Experimental Gerontology 34 (1999) 267–273 Mechanisms of age-related changes in gonadotropinreleasing hormone receptor messenger ribonucleic acid content in the anterior pituitary of male rats Tadashi Shinkai1, George S. Roth* Molecular Physiology and Genetics Section, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute of Health, National Institute on Aging; 5600 Nathan Shock Drive, Baltimore, MD, 21224, USA Manuscript received September 7, 1998; manuscript accepted November 10, 1998 Abstract To determine the mechanism(s) of age-related changes in gonadotropin release from pituitary gonadotrophs in male rats, we measured the number of GnRH (gonadotropin-releasing hormone) receptor containing cells and expression of GnRH receptor mRNA per cell in the anterior pituitary. An in situ hybridization procedure was performed using young (six months) and old (24 –25 months) Wistar rats. An image analysis system was employed for the autoradiographic analysis. The number of pituitary cells increased during aging (approximately 45%, p Ͻ 0.01). On the other hand, the number of GnRH receptor mRNA-containing cells decreased (approximately 25%, p Ͻ 0.05). The percentage of these cells in old rats decreased to less than a half of that in young animals ( p Ͻ 0.01). GnRH receptor mRNA per cell in old rats was only 7% lower than in young ( p Ͻ 0.01). These results suggest that loss of pituitary gonadotroph GnRH receptors and response is primarily due to the loss of gonadotrophs, and that the death mechanism(s) are responsible for decreased stimulation of Gn release during aging. © 1999 Elsevier Science Inc. All rights reserved. Keywords: GnRH receptor; Gene expression; Pituitary; Aging * Corresponding author. Tel: (410) 558-8178; Fax: (410) 558-8323; E-mail: geor@vax.grc.nia.nih.gov. 1 Present address: Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan. 0531-5565/99/$–see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 8 ) 0 0 0 7 4 - 6 268 T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273 Changes in hormone/neurotransmitter action are responsible for many altered physiological functions during aging (Roth and Hess, 1992; Miyamoto & Ruth, 1995; Roth, 1997). Such changes are primarily due to alterations in signal transduction components/events that mediate these functional responses, and include receptors, effectors, second messengers, and gene expression (Roth and Hess, 1992; Miyamoto & Roth, 1995; Roth, 1997). A second level of alteration occurs during the aging of complex tissues that contains various cell types (e.g., the brain) (Zhang et al., 1995). In addition to changes in signal transduction within individual cells, numbers of particular hormone/neurotransmitter responsive cells may be altered, especially due to cell loss. A prime example is the age-related impairment in dopaminergic motor control, which is at least partially due to loss of striatal D2-dopamine receptors (Joseph & Roth, 1993; Roth & Joseph, 1994). Receptor loss is the consequence of both the death of receptor-containing neurons (Han et al., 1989; Zhang et al., 1995) and decreased expression of the receptor gene in the surviving neurons (Mesco et al., 1991). Another important physiological system shown to exhibit impaired neuroendocrine responsiveness during aging is the stimulation of gonadotropin (Gn) release from pituitary cells by gonadotropin releasing hormone (GnRH) (Miyamoto et al., 1992). Such reduced responsiveness appears to be at least partially due to loss of GnRH receptors (Limonta et al., 1988; Miyamoto et al., 1992). However, it is not clear whether all the receptor loss is restricted to decreases within individual gonadotropic cells (gonadotrophs), or is additionally the result of gonadotropic loss during aging. The present study, therefore, has attempted to quanitate the numbers of GnRH receptorcontaining cells in pituitaries of rats of various ages as well as the concentration of receptors per cell. 1. Materials and methods 1.1. Experimental animals and tissue preparation Male Wistar rats aged 6 months (n ϭ 12) and 24 –25 months (n ϭ 12) obtained from the animal colony of the Gerontology Research Center (GRC, National Institute on Aging/ National Institute of Health) were used. The GRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care. The animals were killed by a carbon dioxide overdose. After decapitation, pituitaries were immediately removed and frozen, and stored at Ϫ70°C until required for in situ hybridization. Frozen pituitaries were sectioned in a cryostat (25 m), and the sections were mounted onto 3-aminopropyltriethoxy-silane (Sigma)-coated glass slides. After air drying, tissues were fixed in 4% paraformaldehyde solution (pH 7.6) for 5 min at room temperature. Each pituitary resulted in 150 –170 cross-sections, and every 10 sections were mounted on each slide. 1.2. In situ hybridization A plasmid vector pBluescript containing a 2219-base pair fragment from the rat GnRH receptor complementary DNA (gift from Drs. R. Sellar and K. L. Eidne, Medical Research T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273 269 Council, UK) was used for the preparation of antisense RNA (complementary to the rat GnRH receptor mRNA). To obtain labeled antisense RNA, the plasmid was linearized using Spe1 restriction endonuclease (Promega), and transcribed with T7 polymerase (Boehringer) in the presence of nucleotides (Promega). The transcription reaction and subsequent probe purification were performed with a riboprobe labeling kit (Promega) and 33P-labeled UTP (50 mCi, Amersham). For labeled sense probe synthesis, we used Xho1 restriction endonuclease (Promega), T3 polymerase (Boehringer), nucleotides, and 33P-labeled UTP. Hybridization was performed as described (Peters et al., 1981; Smith & Reinhart, 1993) with slight modification. After hybridization, the sections were rinsed in 4ϫ standard saline citrate (SSC) and 2 mM dithiothreitol solution at room temperature, treated with RNase (20 mg/mL, Sigma) in RNase buffer [0.01 M Tris/HC1 (pH 8.0), 0.5 M NaCl, 1 mM EDTA (pH 8.0) and distilled water] at 37°C for 30 min, and then rinsed in 4ϫ SSC, 2ϫ SSC, and 0.2ϫ SSC for 30 min each at room temperature. The sections were dehydrated in an alcohol series containing 300 mM ammonium acetate and air dried. For autoradiography, the slides were dipped in an autoradiography emulsion (Kodak NTB-3 photographic emulsion, Eastman Kodak Co.) and exposed in a dark place for 7–10 days at 4°C. After development, the slides were counterstained with hematoxylin. 1.3. Quantitative analysis of GnRH receptor mRNA For the autoradiographic analysis, we randomly measured 250 –300 positive cells in 20 –22 different areas of the pituitary (same for both age groups) using an image analysis system (Image-1, Universal Imaging Corporation, West Chester, PA). Cells were analyzed for the intensity of staining based on the amount of silver grain presence over positive cells. Labeling was determined for cells in given fields after subtracting the average background attributable to cell staining. Data were assessed by analysis of variance (ANOVA), using a personal computer program (Ishii, 1982). 2. Results Fig. 1 shows microscopic pictures of rat anterior pituitary sections hybridized with a P-labeled riboprobe complementary to the rat GnRH receptor mRNA. Reaction of individual cells is particularly obvious in anterior pituitaries of young and old rats. The number of reactive cells in the old pituitary was markedly less than in the young. Posterior and intermediate lobes were devoid of positive reaction, and there were few nonspecific reactions in anterior pituitaries when a labeled sense probe was applied to the sections (data not shown). The age-associated changes in the anterior pituitary cell number and GnRH receptor mRNA-containing cell number per 0.01 mm2 m microscopic field are shown in Fig. 2a. The number of pituitary cells increased during aging (approximately 45%, p Ͻ 0.01). On the other hand, the number of GnRH receptor mRNA-containing cells decreased (approximately 25%, p Ͻ 0.05). The percentage of these cells in 24 –25 month-old rats decreased to about half of that in 6-month-old animals (Fig. 2b) ( p Ͻ 0.01). 33 270 T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273 Fig. 1. Microscopic pictures of rat anterior pituitaries hybridized with 33P-labeled riboprobe complementary to the rat GnRH receptor mRNA. Silver grains are concentrated over the reactive cells. The numbers of reactive cells are less in old pituitary (O) than in young pituitary (Y). Bar ϭ 100 m. To determine the differences of GnRH receptor mRNA levels in individual reactive cells from young and old rats, we measured the optical density of positive cells (Fig. 2c). Expression of GnRH receptor mRNA in cells of old rats is approximately 7% lower than the mRNA level in young rats. This difference is statistically significant ( p Ͻ 0.01). T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273 271 Fig. 2. Comparison of total pituitary cell number and GnRH receptor mRNA-containing cell number per 0.01 mm2 microscopic field (a) and percentage of GnRH receptor containing cells (gonadotrophs) (b) in young (6 month) and old (24 –25 month) rat pituitary cells. (c) Comparison of GnRH receptor mRNA levels in young (6 month) and old (24 –25 month) positive cells. Values shown are means Ϯ SEM. Absence of error bars indicates smaller than would be visible on the figure. *Significantly different, p Ͻ 0.05; **, p Ͻ 0.01. 3. Discussion Results presented here suggest that the number of pituitary GnRH receptor mRNAcontaining cells decreases by 25% during aging. The percentage of these cells in 24 –25month-old rats decreases to less than half of that in 6-month-old animals. We also find that expression of GnRH receptor mRNA per cell is reduced during aging. However, the decrease is only 7%. These results suggest that loss of pituitary gonadotroph GnRH receptors and response is primarily due to the loss of gonadotrophs during aging. Some researchers have reported that GnRH receptors decrease in the pituitary during aging and concluded that the reduced secretion of LH in old rats may be due to this reduction (Limonta et al., 1988). We also found age-related reduction in the number of GnRH receptors in the pituitary (Miyamoto et al., 1992) and suggested that the GnRH receptor decrease might 272 T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273 be caused by gonadotroph loss during aging. On the other hand, Sonntag reported that there were no age-related differences in GnRH receptors in male rats (Sonntag et al., 1984). Such discrepancies may be due to differences in the strain of rats or the preparations employed. The strain issue seems less likely to be the explanation, however, because both Limonta et al. (1988) and Sonntag et al. (1984) used Sprague–Dawley rats. Thus, it is possible that postreceptor events may also contribute to decreased responsiveness during aging. Our study demonstrates that expression of GnRH receptor mRNA per cell decreases only slightly during aging. In female C57bL/6NNia mice, an elevation in the number of GnRH receptors per gonadotroph with advancing age has been reported (Parkening & Pal, 1995). This experiment was performed in cultured cells, while our study is in vivo. Such differences, as well as in species and sex, may account for differences in results. In any case, however, receptor loss per cell is probably not a major cause of altered responsiveness of pituitary cells to GnRH during aging. The loss of receptor containing cells appears to be more important. In this regard, our results are consistent with those of Console et al. (1994), who reported an age-related decrease in the density of Sprague–Dawley rat gonadotrophs as identified by an antibody to LH (Gn). We have extended their findings by examining the Wistar strain, which we have previously characterized for various age changes in receptors and responsiveness (Myamoto et al., 1992; Roth & Hess, 1992; Zhang et al., 1995). Our advances include the use of in situ hybridization to identify cells containing the mRNA for the GnRH receptor, more mature young animals (six months as opposed to four months) to identify senescence rather than possible maturational changes, and much longer numbers of animals per group (12 as opposed to 4 –5). It should also be noted that the larger decrease (ϳ45%) in relative GnRH receptor mRNA-containing cells in our study and similar decrease in gonadotroph cell density reported by Console et al. (1994) appear to be partially due to the increase in total pituitary cells that we observe with age. Console et al. (1993) also observed a decrease in somatotroph cell density with age. In a somewhat analogous system, two mechanisms that may cause loss of D2-dopamine receptors from the rat striatum during aging have reported. One mechanism responsible for such loss is a decreased number of D2-dopamine receptor-containing neurons (Han et al., 1989), and the other is reduction of D2-dopamine receptor gene expression in the surviving neurons (Mesco et al., 1991). Unlike the situation for age changes in D2-dopamine receptors, our present study suggests that loss of pituitary gonadotroph GnRH receptors and response during aging is primarily due to loss of pituitary gonadotroph GnRH receptors and response during aging is primarily due to loss of gonadotrophs rather than altered receptor gene expression, and that mechanism(s) of gonadotroph loss are responsible for decreased stimulation of GnRH during aging. Acknowledgments We thank Ms. Sheila A. Mathias for technical assistance and Drs. Donald K. Ingram and Yongquan Luo for helpful advice. 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