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. The GnRH receptor cDNA was a generous gift from Drs.
R. Sellar and K. L. Eidne, Medical Research Council, UK.

T. Shinkai, G.S. Roth/Experimental Gerontology 34 (1999) 267–273

273

References
Console, G. M., Gomez Dumm, C. L. A., & Goya, R. G. (1993). Impact of aging on the morphology and function
of the somatotroph population in rats. Mech Ageing Dev 70, 45–51.
Console, G. M., Gomez Dumm, C. L. A., & Goya, R. G. (1994). Immunohistochemical and radioimmunological
study of pituitary gonadotrophs during aging in male rats. Mech Ageing Dev 73, 87–95.
Han, Z., Kuyatt, B. L., Kochman, K. A., Desouza, E. B., & Roth, G. S. (1989). Effect of aging on concentrations
of D2-receptor-containing neurons in the rat striatum. Brain Res 498, 299 –307.
Ishi, S. (1982). Tokyo: Programs of Statistical Methods for Biologists by N 88-Basic. Baifukan.
Joseph, J. A., & Roth, G. S. (1993). Hormonal regulation of motor behavior in senescence. J Gerontol 48, 51–55.
Limonta, P., Dondi, D., Maggi, R., Martini, L., & Piva, F. (1988). Effect of aging on pituitary and testicular
luteinizing hormone-releasing hormone receptors in the rat. Life Sci 42, 335–342.
Mesco, E. R., Carlson, S. G., Joseph, J. A., & Roth, G. S. (1993). Decreased striatal D2 dopamine receptor mRNA
synthesis during aging. Mole Brain Res 17, 160 –162.
Mesco, E. R., Joseph, J. A., Blake, M. J., & Roth, G. S. (1991). Loss of D2 receptors during aging is partially
due to decreased levels of mRNA. Brain Res 545, 355–357.
Miyamoto, A., Maki, T., Blackman, M. R., & Roth, G. S. (1992). Age-related changes in the mechanisms of
LHRH-stimulated LH release from pituitary cells in vitro. Exp Gerontol 27, 211–219.
Miyamoto, A., & Roth, G. S. (1995). Changes in transmembrane signaling mechanisms during Aging-Cellular
and molecular aspects. In: A. Macieria-Coelho (Ed.), Molecular Basis of Aging (pp. 293–306). Boca Raton,
FL: CRC Press.
Parkening, T. A., & Pal, D. (1995). GnRH receptor sites increase on the surface of cultured gonadotropes of
senescent C57BL/NNia mice. J Gerontol 50A, B342–B350.
Peters, L. L., Hoefer, M. T., & Ben-Jonathan, N. (1981). The posterior pituitary: Regulation of anterior pituitary
prolactin secretion. Science 213, 659 – 661.
Roth, G. S. (1997). Age changes in signal transduction and gene expression. Mech Ageing Dev 98, 231–238.
Roth, G. S., & Hess, G. D. (1992). Changes in the mechanisms of hormone and neurotransmitter action during
aging: Current status of the role of receptor and post-receptor alterations. A review. Mech Ageing Dev 20,
175–194.
Roth, G. S., & Joseph, J. A. (1994). Cellular and molecular mechanisms of impaired dopaminergic function
during aging. Ann NY Acad Sci 791, 129 –135.
Smith, M. S., & Reinhart, J. (1993). Changes in pituitary gonadotropin-releasing hormone receptor messenger
ribonucleic acid content during lactation and after pup removal. Endocrinology 133, 2080 –2084.
Sonntag, W. E., Forman, L. J., Fiori, J. M., Hylka, V. W., & Meites, J. (1984). Decreased ability of old male rats
to secrete luteinizing hormone (LH) is not due to alterations in pituitary LH-releasing hormone receptors.
Endocrinology 114, 1657–1664.
Zhang, L., Ravipati, A., Joseph, J. A., & Roth, G. S. (1995). Aging related changes in rat striatal D2 receptor
mRNA containing neurons: A quantitative non-radioactive in situ hybridization. J Neurosci 15, 1735–1740.

