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Morphometric analysis of somatotrophs: Effects of age and dietary restriction
Neurobiologyof Aging,Vol. 17, No. 1, pp. 79-86, 1996 Copyright © 1995ElsevierScienceInc. Printed in the USA. All rights reserved 0197-4580/96 $15.00 + .00
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Morphometric Analysis of Somatotrophs: Effects of Age and Dietary Restriction I S A O S H I M O K A W A , * B Y U N G P A L YU,~" Y O S H I K A Z U H I G A M I * A N D T A K A Y O S H I I K E D A *
*Department of Pathology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki City 852 Japan ~Department of Physiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756 Received 18 July 1994; Revised l0 F e b r u a r y 1995; Accepted 9 M a y 1995 SHIMOKAWA, I., B. P. YU, Y. HIGAMI AND T. IKEDA. Morphometricanalysisofsomatotrophs: Effectsof ageanddietary restriction. NEUROBIOL AGING 170) 79-86, 1996.- W e investigated changes in the cell number of somatotrophs in the anterior pituitary of male F344 rats caused by age and lifelong dietary restriction (DR). Morphometric and immunohistochemical techniques were performed on the pituitary gland at 6, 18, and 24 months of age. In ad lib fed (AL) rats, cell density of somatotrophs progressivelydecreased with age. However, when normalized according to the increased volume of the anterior lobe, which occurs during aging, the total number of somatotrophs remained constant until 18 months, then decreased. In contrast, the cell density of DR rats was not affected to the same extent as that of AL rats, and the total number of cells seemed to remain constant through 24 months, thus, retarding the age-related numerical changes. However, the cell number in DR rats, when normalized by body weight, was larger than that found in AL rats. We also found that average cell volume of somatotrophs shows little age or dietary restriction effects, but the nucleus volume significantly increases at 24 months in both groups. The numerical changes which occur in somatotrophs during the aging process may be associated with the aging phenomena found in GH secretion. Aging Somatotroph Growth hormone
Dietaryrestriction
Morphometry
IT IS GENERALLY accepted that the pituitary hormones change significantly during the aging process. One such change includes a reduction in the secretion of growth hormone (GH) (8,22), an important pituitary hormone which regulates metabolism in the body after sexual maturation. Significant changes are also found in the pituitary gland's responsiveness to GHreleasing hormone (GHRH), where sensitivity to G H R H decreases in aged organisms (5,19,20). Recent evidence shows a reduction in GH-mRNA expression in the pituitary glands of aged male mice (6). The importance of age-related decrease in GH is further emphasized by the fact that long-term, low-dose GH treatment in aged mice is reported to improve immune function and prolong life span (10). GH administration has also been clinically tested as a means to prevent physical frailty and promote resistance to physical dysfunction in elderly people (18). When considered together, these findings strongly indicate an important association between GH and the aging process in animals and humans. Some current research is focused on exploring the mechanisms causing impaired response of the pituitary gland to secre-
Cell number
Immunohistochemistry
tagogues at the cellular level in aged animals (1,5,16). However, data which explore the possibility whether these changes with age are accompanied by alterations in the cell number of somatotrophs, or by functional changes of individual somatotrophs, or both, are limited. There is little morphometric data available to elucidate the age-related alterations of the pituitary gland. A primary purpose of this study, therefore, is to determine the influence of age on the cell number of somatotrophs from a morphometric analysis. Concurrently, structural changes in the pituitary gland will be measured because they may also be altered by age and could influence the changes in cell density and the total cell number of somatotrophs. Our analyses will also examine the influence of lifelong dietary restriction on the morphometric aspects of somatotrophs and the pituitary gland, because dietary restriction retards agerelated functional declines (27) and pathogenesis (20) in various organs, including the pituitary gland (3, l 1,25). Our study provides one segment of basic information on the mechanisms which cause the aging phenomena of GH secretion from the pituitary gland.
~Requests for reprints should be addressed to B. P. Yu, Department of Physiology, The University of Texas Health ScienceCenter at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756. 79
80
SHIMOKAWA ET AL.
Our laboratory fully reports rat maintenance and dietary regimens, as well as data, on the longevity and pathology of male Fischer 344 rats in the barrier facility as described by Yu, et. al (26). Male Fischer 344 rats were purchased as weanlings (2630 days of age) from the Charles River Laboratories at Kingston, NY, and maintained under specific pathogen-free conditions (12L: 12D cycle with lights on at 0530). Until 6 weeks of age, all rats were fed a standard semi-synthetic diet ad lib. After 6 weeks, one group (Group DR) was restricted to approximately 6007o of the energy intake of the control rats fed ad lib (Group AL). Preparation and composition of the diets was described in a previous report (26). Pituitary glands were collected from rats sacrificed between 0900 and 1200 under ether anesthesia at 6, 18, 24 months of age (too). Rats with pituitary tumors were excluded from this study.
tions for 5 min in a mixture of 2.0 g KOH in 10 ml absolute alcohol and 5 ml propylene oxide using Maxwell's method (12). Endogenous peroxidase activity was suppressed by 0.307o HzO2 in absolute methyl alcohol for 20 min. The primary antibody, antiserum-rat GH (NIDDK-anti-rGH-IC-1), was diluted to 1:100 and applied to sections overnight at 4°C. The secondary antibody, rabbit serum to monkey IgG (United States Biochemical Corporation, OH), was biotinylated using a Biotinylation kit (Amersham, UK), diluted to 1:100, and applied for 1 h at room temperature. Strept-avidin biotin complex was made using an ABC kit (Dako Corporation, CA) and was applied to the sections for 1 h. The substrate for peroxidase was 0.5070 3,3'diaminobenzidine-tetrahydrochloride and 0.01070 hydrogen peroxide in 0.01 M PBS (pH 7.4). Negative control was achieved by replacing the primary antiserum with nonimmune monkey serum. Sections were washed twice in 0.01 M PBS for 10 min between steps. Immunostained sections were counterstained with hematoxylin, washed, dehydrated, and mounted.
Tissue Processing
Morphometric Analysis
METHOD
Rat Maintenance and Dietary Regimen
To analyze the anterior lobe, the resected pituitary glands were fixed in 10% buffered formalin (pH 7.4) overnight. Samples were then embedded in paraffin and cut serially into 2 #m-thick sections. Every 50th section was stained with hematoxylin-eosin (HE). Five to eight HE sections were obtained from each pituitary gland. For the morphometric study of somatotrophs, the anterior lobe was dissected from the pituitary gland and cut into cubes. The cubes were fixed in 107o glutaraldehyde-0.1 M phosphate buffer (PB, pH 7.4) at ice cold temperature for 2 h, then washed in 0.1 M PB. The cubes were then postfixed in 107o osmium tetroxide for 1 h, routinely processed, and embedded in Epon resin. Two blocks were chosen from each anterior lobe. One ~mthick sections were cut from each block and placed on albumincoated slides.
Immunohistochemical Procedures The avidin-biotin complex method (9) was used to mark somatotrophs. Epon resin was removed by immersion of sec-
Morphometric evaluation was done at two different levels by the point-counting method as described by Weibel (24). The morphometric procedures used are shown in Fig. 1, and morphometric parameters, symbols of the technical terms, are presented in Table 1. Following are definitions of the morphometric terms used in this study. The volume density refers to the proportion of the volume of each component in comparison to that of the reference compartment. The volume density of the anterior lobe in the pituitary gland [Vv(Ant/PG)] is the proportion of the anterior lobe to that of the pituitary gland as a whole. The volume density of nonpituitary cell components in the anterior lobe [Vv(NP/Ant)] are the proportion of the aggregate volume of nonpituitary cell components to the total volume of the anterior lobe. The volume density of somatotrophs in the anterior lobe [Vv(GH/Ant)] is the proportion of the aggregate volume of immunostained somatotrophs to the total volume of the anterior lobe. The volume density of nuclei in the cytoplasm of somatotrophs [Vv(NU/GH)] represents the proportion of the
Pituitary gland (PG) I
~
Anterior lobe (AL)
Pituitary cells
I
Immunostained somatotrophs (GH)
I Nucleus (NU)
I Intermediate lobe Posterior lobe
I Non-pituitary cell components (NP) (blood vessels, interstitial cells) I Unstained pituitary cells
, Cytoplasm
FIG. 1. Morphometric model of the pituitary gland.
81
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS TABLE 1 MORPHOMETRICPARAMETERSAND TERMSUSED 1N THE TEXT Term
Symbol
Volume density
Numerical area density Numerical volume density Average volume Total cell number* Cell number/unit BW*
Component
-
Anterior lobe Nonpituitary cells (blood vessels, interstitial cells) Somatotrophs Somatotroph nuclei Somatotroph nuclei Somatotroph nuclei Somatotrophs Somatotroph nuclei Somatotroph nuclei
Vv(Ant/PG) Vv(NP/Ant) Vv(GH/Ant) Vv(NU/GH) NA(GH/Ant) Nv(GH/Ant) V(GH) N(GH/Ant) N(GH/unit BW)
Reference Compartment
Dimension
mm -2 mm -3 ktm3 -
Pituitary gland Anterior lobe Anterior lobe Somatotrophs Anterior lobe Anterior lobe Anterior lobe Unit of BW
Density is defined as "the quantity per unit volume, unit area or unit length". In the morphometric analysis, volume density refers the proportion of the volume of each component to that of reference compartment. The numerical area or volume density of somatotrophs represents the cell number of somatotrophs per unit area or volume of the anterior lobe (i.e., the cell density). The definition of each term is described in the Method section. *These two parameters were calculated as values relevant to the values of Group AL at 6 months. The detailed description can be found in the Method section.
volume of nucleus to the volume of somatotroph. The numerical area or volume density of somatotrophs [Na(GH/Ant), Nv(GH/Ant)] represents the cell number of somatotrophs per unit area or volume of the anterior lobe or the cell density. Vv(Ant/PG) was estimated using the fraction of test points lying in the anterior lobe and the pituitary gland classified as Level 1 in Fig. 1. To get the estimate, we used objective lens: x2, eye piece: x 10 with a square test grid (100 test points per cm2). This procedure was performed on the HE sections from each pituitary gland, and the data was compiled using Simpson's formula as described as Ahrne (2). Vv(NP/Ant), Vv(GH/Ant), Vv(NU/GH), and Na(GH/Ant) were determined using 1/~m semi-thin sections. Eight fields were systematically selected for each anterior lobe, utilizing Level 2, as shown in Fig. 1, with objective lens: ×100, eye piece: ×10 with a square test grid (100 test points per cm2). At this level, we counted the number of points over the nucleus and the cytoplasm of immunostained cells, the number of points over the blood vessels and connective tissue, and the number of nuclei of immunostained cells. Nv(GH/Ant) was calculated using the Weibel and Gomez formula (24): K (Na(GH/Ant)) 3/2 Nv(GH/Ant) = - - . f~ (Vv(GH/Ant)) I/2
For this estimate, the size distribution coefficient K of 1.00 and the shape coefficient/~ of 1.38 in the formula were used. We assumed that the nuclei of the respective cell types in the pituitary gland are spherical, as did Poole et al. (15). The average volume of somatotrophs is calculated by the following formula (24): V(GH) -
Vv(GH/Ant) × 109 (/~m 3 ) Nv(GH/Ant)
Relative changes in the total cell number of somatotrophs in the anterior lobe were expressed based on changes in pituitary weight. As described later, this morphometric analysis indicated
an increase in the anterior lobe volume that was in proportion to the increase of the pituitary weight during aging of both dietary groups of rats. This increase was not due to a disproportional growth of the nonpituitary cell components. Hence, changes in the volume of the anterior lobe can be estimated as changes in the weight of the pituitary gland. From this information, we approximated relative changes in the total cell number of somatotrophs in the anterior lobe [N(GH/Ant] and in the cell number of somatotrophs per unit body weight [N(GH/unit BW)] by using the following formulas: Nv(x) N(x) =
PW(x) •
Nv(AL-6mo) PW(AL-6mo) N(x) = Value of N(GH/Ant) of each Group at age x, relevant to the value of Group AL at 6 mo. Nv(x) = Mean numerical volume density of somatotrophs in each Group at age x. Nv(AL6mo) = Mean numerical volume density of somatotrophs in Group AL at 6 mo. PW(x) -- Mean pituitary weight of each Group at age x. PW(AL-6mo): Mean pituitary weight of Group AL at 6 mo.
N(x) =
Nv(x)
PW/100 g BW(x) •
Nv(AL-6mo) PW/100 g BW(AL-6mo)
N(x) = Value of N(GH/unit BW) of each Group at age x, relevant to the same value of Group AL at 6 too. Nv(x) -- Mean numerical volume density of somatotrophs in each Group at age x. Nv(AL-6mo) = Mean numerical volume density of somatotrophs in Group AL at 6 mo. PW/100 g BW(x) = Mean PW/ 100 g BW of each Group at age x. PW/100 g BW(AL-6mo): Mean PW/100 g BW of Group AL at 6 mo.
Statistical .4 nalysis Repeated measures using the two-way analysis of variance (ANOVA) tested the data for the main effects of age (AGE) and diet (DIET) and the interaction between age and diet (AGE x DIET)• Statistical difference in mean values was further exam-
82
S H I M O K A W A ET AL. TABLE 2 PITUITARY WEIGHT NORMALIZED BY BODY WEIGHT IN MALE FISCHER 344 RATS Pituitary Weight (mg)a~ Age (mo) 6 18 24
Body Weight (g)~2
PW/100 g BW#3
AL
DR
AL
DR
AL
DR
8.18 _+ 0.51 (5) 10.37 _+ 1.85 a (5) 15.51 _+ 1.78 b'c (4)
6.56 _+ 1.05 (5) 8.46 _+ 1.28 (4) 10.82 + 1.19 ~'f (5)
354 -+ 32 (1 l) 515 -+ 32 a (1 I) 512_+ 42 b (8)
214 _+ 13d (10) 275 _+ 24 a'e (l 1) 302_+ 11 b'c'f (10)
2.33 _+ 0.25 (5) 2.10 _+ 0.29 (5) 3.08 -+ 0.45 c (4)
3.11 _+ 0.46 (4) 3.28 _+ 0.30 e (4) 3.61 _+0.35 (5)
Numbers in parentheses indicate numbers of rats examined. The numbers of rats examined are different among PW and BW because PW was measured in rats for Level 1 of the present morphometric analysis but not in rats used for Level 2. Values represent the arithmetic mean _+ SD. The statistical analyses by ANOVA are as follows; ~tAge: F(2, 22) = 45.123; p < 0.01, Diet: F(1,22) = 29.189; p < 0,01, Age x Diet: F(2, 22) = 3.661;p < 0.05, #2Age: F(2, 55) = 128.26; p < 0.01, Diet: F(I, 55) = 795; p < 0.01, Age x Diet: F(2, 55) = 18.78; p < 0.01, ~3Age: F(2, 21) = 9.846; p < 0.01, Diet: F(1,21) = 36.837; p < 0.01, Age x Diet: F(2, 21) = 1.971; p > 0.05. The superscript letters represent statistical significance at p < 0.05 as determined by a posthoc test for multiple comparisons; aversus 6 mo, bversus 6 mo, and Cversus 18 mo at each dietary group, dversus AL at 6 mo, eversus AL at 18 mo, fversus AL at 24 mo.
ined by a posthoc method, the Games-Howell test, where appropriate. A specified hypothesis o n N a ( G H / A n t ) a n d N v ( G H / A n t ) at 24 m o was tested with a n u n p a i r e d t test, because we considered the posthoc test s o m e w h a t conservative in detecting the statistical significance in this situation. Statistical analyses were p e r f o r m e d using the statistical c o m p u t e r s o f t w a r e s SuperA N O V A a n d StatView SE + G r a p h i c s for M a c i n t o s h ( A b a c u s Concepts, Inc., Berkeley, CA). The volume density, or proportion, was analyzed after the arcsine t r a n s f o r m a t i o n o f individual values. The statistical significance was accepted at p < 0.05. RESULTS
Pituitary Weights and Body Weights P i t u i t a r y weights ( P W ) a n d b o d y weights (BW) increased with age in b o t h dietary groups (Table 2). The increase rate o f PW, however, was greater in G r o u p A L t h a n in G r o u p D R after 18 mo. G r o u p D R s h o w e d a progressive increase in B W f r o m 6 to 24 too; G r o u p A L reached a plateau at 18 mo. As one might expect, b o t h P W a n d B W were heavier in g r o u p A L t h a n in g r o u p DR. Interestingly, the average values o f P W were not as widely varied between group A L a n d D R as those o f BW. Therefore, when P W was factored with BW ( P W / 1 0 0 g BW), P W was higher in g r o u p D R t h a n in g r o u p A L ( T a b l e 2). The P W / 1 0 0 g B W was also increased with age.
Volume Density of the Anterior Lobe in the Pituitary Gland [Vv(Ant/PG)] and the Volume Density of Nonpituitary Cell Components in the Anterior Lobe [Vv(NP/Ant)] We e x a m i n e d structural alterations due to age a n d the possible influence diet h a d o n the a n t e r i o r lobe o f the pituitary gland. V v ( A n t / P G ) was approximately 0.82, meaning the anterior lobe occupied 8207o o f the volume o f the pituitary gland. V v ( A n t / P G ) did n o t change significantly with age or dietary restriction (Table 3). V v ( N P / A n t ) or n o n p i t u i t a r y cell c o m p o nents was approximately 0.03, comprised mainly connective tissues a n d b l o o d vessels a n d occupied a b o u t 3070 o f the a n t e r i o r lobe. V v ( N P / A L ) s h o w e d n o significant change a m o n g age groups or between dietary groups (Table 3).
Volume Density of Somatotrophs in the Anterior Lobe [Vv(GH/Ant)] V v ( G H / A n t ) was estimated at 0.451 in A L rats a n d 0.414 in D R rats at 6 m o o f age (Table 4). Age-related decreases in V v ( G H / A n t ) were noted in both dietary groups. Although there was no significant dietary effect in V v ( G H / A n t ) as a whole, the decrease rate was smaller in DR rats.
Numerical Area and Volume Density of Somatotrophs & the Anterior Lobe [Na(GH/Ant)], Nv(GH/Ant)] N a ( G H / A n t ) ] a n d N v ( G H / A n t ) decreased progressively with age in b o t h dietary groups (Table 5). There were no significant dietary effects evident in N a ( G H / A n t ) a n d N v ( G H / A n t ) . However, a further analysis for N a ( G H / A n t ) a n d N v ( G H / A n t ) at 24 m o revealed significant differences in the cell density between G r o u p A L a n d D R ( p < 0.05 for N a ( G H / A L ) , p = 0.0643 for N v ( G H / A L ) evaluated by unpaired t test, in this case, a specific hypothesis t h a t diet has n o effect o n N a ( G H / A n t ) or N v ( G H / A n t ) at 24 m o was tested).
TABLE 3 VOLUME DENSITIES OF THE ANTERIOR LOBE IN THE PITUITARY GLAND AND NONPITUITARY CELL COMPONENTS IN THE ANTERIOR LOBE Vv(Ant/PG)#~ Age (mo) 6 18 24
Vv(NP/Ant)~2
AL
DR
AL
DR
0.815 + 0.020 (5) 0.821 _+ 0.031 (5) 0.839 ___0.050 (4)
0.772 _+ 0.027 (4) 0.823 _ 0.039 (5) 0.819 ___0.035 (5)
0.032 _+ 0.009 (6) 0.029 _+ 0.013 (6) 0.025 + 0.004 (3)
0.031 + 0.012 (6) 0.036 + 0.012 (6) 0.030 + 0.017 (5)
Values represent the arithmetic mean :t: SD. Numbers in parentheses indicate number of rats examined. The statistical analyses by ANOVA are as follows; #~Age: F(2, 22) = 2.702; p > 0.05, Diet: F(1, 22) = 2.385; p > 0.05, Age x Diet: F(2, 22) = 0.957; p > 0.05, #2Age: F(2, 26) = 0.462; p > 0.05, Diet: F(1,26) = 0.644; p > 0.05, Age x Diet: F(2, 26) = 0.371; p > 0.05.
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS TABLE 4 VOLUME DENSITY OF SOMATOTROPHS IN THE ANTER1OR LOBE Vv(GH/Ant) Age (mo)
6 18 24
AL
DR
0.451 _+ 0.086 (6) 0.335 _+ 0.075 (6) 0.174 +_ 0.040 b'c (3)
0.414 +_ 0.031 (6) 0.346 _+ 0.050 (6) 0.297 _+ 0.078 (5)
Values represent the arithmetic mean _+SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; Age: F(2, 26) = 22.236; p < 0.01, Diet: F(1,26) = 2.514; p > 0.05, Age x Diet: F(2, 26) = 3.972; p < 0.05. The superscript letters represent statistical significance at p < 0.05 by a posthoc test for multiple comparisons; bversus 6 mo, Cversus 18 mo at Group AL.
~
83
1.20 1.00
~ 0.80 o O.fiO
I~ .~
E 0.40 ¢...
>
0.20
~- 0.00
0
~ 6
/.,'
I 18 Age (months)
~ 24
FIG. 2. Relative changes in total cell number of somatotrophs in the anterior lobe as normalized to the level of the total number in Group AL at 6 months.
Total Cell Number of Somatotrophs in the Anterior Lobe [N(GH/Ant)] and Cell Number of Somatotrophs per Unit B W [N(GH/unit BW)] The N ( G H / A n t ) , which showed n o decrease until 18 m o in G r o u p A L showed a reduction by a p p r o x i m a t e l y 2 5 % thereafter. In contrast, the rats in G r o u p DR, N ( G H / A n t ) were nearly c o n s t a n t t h r o u g h the 24 m o m a r k (Fig. 2). Data in Fig. 2 shows t h a t the total cell n u m b e r o f s o m a t o t r o p h s was greater in G r o u p A L ; however, when n o r m a l i z e d to unit b o d y weight, N ( G H / u n i t B W ) was higher in G r o u p DR. N ( G H / u n i t B W ) progressively decreased after 6 m o in b o t h dietary groups (Fig. 3).
Estimates of Average Single Cell Size (Volume) of Somatotrophs [V(GH)] and Volume Density of the Nucleus in the Cytoplasm [Vv(NU/GH)] V ( G H ) was estimated at 900-1000 ~tm 3 a n d n o statistical differences were seen between dietary groups or a m o n g age
groups (Table 6). However, the nucleus volume of somatotrophs significantly increased at 24 m o in b o t h dietary groups.
Morphological Changes of Somatotrophs Light microscopic prints of the immunostained somatotrophs in A L a n d D R rats at 6 m o a n d 24 m o are s h o w n in Fig. 4 a n d Fig. 5. Cell density was reduced in b o t h dietary groups at 24 m o (Fig. 4). Cytoplasmic v a c u o l a t i o n (See Fig. 5 at high magnification) b e c a m e noticeable in s o m a t o t r o p h s o f b o t h dietary groups at 24 too. No a p p a r e n t difference in the degree o f cytoplasmic v a c u o l a t i o n between G r o u p A L a n d DR was detected by light microscopic o b s e r v a t i o n .
TABLE 5 NUMERICAL AREA AND VOLUME DENSITIES OF SOMATOTROPHS IN THE ANTERIOR LOBE N a ( G H / A n t ) #1
Age (mo)
6 18 24
AL
DR
i
N v ( G H / A n t ) #2
AL
1.00
DR
3597 _+ 559 3488 + 271 4.52 _+ 0.78 4.51 _+ 0.48 (6) (6) (6) (6) 2895 _+ 558 2958 + 450 3.83 + 0.66 3.85 +_ 0.81 (6) (6) (6) (6) 1551 _+ 434 t~'~ 2426 + 487 b'g 1.80 +_ 0.43 b'c 2.80 + 0.68 b'h (3) (5) (3) (5)
Na(GH/Ant): cells/mm 2. Nv(GH/Ant): cells x 105/mm 3. Values represent the arithmetic mean +_ SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; ~JAge: F(2, 26) = 24.875; p < 0.01, Diet: F(1,26) = 2.559; p > 0.05, Age x Diet: F(2, 26) = 2.672; p > 0.05, #2Age: F(2, 26) = 24.905; p < 0.01, Diet: F(I, 26) = 1.832; p > 0.05, Age x Diet: F(2, 26) = 1.566; p > 0.05. The superscript letters represent statistical significance at p < 0.05 as determined by a posthoc test for multiple comparisons; bversus 6 mo, and Cversus 18 mo at each dietary group, g'hAt 24 mo, a specific hypothesis that diet had no effect on Na(GH/Ant) or Nv(GH/Ant) was tested by unpaired t test; gp < 0.05, hp = 0.0643.
1.40 1.20 0.80 0.60
~ 0.20 rr
0.00
I
6
,//
I
18 Age (months)
I
24
FIG. 3. Relative changes in cell number of somatotrophs per unit body weight. Each value was normalized to the level of cell number of somatotrophs per unit body weight in Group AL at 6 months.
84
S H I M O K A W A ET AL.
FIG. 4. Immunostained somatotrophs in Group AL and Group DR. A: Group AL at 6 mo; B: Group AL at 24 too; C: Group DR at 6 too; D: Group DR at 24 mo (phase contrast optics, original magnification: x40). Note the reduction in cell density of immunostained somatotrophs at 24 mo, compared to that seen at 6 mo in both dietary groups. The cytoplasmic vacuolation is also noticeable in somatotrophs at 24 mo of both dietary groups.
DISCUSSION
There are few reports connecting structural changes of the pituitary gland with age and lifelong dietary restriction, even though information on those structural changes is important to understanding the physiological changes in pituitary function (3). Our analysis indicates that aging and dietary restriction have
TABLE 6 AVERAGE SINGLE CELL VOLUME OF S O M A T O T R O P H S AND VOLUME DENSITY OF THE NUCLEUS IN CYTOPLASM OF S O M A T O T R O P H S V(GH) #~ Age (mo)
6 18 24
V v ( N U / G H ) #2
AL
DR
AL
DR
1002 + 110 (6) 879 + 169 (6) 965 -+ 65 (3)
921 _+ 42 (6) 914 + 99 (6) 1059 _+ 144 (5)
0.269 + 0.027 (6) 0.260 + 0.020 (6) 0.343 +- 0.070 (3)
0.269 _+ 0.044 (6) 0.271 + 0.033 (6) 0.333 -+ 0.048 (5)
V(GH): #m 3. Values represent the arithmetic mean +_SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; #1Age: F(2, 26) = 2.392; p > 0.05, Diet: ~F(21,26) = 0.156; p > 0.05, Age x Diet: F(2,26) = 1.446; p > 0.05, AGE: F(2, 26) = 7.649; p < 0.01, Diet: F(1,26) = 0.285; p > 0.05, Age x Diet: F(2, 26) = 0.725; p > 0.05.
no direct influence on the structure, except on the absolute size, and that changes in the absolute size of the anterior lobe are not caused by disproportional growth of the connective tissue in that lobe. We can say that aging increases the total number of pareno chymal cells in the anterior lobe, while dietary restriction decreases the total cell number in proportion to the pituitary weight. Note, however, that the number of parenchymal cells is larger in DR rats than in A L rats when compared by body weight. Our primary findings during this study were loss in N a ( G H / Ant) and N v ( G H / A n t ) , or cell density, and total cell number of immunostained somatotrophs during aging. These changes may provide morphological bases for observed reductions in G H concentration (22) and G H - m R N A expression (6) in pituitary glands o f aged animals. Such reductions could partly be due to losses in cell numbers if immunostained somatotrophs represent most cells containing GH-peptide or G H - m R N A . Some reports suggest functional impairments in somatotrophs in in vitro studies. Ceda et al. (5) indicated a diminished production of c A M P by pituitary cells after G H R H stimulation in aged rats. Robberecht et al. (16) also reported reductions in the G H R H - s t i m u l a t e d adenylate cyclase activity in aged rats. Abribat et al. (1) reported alterations o f G H R H binding sites. These findings are, however, obviously dependent on the cell density of somatotrophs in the pituitary tissue. The response of individual somatotrophs to G H R H may not be impaired as we expected. G H is released from the anterior pituitary in an intermittent, pulsatile fashion in rats (23), and this secretion is attenuated in
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS
FIG. 5. High power microphotographs of somatotrophs. A: Group AL at 6 mo; B: Group AL at 24 mo (phase contrast optics, original magnification: x 100). Note the cytoplasmic vacuolation of somatotrophs at 24 mo. older rats (22). An age-related impairment in pituitary responsiveness to GHRH in vivo was also reported (3,5,6). In this study, morphometric analysis suggests a decrease in the cell number of somatotrophs per body weight, by 25°7o at 18 mo and 5007o at 24 mo in group AL. The relative change in the cell number per unit of body weight seems closely correlated to the reported reduction in amplitude of GH secretion and responsiveness to GHRH in in vivo experiments, although the reduced frequency of spontaneous GH pulsatile secretion in aged animals is probably the result of changes in hypothalamic regulation. Our findings on somatotrophic reduction in the cell density do not agree with the earlier results of Rossi et al. (17), who observed that somatotrophic cell numbers decreased in female rats but increased in aged male Sprague-Dawley (SD) rats. Because immunoreactivity and expression of mRNA for GHRH in the hypothalamus, which stimulates the proliferation of somatotrophs (4), decreased in aged male SD rats (7,14), it is unclear why the number of somatotrophs increased in male rats. At this time, we can offer no experimental data to explain this discrepancy.
85
Another unanswered question relates to our observation of reductions in the total cell numbers of somatotrophs in AL rats after 18 too. The total number of somatotrophs is influenced by the cell turnover rate, which is determined by removal of aged somatotrophs and addition of newly formed somatotrophs. Our findings did not provide definitive information regarding an agerelated change in either reduced cell renewal or accelerated cell loss. However, when considering reports of reduced GHRHimmunoreactivity and GHRH-mRNA expression in aged male rats (7,14), one might conclude that cell renewal decreases in that group. To investigate that possibility, however, further analysis on the cell turnover of somatotrophs, particularly cell loss, is needed. To our knowledge, there are no reports on morphologic changes in somatotrophs of aged animals. This study revealed cytoplasmic vacuolation and a relative increase in the nucleus volume, which are possible signs of cellular aging, in most somatotrophs after 18 mo. We are now examining pituitary tissues by electron microscopy to identify subcellular structure(s) showing vacuolation. Little is known about the long-term effects of dietary restriction on GH secretion although DR has been reported to have a GH lowering effect (3,13). Our morphometric tests demonstrated that the cell number of somatotrophs per body weight was significantly higher in Group DR than in Group AL. This provides the restricted rats with greater capacity for GH secretion from the pituitary gland, assuming that each somatotroph in both dietary groups of rats has a similar capacity for GH secretion. Additionally, the relatively large cell number of somatotrophs in Group DR could attenuate the deterioration processes in somatotrophs because of overreactivity, because demand for GH synthesis/secretion per cell should be less under dietary restriction. It is worth noting that in Group DR, the total number of somatotrophs in the anterior lobe remained at the same level from 6 mo through 24 mo, which could mean that GH cell turnover remains constant during senescence. This suggests that age-related changes in hypothalamic signals involved in somatotrophic cell turnover are retarded by dietary restriction. These data are consistent with well-established anti-aging actions of dietary restriction (25,27), but the effective modification by dietary restriction of age-related changes in cell turnover and morphology of somatotrophs warrants further investigation. ACKNOWLEDGEMENTS
This research was supported by NIH Grant Number AG01188. We are grateful to A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases for providing the antibody for rat GH. We also thank E. J. Masoro for his useful comments. Thanks also goes to W. R. Mejia, Y. Suh, and F. Perkins, Jr. at the University of Texas Health Science Center at San Antonio for their invaluable technical assistance.
REFERENCES
1. Abribat, T.; Deslauriers, P.; Brauzeau, P.; Gaudreau, P. Alterations of pituitary growth hormone-releasingfactor binding sites in aging rats. Endocrinology 128:633-635; 1990. 2. Aherne, W. A.; Dunnil, M. S. Morphometry, London: Edward Arnold Ltd.; 1982. 3. Bertrand, H. A. Modulation of aging in endocrine systems by dietary restriction. In B. P. Yu, ed., Modulation of aging processes by dietary restriction, Boca Raton, FL: CRC Press; 1994:89-120. 4. Billestrup,N.; Swanson, L. W.; Vale, W. Growth hormone-releasing
factor stimulatesproliferation of somatotrophs in vitro. Proc. Natl. Acad. Sci. USA 83:6854-6857; 1986. 5. Ceda, G. P.; Valenti,G.; Butturini, U.; Hoffman, A. R. Diminished pituitary responsivenessto growth hormone-releasingfactor in aging male rats. Endocrinology 118:2109-2114; 1986. 6. Crew, M. D.; Spindler, S. R.; Walford, R. L.; Koizumi, A. Agerelated decrease of growth hormone and prolactin gene expression in the mouse pituitary. Endocrinology 121:1251-1255; 1987. 7. de Gennaro Colonna, V.; Zoli, M.; Cocchi, D.; Maggi, A.; Mar-
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S H I M O K A W A ET AL. rama, P.; Agnati, L. F.; Mueller, E. E. Reduced growth hormone releasing factor (GHRF)-Iike immunoreactivity and GHRF gene expression in the hypothalamus of aged rats. Peptides 10:705-708; 1989. Finkelstein, J. W.; Roffwarg, H. P.; Boyer, R. M.; Kream, J.; Hellman, L. Age-related change in the twenty-four hour spontaneous secretion of growth hormone. J. Clin. Endocrinol. Metab. 35:665-670; 1972. Hsu, S. M.; Raine, L.; Fanger, H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol. 75:734-738; 1981. Khansari, D. N.; Gustad, T. Effects of long-term, low-dose growth hormone therapy on immune function and life expectancy of mice. Mech. Aging Develop 57:87-100; 1991. Masoro, E. J. Food restriction in rodents: an evaluation of its role in the study of aging. J. Gerontol. 43:59-64; 1988. Maxwell, M. H. Two rapid and simple methods used for the removal of resins from 1.0 m thick epoxy sections. J. Microscopy. 112:253-255; 1978. Merry, B. J.; Holeham, A. M. The endocrine responses to dietary restriction in the rat. In A. D. Woodhead; A. D. Blackett; Hollaender, A., eds. Molecular biology of aging. New York, Plenum Press; 1985:117-142. Morimoto, N.; Kawakami, F.; Makino, S.; Chihara, K.; Hasegawa, M.; Ibata, Y. Age-related changes in growth hormone-releasing factor and somatostatin in the rat hypothalamus. Neuroendocrinology 47:459-464; 1988. Poole, M. C.; Kornegay, 11I, W. D. Cellular distribution within the rat adenohypophysis: A morphometric study. Anat. Rec. 204:4553; 1982. Robberecht, P.; Gillard, M.; Waelbroeck, M.; Camus, J.-C.; De Neef, P.; Christophe, J. Decreased stimulation of adenylate cyclase by growth hormone-releasing factor in the anterior pituitary of old rats. Neuroendocrinology 44:429-432; 1986.
17. Rossi, G. L.; Bestetti, G. E.; Galbiati, E.; Muller, E. E.; Cocchi, D. Sexually dimorphic effects of aging on rat somatotropes and lactotropes. J. Gerontol. Biol. Sci. 46:B152-158; 1991. 18. Rudman, D.; Feller, A. G.; Nagraj, H. S.; Gergans, G. A.; Lalitha, P. Y.; Goldberg, A. F.; Schlenker, R. A.; Cohen, L.; Rudman, I. N.; Mattson, D. E. Effects of human growth hormone in men over 60 years old. New Eng. J. Med. 323:1-6; 1990. 19. Shibasaki, T.; Shimuzu, K.; Makahara, M.; Masuda, A.; Ibiki, K.; Demura, H.; Wakabayashi, I.; Ling, N. Age-related changes in plasma growth hormone response to growth hormone-releasing hormones in man. J. Clin. Endocrinol. Metab. 58:212-214; 1984. 20. Shimokawa, I.; Higami, Y. Effect of dietary restriction on pathologic processes. In B. P. Yu, ed., Modulation of aging processes by dietary restriction. Boca Raton, FL: CRC; 1994:247-265. 21. Sonntag, W. E.; Hylka, V. W.; Meites, J. Impaired ability of old male rats to secrete growth hormone in vivo but not in vitro in response to hpGRF (I-44). Endocrinology 113:2305-2307; 1983. 22. Sonntag, W. E.; Steger, R. W.; Forman, L. J.; Meites, .1. Decreased pulsatile release of growth hormone in old male rats. Endocrinology 107:1875-1879; 1980. 23. Tannenbaum, G. S.; Martin, Jo B. Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98;562-570; 1976. 24. Weibel, E. R. Stereological methods vol. 1, Practical methods for biological morphometry. San Diego, CA: Academic Press lnc; 1978. 25. Weindruch, R.; Walford, R. L. The retardation of aging and disease by dietary restriction, Springfield, IL: Charles C. Thomas; 1988. 26. Yu, B. P.; Masoro, E. J.; McMahan, C. A. Nutritional influences on aging of Fischer 344 rats: 1. Physical, metabolic, and longevity characteristics. J. Gerontol. 40:657-670; 1985. 27. Yu, B. P. Modulation of aging processes by dietary restriction. Boca Raton, FL: CRC Press, 1994.
~' ~°~, 9 ELSEVIER
0197-4580(95)02028-4
Morphometric Analysis of Somatotrophs: Effects of Age and Dietary Restriction I S A O S H I M O K A W A , * B Y U N G P A L YU,~" Y O S H I K A Z U H I G A M I * A N D T A K A Y O S H I I K E D A *
*Department of Pathology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki City 852 Japan ~Department of Physiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756 Received 18 July 1994; Revised l0 F e b r u a r y 1995; Accepted 9 M a y 1995 SHIMOKAWA, I., B. P. YU, Y. HIGAMI AND T. IKEDA. Morphometricanalysisofsomatotrophs: Effectsof ageanddietary restriction. NEUROBIOL AGING 170) 79-86, 1996.- W e investigated changes in the cell number of somatotrophs in the anterior pituitary of male F344 rats caused by age and lifelong dietary restriction (DR). Morphometric and immunohistochemical techniques were performed on the pituitary gland at 6, 18, and 24 months of age. In ad lib fed (AL) rats, cell density of somatotrophs progressivelydecreased with age. However, when normalized according to the increased volume of the anterior lobe, which occurs during aging, the total number of somatotrophs remained constant until 18 months, then decreased. In contrast, the cell density of DR rats was not affected to the same extent as that of AL rats, and the total number of cells seemed to remain constant through 24 months, thus, retarding the age-related numerical changes. However, the cell number in DR rats, when normalized by body weight, was larger than that found in AL rats. We also found that average cell volume of somatotrophs shows little age or dietary restriction effects, but the nucleus volume significantly increases at 24 months in both groups. The numerical changes which occur in somatotrophs during the aging process may be associated with the aging phenomena found in GH secretion. Aging Somatotroph Growth hormone
Dietaryrestriction
Morphometry
IT IS GENERALLY accepted that the pituitary hormones change significantly during the aging process. One such change includes a reduction in the secretion of growth hormone (GH) (8,22), an important pituitary hormone which regulates metabolism in the body after sexual maturation. Significant changes are also found in the pituitary gland's responsiveness to GHreleasing hormone (GHRH), where sensitivity to G H R H decreases in aged organisms (5,19,20). Recent evidence shows a reduction in GH-mRNA expression in the pituitary glands of aged male mice (6). The importance of age-related decrease in GH is further emphasized by the fact that long-term, low-dose GH treatment in aged mice is reported to improve immune function and prolong life span (10). GH administration has also been clinically tested as a means to prevent physical frailty and promote resistance to physical dysfunction in elderly people (18). When considered together, these findings strongly indicate an important association between GH and the aging process in animals and humans. Some current research is focused on exploring the mechanisms causing impaired response of the pituitary gland to secre-
Cell number
Immunohistochemistry
tagogues at the cellular level in aged animals (1,5,16). However, data which explore the possibility whether these changes with age are accompanied by alterations in the cell number of somatotrophs, or by functional changes of individual somatotrophs, or both, are limited. There is little morphometric data available to elucidate the age-related alterations of the pituitary gland. A primary purpose of this study, therefore, is to determine the influence of age on the cell number of somatotrophs from a morphometric analysis. Concurrently, structural changes in the pituitary gland will be measured because they may also be altered by age and could influence the changes in cell density and the total cell number of somatotrophs. Our analyses will also examine the influence of lifelong dietary restriction on the morphometric aspects of somatotrophs and the pituitary gland, because dietary restriction retards agerelated functional declines (27) and pathogenesis (20) in various organs, including the pituitary gland (3, l 1,25). Our study provides one segment of basic information on the mechanisms which cause the aging phenomena of GH secretion from the pituitary gland.
~Requests for reprints should be addressed to B. P. Yu, Department of Physiology, The University of Texas Health ScienceCenter at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756. 79
80
SHIMOKAWA ET AL.
Our laboratory fully reports rat maintenance and dietary regimens, as well as data, on the longevity and pathology of male Fischer 344 rats in the barrier facility as described by Yu, et. al (26). Male Fischer 344 rats were purchased as weanlings (2630 days of age) from the Charles River Laboratories at Kingston, NY, and maintained under specific pathogen-free conditions (12L: 12D cycle with lights on at 0530). Until 6 weeks of age, all rats were fed a standard semi-synthetic diet ad lib. After 6 weeks, one group (Group DR) was restricted to approximately 6007o of the energy intake of the control rats fed ad lib (Group AL). Preparation and composition of the diets was described in a previous report (26). Pituitary glands were collected from rats sacrificed between 0900 and 1200 under ether anesthesia at 6, 18, 24 months of age (too). Rats with pituitary tumors were excluded from this study.
tions for 5 min in a mixture of 2.0 g KOH in 10 ml absolute alcohol and 5 ml propylene oxide using Maxwell's method (12). Endogenous peroxidase activity was suppressed by 0.307o HzO2 in absolute methyl alcohol for 20 min. The primary antibody, antiserum-rat GH (NIDDK-anti-rGH-IC-1), was diluted to 1:100 and applied to sections overnight at 4°C. The secondary antibody, rabbit serum to monkey IgG (United States Biochemical Corporation, OH), was biotinylated using a Biotinylation kit (Amersham, UK), diluted to 1:100, and applied for 1 h at room temperature. Strept-avidin biotin complex was made using an ABC kit (Dako Corporation, CA) and was applied to the sections for 1 h. The substrate for peroxidase was 0.5070 3,3'diaminobenzidine-tetrahydrochloride and 0.01070 hydrogen peroxide in 0.01 M PBS (pH 7.4). Negative control was achieved by replacing the primary antiserum with nonimmune monkey serum. Sections were washed twice in 0.01 M PBS for 10 min between steps. Immunostained sections were counterstained with hematoxylin, washed, dehydrated, and mounted.
Tissue Processing
Morphometric Analysis
METHOD
Rat Maintenance and Dietary Regimen
To analyze the anterior lobe, the resected pituitary glands were fixed in 10% buffered formalin (pH 7.4) overnight. Samples were then embedded in paraffin and cut serially into 2 #m-thick sections. Every 50th section was stained with hematoxylin-eosin (HE). Five to eight HE sections were obtained from each pituitary gland. For the morphometric study of somatotrophs, the anterior lobe was dissected from the pituitary gland and cut into cubes. The cubes were fixed in 107o glutaraldehyde-0.1 M phosphate buffer (PB, pH 7.4) at ice cold temperature for 2 h, then washed in 0.1 M PB. The cubes were then postfixed in 107o osmium tetroxide for 1 h, routinely processed, and embedded in Epon resin. Two blocks were chosen from each anterior lobe. One ~mthick sections were cut from each block and placed on albumincoated slides.
Immunohistochemical Procedures The avidin-biotin complex method (9) was used to mark somatotrophs. Epon resin was removed by immersion of sec-
Morphometric evaluation was done at two different levels by the point-counting method as described by Weibel (24). The morphometric procedures used are shown in Fig. 1, and morphometric parameters, symbols of the technical terms, are presented in Table 1. Following are definitions of the morphometric terms used in this study. The volume density refers to the proportion of the volume of each component in comparison to that of the reference compartment. The volume density of the anterior lobe in the pituitary gland [Vv(Ant/PG)] is the proportion of the anterior lobe to that of the pituitary gland as a whole. The volume density of nonpituitary cell components in the anterior lobe [Vv(NP/Ant)] are the proportion of the aggregate volume of nonpituitary cell components to the total volume of the anterior lobe. The volume density of somatotrophs in the anterior lobe [Vv(GH/Ant)] is the proportion of the aggregate volume of immunostained somatotrophs to the total volume of the anterior lobe. The volume density of nuclei in the cytoplasm of somatotrophs [Vv(NU/GH)] represents the proportion of the
Pituitary gland (PG) I
~
Anterior lobe (AL)
Pituitary cells
I
Immunostained somatotrophs (GH)
I Nucleus (NU)
I Intermediate lobe Posterior lobe
I Non-pituitary cell components (NP) (blood vessels, interstitial cells) I Unstained pituitary cells
, Cytoplasm
FIG. 1. Morphometric model of the pituitary gland.
81
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS TABLE 1 MORPHOMETRICPARAMETERSAND TERMSUSED 1N THE TEXT Term
Symbol
Volume density
Numerical area density Numerical volume density Average volume Total cell number* Cell number/unit BW*
Component
-
Anterior lobe Nonpituitary cells (blood vessels, interstitial cells) Somatotrophs Somatotroph nuclei Somatotroph nuclei Somatotroph nuclei Somatotrophs Somatotroph nuclei Somatotroph nuclei
Vv(Ant/PG) Vv(NP/Ant) Vv(GH/Ant) Vv(NU/GH) NA(GH/Ant) Nv(GH/Ant) V(GH) N(GH/Ant) N(GH/unit BW)
Reference Compartment
Dimension
mm -2 mm -3 ktm3 -
Pituitary gland Anterior lobe Anterior lobe Somatotrophs Anterior lobe Anterior lobe Anterior lobe Unit of BW
Density is defined as "the quantity per unit volume, unit area or unit length". In the morphometric analysis, volume density refers the proportion of the volume of each component to that of reference compartment. The numerical area or volume density of somatotrophs represents the cell number of somatotrophs per unit area or volume of the anterior lobe (i.e., the cell density). The definition of each term is described in the Method section. *These two parameters were calculated as values relevant to the values of Group AL at 6 months. The detailed description can be found in the Method section.
volume of nucleus to the volume of somatotroph. The numerical area or volume density of somatotrophs [Na(GH/Ant), Nv(GH/Ant)] represents the cell number of somatotrophs per unit area or volume of the anterior lobe or the cell density. Vv(Ant/PG) was estimated using the fraction of test points lying in the anterior lobe and the pituitary gland classified as Level 1 in Fig. 1. To get the estimate, we used objective lens: x2, eye piece: x 10 with a square test grid (100 test points per cm2). This procedure was performed on the HE sections from each pituitary gland, and the data was compiled using Simpson's formula as described as Ahrne (2). Vv(NP/Ant), Vv(GH/Ant), Vv(NU/GH), and Na(GH/Ant) were determined using 1/~m semi-thin sections. Eight fields were systematically selected for each anterior lobe, utilizing Level 2, as shown in Fig. 1, with objective lens: ×100, eye piece: ×10 with a square test grid (100 test points per cm2). At this level, we counted the number of points over the nucleus and the cytoplasm of immunostained cells, the number of points over the blood vessels and connective tissue, and the number of nuclei of immunostained cells. Nv(GH/Ant) was calculated using the Weibel and Gomez formula (24): K (Na(GH/Ant)) 3/2 Nv(GH/Ant) = - - . f~ (Vv(GH/Ant)) I/2
For this estimate, the size distribution coefficient K of 1.00 and the shape coefficient/~ of 1.38 in the formula were used. We assumed that the nuclei of the respective cell types in the pituitary gland are spherical, as did Poole et al. (15). The average volume of somatotrophs is calculated by the following formula (24): V(GH) -
Vv(GH/Ant) × 109 (/~m 3 ) Nv(GH/Ant)
Relative changes in the total cell number of somatotrophs in the anterior lobe were expressed based on changes in pituitary weight. As described later, this morphometric analysis indicated
an increase in the anterior lobe volume that was in proportion to the increase of the pituitary weight during aging of both dietary groups of rats. This increase was not due to a disproportional growth of the nonpituitary cell components. Hence, changes in the volume of the anterior lobe can be estimated as changes in the weight of the pituitary gland. From this information, we approximated relative changes in the total cell number of somatotrophs in the anterior lobe [N(GH/Ant] and in the cell number of somatotrophs per unit body weight [N(GH/unit BW)] by using the following formulas: Nv(x) N(x) =
PW(x) •
Nv(AL-6mo) PW(AL-6mo) N(x) = Value of N(GH/Ant) of each Group at age x, relevant to the value of Group AL at 6 mo. Nv(x) = Mean numerical volume density of somatotrophs in each Group at age x. Nv(AL6mo) = Mean numerical volume density of somatotrophs in Group AL at 6 mo. PW(x) -- Mean pituitary weight of each Group at age x. PW(AL-6mo): Mean pituitary weight of Group AL at 6 mo.
N(x) =
Nv(x)
PW/100 g BW(x) •
Nv(AL-6mo) PW/100 g BW(AL-6mo)
N(x) = Value of N(GH/unit BW) of each Group at age x, relevant to the same value of Group AL at 6 too. Nv(x) -- Mean numerical volume density of somatotrophs in each Group at age x. Nv(AL-6mo) = Mean numerical volume density of somatotrophs in Group AL at 6 mo. PW/100 g BW(x) = Mean PW/ 100 g BW of each Group at age x. PW/100 g BW(AL-6mo): Mean PW/100 g BW of Group AL at 6 mo.
Statistical .4 nalysis Repeated measures using the two-way analysis of variance (ANOVA) tested the data for the main effects of age (AGE) and diet (DIET) and the interaction between age and diet (AGE x DIET)• Statistical difference in mean values was further exam-
82
S H I M O K A W A ET AL. TABLE 2 PITUITARY WEIGHT NORMALIZED BY BODY WEIGHT IN MALE FISCHER 344 RATS Pituitary Weight (mg)a~ Age (mo) 6 18 24
Body Weight (g)~2
PW/100 g BW#3
AL
DR
AL
DR
AL
DR
8.18 _+ 0.51 (5) 10.37 _+ 1.85 a (5) 15.51 _+ 1.78 b'c (4)
6.56 _+ 1.05 (5) 8.46 _+ 1.28 (4) 10.82 + 1.19 ~'f (5)
354 -+ 32 (1 l) 515 -+ 32 a (1 I) 512_+ 42 b (8)
214 _+ 13d (10) 275 _+ 24 a'e (l 1) 302_+ 11 b'c'f (10)
2.33 _+ 0.25 (5) 2.10 _+ 0.29 (5) 3.08 -+ 0.45 c (4)
3.11 _+ 0.46 (4) 3.28 _+ 0.30 e (4) 3.61 _+0.35 (5)
Numbers in parentheses indicate numbers of rats examined. The numbers of rats examined are different among PW and BW because PW was measured in rats for Level 1 of the present morphometric analysis but not in rats used for Level 2. Values represent the arithmetic mean _+ SD. The statistical analyses by ANOVA are as follows; ~tAge: F(2, 22) = 45.123; p < 0.01, Diet: F(1,22) = 29.189; p < 0,01, Age x Diet: F(2, 22) = 3.661;p < 0.05, #2Age: F(2, 55) = 128.26; p < 0.01, Diet: F(I, 55) = 795; p < 0.01, Age x Diet: F(2, 55) = 18.78; p < 0.01, ~3Age: F(2, 21) = 9.846; p < 0.01, Diet: F(1,21) = 36.837; p < 0.01, Age x Diet: F(2, 21) = 1.971; p > 0.05. The superscript letters represent statistical significance at p < 0.05 as determined by a posthoc test for multiple comparisons; aversus 6 mo, bversus 6 mo, and Cversus 18 mo at each dietary group, dversus AL at 6 mo, eversus AL at 18 mo, fversus AL at 24 mo.
ined by a posthoc method, the Games-Howell test, where appropriate. A specified hypothesis o n N a ( G H / A n t ) a n d N v ( G H / A n t ) at 24 m o was tested with a n u n p a i r e d t test, because we considered the posthoc test s o m e w h a t conservative in detecting the statistical significance in this situation. Statistical analyses were p e r f o r m e d using the statistical c o m p u t e r s o f t w a r e s SuperA N O V A a n d StatView SE + G r a p h i c s for M a c i n t o s h ( A b a c u s Concepts, Inc., Berkeley, CA). The volume density, or proportion, was analyzed after the arcsine t r a n s f o r m a t i o n o f individual values. The statistical significance was accepted at p < 0.05. RESULTS
Pituitary Weights and Body Weights P i t u i t a r y weights ( P W ) a n d b o d y weights (BW) increased with age in b o t h dietary groups (Table 2). The increase rate o f PW, however, was greater in G r o u p A L t h a n in G r o u p D R after 18 mo. G r o u p D R s h o w e d a progressive increase in B W f r o m 6 to 24 too; G r o u p A L reached a plateau at 18 mo. As one might expect, b o t h P W a n d B W were heavier in g r o u p A L t h a n in g r o u p DR. Interestingly, the average values o f P W were not as widely varied between group A L a n d D R as those o f BW. Therefore, when P W was factored with BW ( P W / 1 0 0 g BW), P W was higher in g r o u p D R t h a n in g r o u p A L ( T a b l e 2). The P W / 1 0 0 g B W was also increased with age.
Volume Density of the Anterior Lobe in the Pituitary Gland [Vv(Ant/PG)] and the Volume Density of Nonpituitary Cell Components in the Anterior Lobe [Vv(NP/Ant)] We e x a m i n e d structural alterations due to age a n d the possible influence diet h a d o n the a n t e r i o r lobe o f the pituitary gland. V v ( A n t / P G ) was approximately 0.82, meaning the anterior lobe occupied 8207o o f the volume o f the pituitary gland. V v ( A n t / P G ) did n o t change significantly with age or dietary restriction (Table 3). V v ( N P / A n t ) or n o n p i t u i t a r y cell c o m p o nents was approximately 0.03, comprised mainly connective tissues a n d b l o o d vessels a n d occupied a b o u t 3070 o f the a n t e r i o r lobe. V v ( N P / A L ) s h o w e d n o significant change a m o n g age groups or between dietary groups (Table 3).
Volume Density of Somatotrophs in the Anterior Lobe [Vv(GH/Ant)] V v ( G H / A n t ) was estimated at 0.451 in A L rats a n d 0.414 in D R rats at 6 m o o f age (Table 4). Age-related decreases in V v ( G H / A n t ) were noted in both dietary groups. Although there was no significant dietary effect in V v ( G H / A n t ) as a whole, the decrease rate was smaller in DR rats.
Numerical Area and Volume Density of Somatotrophs & the Anterior Lobe [Na(GH/Ant)], Nv(GH/Ant)] N a ( G H / A n t ) ] a n d N v ( G H / A n t ) decreased progressively with age in b o t h dietary groups (Table 5). There were no significant dietary effects evident in N a ( G H / A n t ) a n d N v ( G H / A n t ) . However, a further analysis for N a ( G H / A n t ) a n d N v ( G H / A n t ) at 24 m o revealed significant differences in the cell density between G r o u p A L a n d D R ( p < 0.05 for N a ( G H / A L ) , p = 0.0643 for N v ( G H / A L ) evaluated by unpaired t test, in this case, a specific hypothesis t h a t diet has n o effect o n N a ( G H / A n t ) or N v ( G H / A n t ) at 24 m o was tested).
TABLE 3 VOLUME DENSITIES OF THE ANTERIOR LOBE IN THE PITUITARY GLAND AND NONPITUITARY CELL COMPONENTS IN THE ANTERIOR LOBE Vv(Ant/PG)#~ Age (mo) 6 18 24
Vv(NP/Ant)~2
AL
DR
AL
DR
0.815 + 0.020 (5) 0.821 _+ 0.031 (5) 0.839 ___0.050 (4)
0.772 _+ 0.027 (4) 0.823 _ 0.039 (5) 0.819 ___0.035 (5)
0.032 _+ 0.009 (6) 0.029 _+ 0.013 (6) 0.025 + 0.004 (3)
0.031 + 0.012 (6) 0.036 + 0.012 (6) 0.030 + 0.017 (5)
Values represent the arithmetic mean :t: SD. Numbers in parentheses indicate number of rats examined. The statistical analyses by ANOVA are as follows; #~Age: F(2, 22) = 2.702; p > 0.05, Diet: F(1, 22) = 2.385; p > 0.05, Age x Diet: F(2, 22) = 0.957; p > 0.05, #2Age: F(2, 26) = 0.462; p > 0.05, Diet: F(1,26) = 0.644; p > 0.05, Age x Diet: F(2, 26) = 0.371; p > 0.05.
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS TABLE 4 VOLUME DENSITY OF SOMATOTROPHS IN THE ANTER1OR LOBE Vv(GH/Ant) Age (mo)
6 18 24
AL
DR
0.451 _+ 0.086 (6) 0.335 _+ 0.075 (6) 0.174 +_ 0.040 b'c (3)
0.414 +_ 0.031 (6) 0.346 _+ 0.050 (6) 0.297 _+ 0.078 (5)
Values represent the arithmetic mean _+SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; Age: F(2, 26) = 22.236; p < 0.01, Diet: F(1,26) = 2.514; p > 0.05, Age x Diet: F(2, 26) = 3.972; p < 0.05. The superscript letters represent statistical significance at p < 0.05 by a posthoc test for multiple comparisons; bversus 6 mo, Cversus 18 mo at Group AL.
~
83
1.20 1.00
~ 0.80 o O.fiO
I~ .~
E 0.40 ¢...
>
0.20
~- 0.00
0
~ 6
/.,'
I 18 Age (months)
~ 24
FIG. 2. Relative changes in total cell number of somatotrophs in the anterior lobe as normalized to the level of the total number in Group AL at 6 months.
Total Cell Number of Somatotrophs in the Anterior Lobe [N(GH/Ant)] and Cell Number of Somatotrophs per Unit B W [N(GH/unit BW)] The N ( G H / A n t ) , which showed n o decrease until 18 m o in G r o u p A L showed a reduction by a p p r o x i m a t e l y 2 5 % thereafter. In contrast, the rats in G r o u p DR, N ( G H / A n t ) were nearly c o n s t a n t t h r o u g h the 24 m o m a r k (Fig. 2). Data in Fig. 2 shows t h a t the total cell n u m b e r o f s o m a t o t r o p h s was greater in G r o u p A L ; however, when n o r m a l i z e d to unit b o d y weight, N ( G H / u n i t B W ) was higher in G r o u p DR. N ( G H / u n i t B W ) progressively decreased after 6 m o in b o t h dietary groups (Fig. 3).
Estimates of Average Single Cell Size (Volume) of Somatotrophs [V(GH)] and Volume Density of the Nucleus in the Cytoplasm [Vv(NU/GH)] V ( G H ) was estimated at 900-1000 ~tm 3 a n d n o statistical differences were seen between dietary groups or a m o n g age
groups (Table 6). However, the nucleus volume of somatotrophs significantly increased at 24 m o in b o t h dietary groups.
Morphological Changes of Somatotrophs Light microscopic prints of the immunostained somatotrophs in A L a n d D R rats at 6 m o a n d 24 m o are s h o w n in Fig. 4 a n d Fig. 5. Cell density was reduced in b o t h dietary groups at 24 m o (Fig. 4). Cytoplasmic v a c u o l a t i o n (See Fig. 5 at high magnification) b e c a m e noticeable in s o m a t o t r o p h s o f b o t h dietary groups at 24 too. No a p p a r e n t difference in the degree o f cytoplasmic v a c u o l a t i o n between G r o u p A L a n d DR was detected by light microscopic o b s e r v a t i o n .
TABLE 5 NUMERICAL AREA AND VOLUME DENSITIES OF SOMATOTROPHS IN THE ANTERIOR LOBE N a ( G H / A n t ) #1
Age (mo)
6 18 24
AL
DR
i
N v ( G H / A n t ) #2
AL
1.00
DR
3597 _+ 559 3488 + 271 4.52 _+ 0.78 4.51 _+ 0.48 (6) (6) (6) (6) 2895 _+ 558 2958 + 450 3.83 + 0.66 3.85 +_ 0.81 (6) (6) (6) (6) 1551 _+ 434 t~'~ 2426 + 487 b'g 1.80 +_ 0.43 b'c 2.80 + 0.68 b'h (3) (5) (3) (5)
Na(GH/Ant): cells/mm 2. Nv(GH/Ant): cells x 105/mm 3. Values represent the arithmetic mean +_ SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; ~JAge: F(2, 26) = 24.875; p < 0.01, Diet: F(1,26) = 2.559; p > 0.05, Age x Diet: F(2, 26) = 2.672; p > 0.05, #2Age: F(2, 26) = 24.905; p < 0.01, Diet: F(I, 26) = 1.832; p > 0.05, Age x Diet: F(2, 26) = 1.566; p > 0.05. The superscript letters represent statistical significance at p < 0.05 as determined by a posthoc test for multiple comparisons; bversus 6 mo, and Cversus 18 mo at each dietary group, g'hAt 24 mo, a specific hypothesis that diet had no effect on Na(GH/Ant) or Nv(GH/Ant) was tested by unpaired t test; gp < 0.05, hp = 0.0643.
1.40 1.20 0.80 0.60
~ 0.20 rr
0.00
I
6
,//
I
18 Age (months)
I
24
FIG. 3. Relative changes in cell number of somatotrophs per unit body weight. Each value was normalized to the level of cell number of somatotrophs per unit body weight in Group AL at 6 months.
84
S H I M O K A W A ET AL.
FIG. 4. Immunostained somatotrophs in Group AL and Group DR. A: Group AL at 6 mo; B: Group AL at 24 too; C: Group DR at 6 too; D: Group DR at 24 mo (phase contrast optics, original magnification: x40). Note the reduction in cell density of immunostained somatotrophs at 24 mo, compared to that seen at 6 mo in both dietary groups. The cytoplasmic vacuolation is also noticeable in somatotrophs at 24 mo of both dietary groups.
DISCUSSION
There are few reports connecting structural changes of the pituitary gland with age and lifelong dietary restriction, even though information on those structural changes is important to understanding the physiological changes in pituitary function (3). Our analysis indicates that aging and dietary restriction have
TABLE 6 AVERAGE SINGLE CELL VOLUME OF S O M A T O T R O P H S AND VOLUME DENSITY OF THE NUCLEUS IN CYTOPLASM OF S O M A T O T R O P H S V(GH) #~ Age (mo)
6 18 24
V v ( N U / G H ) #2
AL
DR
AL
DR
1002 + 110 (6) 879 + 169 (6) 965 -+ 65 (3)
921 _+ 42 (6) 914 + 99 (6) 1059 _+ 144 (5)
0.269 + 0.027 (6) 0.260 + 0.020 (6) 0.343 +- 0.070 (3)
0.269 _+ 0.044 (6) 0.271 + 0.033 (6) 0.333 -+ 0.048 (5)
V(GH): #m 3. Values represent the arithmetic mean +_SD. Numbers in parentheses indicate numbers of rats examined. The statistical analyses by ANOVA are as follows; #1Age: F(2, 26) = 2.392; p > 0.05, Diet: ~F(21,26) = 0.156; p > 0.05, Age x Diet: F(2,26) = 1.446; p > 0.05, AGE: F(2, 26) = 7.649; p < 0.01, Diet: F(1,26) = 0.285; p > 0.05, Age x Diet: F(2, 26) = 0.725; p > 0.05.
no direct influence on the structure, except on the absolute size, and that changes in the absolute size of the anterior lobe are not caused by disproportional growth of the connective tissue in that lobe. We can say that aging increases the total number of pareno chymal cells in the anterior lobe, while dietary restriction decreases the total cell number in proportion to the pituitary weight. Note, however, that the number of parenchymal cells is larger in DR rats than in A L rats when compared by body weight. Our primary findings during this study were loss in N a ( G H / Ant) and N v ( G H / A n t ) , or cell density, and total cell number of immunostained somatotrophs during aging. These changes may provide morphological bases for observed reductions in G H concentration (22) and G H - m R N A expression (6) in pituitary glands o f aged animals. Such reductions could partly be due to losses in cell numbers if immunostained somatotrophs represent most cells containing GH-peptide or G H - m R N A . Some reports suggest functional impairments in somatotrophs in in vitro studies. Ceda et al. (5) indicated a diminished production of c A M P by pituitary cells after G H R H stimulation in aged rats. Robberecht et al. (16) also reported reductions in the G H R H - s t i m u l a t e d adenylate cyclase activity in aged rats. Abribat et al. (1) reported alterations o f G H R H binding sites. These findings are, however, obviously dependent on the cell density of somatotrophs in the pituitary tissue. The response of individual somatotrophs to G H R H may not be impaired as we expected. G H is released from the anterior pituitary in an intermittent, pulsatile fashion in rats (23), and this secretion is attenuated in
EFFECTS OF DIETARY RESTRICTION ON AGED SOMATOTROPHS
FIG. 5. High power microphotographs of somatotrophs. A: Group AL at 6 mo; B: Group AL at 24 mo (phase contrast optics, original magnification: x 100). Note the cytoplasmic vacuolation of somatotrophs at 24 mo. older rats (22). An age-related impairment in pituitary responsiveness to GHRH in vivo was also reported (3,5,6). In this study, morphometric analysis suggests a decrease in the cell number of somatotrophs per body weight, by 25°7o at 18 mo and 5007o at 24 mo in group AL. The relative change in the cell number per unit of body weight seems closely correlated to the reported reduction in amplitude of GH secretion and responsiveness to GHRH in in vivo experiments, although the reduced frequency of spontaneous GH pulsatile secretion in aged animals is probably the result of changes in hypothalamic regulation. Our findings on somatotrophic reduction in the cell density do not agree with the earlier results of Rossi et al. (17), who observed that somatotrophic cell numbers decreased in female rats but increased in aged male Sprague-Dawley (SD) rats. Because immunoreactivity and expression of mRNA for GHRH in the hypothalamus, which stimulates the proliferation of somatotrophs (4), decreased in aged male SD rats (7,14), it is unclear why the number of somatotrophs increased in male rats. At this time, we can offer no experimental data to explain this discrepancy.
85
Another unanswered question relates to our observation of reductions in the total cell numbers of somatotrophs in AL rats after 18 too. The total number of somatotrophs is influenced by the cell turnover rate, which is determined by removal of aged somatotrophs and addition of newly formed somatotrophs. Our findings did not provide definitive information regarding an agerelated change in either reduced cell renewal or accelerated cell loss. However, when considering reports of reduced GHRHimmunoreactivity and GHRH-mRNA expression in aged male rats (7,14), one might conclude that cell renewal decreases in that group. To investigate that possibility, however, further analysis on the cell turnover of somatotrophs, particularly cell loss, is needed. To our knowledge, there are no reports on morphologic changes in somatotrophs of aged animals. This study revealed cytoplasmic vacuolation and a relative increase in the nucleus volume, which are possible signs of cellular aging, in most somatotrophs after 18 mo. We are now examining pituitary tissues by electron microscopy to identify subcellular structure(s) showing vacuolation. Little is known about the long-term effects of dietary restriction on GH secretion although DR has been reported to have a GH lowering effect (3,13). Our morphometric tests demonstrated that the cell number of somatotrophs per body weight was significantly higher in Group DR than in Group AL. This provides the restricted rats with greater capacity for GH secretion from the pituitary gland, assuming that each somatotroph in both dietary groups of rats has a similar capacity for GH secretion. Additionally, the relatively large cell number of somatotrophs in Group DR could attenuate the deterioration processes in somatotrophs because of overreactivity, because demand for GH synthesis/secretion per cell should be less under dietary restriction. It is worth noting that in Group DR, the total number of somatotrophs in the anterior lobe remained at the same level from 6 mo through 24 mo, which could mean that GH cell turnover remains constant during senescence. This suggests that age-related changes in hypothalamic signals involved in somatotrophic cell turnover are retarded by dietary restriction. These data are consistent with well-established anti-aging actions of dietary restriction (25,27), but the effective modification by dietary restriction of age-related changes in cell turnover and morphology of somatotrophs warrants further investigation. ACKNOWLEDGEMENTS
This research was supported by NIH Grant Number AG01188. We are grateful to A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases for providing the antibody for rat GH. We also thank E. J. Masoro for his useful comments. Thanks also goes to W. R. Mejia, Y. Suh, and F. Perkins, Jr. at the University of Texas Health Science Center at San Antonio for their invaluable technical assistance.
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