Summary
The purpose of the present study was to localize luteinizing hormone-releasing hormone (LHRH) mRNA within the male rat forebrain using an in situ hybridization approach. The expression of LHRH mRNA was compared in castrate and intact males to approach questions on the chronic influences of circulating testicular steroids on the gene expression of the peptide. Frozen 10 μm sections fixed in paraformaldehyde were obtained from the forebrain region of intact and 2 week post-castrate adult male rats. LHRH mRNA was autoradiographically detected using an oligomer (59mer) complementary to the mRNA coding for amino acids-5 to 15 of the human LHRH preprohormone. Individual brain sections were incubated in prehybridization buffer for 2 h to reduce nonspecific binding. Following this, 20 μl of hybridization buffer containing 65,000–120,000 cpm of the 59mer were applied to sections and hybridized at 37° C for 3 days. The sections were then rinsed over a 48 h period, dehydrated, dipped in Kodak NTB2 liquid emulsion and exposed for 22 days. Autoradiograms were developed and counterstained with fast green and cresyl violet. As reported in the female, LHRH message-containing cells were localized in ventral septal regions, the diagonal bands of Broca, preoptic area and anterior hypothalamus. On occasion, LHRH gene expressing cells were found to appear in loose clusters. Labeled cells were never found in control sections treated with hybridization buffer lacking the 59mer. The total number of LHRH mRNA-containing cells localized in intact rats did not differ significantly from the castrate group. The mean grain counts per cell (±SEM) for the intact (30.1±1.2) and castrate (24±1.1) groups were found to differ, as did the histogram distribution of these two populations. These results are in contrast to those expected on the basis of a negative feedback effect, and instead suggest that long term exposure to testicular steroids can actually increase the content of LHRH mRNA within individual neurons.
Similar content being viewed by others
References
Adelman JP, Mason AJ, Hayflick JS, Seeburg PH (1986) Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat. Proc Natl Acad Sci USA 83: 179–183
Barr GD, Barraclough CA (1978) Temporal changes in medial basal hypothalamic LH-RH correlated with plasma LH during the rat estrous cycle and following electrochemical stimulation of the medial preoptic area in pentobarbital-treated proestrous rats. Brain Res 148: 413–423
Bently JB, Singer RH (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucl Acids Res 12: 1777–1799
Childs (Moriarty) GV, Ellison DG, Lorenzen JR, Collins TJ, Schwartz NB (1982) Immunocytochemical studies of gonado-tropin storage in developing castration cells. Endocrinology 111: 1318–1328
Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in site hybridization using asymmetrical RNA probes. Dev Biol 101: 485–502
Daniel WW (1978) Applied nonparametric statistics. Houghton Mifflin Company, London
Diuzen DE, Ramirez VD (1983) In vivo LHRH release from the median eminence of conscious, unrestrained intact, acutecastrate and long-term castrate males as determined with push-pull perfusion (PPP). Abstract 65th Annual Meeting. Endocrine Society, p 156
Drouin J, Labrie F (1976) Selective effects of androgens on LH and FSH release in anterior pituitary cells in culture. Endocrinology 98: 1528–1534
Drouva SV, Gauton JP, Pattou E, Laplante E, Kordon C (1986) Effects of estradiol and progesterone on immunoreactive forms of hypothalamic luteinizing hormone-releasing hormone. Neuroendocrinology 43: 32–37
Ellis GB, Desjardins C, Fraser HM (1983) Control of pulsatile LH release in male rats. Neuroendocrinology 37: 177–183
Ellis GB, Desjardins C (1984) Orchidectomy unleashes pulsatile luteinizing hormone secretion in the rat. Biol Reprod 30: 619–627
Eskay RL, Mical RS, Porter JC (1977) Relationship between luteinizing hormone concentration in hypophysial portal blood and luteinizing hormone release in intact, castrated, and electrochemically-stimulated rats. Endocrinology 100: 263–270
Fukuda-Schwanzel M, Morrell JI, Pfaff DW (1985) Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat. J Comp Neurol 238: 348–364
Gay VL, Midgley AR Jr (1969) Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioim-munoassay. Endocrinology 84: 1359–1364
Giguere V, Lefebvre F, Labrie F (1981) Androgens decrease LHRH binding sites in the rat pituitary cell in culture. Endocrinology 108: 350–352
Gross DS (1980) Effect of castration and steroid replacement on immunoreactive gonadotropin-releasing hormone in the hypothalamus and preoptic area. Endocrinology 106: 1442–1450
Gross DS, Rothfeld JM (1985) Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system. J Histochem Cytochem 33: 11–20
Kalra PS, Kalra SP (1978) Effect of intrahypothalamic testosterone implants on LHRH levels in the preoptic area and medial basal hypothalamus. Life Sciences 23: 65–68
Kalra PS, Kalra SP (1980) Modulation of hypothalamic luteinizing hormone-releasing hormone levels by intracranial and subcutaneous implants of gonadal steroids in castrated rats: effects of androgen and estrogen antagonists. Endocrinology 106: 390–397
Kalra SP, Kalra PS (1983) Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4: 311–351
Kalra PS (1985) Further studies on the effects of testosterone on hypothalamic LH-RH and serum LH levels: castrationinduced delayed response. Neuroendocrinology 41: 219–223
Kelly MJ, Ronnekleiv OK, Eskay RL (1982) Immunocytochemical localization of luteinizing hormone-releasing hormone in neurons in the medial basal hypothalamus of the female rat. Exp Brain Res 48: 97–106
King JC, Anthony ELP (1983) Biosynthesis of LHRH inferences from immunocytochemical studies. Peptides 4: 963–970
Kozlowski GP, Dees Les W (1984) Immunocytochemistry for LHRH neurons in the arcuate nucleus of the rat: fact or artifact. J Histochem Cytochem 32: 83–91
McCabe JT, Morrell JI, Pfaff DW (1987) In situ hybridization as a quantitative autoradiographic method: an example from vasopressin and oxytocin gene transcription in the Brattleboro rat. In: Uhl C (ed) In situ hybridization in brain. Plenum Press, New York (in press)
McCabe JT, Morrell JI, Richter DW, Pfaff DW (1986) Localization of neuroendocrinologically-relevant RNA in brain by in situ hybridization. Frontiers Neuroendocr 9: 149–167
Merchenthaler I, Gores T, Setalo G, Petrusz P, Flerko B (1984) Gonadotropin-releasing hormone (GnRH) neurons and path-ways in the rat brain. Cell Tissue Res 237: 15–29
Morrell JI (1986) Symposium on in situ hybridization with nucleotide probes: a histochemical tool — introduction. J Histochem Cytochem 34: 25–26
Naik DV (1976) Immunohistochemical localization of LH-RH during different phases of estrus cycle of rat with reference to the preoptic and arcuate neurons, and the ependymal cells. Cell Tissue Res 173: 13
Rothfeld JM, Gross DS (1985) Gonadotropin-releasing hormone within the organum vasculosum lamina terminalis in the ovariectomized, estrogen progesterone-treated rat. A quantitative immunocytochemical study using image analysis. Brain Res 338: 309–315
Rudenstein RS, Bigdell H, McDonald MH, Snyder PJ (1979) Administration of gonadal steroids to the castrated male rat prevents a decrease in the release of gonadotropin-releasing hormone from the incubated hypothalamus. J Clin Invest 63: 262–267
Schwartz JP, Costa E (1986) Hybridization approaches to the study of neuropeptides. Ann Rev Neurosci 9: 277–304
Sherwood NM, Chiappa SA, Fink G (1976) Immunoreactive luteinizing hormone releasing factor in pituitary stalk blood from female rats: sex steroid modulation of response to electrical stimulation of preoptic area of medial eminence. J Endocr 70: 501–511
Shin SH, Howitt CJ (1976) Effect of testosterone on hypothalamic LH-RH content. Neuroendocrinology 21: 165–174
Shivers BD, Harlan RE, Morrell JI, Pfaff DW (1983) Immunocytochemical localization of luteinizing hormone-releasing hormone in male and female rat brains. Neuroendocrinology 36: 1–12
Shivers BD, Harlan RE, Hejtmancik JF, Conn PM, Pfaff DW (1986a) Localization of cells containing LHRH-like mRNA in rat forebrain using in situ hybridization. Endocrinology 118: 883–885
Shivers BD, Schachter BS, Pfaff DW (1986b) In situ hybridization for the study of gene expression in the brain. In: Conn PM (ed) Methods in enzymology, Vol 124. Academic Press, New York, pp 497–510
Wise PM, Rance N, Selmanoff M, Barraclough CA (1981) Changes in radioimmunoassayable luteinizing hormone-releasing hormone in discrete brain areas of the rat at various times on proestrus, diestrus day 1, and after phénobarbital administration. Endocrinology 108: 2179–2185
Witkin JW, Paden CM, Silverman A-J (1982) The luteinizing hormone-releasing hormone (LHRH) systems in the rat brain. Neuroendocrinology 35: 429–438
Young III WS, Mezey E, Siegel R (1985) Quantitation of vasopressin and oxytocin mRNA: an in situ hybridization histochemical study of normal adrenalectomized and Brattleboro rats. Soc Study Neurosci Abstr 11: 142
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rothfeld, J.M., Hejtmancik, J.F., Conn, P.M. et al. LHRH messenger RNA in neurons in the intact and castrate male rat forebrain, studied by in situ hybridization. Exp Brain Res 67, 113–118 (1987). https://doi.org/10.1007/BF00269459
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00269459