Age-related changes in growth hormone (GH) cells in the pituitary gland of male mice are mediated by...

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2004) 164–173
Brain Research 998 (
0006-8

doi:10.

* C

9918.

E-m

Research report

Age-related changes in growth hormone (GH) cells in the pituitary gland

of male mice are mediated by GH-releasing hormone but not by

somatostatin in the hypothalamus

Sachi Kuwaharaa, Dwi Kesuma Saria, Yasuhiro Tsukamotoa, Shin Tanakab, Fumihiko Sasakia,*

aLaboratory of Veterinary Anatomy, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho,

Sakai, Osaka 599-8531, JapanbLaboratory Animal Research Facilities, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, 474-8522, Japan

Accepted 21 October 2003

Abstract

Using immunocytochemical and morphometric methods, we examine changes with age of growth hormone-releasing hormone (GHRH)

in the arcuate nucleus (ARC), changes of somatostatin (SS) in the periventricular nucleus (PeN) of the hypothalamus, and changes of growth

hormone (GH) cells in the anterior pituitary in male C57BL/6J mice at 2 months old (2 M), 4 M, 12 M and 24 M. The number of GHRH-ir

neurons decreased significantly with age. The number of SS-ir neurons did not differ significantly between these all age groups. The volume

of the anterior pituitary and the number of adenohypophysial parenchymal cells fell dramatically from 4 to 12 M. The proportion of GH-ir

cells decreased significantly with age, and in absolute number from 4 to 12 M and in size from 2 to 4 M and from 4 to 12 M. These results

suggest that the reduction in GH-ir cells in male mice is modulated by the reduction in GHRH-ir neurons, but not by SS-ir neurons.

D 2003 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Aging process

Keywords: GH-ir cell; GHRH-ir neuron; SS-ir neuron; Immunocytochemistry; Morphometry

1. Introduction

It is widely acknowledged that secretion of growth

hormone (GH) declines with age in animals [22] and in

humans [3]. Pulsatile release and the mean concentration of

GH in the anterior pituitary are attenuated in male rats with

age [32]. This decrease in GH may be responsible for a loss

of muscle mass, increase in adipose tissue mass, and a

deterioration in several tissue and organ functions [3].

Release of GH from the anterior pituitary is regulated by

at least two hypothalamic neuropeptides: growth hormone-

releasing hormone (GHRH), which stimulates GH synthesis

and release, and somatostatin (SS), which inhibits GH

synthesis and release [10]. GHRH neurons are located in

993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

1016/j.brainres.2003.10.060

orresponding author. Tel.: +81-72-254-9475; fax: +81-72-254-

ail address: [email protected] (F. Sasaki).

the arcuate nucleus (ARC) of humans [1,2], rats [6,9,24,31]

and mice [25,29,30] and SS neurons are located in the

periventricular nucleus (PeN) of rats [9,12,14,19] and mice

[25]. They directly secrete each neuropeptide to the median

eminence (ME) so as to modulate GH secretion. The age-

related decline in GH secretion may therefore be responsible

for neuropeptides from GHRH and SS neurons. It has been

suggested that the age-related decline of GH secretion is a

result of more reduced responsiveness of GH cells to GHRH

[3,32] than any defect in the mechanisms of GH release

[35]. However, the contribution of SS to the reduced

secretion of GH from GH cells remains controversial

[11,21,32,34]. We have already reported that pituitary GH

immunoreactive (-ir) cells [27,30] and ARC GHRH-ir

neurons and PeN SS-ir neurons [25] mature in differently

during postnatal development. It is important to study the

hypothalamo (GHRH/SS)-pituitary (GH)-somatic axis in

mature animals in order to understand the physiological

mechanism of aging; we believe that this has not been

S. Kuwahara et al. / Brain Research 998 (2004) 164–173 165

studied in detail. However, there is little information about

age-related changes in hypothalamic ARC GHRH neurons

and PeN SS neurons [21]. An immunocytochemical and

morphometric study was therefore conducted to examine

age-related changes in ARC GHRH neurons, PeN SS

neurons and pituitary GH cells.

2. Materials and methods

2.1. Animals

Adult male C57BL/6J mice were obtained from the

National Institutes on Aging (NIA, MD, USA). These mice

were in four age groups: 2 months old (2 M), 4 months old

(4 M), 12 months old (12 M) and 24 months old (24 M).

Five mice from each group were anaesthetized by ether

inhalation, and killed in the morning between 1000 and

1200 h. The body weight in 2, 4, 12 and 24 M mice was

30.31F 0.86 (meanF S.E.), 30.79F 1.22, 29.98F 1.12

and 33.41F 0.92 g, respectively [the body weight in 24

M mice was significantly heavier (F = 2.26, P < 0.05) than

that in 2 and 12 M mice].

After immersion in Bouin’s solution for 30 min, the

hypothalamus was taken from the brain as follows (Fig. 1):

their brains were cut frontally at the anterior part of the optic

chiasma (a) and at the anterior part of the cerebellum (b), cut

longitudinally at both lateral sides (c,d) of hypothalamus,

Fig. 1. Ventral view of hypothalamus with the frontal plane of the anterior

part of the optic chiasma. Their brains were cut frontally at the anterior part

of the optic chiasma (a) and at the anterior part of the cerebellum (b), cut

longitudinally at both lateral sides (c,d) of hypothalamus, and cut

horizontally at the part of the corpus callosum (e) at the plane of the

anterior part of the optic chiasma. cc: corpus callosum, LV: lateral ventricle,

on: optic nerve, oc: optic chiasma.

and cut horizontally at the part of the corpus callosum (e) at

the plane of the anterior part of the optic chiasma. It was

further placed in the same fixative for 24 h. The pituitary

glands from mice of each age were fixed in 10% neutral

formalin for 24 h. All specimens were dehydrated in a

graded series of ethanol solutions, cleared in xylene, and

embedded in Tissue Prep (Fisher Scientific, Pittsburgh, PA,

USA).

2.2. Experimental procedures

The parts of ARC and PeN in all hypothalami were

frontally cut in serial sections of 10 Am as described

previously [25] and were mounted on poly-L-lysine (50

Ag/ml)-coated slides. In all immunocytochemical proce-

dures, sections were processed by the avidin–biotin–per-

oxidase complex (ABC) method, using either anti-mouse

GHRH rabbit serum (1:5000) or anti-mouse SS rabbit serum

(1:1000), both of which were donated by Dr. Miki (Tokyo

Women’s Medical College) as described previously [25].

The absorption tests for the GHRH antiserum and SS

antiserum are described in Refs. [20,25], respectively.

Sections from the lateral, parasagittal, and midsagittal

parts of the pituitary gland were stained immunocytochem-

ically by ABC method, using anti-rat GH rabbit serum

(1:8000), which was donated by Dr. A. F. Parlow (NIDDK,

National Hormone and Pituitary Program) as described

previously [15]. The specificity of this antiserum was

verified by absorption testing using antigen. The absorp-

tion test for the GH antiserum was performed by adding

100 Ag/ml rGH (Biogenesis, UK) to the corresponding

antiserum.

Proliferating cell nuclear antigen (PCNA) and in situ

apoptosis tests were performed to observe proliferation of

cells and nuclear DNA fragmentation in ARC and PeN

neurons and adenohypophysial cells, respectively, as de-

scribed previously [25]. Furthermore, the in situ method

[antibody against single-stranded DNA (Dako, Tokyo, Ja-

pan)] by ABC method was used to observe apoptic cells of

ARC and PeN neurons and adenohypophysial cells.

2.3. Morphometry

2.3.1. Hypothalamic GHRH-ir and SS-ir neurons

Serial sections of the ARC [total number of sections per

mouse: 71.2F 1.0 (meanF S.E.)] were cut along the ante-

rior–posterior axis of the ARC and selected systematically

at intervals of four sections from all mice. The number of

sections for ARC GHRH-ir neurons selected from each

animal was 18.4F 0.5 for 2 M, 17.6F 0.2 for 4 M,

18.2F 0.6 for 12 M and 17.0F 0.3 for 24 M mice. From

serial sections of the PeN (total number of sections per

mouse: 78.9F 1.1), which were cut along the anterior–

posterior axis of the PeN, sections were selected from each

mouse at intervals of four sections in order to count the PeN

SS-ir neurons. The number of sections obtained per animal

Fig. 2. Number of GHRH-ir neurons in the ARC of male mice versus age.

The number falls significantly with age. Differences were significantly

tested with the previous younger group. *P< 0.05.

S. Kuwahara et al. / Brain Research 998 (2004) 164–173166

was 20.3F 0.9 for 2 M, 19.2F 0.6 for 4 M, 19.4F 0.6 for

12 M and 19.6F 0.7 for 24 M mice.

The total number (T) of immunoreactive cells in the ARC

or PeN was derived by counting the number of neurons

(SNı) in the number of the sections (ı) selected from the

A

VV

C

V

V

Fig. 3. GHRH-ir neurons in the ARC of male mice at 2 M (A), 4 M (B), 12 M (C) a

from 2 to 4 M and only a few GHRH-ir neurons were detected at 12 and 24 M

represents 50 Am (A–D).

number of total sections (n) in the nucleus in each mouse,

and then multiplying by SNı by n/ı according to the

formula: T= n/ı�SNı [25,26].

2.3.2. Adenohypophysial GH-ir cells

To estimate the volume of the anterior pituitary, pituitaries

from 20 mice (5 per group) were sagittally and serially cut 5

Am thick, and sections weremounted on poly-L-lysine (50 Ag/ml)-coated slides. The areas (A1, A2,. . ., An) of the anterior

pituitary were measured in every 10th section with an image

analyzer (Cosmozone, 1SB, Nikon, Tokyo, Japan). The

volume (V) of the anterior pituitary was calculated as

V= 5� 10�SAı Am3.

The proportion, number and size of GH-ir cells were

evaluated as follows. A square of side 40 Am was drawn

at a magnification of � 400 on the screen of a personal

computer. For each animal, about 2000 nuclei of the

adenohypophysial cells in these squares were counted

with a rule in the lateral, parasagittal and midsagittal

parts of the anterior lobe [28]. The nuclei of cells

immunoreactive with GH antiserum were also counted.

The numbers of cells immunoreactive with GH antiserum

B

D

nd 24 M (D). The number of GHRH-ir neurons in the ARC fell dramatically

. GHRH-ir neurons are indicated by arrows. V: third ventricle. Scale bar

Fig. 4. The ME stained for GHRH in male mice at 2 M (A), 4 M (B), 12 M

(C) and 24 M (D). The numbers of immunoreactive fibers were clearly

diminished. V: third ventricle. Scale bar represents 100 Am (A–D).

Fig. 5. Number of SS-ir neurons in PeN of male mice versus age. There is

no significant difference in number between all age groups.


were expressed as percentages of a total of about 2000

nuclei.

For each animal, the mean number (N) of nuclei of

adenohypophysial cells in these squares (402 Am2) was

calculated, and was transformed into the mean number

(N3/2 Am3) of nuclei of adenohypophysial cells (T) using

the formula: T= N3/2�V/403 [29]. The numbers of GH-ir

cells were then calculated by multiplying this quantity by

the proportion of GH-ir cells.

About 100 cells per mouse with nuclei having entire cells

inside the squares were randomly chosen from each prepara-

tion of cells immunoreactive to GH antiserum, and their areas

were calculated using the image analyzer.

2.4. Statistical analysis

Results were expressed as meanF S.E. for five mice. The

data were analyzed by one-way ANOVA followed by Fish-

er’s least significant difference test, with significant differ-

ences, which were assigned at the 5% probability level.

3. Results

3.1. Number of GHRH-ir neurons in the ARC and

immunocytochemical detection of GHRH in the ME

The number of GHRH-ir neurons [2 M (1023F 50

neurons), 4 M (478F 52 neurons), 12 M (299F 52 neu-

rons) and 24 M (155F 12 neurons)] decreased significantly

(F = 98.71, P < 0.05) with age (Figs. 2 and 3).

The immunoreactive of fibers in the ME were similar in

immunoreactive intensity for 2 and 4 M mice. The intensi-

ties of immunoreactive fibers showed a slight reduction at

12 and 24 M compared with those at 2 and 4 M, but the

numbers of immunoreactive fibers were clearly diminished

(Fig. 4).

3.2. Number of SS-ir neurons in the PeN and immunocy-

tochemical detection of SS in the ME

The number of SS-ir neurons did not differ significantly

between 2 M (2163F 38 neurons), 4 M (2091F 69 neu-

rons), 12 M (2358F 152 neurons) and 24 M (2154F 68

neurons in 24 M) (Figs. 5 and 6).

No clear difference was observed between 2, 4, 12 and

24 M for SS reactivity in the ME (Fig. 7).

3.3. PCNA and apoptosis observation in the ARC and PeN

Proliferation cells or DNA fragmentation cells were not

found in the ARC or PeN at any age.

3.4. Volume of the anterior pituitary

The volume of the anterior lobe increased significantly

(F = 24.68, P < 0.05) from 2 M (0.55F 0.02 mm3) to 4 M

(0.71F 0.06 mm3), but 12 M (0.33F 0.02 mm3) decreased

significantly compared to 4 M. There was no significant

difference in the volume of the anterior lobe between 12

and 24 M (0.38F 0.01 mm3) (Fig. 8A).

A

V

B

V

C

V

D

V

Fig. 6. PeN SS-ir neurons of male mice at 2 M (A), 4 M (B), 12 M (C) and 24 M (D). SS-ir neurons are indicated by arrows. The number of SS-ir neurons do

not differ significantly between all age groups. V: third ventricle. Scale bar represents 50 Am (A–D).


3.5. Number of adenohypophysial cells

The number of adenohypophysial cells did not differ

significantly between 2 M [37.01F1.72 (� 105)] and 4 M

[38.75F 2.16 (� 105)], but it decreased significantly by 12

M [22.20F 0.82 (� 105)] (F = 32.41, P < 0.05) compared

to 4 M mice. The number of adenohypophysial cells did

not differ between 12 and 24 M [23.46F 0.47 (� 105)]

mice (Fig. 8B).

3.6. Proportion of GH-ir cells

The proportion of GH-ir cells fell significantly

(F = 29.30, P < 0.05) with age: 2 M (55.36F 0.70%), 4

M (51.42F 1.11%), 12 M (46.75F 0.86%) and 24 M

(43.36F 1.13%) (Figs. 9A and 10).

3.7. Number of GH-ir cells

The number of GH-ir cells did not differ significantly

between 2 M [20.47 F 0.87 ( � 105)] and 4 M

[19.89F 1.02 (� 105)], but the number of GH-ir cells

decreased significantly (F = 53.26, P < 0.05) between 4

and 12 M [10.39F 0.51 (� 105)]. There was no significant

difference in number between 12 and 24 M [10.19F 0.45

(� 105)] (Figs. 9B and 10).

3.8. Size of GH-ir cells

The size of GH-ir cells decreased significantly (F = 39.57,

P < 0.05) between 2 M (46.39F 0.47 Am2) and 4 M

(40.49F 1.03 Am2), and between 4 and 12 M (37.15F 0.51

Am2). GH-ir cells in 24 M (35.13F 1.01 Am2) mice did not

differ in size from those in the 12 M mice (Figs. 9C and 10).

3.9. PCNA and Apoptosis observation in the anterior

pituitary

Many proliferation cells were detected in the anterior

pituitary in 2 M mice. In 4 M mice, they were also detected,

but in smaller numbers than in 2 M mice. Few proliferation

cells were detected in the 12 and 24 M mice (Fig. 11).

DNA fragmentation cells found abundantly in 2 M mice,

but only a few were detected in the older groups (Fig. 12).

4. Discussion

We have found that the number of ARC GHRH-ir

neurons in male mice decreases with age from 2 to 24 M.

In particular, the number of ARC GHRH-ir neurons in 4 M

mice was approximately half that in 2 M mice, and GHRH

immunoreactivity in the ME was slightly reduced in 12 and

24 M, but the number of immunoreactive fibers is clearly

diminished in 24 M. In elderly (24 M) male rats treated with

colchicines, GHRH-ir neurons in the ARC show no change

in distribution or immunoreactive intensities from those in

young (3 M) rats [21], but GHRH-ir intensities in the ME

[7,21] and hypothalamic GHRH mRNA [7] are markedly

reduced in elderly rats. It is reported that colchicines is

necessary to detect perikaryal immunolabeling for GHRH in

the hypothalamus, but colchicine can itself induce the

expression of genes that are not active under physiological

conditions in a cell [4,23]. In our study, no apoptotic cells

were found in the ARC or PeN neurons at any age

examined, so we believe that GHRH neurons do not

themselves reduce in number even in old age; but only

GHRH neurons that played an active function were immu-

nologically stained. In postnatal development, the number

Fig. 8. The volume of the anterior pituitary (A) and the number of

adenohypophysial cells (B) during aging. The volume of the anterior

pituitary and the number of adenohypophysial cells in 12 M are

significantly smaller than those in 4 M. Significant difference (*P< 0.05)

compared with the previous younger group.

Fig. 7. The ME stained for SS in male mice at 2 M (A), 4 M (B), 12 M (C)

and 24 M (D). No clear difference was observed across the four age groups

in their SS—immunoreactivity in the ME. V: third ventricle. Scale bar

represents 100 Am (A–D).


of ARC GHRH-ir neurons decreased dramatically from 5 to

20 days, gradually increased until day 40, then decreased

until day 60 [25]. From our results ([25], present data), it

appears that the number of ARC GHRH-ir neurons in mice

reaches its peak value just after onset of puberty, at day 40,

then decreases with age.

The number of PeN SS-ir neurons was not significantly

different, and the intensities and number of SS-ir fibers in

the ME were almost unchanged with age in our mice. In

elderly (24 M) male rats which were treated with colchicine,

SS-ir neurons in the PeN, and SS-ir intensities in the ME,

showed no change from those of young (3 M) rats [21].

However, Hoffman and Sladek [11] reported that the num-

ber of SS-ir fibers fell markedly in the ME with advancing

aging in male rats at 3, 12, 20 and 30 M. This conflicting

result remains unresolved. SS mRNA expression in the PeN

also showed an age-related decrease [33]. During postnatal

development, the number of SS-ir neurons increased signif-

icantly from days 5 to 30. The number of SS-ir neurons did

not increase significantly between days 30 and 40, and

increased between ages 40 and 60 of days [25]. These data

show that in mice, the number of PeN SS-ir neurons

changes during postnatal development, but does not change

after 2 M.

In our present study, the volume of the anterior

pituitary had decreased dramatically in 12 M mice from

its value in younger mice. In male rats, the weight of the

pituitary showed no significant differences between 6, 12,

and 18 M, but female pituitary glands at 12 and 18 M

were significantly heavier than in 6 M females [34]. In our

mice, many anterior pituitary cells that were stained by

PCNA, which has been used as a marker of cell prolifer-

ation [16,18], were detected at 2 and 4 M, but few PCNA-

ir cells were found in the adenohypophysis at 12 and 24

M. Furthermore, many apoptotic cells were found in 2 M

mice, but only a few apoptotic cells were detected

thereafter. The data presented here indicate that at 12

and 24 M, the proliferation of pituitary cells was extreme-

ly small and the volume and number of adenohypophysial

cells had decreased. Thus, the capability of regeneration of

the adenohypophysial cells may decrease with age. On the

other hand, GHRH is essential for the proliferation of the

somatotrope population during early development of the

pituitary [17]. With increasing age, GHRH synthesis and

secretion in GHRH neurons decreased, so that the anterior

Fig. 9. Proportion (A), number (B) and size (C) of GH-ir cells in the

anterior pituitary of male mice versus age. The proportion of GH-ir cells

(A) decreased with age. The number of GH-ir cells (B) in 4 M was

approximately half that in 12 M. The size of the GH-ir cells (C) fell from 2

to 4 M and from 4 to 12 M. Significant difference (*P < 0.05) compared

with the previous younger group.


pituitary cells decreased: this implies a marked decrease in

GH cells, because about 50% of anterior pituitary cells are

GH cell.

We observed that the proportion of GH-ir cells de-

creased significantly with age, that the number of GH-ir

cells reduced from 4 to 12 M, and the size of GH-ir cells

was smaller at 4 M than 2 M and at 12 M than 4 M. It has

previously been reported that no differences exist in basal

plasma levels of GH between 12 and 28 M in C57BL/6J

male mice [8], but Crew et al. [5] have reported that GH

mRNA levels fall with age and they suggested that

transcriptional activity is reduced in specific genes of

elderly male mice. In old male rats, the amplitude and

duration of GH pulse are significantly decreased, reducing

secretion of GH to about one third of that in young male

rats [32]. In male rats, GH-ir cells and GH cells, which

contain large secretory granules ultrastructurally (Type I

cells), decrease with age from 12 M [13,34], and pituitary

GH level at 18 M is significantly lower than at 6 M [34].

The age-related changes in GH secretion may be not due

to a change in the pituitary response to GHRH [36], but

due to the dysfunction in the synthesis, transport or release

of GHRH [21,35]. In our morphological study, however,

the reduction in the proportion, number and size of GH-ir

cells with age in male mice may therefore be caused by the

decrease in the number of GHRH-ir neurons, with no

involvement of SS-ir neurons.

In conclusion, the fall in the number of GHRH-ir neurons

reduces the number of GH-ir cells.

Acknowledgements

We thank Dr. A.F. Parlow (NIDDK) for the gift of rGH

antiserum and Dr. N. Miki (Tokyo Women’s Medical

College) for the gifts of mGHRH and mSS antisera. This

work was supported by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Science, Sports,

and Culture of Japan to F.S. (No. 13660303).

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anterior pituitary of male mice at 2 M (A, a), 4 M (B, b), 12 M (C, c) and 24 M (D, d). DNA fragmentation cells were found in the anterior pituitary at four age

groups. Many apoptotic cells were detected in 2 M mice, but only a few were detected at 4, 12 and 24 M. Scale bar represents 50 Am (A–D).


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Age-related changes in growth hormone (GH) cells in the pituitary gland of male mice are mediated by...

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