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Growth patterns and hormonal profile of male rats with protein-calorie malnutrition.

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THE ANATOMICAL RECORD 197:339-354 (1980)
Growth Patterns and Hormonal Profile of Male Rats With
Protein-Calorie Malnutrition
DAMON C. HERBERT
Department of Anatomy, The University of Texas, Health Science Center at San
Antonio, San Antonw, Texas 78284
ABSTRACT
A condition of protein-calorie malnutrition was precipitated in
young Sprague-Dawley male rats at 20 days of age using an 8% low protein diet
(LPD). At five-day intervals for up to 50 days of age, the rats were studied to
determine the effect of a n LPD on the reproductive axis of the endocrine system.
Daily monitoring of the body weight, as well a s the consumption of food, kilocalories, and protein was conducted. The same parameters were followed over the
identical time period in a group of animals designated as controls which were fed a
standard laboratory diet (SLD) containing 27% protein. The controls showed a
linear growth rate over the 30-day experimental period. In comparison, the malnourished rats grew more slowly so that by 50 days of age, their mean body weight
was 68.9 5 3.1 g as compared to 249.1 t 6.1 g for the controls. The daily food,
kilocalorie, and protein intake by the experimental animals were also appreciably
less. The pituitary gland, ventral prostate gland, testes and liver were smaller in
the animals fed the LPD. This was observed as early as five days after initiating the
dietary regimes and remained a consistent observation until the end of the experiment. In general, the absolute weights of these organs in the 50 day-old malnourished rats were similar to those found in 25 to 26 day-old animals fed the SLD.
The relative weights of the pituitary gland and liver remained similar between the
two animal groups. The testes and ventral prostate gland, however, were relatively
smaller in the malnourished animals a t nearly every time interval studied. On
light microscopic examination of the testes, it was found that normal maturation of
the germ cells failed to occur in all but one of the experimental animals, whereas
maturation proceeded normally in the rats fed the SLD. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), and testosterone
were lower in the malnourished animals at all ages studied. These hormones did
not exhibit the fluctuations that were seen in the controls and are typical in rats
that are becoming sexually mature. The effect of protein deficiency on the concentration of the pituitary gonadotrophins was more varied. FSH concentrations were
consistently lower, PRL was moderately affected, and LH remained essentially
unchanged. Hypothalamic LH-releasing hormone was measured and found to be
significantly less in the rats fed the LPD a t most of the time intervals examined.
axis is impaired
These results indicate that the hypothalamo-hypophyseal-gonadal
when the consumption of proteins and calories is decreased. The possible involvement of extrahypothalamic centers in the control of hormone secretion in the
protein-deficient rat is discussed.
A large number of reports have been published describing the complications and deleterious effects of protein-calorie malnutrition
(PCM) in man. It is well established that this
problem abounds in countries that are overpopulated. Despite the apparent paradox, reproductive function is impaired in individuals
with PCM (Brasel, '78).
0003-276x/80/1973-0339$02.90 0 1980 ALAN R. LISS. INC.
The relationships between reproduction and
nutrition have been studied in laboratory animals by employing diets deficient in or totally
devoid of protein, or under conditions where
animal subjects have been chronically underfed (semistarved). Early reports from the work
Received May 23, 1979; accepted October 8, 1979.
339
340
DAMON C. HERBERT
of Evans and Bishop ('22) established the need
for adequate dietary protein in order to initiate
and maintain regular estrous cycles in the
female rat. In the male, puberty was found to be
delayed when animals were given a proteinfree diet (Horn, '55). If, however, they were
refed a diet containing either 6%or 1810 casein
for 30 days, 25% and Two, respectively, of the
animals became sexually mature.
The absolute weights of the gonads and accessory reproductive organs were consistently
lower in male rats a) given a protein-free diet
(Horn, '55; Srebnik and Nelson, '62; Srebnik,
'641, b) starved for seven days (Mulinos and
Pomerantz, '411, or c) chronically underfed
(Mulinos and Pomerantz, '40, '41; Grewal e t al.,
'71; Howland, '75). The most severe changes
were noted in the weights of the accessory organs (Mulinosand Pomerantz, '41; Srebnik and
Nelson, '62; Srebnik, '64). Moreover, it was reported by Srebnik and Nelson ('62) that the
debilitating effects of a protein-free diet were
more pronounced i n younger than in older animals. Pomerantz and Mulinos ('39), (Mulinos
and Pomerantz, '40) termed the malnourished
condition associated with chronic underfeeding
"pseudo-hypophysectomy." Atrophy of the reproductive organs was quite pronounced in
their animals but to a lesser degree than that
observed in hypophysectomized rats. This observation was later substantiated by Srebnik
('64) who found larger testes, seminal vesicles,
and prostates in males fed a protein-free diet as
compared to hypophysectomized rats. These
findings indicated that the anterior pituitary
gland was exerting a trophic effect on the reproductive tract, even in the absence of dietary
protein.
Factors controlling the secretion of the anterior pituitary gonadotrophins and the gonadal
hormones have been shown to be sensitive to
dietary manipulations. The pituitary content of
luteinizing hormone (LH) and folliclestimulating hormone (FSH) was reduced a s a result of
protein deprivation (Srebnik and Nelson, '62),
but was more variably affected by chronic underfeeding or starvation (Meites and Reed, '49;
Root and RUSS,'72; Howland and Skinner, '73;
Howland, '75; Nakanishi et al., '76). Feed restriction for periods of 30 or 60 days has been
reported to decrease pituitary prolactin (PRL)
levels (Nakanishi et al., '76). All three gonadotrophins were lower in the serum of rats either
starved for seven days (Howland and Skinner,
'73; Campbell et al., '77) or underfed for varying
Deriods of time (Howland, '75; Stewart et al..
'75; Campbell e t al.,'77). Similarly, these two
forms of malnutrition also had an adverse effect
on testosterone secretion (Grewal et al., '71;
Howland, '75).
The most prevalent forms of malnutrition in
today's world are thought to be related to a
deficiency of protein in the diet and not due to
starvation or consumption of a protein-free
diet. In addition, the largest single group of
undernourished persons are children (WHO,
'72). In view of this, it was of interest to examine the effect of a low protein diet on the development of the reproductive system in animals
that were maturing sexually in a n effort to gain
information that could be related to preadolescent children suffering from PCM. Such studies
have been conducted using female rats (Evans
and Bishop, '22) but, heretofore, have not been
thoroughly examined in the male. The present
report details changes that have occurred in the
hypothalamo-hypophyseal-gonadalaxis of protein-calorie malnourished male rats a t fiveday
intervals between 20 and 50 days of age.
MATERIALS AND METHODS
Twenty day-old Sprague-Dawley male rats
were purchased from Simonsen Laboratories,
Inc., Gilroy, CA. They were housed four animals per cage in air-conditioned quarters with
a light-dark cycle of 14:10, controlled temperature of 22-24"C, and humidity regulated a t
500/0.Food and water were provided ad libitum'.
The animals were separated into 13 experimental groups with each group containing
eight animals. Animals i n the first seven
groups served a s controls and were fed a standard laboratory diet (SLD) while rats in the
remaining six groups were given a low protein
diet (LPD). Both diets were purchased in pellet
form from ICN Pharmaceuticals, Inc., Cleveland, OH. The SLD consisted of 27%protein in
the form of vitamin free casein, 5w0 starch,
10% vegetable oil, and 4% salt mixture USP
XIV. The experimental LPD was composed of
8% protein (vitamin free casein), 7810 starch,
lWo vegetable oil, and 4% salt mixture USP
XIV. The SLD was provided with 4.3 Kcal per
gram and the LPD 4.5 Kcal per gram. A special
vitamin fortification mixture, prepared by ICN
'Since the main objective ofthe study was to induce protein-ealorie
malnutrition and nottostudytheefleetsofdeficiency ineitherproteins
or calories alone, the rats were not pair-fed. Moreover, when pairfeeding is employed. the control animals are fed less than their normal
amount of food and are, therefore, partially protein and/or calorie
malnourished,aemistarved, and ntrensed. Both stress (Eukereta].. 75)
and semistarvation (Campbell et el.. 7 7 ) have been reported to atTed
pituitary hormone secretion
341
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
Pharmaceuticals, Inc., was incorporated into
each diet in an amount of 1 kg per 45.5 kg of
diet.
The changes in body weight and the amount
of food consumed by each rat were monitored on
a daily basis. The latter calculation was made
by weighing the amount of food present each
morning, subtracting this from the amount of
food measured at the same time on the previous
day, and dividing by eight. Additional computations were made from these data to determine
the daily intake of protein and kilocalories
(Kcal)by each rat. These data were statistically
analyzed by analysis of variance and by the “t”
test for comparison of differences between
means.
One group of control animals was sacrificed
immediately upon arrival. The remaining control and the experimental rats were sacrificed
a t five-day intervals beginning a t 25 days of
age and continuing through 50 days of age. At
each of these intervals, eight control and eight
experimental animals were sacrificed. This
provided exposure of the rats to either the SLD
or the LPD for periods of 0 to 30 days over the
ages of 20 to 50 days. All animals were sacrificed by decapitation between 0800 and 1100
hours. Trunk blood was collected, allowed to
clot, and the serum collected and stored a t
-20°C. The following organs were then removed from each animal and weighed: the
hypothalamus, pituitary gland, testes, ventral
prostate gland, and the liver. The testes were
fixed for 72 hours in Bouin-Hollande solution,
embedded in paraffin wax, and stained with
hematoxylin and periodic acid-Schiffs reagent.
The hypothalamus was placed in 1m12 N acetic acid and homogenized. This was subsequently heated in boiling water for five minutes, centrifuged, and the supernatant collected for lyophilization. The pituitary gland
from each rat was hemisected along the midline. Half was homogenized in a 0.01 M phosphate-saline buffer, pH 7.0, and the other half
fixed for histologic examination (the details of
these findings will be the subject of a future
report).
The pituitary homogenates along with a portion of the sera samples were assayed for LH,
FSH, and PRL, using double antibody radioimmunoassays. The LH assay procedure was
similar to the one described by Niswender et al.
(’68),while the FSH and PRL assays were carried out using reagents supplied by the
NIAMDD. The lyophilized hypothalami were
reconstituted in 1ml distilled water, and the
content of LH-releasing hormone (LHRH)
measured using a modification of the procedures described by Arimura et al. (’73)and Nett
and Adams (’77).The LHRH antibody was generously supplied by Dr. Terry Nett. Serum total
testosterone were quantified using a modification of the assay procedure employed by
Pauerstein et al. (’78).The antibody to testosterone was purchased from Endocrine Sciences. The coefficient of variation in the testosterone assay was 2.0%. All assay and organ
weight data were pooled according to experimental grouping and statistically evaluated as
previously described.
RESULTS
Body growth and development
The initial body weight of the male rats a t 20
days of age ranged from 44.5 to 46.6 g, with a n
average of 45.3 ? 0.2 g. No statistical differences were present among the 13 animal
groups. The changes in the body weight of the
control rats fed the 27% protein diet were linear
over nearly the entire 30-day experimental
period (Fig. 1).In contrast, the animals given
the LPD gained weight a t a much slower rate,
which was neither linear nor parallel to that of
the controls. These animals had an irregular
growth pattern, gaining a small amount of
weight over a few days, followed by a brief
250 -
200 -
-E
2
150-
Em
.-
2”
s” loo-
m
0
J , , , , , , ,
x)
25
30 35 40
Age(doys)
45
50
Fig. 1. Mean body weight (-t SE) of the control (solid
circles) and experimental (open circles) rats at fiveday time
intervals between 20 and 50 days of age.
342
DAMON C. HERBERT
Fig. 2. A control and an experimental rat at 50 days of age. Patchy hair loss can be noted on the neck of the
malnourished rat.
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
w
701
60 -
I
v
50-
-5
40-
-4 0
3
8
Y
u)
3020 -
-3
a
r
-
'"1, *
p-
0
20
?
0,
8.
,
25
-2
-
-=:
- *--*--P--c---a
x)
,
35
,
40
,
45
,
50
Age (days)
Fig. 3. Mean ( 2 SE) intake of kilocalories (solidline) and
grams protein (broken line) of animals fed either the SLD
(solid circles) or the LPD (open circles).
period of no weight gain, and then another
episode in which there was a n increase in body
weight. At the end of the experimental period,
the average body weight of the animals fed LPD
was 68.9 +- 3.1 g which was similar to the
weight of 24 day-old control rats (67.5 +- 1.4 g).
Figure 2 illustrates a control and an experimental animal which had been fed their respective diets for 30 days. A small amount of
hair loss is apparent on the dorsal and lateral
sides of the neck of the malnourished rat. This
was noted in 23 of 48 animals fed the LPD. In
general, it was confined to the dorsal or the
ventral aspects of the trunk of the animal and
was typically seen in patches. No external parasites could be found on these animals that
would have resulted in the alopecia. Thinning
of the hair has previously been reported in
animals fed an LPD (Tulp et al., '79). No hair
loss was observed in the controls.
Food, kilocalorie, and protein consumption
The Kcal and grams protein consumed by
each rat in its respective experimental group
are shown in Figure 3. Curves representing the
daily food intake are not illustrated, but were
found to nearly parallel those depicting the
Kcal consumed by both the control and the
malnourished rats. The animals fed the SLD
consumed food, and therefore had an intake of
Kcal and proteins, at a rate that was distinctly
different from that observed in the experimental rats. These parameters were linear in
the controls for up to ten days (30 days of age)
after which the rate of food consumed and intake of Kcal and grams protein began to de-
343
crease. Despite this, the weight of the rats continued to rise linearly (Fig. 1). In the rats fed
the LPD, there was a n initial decline in the
amount of food, Kcal, and grams protein consumed which was then followed by a 10-day
period in which there was no change. After 35
days of age, however, a linear increase in the
daily intake of food, Kcal, and protein was
noted. At all time intervals examined, these
parameters were consistently lower in the rats
fed the LPD as compared to the controls. At the
end of the experiment, the food and Kcal consumed by the malnourished rats was approximately the same as that observed in 22 day-old
rats maintained on the SLD.
Absolute and relatiue organ weights
Table 1 contains the absolute weights of the
pituitary gland, testes, ventral prostate gland,
and liver in both the control and experimental
animals. All four organs increased consistently
in weight over the 30-day period in the control
animals. In most instances, the increases were
significantly greater a t each successive time
interval. The major exception to this was in the
pituitary gland weight where the changes,
while greater at each five-day time interval,
were statistically significant only when comparing the values measured on day 25 with day
30, or day 30 with day 35. In the malnourished
animals, consumption of the LPD led to more
erratic changes in the pituitary gland, testes,
ventral prostate, and liver during the 30-day
experimental period. Moreover, the weights of
these organs were always significantly less (P
< 0.001) than those recorded for the controls a t
all of the time intervals examined. A significant reduction (P < 0.02) in pituitary gland
weight occurred in the experimental animals
between 20 and 40 days of age. Over the remaining 10 days of the study, a slight increase
was noted, so that by 50 days of age, the pituitary gland had obtained a weight which was
similar to that found in the 25 day-old control
rats. There was no significant change in testicular weight of the malnourished animals for
the first 20 days of the study. After this period
of time, a marked increase was recorded; however, the growth rate was less than that observed in the controls. At the end of the experiment, the testes were similar in weight to those
of a 26 day-old rat maintained on the SLD. The
absolute weight of the ventral prostate gland in
all of the experimental animals was consistently less than the value recorded in the 20
day-old rats. Between 20 and 30 days of age,
there was a significant decrease (P < 0.005) in
prostatic weight, which was then followed by a
344
DAMON C. HERBERT
TABLE 1. Absolute organ weights in protein-calorie malnourished male rats
Pituitary gland
(mg)
Age
(days)
20
C
M
C
2.3
t 0.2"
-
303
t 16
25
2.5
t 0.2
30
35
5.6
0.2
540
265
48.1
28.9
i24'
t 25
t 2.5'
t 1.7
1.6
0.1
855
f42
t 31
?
1.6
297
66.5
5.27
t 0.9
2
94.4
7.9"
t 1.9
137.9
i 9.5'
f
261
t 28
1.6
0.1
1648
t4w
t28e
6.8
1.9
201.5
t 0.2
2089
t 33'
457
t 0.5
t 486
t- 16.01
2256
556
611
47d
260.0
21.1
i-
i 13.6
t 2.8
i-
7.1
t 0.4
5
2.5
0.26
354
*
M
-
C
2.5
i 0.1
3.5
2.0
t 0.1h
21.1
2
1235
6.1
Liver
(PI
M
-
C
31.3
t 1.6
t 4of
i 0.3
50
M
-
* 0.1
?
45
1.7
Ventral prostate
gland (mg)
* 0.1
3.7
t 0.2
40
Testes
(mg)
f 0.1
6.0
* 0.1'
23.2
k
7.1
_~0.3~
t 0.1
10.3
0.4'
t 0.1h
20.1
2.6
2
23.9
3.3
t 0.4b
2.5
2.9
11.3
2
2.3
0.1
2
12.0
2.7
0.2
3.3
* 0.4
f 0.2e
C = control rats fed the SLD
M = malnourished rats fed the LPD
=mean 2 SE
P .C 0.05 (statistical comparisons were made between the mean for each organ welght and the weight of t h a t organ at the previous time Interval)
'P < 0.02
"P < 0.01
*P < 0.005
' P < 0.001
TABLE 2. Relative organ weights in protein-calorie malnourished male rats
Pituitary gland
(mg)
C
M
C
M
4.6
-
624
t 34
-
i 0.4'
25
30
3.4
3.6
757
512
67.3
f 0.2
t 29
t 56''
t 2.4
788
43
596
61.1
t63
t 4.9
876
38
510
66.9
i 53g
t 5.4
866
29
t
629
49
t 6.6
739
144d
t 7.9
2
822
45
?
104.6
5.6
t 4.F
3.4
50
=
M
=
t
2.9
0.2
?
2
k
3.2
948
f 0.4
t 30
2.4
f 0.2
C
3.2
3.3
* 0.3
3.1
0.3
t 0.1h
3.2
t 0.1
45
5
3.9
i 0.2
40
M
-
C
63.5
t 3.8
-c 0.3
t 0.1
35
Ventral prostate
gland (mg)
Testes
(mg)
5
3.6
0.3"
907
21
k
t
t
72.8
95.9
62.7
3.7
2
Liver
(g)
C
M
5.0
t 0.2
-
2
42.8
4.9
0.1
5.6
4.5
f 0.2
4.7
* 2.P
t 0.1
45.3
2.7'
5.0
4.9
i:0.1
t 0.1
t
35.9
t 0.2':
* 4.8
5.4
5.2
t 0.2
2 0.2
37.2
4.3
t 0.1
5.3
30.7
t
4.8
0.1
4.4
t 0.18
4.8
t 0.2
control rats fed t h e SLD
malnourished rats fed the LPD
dmean i SE. Organ weights are expressed per 100 g body weight.
DP< 0.05 (statistical comparisons were made between means from control and experimental animals at each time interval for each organ)
P < 0.025
dP < 0.02
e P c 0.01
I P < 0.005
'P c 0.001
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
period in which there was no additional change.
An appreciable reduction (P < 0.005) in liver
weight also occurred over the first five days in
the animals fed the LPD. However, between 25
and 50 days of age, the liver enlarged and
reached a weight which was very similar to
that observed in the 25 day-old control rats.
The relative organ weights are summarized
in Table 2. The weight of the pituitary gland
gradually decreased in the controls as they
aged. A similar observation was made in the
malnourished rats between 20 and 40 days of
age. For the remaining period of the study, an
increase in pituitary weight was recorded. In
spite of this, the pituitary gland of the 50 dayold malnourished r a t s was significantly
smaller (P < 0.02) than that found in the controls a t the onset of the study. When comparing
the values for pituitary gland weights in the
controls with the experimental animals at each
of the ages studied, a significant difference was
found only at 35 and 50 days of age. At a majority of the time intervals sampled, the relative
weights of the testes and ventral prostate
glands were statistically greater in the rats
maintained in the SLD. Moreover, these two
organs continued to enlarge relative to the body
weight in the controls over the 30-day experimental period. There were no significant
changes in testicular weights in the protein
deficient animals until after 40 days of age.
This is in marked contrast t o the decrease
recorded for the weight of the ventral prostate
gland which occurred throughout the course of
the study. There were fluctuations in liver
weight in both the control and experimental
animals. Significant differences between the
two animal groups were found at only two time
intervals. At 50 days of age, the weight of the
liver was essentially the same in both animal
groups, and was not significantly different from
the weight recorded in the control rats a t the
beginning of the experiment.
Histology of the testes
Histologic examination of the testes revealed
that the diameter of the seminiferous tubules
was less, and the size and number of Leydig
cells were decreased in the animals fed the LPD
(Figs. 6-11). In addition, a disruption of the
overall integrity of the tubular epithelium was
evident. These changes were first observed as
early as five days following the onset of the
study (Figs. 6,7),and continued to be prevalent
as long as the LPD was administered. Spermatids in the Golgi phase could first be differentiated in the controls at 25 days of age (Fig.
6). Five days later, more mature spermatids
345
were easily distinguished in all of the animals
maintained on the SLD. Spermiogenesis progressed normally in the controls (Figs. 8, lo),
with immature spermatozoa (stage 19 spermatids) being observed in the seminiferous
tubules a t 45 and 50 days of age (Fig. 10). All
but one of the animals maintained on the LPD
did not undergo puberty. Golgi phase spermatids predominated in the testes of the experimental animals from 2 5 4 0 days (Figs. 7,9).At
45 days of age, after being on the LPD for 25
days, acrosomal phase spermatids were present
in one animal, mature phase in another, and
either cap or Golgi phase spermatids in the
remaining six. By 50 days of age, five of the
eight rats had mature phase spermatids in
their seminiferous tubules (Fig. 11).A large
number of stage 19 spermatids were observed
in only one of the five, indicating that it was
sexually mature. The remaining 50 day-old
animals displayed more immature forms of the
spermatids in their seminiferous tubules.
Serum gonadotrophins
The concentration of circulating LH, FSH,
and PRL are shown in Figure 4. LH increased
by 107% in the control animals during the first
five days of the study and rose again by 116%
between 35 and 40 days of age. No significant
changes were found from days 25 to 35 or after
40 days of age. Fluctuations in serum LH occurred in the malnourished rats throughout the
entire experimental period; however, no two
values were found to be statistically different.
LH in these animals was consistently lower
than those values recorded for the rats fed the
LPD.
Serum concentrations of FSH increased from
370
20 to 526 2 47 ngiml in the control
animals during the five day period between 25
and 30 days of age. No additional vacillations
occurred until after day 40. Thereafter, a significant decrease was recorded. In contrast, FSH
declined very rapidly in the protein deficient
animals from the onset of the study until day
25. The hormone levels then plateaued and remained essentially unchanged for the duration
of the experiment. By 50 days of age, FSH in the
animals was 5wo less than the value measured
in the age-matched controls.
PRL rose throughout nearly the entire 30day experimental period in the rats maintained
in the SLD. Likewise, an increase in hormone
levels occurred between 20 and 40 days of age in
the malnourished rats. However, the rate of
changes in serum PRL in these animals was
significantly less than that which transpired in
the control animals.
*
346
DAMON C. HERBERT
20
25
30
35
45
40
50
T
0 '
$0
215
40
I
I
T
I
35
40
45
50
Age (days)
Fig. 4. Serum concentration of LH, FSH, and PRL in
control (solid circles) and malnourished (open circles) rats
from 20 to 50 days of age. Each point representsthe mean ?
SE. Statistical comparisons were made between means from
control and malnourished animals at each time interval.
Pituitary gonadotrophins
The content of the pituitary gonadotrophins
was, with one exception (LH on day 251, statistically greater in the control animals a t each
time interval sampled. The mean hormone values in the two animal groups on day 50 were:
LH, 79.1 2 14.5 vs. 12.2 5 1.0 pg (controls vs.
experimentals); FSH, 55.5 t 5.3 vs. 11.4 t 2.1
pg; PRL, 610.1 t 84.9 vs. 97.4 21.6 ng.
*
Table 3 summarizes the concentrations of
pituitary LH, FSH and PRL. There was approximately a two and one half fold increase in LH
levels in the pituitary glands of the rats fed the
protein-enriched SLD from the beginning of the
experiment until day 35. Five days later, a
sharp decline was detected which coincided
with a marked increase in circulating LH (Fig.
4). At the last two sampling times, LH values
were similar to those recorded on day 35. In the
experimental rats, pituitary LH concentrations
displayed minimal variation from one five-day
interval to another. The one age at which there
was a deviation from this was on day 45 when
hormone values increased by 88%.In comparison to the control rats, pituitary LH differed
significantly only at 35 and 50 days of age.
The concentration of pituitary FSH in the
rats fed the SLD followed a similar pattern as
that described for serum FSH. Higher values
were present in the middle of the experiment
than a t either the onset or a t the termination of
the study. Aside from day 40 (P < 0.021, FSH
remained essentially unchanged in the malnourished rats. However, the FSH values in
these animals were consistently and, with one
exception, significantly different from those
measured in the controls.
During the first half of the experiment, PRL
rose by 338% in the pituitary glands of the
control animals. Thereafter, hormone levels
vacillated considerably. By day 50, they had
reached their peak value of 105.3 ? 18.7ng/mg.
Significant changes in pituitary PRL were not
measured in the protein deficient experimental
animals until after 40 days of age, at which
time a two-fold increase was observed. At three
of the six intervals studied between days 25 and
50, PRL concentrations were statistically different from the control values.
Serum testosterone
The levels of total testosterone in the sera of
the control and malnourished rats a r e
presented in Table 4. Hormone concentrations
were similar in the control animals until after
40 days of age. The values recorded on days 45
and 50 differed significantly from those measured a t 40 (P < 0.025) and 45 (P < 0.001)days of
age, respectively. Total testosterone was detectable only on days 25,45, and 50 in the sera
of the protein deficient rats. At the other ages
sampled, hormone levels were below the
minimum level of sensitivity for the radioimmunoassay. No statistical differences were
observed between the two animal groups that
were 25 days old, but the values measured on
days 45 and 50 were significantly greater in the
rats given the SLD.
347
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
Hypothalamic LH-releasing hormone
There was a general increase in the content
of LHRH in the control hypothalami as the
animals aged (Fig. 5 ) . In comparison, sharp
fluctuations were recorded in hypothalamic
LHRH in the malnourished rats; the only
highly significant change (P < 0.001) was a
71% rise between 45 and 50 days. A greater
amount of LHRH was consistently found after
day 25 in all animals fed the SLD. When these
results are expressed in terms of hormone concentration (Pg LHRH/mg hypothalamus), a
significant difference between the two animal
groups was detected from 30 to 45 days of age
(Fig. 5 ) .
DISCUSSION
In the present report, the reproductive axis of
the endocrine system was studied in malnourished male rats over a period in which
marked hormonal changes have been shown to
occur (Negro-Vilar et al., '73; Dohler and
Wuttke, '75) and when the onset of puberty was
expected. The observations represent the first
comprehensive analysis of the interrelationships between hormone secretion and nutrition
in sexually maturing animals. Previous investigators have focused their endocrine nutrition
studies almost entirely on the adult r a t
(Mulinos and Pomerantz, '40, '41; Srebnik and
Nelson, '62; Srebnik, '64, '70; Root and Russ,
'72; Howland and Skinner, '73; Howland, '75;
Campbell et al., '77).
The diet that was fed our experimental animals contained a similar amount of energy (4.5
Kcal per gram) as that present in the control
SLD (4.3 Kcal per gram), but provided 70.4%
less protein. Due to the differences in the
amounts of food consumed by the two groups of
rats, the malnourished animals also became
deficient in their daily intake of calories within
a very short period of time. The resultant effect
was a malnourished state precipitated by insufficient calorie and protein consumption. This
led to a dramatic decrease in the growth rate
and pattern of the developing animals. By 50
days of age and after having been fed the LPD
for 30 days, the weight of the malnourished rats
was less than one third that of the controls. In a
similar study, Tulp and coworkers ('79)
recorded a 730decrease in body weight in male
Fig. 5. Mean (t SE) LHRH content (top) and concentration (bottom) of hypothalami from 20 to 50 day old control
(solid circles) and malnourished (open circles) rats.StatisticaI comparisons were made as described in the legend for
Figure 4.
I
I
I
I
I
I
I
20 25 30 35 40 45 50
Age (days)
348
DAMON C. HERBERT
TABLE 3. Concentration of the pituitary gonodotrophins
LH
M
C
5.3
1.5"
4.7
f 0.6
30
f
2
7.5
1.0
5.7
12.0
4.3
43.6
f 8.0
t 0.5'
i 2.8
2 12.3
?
4.1
0.7'
t 1.0
?
5.9
0.4
?
9.5
0.8
?
2.4
0.7"
12.7
2.0
?
11.1
1.5
?
8.1
1.0
6.1
r 1.3
11.1
1.8
?
5.1
0.5"
?
7.7
0.5
f 0.6"
7.6
t
11.2
4.3
21.7
36.1
9.3
i 5.9
?
f 0.3
t 0.8
40
50
3.7
i 0.5e
t 1.7
5.9
0.5d
?
45
8.4
5.3
0.4
M
C
16.0
? 3.4t'
?
10.7
0.9
35
M
C
4.1
t 0.5"
25
PRL
FSH
20.3
4.7e
?
70.2
16.1
3.4e
?
23.4
27.4
3.7
t 3.2
2
61.7
49.9
2 8.5
t 7.8
105.3
45.9
5 18.7
f 9.6'
C = control rats fed the SLD
M = malnourished rats fed the LPD
'mean 2 SE, pgimg
%ghg
P < 0.02 (statistical comparisons were made between means from control and experimental animals at each time interval for each hormone)
dP < 0.005
( P < 0.001
(.
TABLE 4 . Total serum testosterone
Age
Control
rats
Malnourished
rats
20
0.74
t 0.08"
-
25
30
35
40
0.31
0.36
0.38
f 0.03
i 0.03
t 0.04
?
0.25
i
0.20
< 0.20
< 0.20
i 0.01
0.79
0.10
45
50
1.65
* 0.30
0.27
f 0.01b
2
3.42
0.77
0.27
2 0.02"
"mean 2 SE, nglml
bP< 0.001. Statistical comuarisons were made as described in Table 3.
rats fed a n 8%protein diet from 21 to 53 days of
age. Widdowson and McCance ('63) studied the
growth patterns of male rats that were underfed between three and six weeks of age. At the
end of their experimental period, they reported
that the malnourished rats were ". . . over 60
grams behind the well nourished ones and were
less than half their weight." In spite of their
reduced food intake, these animals grew at
what appeared to be a steady and rather constant rate over the three-week period (Widdowson and McCance, '63). This was not true for
rats fed a n LPD (Fig. 1;Tulp et al., '79).For the
first 15 days of our study, the total increase in
body weight for each rat was 6.2 grams, while
over the latter phase of the experiment, each
Figs. 6, 7. Sections of testes from a control (Fig. 6) and an experimental rat (Fig. 7) at 25 days of age. The number of
developing germ cells is less in the seminiferoustubules of the malnourished rat. In addition, many of these cells have become
detached from the Sertoli cells and appear "freefloating" individually or in clusters within the lumen of the tubules. x 200.
(Insert x 375.)
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
349
350
DAMON C. HERBERT
Fig. 8, 9. Sections of testes from a control (Fig. 8)and an experimental rat (Fig. 9) at 40 days of age. Maturation phase
spermatids are present in the control testis (asterisk)but are absent in the seminiferous tubules of the rats fed the LPD. In
these animals, the most mature spermatids were in the Golgi phase. x 200. (Insert x 300.)
PROTEIN-CALORIEMALNUTRITION IN W
E RATS
351
Fig. 10, 11. Sections of the testes from a control (Fig. 10) and an experimental rat (Fig. 11) at 50 days of age. Step 19
spermatids are only present in the seminiferous tubules of the control animal. X 200. (Insert x 300.)
352
DAMON C. HERBERT
rat gained 17.7 grams. These changes can be
attributed to the differences in eating habits in
each of the two halves of the 30-day study. A
significant increase was not recorded in the
amount of food, proteins, or calories consumed
between 20 and 35 days of age. In contrast, a
rather marked rise in all three parameters was
noted between 35 and 50 days of age. The factors controlling these changes are not fully understood.
The differences between the two animal
groups in the absolute weights of the pituitary
gland, gonads, ventral prostate gland, and the
liver were striking. All four organs were consistently smaller in the experimental rats. One
intriguing aspect was the rapid onset of the
malnourished state which was accompanied by
an initial decrease in all of the organ weights
recorded. With the exception of the ventral
prostate gland, there was a recovery from the
initial weight loss. The time span preceding the
onset of the recovery was the shortest in regard
to the liver weight and the longest for the
pituitary gland. At the end of the study, the
weight of the pituitary gland, gonads, and liver
was similar to that of normal rats that were 26
days of age or younger. The ventral prostate
gland was severely atrophic and weighed 32.6%
less than that measured for the 20 day-old controls.
The effect of protein deficiency on the hypothalamo-hypophyseal-gonadalaxis cannot be
ascribed solely to inadequate dietary proteins
and calories. The relative liver weight, which
was used as a n index to determine the general
systemic effects of the two diets, was similar in
both animal groups throughout a majority of
the study. Rather, the data collected from the
experimental animals suggest the existence of
multiple hormonal deficiencies which commenced almost immediately after the introduction of the LPD and were sustained as long as
the malnourished condition existed. The patterns of gonadotrophin secretion during the
30-day period between 20 and 50 days of age
were markedly different in the protein-calorie
malnourished rats as compared to normal animals of the same age. There was a n elevation in
LH, FSH, and PRL in the serum of the rats fed
the SLD between 20 and 40 days of age; thereafter, either no major changes transpired or, as
was the case with FSH, hormone levels decreased. Similar fluctuations in peripheral
gonadotrophins have been reported in the male
rat by Dohler and Wuttke ('75). Malnutrition,
on the other hand, caused a fall in serum FSH,
very pronounced vacillations in LH levels and
only a moderate increase in peripheral PRL
concentrations. By 50 days of age, only PRL
was found to be elevated above values measured a t the onset of the study. LH was 39%
lower and FSH had decreased by 5wo. As a
consequence, the circulating levels of the
gonadotrophins were too low to initiate and
maintain normal growth and development of
the gonads in the malnourished rats. The relative testicular weights, the diameter of the
seminiferous tubules, and the size of the interstitial cells of Leydig were all smaller. The
physiologic activity of these cells was severely
depressed which was reflected not only by lowered serum testosterone values but also by a
marked atrophy of the ventral prostate gland.
Ultimately, a condition of hypopituitarism
evolved which resulted in a failure of these
animals to become sexually mature.
Other investigators have found one or more
of the gonadotrophins, as well as testosterone,
to be affected by starvation (Grewal et al., '71;
Howland and Skinner, '73; Campbell et al., '771,
chronic underfeeding (Howland, '75; Stewart et
al., '75; Campbell et al., '771, or protein deprivation (Srebnik, '70). Glass et al. ('79) have recently reported that a 90protein-containing
diet fed to rats from 21 to 50 days of age failed to
significantly affect either serum LH or testosterone, but did reduce circulating FSH levels. They were able to measure a decrease in the
release of testosterone by gonads from malnourished rats that were cultured either alone
or in the presence of human chorionic gonadotrophin. The morphology of the seminiferous
tubules was normal in their animals, which is
in contrast t o our findings and those of Horn
('55). Glass and colleagues ('79) did note a n
effect of their diet on sex accessory organ
weights. The reason(s) for the discrepancies between the present report and that of Glass et al.
('79) is (are)unclear. It seems unlikely that one
can account for these differences solely on the
basis of the strain of animal studied [SpragueDawley (ours) vs. Wistar (Glass et al., '7911.
Another possible explanation may reside in the
percentage of protein within the two diets. The
additional 1% they employed may have exceeded a minimal threshold of dietary protein
needed to sustain "normal" reproductive function and to stimulate puberty to occur, even
though FSH levels were reduced and sex accessory organ weights were well below those found
in the controls.
The events controlling hormone synthesis
and its storage were not as markedly affected
by protein deficiency as were those regulating
PROTEIN-CALORIE MALNUTRITION IN MALE RATS
gonadotrophin release. The concentration of
pituitary LH was, in general, similar in both
animal groups. Pituitary PRL, on the other
hand, tended to be elevated in the controls
while FSH was, with one exception, significantly lower in the experimental rats. Other
forms of malnutrition have no effect upon
pituitary LH, FSH, and PRL (Meites and Reed,
'49; Howland, '79, or have been reported to
reduce (Meites and Reed, '49; Srebnik and Nelson, '62; Nakanishi et al., '76) or even elevate
(Root and Russ, '72; Howland, '75; Nakanishi,
'76) pituitary LH, FSH and PRL. Likewise, a
variable pattern of LH and FSH secretion by
the anterior pituitary gland of malnourished
rats was observed in animals challenged by
provocative stimuli, such as hemigonadectomy
(Stewart et al., '73, '75) and total castration
(Srebnik, '70; Root and Russ, '72; Howland and
Skinner, '73; Glass et al., '79) or LHRH administration (Root and Duckett, '75; Glass et al.,
'79). In some instances, the response was
greater than that observed in the controltreated animals (Root and Russ, '72; Stewart et
al., '75; Glass et al., '791, suggesting that while
sufficient levels of gonadotrophins were contained within the pituitary gland which could
be released in response to a given challenge,
inadequate input from the hypothalamus
existed, which over a prolonged period of time,
could not maintain normal pituitary function.
Our findings of reduced LHRH in the hypothalami of the rats fed the LPD would support
this premise. Chronic underfeeding (Piacsek
and Meites, '67) and starvation (Negro-Vilar et
al., '71) also reduced bioassayable LHRH (Piacsek and Meites, '67) and FSH-RF (Negro-Vilar
et al., '71); however, hypothalamic LHRH, a s
measured by radioimmunoassay, was unchanged in rats deprived of food for seven days
(Root et al., '75). The question then arises as t o
why the hypothalamo-hypophyseal-gonadal
axis does not give a "castration-response" in the
protein-deficient rat. The answer may in part
be related to the extrahypothalamic control
over reproductive physiology. Pineal gland activity seems to be enhanced by underfeeding
(Piacsek and Meites, '67; Sorrentino et al., '71;
Walker and Bethea, '77; Walker and Frawley,
'77). Exposing malnourished rats to constant
light (Piacsek and Meites, '67) or pinealectomizing blinded underfed animals (Sorrentino
et al., '71) increased gonadal and sex accessory
organ weight. Moreover, a greater compensatory ovarian hypertrophy response (Walker
and Bethea. '77) and higher serum LH values
(Walker and Frawley, '75) were recorded in rats
353
that were chronically underfed and exposed to
constant light or pinealectomized than were
found in malnourished, untreated animals.
These findings suggest that the putative antigonadotrophic hormone(s) secreted by the
pineal gland is (are) exerting a greater regulatory effect over the hypothalamus, pituitary
gland, and gonads than what generally occurs
in the normal, well-fed animal.
ACKNOWLEDGMENTS
The author wishes to thank Donna L. Kenneally for her expert technical assistance, Dr.
T. Siler-Khodr and the members of the Radioimmunoassay Core of the Center for Research
in Reproductive Biology for conducting some of
the radioimmunoassays, and the NIAMDD for
generously supplying the gonadotrophin radioimmunoassay kits,
This work was supported by NIH grants
HD10914 and P30 HD10202.
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