The difference of response of the pituitary glands of male and female albino rats treated with the growth hormone.

THE IUIFFEBENCE OE’ BESPONXE O F THE PITUITARY GLAKDS O F MALE AND FEMALli:
ALBISO RATS TREATED WITH
THE GROWTH HORMONE
EI. S. RUBINSTEIN
Nezcro-A~~atomzcal
Laboratory, Department of Anatomy, Cniversity o f Maryland
X e d i c a l School
I n the course of studies involving the growth hormone, it
was noted that the pituitary glands of artificially enlarged
animals showed a distinct reactive difference depending upon
the sex of the animal, This difference was so striking and
interesting that the data were subjected to statistical treatment in order to test their significance. Because in the albino
rat differences in pituitary weight occur normally between the
sexes, it was deemed advisable to compare the end result with
the normal and t o report the findings which appeared to be
significant.
For this study seventy-four albino rats (Mus norvegicusvar. albinus) from seventeen litters were divided into two
large groups, according to s e x Each group was further subdivided into three subgroups, namely, test animals and two
sets of controls. The test subgroup was composed of the
lightest animals of each litter while the heaviest animals were
inclnded in the control subgroup which remained uninjected.
I n addition, animals intermediate in weight were placed in the
control subgroup which ~ 7 a sto receive a non-specific meat
extract. T h e n thus divided the seventy-€our animals were
arranged as shown in column 1 of table 1. All animals were
kept under laboratory conditions similar to those previously
described ( Rubinstein, ’32).
The differences in initial body weight may also be seen in
table 1, from which it may be noticed that both the test ani131
132
H. S. RUBINSTEIS
mals and the meat injected controls were significantly lighter
than the uninjected controls, although, as may be judged from
the critical ratios,1 this significance was greater f o r the test
animals.
At the beginning of the experiment all animals had already
reached the adult state, being 6 to 8 months of age. They
were thus chosen because it had previously been found that
rats growing normally in the early growth period failed to
respond markedly to the added growth hormone (Rubinstein,
'33).
TABLE 1
Slaowmg the grouping of the animals and the tnitial body weights for all groups
MEAN I N I T I A L
BODY WEIQHT
3 1 aiiimals
Males :
11 test animals
10 uiiiiijected controls
10 meat injeeted eontrols
247.9 & 3.32
278.7 k 3.30
266.1 If: 3.32
43 aiiimals
Females :
16 test aiiimals
1 2 uniqjeeted coiitrols
16 meat injected controls
183.3 k 2.30
201.5 f 2.60
190.2 f 1.58
DEVIATION FROM
UNTNJECTED
COXTROI;
CRITICAL
RATIO
- 30.8 2 4.7
6.56
- 12.6 f 4.7
2.68
- 18.2 f 3.4
5.35
- 11.3 k 3.1
3.64
The test animals mere injected intraperitoneally daily (except Sunday) with 2 ec. of growth hormone prepared as an
aqueous alkaline extract from the anterior lobe of fresh beef
pituitaries. This extract was similar to that employed in
other growth studies, and the details of its preparation have
been reported elsewhere (Rubinstein and Fox, '34).
The injections were continued for 22 weelis, but active differential growth response was obtained only after the ninth
week because of the impotence of the extract used during the
'By critical ratio is meant t h a t figure which results froin dividing the differeiice between the coiitrol aiid test observations by the probable error of this
difference. A critiral ratio of 3 or more has been coiisiderec? as evideiice o f a
significant difference.
DIFFERENTIAL BESPONSE O F PITUITAEY
133
earlier period of the experiment. The meat extract used was
prepared exactly as the pituitary extract and was merely employed to prove that the growth response noted in the test
animals was not due to a food factor but represented a specific
reaction t o the growth stimulating factor of the pituitary
gland.
At the end of the experiment all animals were anesthetized
with ether and sacrificed by bleeding through the carotid
arteries. This method of exitns was used because it had
been found advantageous in other experiments (Rubinstein,
'32) where a minimal quantity of blood was desired in ihe
TABLE
2
Showing t h e mean final body weights f o r all grou,ps
N E A N FINAL
BODY W E I G H T
D ETI A TI ON FROM
UNINJECTED
CONTROL
CRITICAL
RATIO
gm.
Males :
Test animals
Uiiiiijeeted controls
Meat iiijccted controls
365.7 f 3.98
322.6 k 4.43
309.7 & 4.50
Females :
Test animals
TJniiijected controls
Meat injected controls
296.1 & 4.48
220.4 f 3.18
208.9 f 1.97
43.1 t 5.92
7.3
- 12.9 f 6.31
2.1
75.7 -t- 5.48
13.8
- 11.5 f 3.74
3.1
tissue to be studied. The skulls were opened from above and
the brains removed. The pituitary glands were then recovered from the sclla turcica and immediately weighed in a
weighing bottle to within a tenth of a milligram.
RESULTS
A t the end of the experiment all tesl anirrials had become
definitely heavier than their two sets of controls, as may be
judged from table 2 where it may be noted that while the
initial difference between the two sets of controls remained
the same (compare with table 1)at the end of the experiment,
a marked change had occurred between the test animals and
134
H. S. RUBIFSTEIN
both controls. The unchanged differences and critical ratios
of the control groups suggest a parallelism in the slope of
the growth curve for these groups and, indeed, when plotted
out such was found to be the case. On the other hand, the
change in the sign of the deviation from a minus to a plus
for the test animals shows how much larger the slopes of their
growth curpes had become. The significance of this difference may be noted from the critical ratios of this table (2).
These differences in growth may be more immediately appreciated from table 3, which gives the average body weight
changes f o r all groups during the course of the experiment.
TABLE 3
Showing the mean body wright increases for all groups
MEA?S QAIW
IN GRAMS
Males :
Test animals
Uuiiijected controls
Meat injected controls
117.80 f4.87
43.90 e 4.10
43.56 -t- 3.66
Females :
Test animals
Uniiijected controls
Meat injected controls
113.5 f 4.49
18.9 f 1 . 9 i
18.7 2 1.33
D E V I A T I O N FROM
UNJNJECTED
CONTROL
CRITIC.4L
RATIO
-+ 6.40
11.53
- 0.34 k 5.48
0.06
94.6
+- 4.89
19.34
- 0.20
2 2.38
0.84
73.9
It may be noted that while the male and female test animals
gained about 74 and 95 gm. over their respective controls,
the differences noted between the controls are insignificant.
The larger gain on the part of the test females is not mere
chance since it has been consistently noted in all growth experiments in this and other laboratories ( Rubinstein, '32 ;
Rubinstein and Kolodner, '34; Johnson and Hill, '30, in the
mouse).
In addition to the increase noted in body weight, measurements taken of body and tail lengths showed a significant
increase in length of the test animals as compared to their
controls (Rubinstein, '34). The linear measurerncnts were
deemed advisable since it had been previously shown (Downs
135
DIFFERENTIAL RESPONSE O F PITUITARY
and Geiling, '29) that some of the increase in body weight
(about 8 per cent) was duc to the accumulation of water.
Table 4 presents these results quantitatively. Incidently, it
may be noted from this table that the tail lengths of the
meat injected controls mere significantly shorter than those of
the uninjected controls. At any rate, the total length of the
test group was definitely increased.
A stndy of the pituitary weights R S shown in table 5 disclosed that these averaged 0.0033 gm. heavier in the male test
animals than in the uninjected controls. The difference beTABLE 4
Showing meon final body and tail lengths for all groups
MEAN FTNATi
BODY LENGITII
DETIATION
FROM
CONTROT,
2RITICAL
R-4TIO
cm.
Males :
T e s t animals 23.1 2 0.11
Uniiijectcd
21.8 f 0.08
controls
Meat injected
controls
21.6 f 0.16
Females :
Test animals 20.9 f 0.18
Uninjected
controls
19.5 f 0.15
Meat injected
co~itrols
19.2 f 0.05
DXVIATIOII
XEAN F I F A L
FROM
CONTROL
TAIL LENGTH
2RITICAD
RATIO
em.
1.3 & 0.14
9.30
0.3 f 0.14
19.0 f 0.13
18.7
2.86
t 0.04
- 0.2 2 0.18
1.11
18.6 & 0.04
1.4 2 0.23
6.10
18.9 2 0.05
- 0.1
0.06
1.66
1.0 f 0.11
9.10
0.7 f 0.11
6.36
Z?
17.9 k 0.10
- 0.3 f 0.16
1.88
17.2 k 0.04
-
tween the two control groups was insignificant. I n the females, on the other hand, the test animals possessed pituitary
glands which averaged 0.0003 gm. below those of the nninjected controls. This difference, as shown by the small
critical ratio (0.43) is insignificant.
Howcver, when the pituitary glands are considered between
the two sets of female controls it may be noted that the difference is 0.0021 gm. in favor of the uninjected controls.
0.00073
This difference divided by its probable error of
leads to the significant ratio of 2.88 (almost 3), SO that the
difference is significant.
136
H. S. RUBINSTEIN
I n order to better appreciate the relations of the initial
and final body weights t o the pituitary weights, table 6 has
been constructed in which these are tabulated together with
TABLE 5
Showing the mean pituitary weight for all groups. A f t e r 40 to 50 days the
f e n a l e hypophysis norrtmlly becomes heavoer than the males
and the diferences gradually increase
DEVIATION FROBS
UNINJECl’SD
CONTROL
MEAN PITUITARY
WEIGHT
CRIT,OAIi
RATIO
gm.
Males :
Test animals
Uniiijected eoiitroh
Meat injected controls
0.0134 f 0.00041
0.0101
0.00022
0.0102 f 0.00037
*
0.0033 -C 0.00047
7.03
0.0001 t 0.00043
0.23
- 0.0003 & 0.00069
0.43
- 0.0021
2.88
Females :
% 0.00073
TABLE 6
Showing the relations of the initial and final body weights t o the observed and
tabular pituitary weights. The observed pittiitary weights correspond t o
the final body weights while the tabular pituitary weights are t a k m
from Donaldson’s table 148 and are tabulated t o correspond with
the initial and final body weights. T h i s table 6 seems t o denote
a retardation in the growth of the hypophysis of the test
female and also suggests a retardation i n the meat
injected female
~
BODY W E I G H T I N GRAMS
PITUITARY W E I G H T I N GRAMS
Table 148 (Donaldson)
Initial
Final
_
_
Observed
_
~
Males :
Test animals
Uidnjected coiitrols
Meat injected controls
247
278
266
366
323
310
0.0134
0.0101
0.0102
0.0097
0.0094
Females :
Test animals
Uiiiiijected controls
Meat injected controls
153
201
190
296
220
209
0.0152
0.0155
0.0134
0.0124
0.0139
0.0131
;::::,0
~
0.0212
0.0163
0.0145
DIFFERENTIAL EESPONSE O F PITUITARY
137
€he approximate tabular pituitary weights corresponding to
these body weights. The tabular pituitary weights were taken
from table 148 of Donaldson’s ‘The Rat.’ From this table it
may be seen that the observed mean pituitary weight of the
male test animals (0,0134) is definitely heavier than that
which is computed as normal, (0.0122) while the observed
pituitary weights of the two sets of controls are below the
corresponding final tabular weights.
I n the female groups, the observed pituitary weights of the
test and meat injected animals appear to be retarded in comparison with the final tabular normals. The mean observed
pituitary weight of the uninjected controls (0.0155) agrees
quite closely with that considered normal (0.0153) for the
observed final body weight.
DISCUSSION
The pituitary gland of the albino rat for the first 40 or
50 days of life has the same weight in the two sexes. Thereafter, the hypophysis of the female becomes progressively
heavier than that of the male so that the difference becomes
more and more marked until the plateau of the growth curve
is reached. At this time (i.e., at like ages) the pituitary
gland of the female is 4 or 5 mg. heavier than that of the male
(Donaldson, ’24). That the males of this series compare
favorably with these accepted normals may be seen from
table 5 from which it may be noted that the hypophysis of
the uninjected control females average 5.4 mg. more than that
of the males.
Whatever the cause for this relatively heavy pituitary gland
normal for the female albino rat is, it can only be said at
present that it is characteristic for the female albino rat.
It does not exist in the wild Norway rat (Mus norvegicns).
Noreover, it cannot be correlated purely with albinism since
it is absent in the female albino rabbit and guinea pig.
Hence, at present this phenomenon cannot be explained,
although since Donaldson holds the ‘very heavy hypophvsis
in the female’ to be the only outstanding characteristic of the
albino rat as an albino-it may be a form of mutation.
138
H. S. RLZBINSTEIN
It is of striking interest that the normally smaller pituitary gland of the male, the sex which responds less to the
added growth hormone, reacts under test conditions by becoming heavier. On the other hand, the naturally heavier
pituitary gland of the normal female, the sex which is more
responsive to growth hormone, fails to increase in size and
in table 6 appears to be retarded in its growth.
Although table 6 shows pituitary weights which are relatively enlarged in the male test animals and retarded in the
female test animals and female meat injected animals, table
5 which treats any differences within the observed strain on
a statistical basis, discloses the fact that these differences
are probably only significant for the male test animals and
the female meat injected controls. Even in the latter group,
however, the retardation may not be as significant as it first
appears. This statement is based on the fact that, as shown
in table 2, the significantly smaller meat injected control, may
be expected to possess a significantly smaller hypophysis,
since normally, pituitary and body weights show a high degree
of correlation.
Hence, one may conclude from the above that while the
pituitary glands of the test males became quite large by thc
treatment described, those of the females remained unaltered.
The differential response of the glands in the tu7o sexes to
similar treatment is quite interesting and has been previously
noted in other glands under other experimental conditions.
Thus Donaldson (’34) found in his studies on exercise that
the thymus became much reduced in the male, not in the
female; and the suprarenals became “very large in the female
and only slightly affected in the male.”
So f a r as experiments carried out with the growth hormone
are concerned, it is perhaps interesting t o reflect upon the
possible significance of this difference in sex response. It
may be that the male albino rat which is growing a t a rapid
rate normally as a result of maximum growth efficiency of
the pituitary gland can undergo supergrowth only if his
pituitary gland is enlarged. I f this is true then the pituitary
DIFFEREFTIAL RESPONSE OF PITUITARY
139
gland of the tcst male probably became enlarged through some
indirect proccss since there is some evidence at hand (Smith
and MacDowcll, '30 ; Collip, '34) which dcmonslrates that
endocrine products fail t o directly affect the glands from
which they arise. On the other hand, it appears that the
normally large pituitary gland of the female albino rat is
normally relatively less productive of growth hormone than
that of the male for which reason the female responds t o
added growth hormone with a greater degree of growth than
does the male.
The reason f o r the larger pituitary gland in the female
is still enigmatic and will only find explanation in the light
of further research.
While the above suggestions are merely specnlative, not
having been proved at this time, it is safe to say that the
growth hormone, so f a r as the pituitary size is concerned,
tends to restore the predomesticated condition.
CONCLUSIONS
1. The pituitary glands of male and female albino rats
respond differently to the administration of the growth
hormone.
2. I n the adult male, which normally has a smaller pituitary gland than the female, intra-peritoneal administration
of the growth hormone enhances body growth and significantly increases the weight of the pituitary gland.
3. I n the adult female in whom the pituitary gland is
naturally heavier than that of the male, the growth hormone,
similarly administered, leads to a more marked body weight
increase than that produced in the male. The pituitary glands
of these experimentally enlarged females, however, fail t o
increase in weight aiid are retarded in growth.
4. The probable explanations of these phenomena are discussed but their conjectural status f o r the time being is
stressed.
140
H. S. RUBINSTEIN
ACKNOWLEDGMENTS
The author is greatly indebted to Prof. Carl L. Davis, head
of this department, who has so generously offered his advice
and cooperation throughout this work and to Dr. Henry H.
Donaldson f o r his valuable suggestions.
L I T E R A T U R E CITED
COLLIP,J. B. 1934 Some recent advaiiecs i n the physiology of the anterior
pituitary. J. Mt. Sinai Hosp., 1701. 1 , p. 28.
DONALDSON,
H. II. 1924 The rat. Memoirs of The Wistar Institute of Siiatorny
and Biology No. 6. Philadelphia Pa.
DONALDSON,
H. H. 1934 Persoual communication.
DOWNS,W. G., AND E. M. K. GEILING 1929 Possible water balance: The effects
of alkaline anterior pituitary extracts. Proc. SOC. Exp. Biol. mid
Me&, vol. 27, p. 63.
JOHNSON,G. E., AND R. T. HILL 1930 The effect of anterior pituitary extraet
011 the developing albino mouse. Eiidocrinology, vol. 14, p. 400.
RUBINSTEIN,
H. 8. 1932 The effect of the growth hormone on the brain weightbody weight ratio. Auat. Rec., 1701. 53, p. 265.
1933 The effect of the growth hormone on the early g r o v t h period
of the albino rat. Bull. Sch. of Med., U. of Md., vol. 17, p. 163.
1934 The effcct of the growth hormone upon the tail length-body
leiigtli ratio of the albino rat. Bull. Sch. of Med., U. of Md., vol.
18, p. 131.
RUBINSTEIN,
H. S., AND L. 31. Fox 1934 The water and solid contents of the
brains of albino rats treated with t h e growth hormone. J. Comp.
Neur., vol. 60, p. 349.
RUBINSTEIN,
H. S.,AND L. J. KOLODNER1934 The effect of the growth hormone
011 body and tail lengths. Anat. Rec., vol. 58, p. 107.
SMITH, P. E., AND E. A. MACDOWELL1930 On hereditary anterior pituitary
deficiency in the mouse. Anat Rec., vol. 40, p. 249.