 30 min at 37°C) and rinsed at room temperature in decreasing concentrations of SSC containing 1 mM DTT (2X, 1X, 0.5X; 10 min each) to a final stringency of 0.1×SSC at 65°C for 30 min. After dehydration in increasing strengths of ethanol, the sections were exposed to DuPont Cronex X-ray films (DuPont NEN, Boston, MA) before being dipped in NTB-2 liquid emulsion. The dipped autoradiograms were developed 28 days later with Kodak D-19 developer and the sections were counter-stained with thionin through emulsion. No cellassociated autoradiographic signals were obtained in a control trial when sense RNA probes for ERa and ERb were used in identical ISH procedures. In ovarian sections where granulosa cells have a tendency to ‘trap’ non-specific substances, i.e. 35S-labelled nucleotide probes, an additional control was applied. Adjacent sections to those used for ERb and ERa were subjected to in situ hybridization with a 176 nt monkey tyrosine hydroxylase (TH) probe. Immunoreactive TH cells were not found in monkey granulosa cells (Dees et al., 1995). Therefore, TH autoradiographic signals at these cells would be viewed as non-specific trapping.

3. Results The putative rhesus monkey ERb cDNA fragment contained 126 nucleotides which corresponded to the segment between 178 and 303 bases of the human ERb

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Fig. 2. In situ hybridization of ERb and ERa mRNAs in the monkey ovary (panels A and D), mediobasal hypothalamus (panels B and E) and brainstem (panels C and F). With the exception of the top halves of panels A and D, which show brightfield photography of granulosa cells in follicles, all panels are presented in darkfield photography. The magnification of images is indicated by the bar length (400 mm) in the lower left corner of each panel. Insets in the top half panels were magnified as shown in the lower half of the same panel. 3V, the third cerebroventricle; 4V, the fourth cerebroventricle.

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Fig. 3. Expression of ERb and ERa mRNAs in reproductive tissues of female and male monkeys by reverse transcription-polymerase chain reaction (RT-PCR) assays. Negative RT-PCR controls contained no tissue RNA (replaced by RNA-free water). Postive controls contained plasmids with respective ERb and ERa cDNA inserts.

sequence (Fig. 1). There were four mismatched nucleotides at positions 52, 82, 97 and 103 of the monkey ERb sequence (denoted by a dot between the two series), resulting in three predicted coding amino acids (Thr, Ser and Ser) that were different from those (Ala, Gly and Gly) in the human ERb sequence. The validity of the rhesus monkey ERb probe was demonstrated by ISH in the monkey ovary, hypothalamus and brainstem (Fig. 2). Weak to moderate signals of ERb and strong signals of ERa mRNA were observed at the granulosa cell layer of ovarian follicles (Fig. 2A and D). No significant signals were observed when the same follicles were hybridized with TH probes under similar conditions (data not shown), suggesting that the hybridized ERb and ERa signals are specific. In the mediobasal hypothalamus, neurons expressing ERb mRNA were scattered primarily in the lateral hypothalamic nuclei (Fig. 2B), whereas neurons expressing ERa mRNA were located in abundance in the lateral hypothalamic region, ventral medial nuclei and the arcuate nucleus (Fig. 2E). In the brainstem, neurons in the nucleus of the solitary tract (nTS) expressed both ERb and ERa mRNAs (Fig. 2C and F). The expression and distribution of monkey ERb and ERa mRNAs in various tissues were determined by RT-PCR and compared in male and female macaques. Both ERb and ERa mRNAs were expressed in the female reproductive system, including the ovary, endometrium and mammary glands, and in the male reproductive system, including the testis, prostate and mammary tissues (Fig. 3). The myometrium of the uterus contained only ERa mRNA, whereas the epididymis of the male expressed both ERb and ERa

mRNAs (Table 1). Several endocrine glands (i.e. adrenal, thyroid and anterior pituitary), the heart and the spleen expressed both ERb and ERa mRNAs in both the male and female macaques, whereas only ERa mRNA was found in the liver of both genders (Fig. 4). In the brain, regardless of gender, both ERb and ERa mRNAs were expressed in the septum, amygdala, mediobasal hypothalamus and the lateral tegmentum of the brainstem (Fig. 5 and Table 1). Most examined brain regions expressed ERa mRNA, whereas ERb mRNA expression was more selective, particularly in the male (Table 1 and Fig. 5). In all the central or peripheral tissues reported here, none expressed only ERb mRNA. Several tissues, including the thyroid, heart, kidney, liver and pituitary were processed for repeated RT-PCR and the results were consistent with previous trials (data not shown). To evaluate the identity of the RT-PCR signals, selected results were analyzed by Southern blot analysis using ERb or ERa internal primers. Fig. 6 demonstrated that the 126 bp and 363 bp RT-PCR bands contained an internal segment of ERb and ERa sequence, respectively.

4. Discussion The results of this report suggest that both ERb and ERa mRNAs are broadly distributed not only in the female, but also in the male, rhesus monkey, particularly in peripheral tissues. In the brain, however, the number of regions expressing only ERa mRNA is noticeably greater in the male than the female. Collec-

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tively, these results are consistent with the concept that estrogen acts at multiple sites and that estrogenic or antiestrogenic actions may be differentially mediated by ERb and ERa in female and probably male primates. As far as we know, the full length monkey ERb cDNA sequence has not been reported. We are unaware of any study that involves the cloning of a cDNA fragment in the A/B domain of the translated ERb gene in the rhesus macaque. In comparison with human ERa, the A/B domain is the least conserved region within the open reading frame. The specific ERb fragment that we cloned contains 42 amino acids in which only four are matched with ERa. The low homology (9.5%) of this ERb fragment with human ERa was the main reason for its selection for cloning. Other regions such as the DNA binding and ligand binding domains contain 96 and 58% homologous sequences, respectively, between the human ERb and ERa (Mosselman et al., 1996). The selection of the 126 bp fragment for ERb mRNA amplification by RT-PCR is, therefore, ERb-specific. The cRNA probes generated from this 126 bp fragment hybridized with mRNAs in several Table 1 RT-PCR amplification of ERb and ERa mRNAs in rhesus macaques

Tissue Ovary Testus Endometrium Prostate Myometrium Epididymis Mammary tissue Adrenal Thyroid Cardiac muscle Leg muscle Kidney Liver Spleen Anterior pituitary Prefrontal cortex Frontal cortex Putamen Internal capsule Caudate nucleus Septum Hippocampus Amygdala Paraventricular nucleus Preoptic area Mediobasal hypothalamus Raphe/aqueous duct Lateral tegmentum/Pons Locus coeruleus Lateral tegmentum/medulla oblongata Cerebellum

Female

Female

Male

Male

ERb +

ERa +

ERb

ERa

+

+

+

+ +

+

−

+

+ + + + + + − + + + − + + − + + + + + + + − − +

+ + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + − − + + − − − − − + − + + − + − − − +

+ + + + + + − + + + − + + + + + + + + − + + + + +

−

+

−

+

tissues including the ovary, the mediobasal hypothalamus and the brainstem (Fig. 2). However, the relatively short ERb probe produced weaker ISH signals, particularly in the ovary and MBH, as compared to ERa (Fig. 2). To better characterize and quantify ERb expression in future in situ hybridization experiments, multiple ERb-specific probes will be needed to increase the intensity of the mRNA signals in the brain as demonstrated in the mouse and rat (Shughrue et al., 1996; Hrabovszky et al., 1997; Shughrue et al., 1997). The main objective of this report was to determine the distribution of ERb and ERa mRNAs in various tissues of male and female rhesus macaques. No attempt was made to quantify the expression of these fragments. Therefore, adjustment of mRNA quantity by an internal expression of a ‘house-keeping’ gene, i.e. cyclophilin, was not performed. Since each sample was examined for the presence of two separate fragments, they serve as mutual internal controls for the presence of intact mRNA. Thus, data showing the expression of one fragment are valid controls for the non-detected expression of the other fragment. Conversely, data that showed non-detectable expression of either fragment can be questioned without an appropriate mRNA control. As shown in Table 1, only the kidney and the prefrontal cortex in the male monkey failed to show expression of either ERb or ERa mRNA. In these cases, we chose not to make any conclusion in part because only a small portion of the tissue was examined. Furthermore, absence of a signal in the RT-PCR experiments does not imply that a particular tissue will never express the protein, and vice versa. ERb mRNA was detected by RT-PCR in the kidney of human fetus (Brandenberger, et al., 1997), but not in the kidney of 6- to 8-week-old male rats (Kuiper et al., 1997). The identity of the ERb and ERa RT-PCR products in this study was partially verified by Southern blot analysis to contain an internal segment of ERb and ERa sequence, respectively (Fig. 6). Several reports have examined the distribution of ERb and ERa mRNAs by RT-PCR, Northern blotting, ribonuclease protection assay or in situ hybridization in rodents and humans. None was designed to compare directly the distribution of the two ER subtypes between the two genders. Our results suggest that both ERb and ERa mRNAs were similarly distributed in peripheral tissues of male and female. In the human, ERb mRNA was found in the kidney, lung, breast, ovary, uterus, adrenal, prostate and testis (Enmark et al., 1997), although the source of the non-reproductive tissues was not identified by gender. In the male rat, both ERb and ERa mRNAs are present in the epididymis, prostate, testis, pituitary, thymus, adrenal and heart (Kuiper, et al., 1997). In the midgestational human fetus, presumably of both genders, ERb and ERa mRNAs are present in the bone, gut, pancreas, kidney,

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Fig. 4. Expression of ERb and ERa mRNAs in peripheral tissues of female and male monkeys by reverse transcription-polymerase chain reaction (RT-PCR) assays. See Fig. 3 legends for detail description.

muscle, heart, spleen, thymus, pituitary, testis, ovary and uterus (Brandenberger et al., 1997). Interestingly, only ERb mRNA was found in the lung, liver, adrenal and brain in the human fetus (Brandenberger et al., 1997) suggesting that ER subtypes are expressed differentially at different developmental stages. In both male rats (Kuiper, et al., 1997) and male rhesus monkeys (this study), only ERa mRNA was expressed in the liver. In the monkey, both ERb and ERa mRNAs were expressed in gonadotropin-stimulated follicles of the ovary, but only ERb mRNA expression changes following ovulation and during luteinization (Duffy, et al., 1998). In the monkey brain (Table 1), ERa mRNA was detected in almost all the regions examined. The broad distribution of ERa mRNA supports the hypothesis that estrogen plays important roles in the brain. For example, immunoreactive ERa neurons are located in the preoptic area, the bed nucleus of the stria terminalis, the periventricular area, the ventromedial nu-

cleus, the arcuate nucleus and the paraventricular nucleus of adult female African green monkeys (Cercopithecus aethiops) and cynomolgus macaques (Macaca fascicularis) (Herbison et al., 1995). Moreover, our study also showed that ERb mRNA was expressed in many areas, including the cerebral cortex, the basal ganglia, the hypothalamus and the brainstem of the female monkey brain. These results are in general agreement with those in ERa knockout mice which indicate that ERb mRNA is present in the preoptic area, suprachiasmatic nucleus, paraventricular nucleus, premamillary nucleus, medial tuberal nucleus, olfactory bulb, septum, bed nucleus of stria terminalis, amygdala, cerebral cortex, hippocampus and dorsal raphe of the brainstem (Shughrue et al., 1997). An interesting observation in this study is that the expression of ERb mRNA was observed in several brain regions in the female, but not in the male. These regions include the putamen, internal capsule, paraventricular nucleus, hippocampus and the area surrounding

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Fig. 5. Expression of ERb and ERa mRNAs in brain tissues of female and male monkeys by reverse transcription-polymerase chain reaction (RT-PCR) assays. See Fig. 3 legends for detail description. MBH, mediobasal hypothalamus; LT, lateral tegmentum; MO, medulla oblongata.

the aqueductal gray of the brainstem. The significance of this gender difference in the expression of ERb mRNA in primate brain remains illusive and requires confirmation in future studies. It is noteworthy to mention that several brain tissues in the male rat, including the thalamus, hypothalamus, cerebellum, brainstem and spinal cord, express ERb mRNA but no, or very little, ERa mRNA (Kuiper et al., 1997). In the male monkey, we failed to find a tissue that expressed only ERb mRNA. However, since the 363 bp ERa fragment employed in this study corresponds to the ligand binding domain and contains over 50% overlapping nucleotides with the ERb sequence, it is not possible at the present time to conclude that these data represent only ERa mRNA. Further studies are needed to clarify this issue. In summary, although the functional implications of the broad distribution of ERb and ERa mRNAs in both genders of the rhesus macaque are uncertain at this time, the results support the concept that estrogen has a multi-functional impact in normal and pathologi-

cal cells in many tissues of both male and female primates. The expression of ERb in the prostate (Kuiper et al., 1997; Lau et al., 1998; Prins et al., 1998) and bone (Onoe et al., 1997; Arts et al., 1997) reflects an example of the clinical value of ER research. The dynamic changes in ERb and ERa mRNA expressions and their regulation by prolactin in the rat corpus luteum during pregnancy and at term (Telleria et al., 1998) may indicate a functional aspect of different ER subtypes in the mediation of estrogen action in the same tissue at different times. Futher understanding of the regulation and function of different ER subtypes may improve treatments of many diseases, including cancer, osteoporosis, cardiovascular disorders, Alzheimer’s disease and reproductive ailments.

Acknowledgements We thank Cyrus Lee, Nathan Airhart and Jian-hua Yu for their technical assistance. This study was sup-

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Fig. 6. Southern blot analysis of ERb and ERa DNA fragments amplified by reverse transcription-polymerase chain reaction (RT-PCR) with total RNA templates from various tissues of female and male monkeys. See Fig. 3 legends for detail description. N, nucleus; POA, preoptic area; LT, lateral tegmentum; MBH, mediobasal hypothalamus.

ported by NIH grants HD-16631, HD-18185, and RR00163.

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