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TH ESIS

 

 

STUDIES ON THE CONTROL OF GROWTH HORMONE SECRETION
IN THE RAT

ABSTRACT

By Elias Dickerman

I. IN VITRO ASSAY FOR GROWTH HORMONE RELEASING
FACT—‘OR-TGH-RF)

A four hour incubation method was developed for
assaying rat hypothalamic growth hormone releasing
factor (GR-RF). Anterior pituitaries from donor rats
were hemisected and distributed randomly into flasks
containing 4 ml of protein-free medium 199. Six
pituitaries (l2 halves) were used per flask. The
pituitaries were pre-incubated for 1 hour, the
medium was discarded and fresh medium containing
neutralized acid extract of rat hypothalamus or
cerebrum was added. These were incubated for 4
hours under constant gassing with 95% 02-5% CO2, and
the medium was analyzed for GH by the standard tibia
test.

A log-dose relationship was demonstrated when
increasing amounts of hypothalamic extract were
incubated with rat pituitary. Graded doses of
cerebral extract failed to demonstrate such a relation-
ship, indicating that GH-RF exists as a specific

hormone in the hypothalamus. Analysis of pituitary

Elias Dickerman

incubation medium at two dose levels showed log-
dose relationships in all cases, indicating that the
hormone assayed in the medium was GH. This method
is suitable for measuring quantitative changes in
hypothalamic content of GH-RF.
II. EFFECTS OF STARVATION ON PLASMA GH ACTIVITY,

PITUITARY GH, AND HYPOTHALAMIC GH-RF LEVELS

IN THE RAT

The effects of complete food removal for 7 days
were measured on plasma GH activity, hypothalamic
content of growth hormone releasing factor (GH-RF) and
pituitary concentration of growth hormone (GH) in male
rats. Control male rats were starved or fed ad
libitum for 7 days. In another experiment, the
starved rats received replacement doses of L-Na-
thyroxine (2.5 ug/lOO g body weight/day) beginning
on day 2 of starvation. On day 8 all rats were weighed,
anesthetized with ether and bled from the abdominal
aorta. The pituitaries were removed and individually
weighed; the hypothalami were placed in ice cold
O.lN HCl (0.1 ml/hypothalamus). Hypothalamic GH-RF
was assayed by the in_yit§2_incubation method
described above. Lyophylized plasma, anterior
pituitaries, and incubation medium were assayed for
GH by the standard tibia test, using a 4 point assay
procedure. Statistical analysis of the data showed

a significantly lower plasma GH content in starved or

Elias Dickerman

thyroxine-treated starved rats (P<0.0l). Pituitary

GH content and hypothalamic content of GH-RF were

also significantly reduced in the starved rats. These
results indicate that starvation in the rat not

only lowers the levels of GH-RF in the hypothalamus
and of GH in the pituitary, as shown previously,

but also leads to a decrease in plasma GH activity.

STUDIES ON THE CONTROL OF GROWTH HORMONE SECRETION
IN THE RAT

BY

Elias Dickerman

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Physiology

1968

5;. 534; 4/0
.J/CVVéI'

Dedicated
to

my Family

ACKNOWLEDGMENTS

The author wishes to express his gratitude to Dr.
Joseph Meites, Professor of Physiology, for his instruc-
tion and inspiration throughout the course of this study.
Without his patient guidance this manuscript would not
have been possible. Appreciation is eXpressed to Dr.
Andres Negro-Vilar for the informative discussions in—
curred during the course of this study. An expression
of thanks is also extended to the members of the guidance
committee, Drs. W. D. Collings, H. D. Hafs, G. D. Riegle,
and H. A. Tucker for their help in the preparation of

this manuscript.

Special indebtedness is due to Michigan State
University and Dr. J. Meites for the financial assis-
tance during this project. Purified ovine GH was kindly
supplied by the Endocrinology Study Section, National

Institutes of Health, Bethesda, Maryland.

A note of thanks is extended to Mrs. JOan Heaphy
for her kind help in preparing this manuscript. Finally,
a special thanks to my wife, Katherine, and my daughter,
Beyle Sue, for their inspiration, understanding and patience

during the time spent in this study.

iii

   

 

TABLE OF CONTENTS

Page
INTRODUCTION 0 O O 0 O O O O O O O O O O O O O O O O O O O O I O O O O I O O O O O 1
REVIEW OF TI-iE LITERATURE I O O O O O I O O O O O O O O O I O O O O O O 3

I. Nervous Control of Anterior Pituitary
(AP) GH secretionOOOOOOOOOOOOO0.0..0...O 3

A. Body Growth as 3 Parameter of CNS
Control of AP Secretion of GH ...... 3

B. Anterior Pituitary Content or
Release of GH as a Function of

CNS contrOl OOOOOOOOOOOOOOOOOOOOOOO.

II. Factors Affecting GH Secretion .........

A. Environmental Factors ..............

\OCDOD-Q

B. Hormonal Factors ...................

C. Food Intake ........................ lO
EXPERIMENTAL METHODS AND MATERIALS ............. 12
I. Animals ................................ 12

A. Experimental Animals ............... 12

B. Donor Animals ...................... 12

C. Bioassay Animals ................... 12

II. Pre aration of Hypothalamic Extracts
(HE , Pituitaries and Plasma ........... 13

A. Preparation of Hypothalamic
EXtraCtS (HE) 0....000....0...0.0..0 13

B. Preparation of Pituitary Tissue
for Assay .0.00000COOOOOOOOOOOOOOOOO 13

C. Preparation of Plasma for Assay .... 14

III. Incubation 000......OOOOOOOOOOOOOOOOO... 14

iv

Page
IV. GH Bioassay and Statistical Treatment .... 15
EXPERIMENTAL 000......OOOOOOOOOOOOOOOOOOOOOO0.0... 16

I. IN VITRO ASSAY FOR GROWTH HORMONE
RELEASINGFACTOR (GH‘RF) .0.000.00.000.000 16

A. ObjeCtiveS 000.00....OOOOOOOOOOOOOOOOO 16
B. Procedures ........................... 16
CO ReSUltS OOOOOOOOOOOOO0.000000000000000 1?
1. Dose response relationship
between hypothalamic extract and
anterior pituitary (AP) GH
release in vitro ................. l7
2. Comparison of cerebral versus
hypothalamic extract on AP
release of GH OOOOOOOOOOOOOOOOOOOO l7
3. Dose response to medium from
AP incubated with cerebral extract
or medium 199 alone .............. 20
4. Dose response to medium from
AP incubated with cerebral or
hypothalamic extract ............. 23
DO DisCUS81on 0.0.0.0...OOOOOOOOOOOOOOOOO 23
II. EFFECTS OF STARVATION ON PLASMA GH ACTIVITY,
PITUITARY GH, AND HYPOTHALAMIC GH-RF
LEVELS INTI-E RAT OOOOOOOOOOOOOCOOOOOOOOOO 31
A. ObJeCtiveS O0..OOOOOOOOOOOOOOOOOOOOOOO 31

B. Procedures OOOOOOOOOOOOOOOOOOOOOOOO... 31
C. Results 0....OOOOOOOOOO0.0.0.0.0000... 32

1. Plasma GH activity of control
and starved rats ................. 32

2. Effects of starvation and T4 on
plasma GH activity ................ 32

3. Effects of starvation on ,
pituitary GH content ............. 3b

Page

4. Effects of starvation on
hypOthalamiC GH‘RF ...0.00...0.0.0 38

D. DiscuSSion 00.000.000.000...000000.000 38
CONCLUSIONS 000000 ...00......0...0.000..000.000... L*5

REFERENCES .0000.0.00.00.00.0000000.000.000.000.00 46

vi

LIST OF TABLES

Table Page

I. Dose Response Relationship Between
Hypothalamic Extract and Anterior Pituitary
GH ReleaselaVitr00000000000000.000000.00000 18

II. Comparison of Cerebral Extract Versus
Hypothalamic Extract on Anterior Pituitary
Release of GH....0000000000000000000.0000.000 21

-III. Dose Response to Medium from AP Incubated
- with Cerebral Extract or Medium 199 Alone.... 2A

IV. Dose Response to Medium from AP Incubated
with Cerebral or Hypothalamic Extract........ 26

V. Plasma GH Activity of Control and Starved

Rats......................................... 33

VI. Effects of Starvation and T4 on Plasma GH
ACtiVity0000000000000000000000000000.0000.000 35

VII. Effects of Starvation on Relative Pituitary
GH Concentration...000......0000000..0.0..0.. 37

VIII. Effects of Starvation on Hypothalamic
GH-Rf‘ Content...00.000.00.0000...0000000000.. 39

vii

Figure
1.

LIST OF FIGURES

Logarithmic Dose-Response Curve Showing
Pituitary GH Release as a ReSponse to

Hypothalamic Extract......................-

Logarithmic DoseeResponse Curves Showing
Pituitary GH Release as a Response to
Hypothalamic or Cerebral Extract...........

Logarithmic Dose-Response Curves to
Medium from AP Incubated with Cerebral
Extract or Medium 199 Alone................

Logarithmic Dose-Response Curves to
Medium from AP Incubated with Hypothalamic
or Cerebral Extract........................

Logarithmic Dose-Response Curves to Plasma

from Control or Starved Rats. Standard
Curve EXtrapOJ-ated...00.0....0.0..0..000000

viii

Page

19

22

25

27

34

INTRODUCTION

 

The nervous and endocrine systems are responsible
for the integration and coordination of the remaining
systems of the body. The relationship between these
two controlling systems is close and intricate, and
only in the last 10-15 years has information clarified
this relationship.

It is now known that neuroendocrine mechanisms
regulate a wide variety of body functions. The
brain is responsible for control of secretions from
the anterior and posterior pituitary as well as from
the intermediate lobe of the pituitary. Through
hypothalamic control of anterior pituitary secretion,
the brain regulates thyroid, adrenocortical and
gonadal secretions as well as body growth. The
control of anterior pituitary secretions is
exerted through releasing and inhibiting factors
(hormones) in the hypothalamus: corticotropin releasing
factor (CRF), thyrotropin releasing factor (TRF),
follicle stimulating hormone releasing factor (FSH-RF),
luteinizing hormone releasing factor (LRF),
prolactin inhibiting factor (PIF), growth hormone
releasing factor (GH-RF) and possibly a GH inhibiting
factor (GH-IF). Thus, the classical concept of the
pituitary as the master gland of the body needs to be

revised to include a brain-pituitary relationship.

1.

2.
Evidence for a physiological role for GH—RF has

been slow to accumulate. This reflects, primarily,
the lack of good and non-cumbersome methods for the
measure of the hypothalamic releasing factor (GH-RF)
as well as a lack of a sensitive method for the
estimation of GH in the body fluids.

It was of interest, therefore, to attempt to develop
a short term incubation method for assaying rat
hypothalamic GH-RF which would show log-dose reSponse
characteristics and would be sensitive enough to detect
quantitative changes in hypothalamic content of
GH-RF. This study was also concerned with showing
the effects of starvation on plasma GH activity,
pituitary GH and GH—RF levels in the rat, in an
attempt to correlate the previously reported observations
of decreased pituitary content of GH and hypothalamic
GH-RF with plasma GH levels.

REVIEW OF THE LITERATURE

 

I. Nervous Control of Anterior Pituitary(AP)GH
Secretion

 

Although the release of GH from the anterior
pituitary is generally thought to be under hypothalamic
influence, the dependence of GH secretion on the
central nervous system (CNS) has been difficult to
elucidate because of a unique characteristic of GH.
Growth hormone does not have a Specific target organ
nor does it stimulate a gland to secrete hormones, as
is the case with FSH, LH, TSH and ACTH. Growth
hormone acts on general growth processes of the body,
and these include important influence on protein, fat
and carbohydrate metabolism. However, despite this
difficulty, several methods have been used to study
the influence of the CNS on AP secretion of GH. These
methods include one of two categories: (a) those
that measure body growth, (b) those that measure
pituitary GH content or release of GH from the
pituitary. Several methods in each category are
considered below.

A. Body Growth as a Parameter of CNS Control
of AP Secretion of GH

 

 

The description of the hypophyseal portal system
by Popa and Fielding (1933), and the observations by

Houssay gt, Q}, (1935), Wislocki and co-workers

3.

4.
(1936, 1937, 1938) and by Green and Harris (1949) of
the correct direction of blood flow, from the hypo-
thalamus down to the pituitary, led to the suggestion
by Green and Harris (1949) that humoral agents are
secreted into these hypophyseal portal veins and
travel to the pituitary and there exert a regulatory
influence on the secretion of anterior pituitary
hormones. The indirect approaches to answer this
problem consisted in the elimination of hypothalamic
influence on the AP by pituitary stalk section or
pituitary transplants, and by electrical lesions
or stimulation of brain tissue.

Pituitary stalk section in rabbits did not
inhibit growth according to Westman and Jacobsohn
(1940), although it induced marked gonadal atrophy.
A temporary growth delay was observed by Uotila (1939)
in rats following stalk section. Pituitary stalk
section in goats by Daniel and Prichard (1964)
produced severe metabolic disturbances as well as
blunted growth of body and pituitary target organs
during the first few months after Operation. These
observations, rather inconclusive, were explained by
Harris (1950) to be the result of partial sectioning
of the stalk or as a consequence of portal system
regeneration.

The observations on brain lesions are perhaps

better substantiated. Clinically, growth disturbances

5.

due to brain tumors have been known for many years
(Armstrong and Durh, 1922). Cahane and Cahane (1938)
.injured the hypothalamic area in rats and observed
a reduction in body growth. They suggested a possible
role for the nervous system in controlling GH
secretion. However, difficulties arise in the
interpretation of the results obtained. Where
extensive lesions are involved, the possibility of
involving the areas controlling the secretion of the
other AP hormones cannot be excluded. Cerebral
lesions may also interfere with food intake and
temperature regulation (Bogdanove and Lipner, 1952;
Bernardis 23. 31., 1963). Reichlin (1960 a) showed
that lesions of the median eminence and primary
portal plexus of the stalk significantly reduced the
growth rate of rats. Taking into account the
possibility of altered secretions of the remaining AP
hormones, Reichlin (1960 b) in a subsequent study
injected vasopressin, testosterone and thyroxine to
the injured animals. He also used pair-fed controls
to exclude the effect of differences in food intake.
He found that body growth was not restored to normal
following this treatment.

In a more elegant study, where pituitary GH
concentration was measured in injured animals,
Reichlin (1961) showed that GH content in injured

animals was reduced to 15% of that found in non-injured

6.
animals, when measured by the standard tibia test
of Greenspan gt, g1, (1949). These results (Reichlin
1960 a, 1960 b, 1961) and those of Hinton and
Stevenson (1962), Each gt, g}, (1964) and Endroczi gt,
g}, (1956) suggest that lesions involving the anterior
hypothalamus were most effective in retarding growth.
This region comprises the area bounded by the
supraoptic nucleus, the anterior half of the median
eminence and the arcuate nuclei. O'Brien gt, g1. (1964)
found that electrical stimulation of the paraventricular
nuclei of weanling kittens caused acceleration of
growth as measured by body weight and tibial length.

In general, pituitary transplants to hypophy-
sectomized rats have been able to maintain some degree
of growth (Greep, 1936; Martini and de P011, 1956;
Goldberg and Knobil, 1957). Pituitary transplants
were made in different areas with lesser or greater
success: the anterior chamber of the eye (Goldberg
and Knobil, 1957), the kidney capsule (Hertz, 1959),
the abdominal cavity (Swelheim and Wolthius, 1962),
or the subcutaneous tissues (Meites and Kragt, 1964).
The differences in degree of success in part may be
related to the site of transplantation (Halasz gt, g1,,
1962, 1963; Nikitovitch-Winer and Everett, 1958) as well
as to the age of the pituitary donor (Meites gt, gl,,

1962), the delay in transplantation after hypophysectomy

7.
(Smith, 1961), the amount of viable anterior pituitary
tissue present in the grafts, and to post-graft
immunological factors.

B. Anterior Pituitary Content or Release of GH
as a Function of CNSPControl

 

The direct measurement of pituitary GH content
in animals injected with median eminence extracts,
as well as the measurement of pituitary content and
release of GH into medium after incubation of pituitary
tissue with median eminence extracts, constitutes the
best evidence for the control of AP synthesis and
release of GH by neural tissue.

The first attempt to show the existence of a
factor in neural tissue responsible for controlling
the release of anterior pituitary GH was that of
Franz gt, gt, (1962). Although the authors concluded
that there was growth hormone releasing activity in
the hypothalamus of swine, their work and conclusions
have been criticized since standard assay procedures
were not followed and large variations in response
were obtained. Deuben and Meites were the first to
show conclusive evidence in this regard (1963, 1964).
They reported that neutralized acid extracts of rat
hypothalamus produced a 4 to 6-fold increase in GH
release by rat anterior pituitary after a 6 day culture.

Cerebral cortex failed to increase GH secretion upon

 

 

8.

culture. Deuben and Meites (1965) also reported
reinitiation of pituitary GH release 32 ytttg_by
a neutralized acid extract of rat hypothalamus after
release had ceased. These findings have been con-
firmed using tg_ytttg_(Schally gt, g}, 1965; Schally
gt, 2.1-: 1968; Krulich gt. gt, 1967) and 19.2119
methods (Pecile gt, g1,, 1965; Muller gt, g;,, 1965;
Muller and Pecile 1965; Krulich gt, g;,, 1965; Schally
gt, gt. 1966; Dhariwal gt, g1, 1966; Machlin gt, g},
1967).

The specificity of tg_ytyg_assay procedures
for hypothalamic GH-RF has been questioned by Rodger
gt, g1, (1967), although dose-reSponse relationships
have been reported by Meites and Fiel (1965) and
Katz gt, gt. (1967). Dose-response relationships have
not yet been demonstrated for ig_ytttg_methods of
assay for GH-RF (Schally gt, gt, 1965; Schally gt, _1.
1968; Krulich gt. 3;. 1967). It was of interest
therefore, to attempt to develop a short term incu-
bation method for assaying rat hypothalamic GH-RF
which would be sensitive enough to detect quantitative

changes in hypothalamic content of GH-RF.

11. Factors Affecting GH Secretion

 

A. Environmental Factors. The advent of radio-

 

immunological methods for measuring GH in body

9.
fluids greatly advanced our understanding of GH
control in humans. Roth gt, g1, (1963a) showed
that moderate exercise stimulated human growth
hormone (HGH) secretion. The rise in plasma GH could
be blunted by the feeding of glucose prior to the
exercise. Abdominal and chest surgery under ether
anesthesia was frequently followed by an elevation in
HGH (Glick gt, a;, 1965). Takahashi gt, 2;, (1968)
have reported increased HGH associated with the
initiation of sleep, not related to changes in
glucose, insulin or cortisol. It is of interest to note
in this reSpect, that exercise, moderate or severe
hypoglycemia, and cold, all known to elicit GH secretion
in humans, failed to do so in rats (Schalch and
Reichlin, 1967).

B. Hormonal Factors. Administration of

 

insulin in doses large enough to lower blood glucose
resulted in increased HGH (Roth et. a1. 1963 b). The
increased HGH with insulin induced hypoglycemia has
been confirmed by Hunter and Greenwood (1964), Frantz
and Rabkin (1964) and others. It is of interest that
ethanol induced hypoglycemia does not increase HGH
(Arky and Freinkel, 1954).

Thyroidectomy in the rat resulted in a degranulation
of pituitary acidophiles (Purves and Griesbach, 1946;

IO.
Schooley gt, gt,, 1966), a decrease in growth rate
(Koneff gt, gt, 1949; Schooley gt, gt, 1966) and
a decrease in pituitary GH content (Contopoulos gt.
gt,, 1958; Knigge, 1958; and Schooley gt, gt., 1966).
Hyperthyroidism in rats also produced blunting of
body growth, degranulation of pituitary acidophiles and
a reduction in pituitary content of GH (Solomon and
Greep, 1959). The interference to growth by
estrogens has been suspected for some time
(Gaarenstrom and Levie, 1939; Reece and Leonard, 1939)
although the mechanism of action remains uncertain
(Meites, 1949a; Sullivan and Smith, 1957; Josimovich
gt, gt, 1967; and Birge gt, gt,, 1967). The study by
Birge gt, gt. (1967) suggests that diethylbesterol
incubated with rat anterior pituitary caused suppression
of GH release, but had no effect on the amount stored
in the pituitary. Androgens, on the other hand,
stimulate growth when given in small doses (Rubinstein
and Solomon, 1941). The effects of corticosteroids
may be similar to those observed with high doses of
androgens (Evans gt, gt,, 1943; Marx gt, gt,, 1943;
and Geschwind and Li, 1955), in which growth of long
bones ceases by the closing of the epiphyses.

C. Food Intake. Reduced food intake or

 

starvation have been reported to decrease pituitary,

thyroid, and gonadal weights in the rat (Jackson, 1916;

11.

Jackson, 1917; and Mulinos and Pomerantz, 1941). The
resultant decreases in gonadal (Mulinos gt, gt,, 1939)
and thyroid functions (Stephens, 1940; Meites 1949 b;
and Meites and Wolterink, 1950) apparently are due
to a fall in circulating gonadotropins and thyrotropin.
The decrease in gonadotropins may be due in part to
a direct action of under-nutrition on the hypothalamus,
resulting in reduced synthesis and release of hypo-
thalamic releasing factors (Piacksek and Meites, 1967;
Negro-Vilar, Dickerman and Meites, 1968). Underfeeding
has also been reported to result in reduced absolute
adrenal weight in the rat (Quimby, 1948).

The decrease in body growth and skeletal length
that occurs during starvation in the rat may be due
in part to a reduced level of pituitary GH (Meites
and Fiel, 1965; Friedman and Reichlin, 1965) and in
hypothalamic growth hormone releasing factor (Meites
and Fiel, 1965). Since these observations can be
interpreted as indicative of increased as well as of
decreased release of GH from the pituitary and of
GH-RF from the hypothalamus, it was of interest to
determine plasma GH activity in addition to hypo-
thalamic GH—RF content and pituitary GH concentration

during starvation in the rat.

 

 

 

EXPERIMENTAL METHODS AND MATERIALS

1. Animals

A.

Experimental Animals

 

Intact male rats of the Sprague-Dawley
strain (Holtzman, Madison, Wisc.) weighing
about 290-310g each, were used as control and
experimental animals.

Control animals were maintained on a
diet of Wayne Lab Blox pellets (Allied Mills,
Chicago, Ill.) and fed gg_libitum. Food, but
not water, was removed from the experimental
animals for 7 days. The rats were weighed at
the beginning and end of each experiment.

Donor Animals

 

Mature male rats of the Sprague-Dawley
strain (Holtzman, Madison, Wisc.) weighing
200-220g each, were used as hypothalamic and
pituitary donors. All donor animals were fed
gg_libitum.

Bioassay Animals

 

Animals for GH bioassays were immature
female rats (Charles River Breeding Laboratories,
Wilmington, Mass.) hypophysectomized at 26 days
of age. They were used for GH bioassay 15-16

days after operation. The regular diet of

12.

13.
these animals was supplemented daily with
orange slices, carrots and sugar cubes. All
bioassay as well as experimental animals
were housed in a temperature controlled room
(25:10C) with automatically controlled

lighting (14 hr light daily).

II. Preparation of Hypothalamic Extracts (HElL,
Pituitaries and Plasma

 

A.

Preparation of Hypothalamic Extracts (HE)
Donor rats were killed by decapitation and
their hypothalami and/or pituitaries were
removed. The hypothalami were placed in ice
cold 0.1N HCl (0.1 ml per hypothalamus) and
homogenized in a total volume of 0.15 ml per
hypothalamus. The homogenate was centrifuged
at 12,000g for 40 minutes at 400. Just prior
to use, the supernatants were placed in
protein free medium 199 (Difco Labs.,
Detroit, Mich.) and the pH was adjusted to 7.4
by adding 1N NaOH 3 drop at a time and testing
with glass electrodes. The same procedure was
used in preparing cerebral extracts (CE).

Preparation of Pituitary Tissue for Assgy

 

The pituitaries were removed from the
control and experimental animals, individually

weighed and stored at -20°C. Prior to assay,

III.

14.
the pituitaries were thawed, homogenized in
saline and injected as an aqueous suspension
into assay rats.

C. Prgparation of Plasma for Assay

 

Blood was collected from the control and
experimental animals via the abdominal aorta
with a heparinized syringe. The blood from
each group was pooled, centrifuged at 4 C
and the plasma obtained immediately lyophylized
and stored at -20 C. Lyophylized plasma was
reconstituted to the desired volume with
saline prior to being injected into the assay
animals. A volume of 2 ml daily was injected

intraperitoneally into each assay rat.

Incubation

 

The posterior lobe was removed from each

donor pituitary and the anterior lobe was hemisected.
The halves were randomly distributed in the incubation
flasks. A total of 6 anterior pituitaries (12 halves)
were placed in each flask. In preliminary experiments
it was observed that pre-incubation of the pituitaries
increased the precision of the assay, suggesting that
during this time release of GH occurred as a result

of injury to the tissue. After a number of preliminary

IV.

15.
trials to determine the optimal pre—incubation and
incubation times, the following procedure was adopted.
The pituitaries were pre-incubated in 4 m1 of medium
199/flask for 1 hour and the medium was discarded.
Four m1 of fresh medium containing the neutralized acid
extract of hypothalamus or cerebrum was rapidly added
to the flasks. and 4 hours later the incubation was
terminated. The pituitary tissue was weighed and the
medium stored at -20 C until assayed. Incubations were
carried out in a Dubnoff metabolic shaker (60 cycles per
minute) under constant gassing with 95% 02-5% C02 at 37 C.

GH Bioassay and Statistical Treatment

 

Growth hormone activity was measured by the
standard tibia test of Greenspan gt. gt.{l949).
Aqueous solutions of incubation medium, pituitary homo-
genate, or reconstituted plasma were injected intra—
peritoneally once a day for 4 days. Each assay included
two or more doses of the control and experimental unknown
solutions, as well as two doses of NIH-GH-SB for refe-
rence standards. NIH—GH—SB was kindly supplied by the
Endocrinology Study Section, NIH.

The data were analyzed by linear regression or one
way analysis of variance. Breakdown of significance was

determined by Duncan's Multiple range test (Bliss, 1967).

EXPERIMENTAL

I. IN VITRO ASSAY FOR GROWTH HORMONE RELEASING FACTOR
(GH-RF)

A.

Objectives

 

The purpose of these experiments was to
develop a short term incubation method for
assaying rat hypothalamic GH-RF which would
show log-dose reSponse characteristics and be
sensitive enough to detect quantitative changes
in hypothalamic content of GH-RF.

Procedures

 

Donor rat pituitary tissue was pre-
incubated in medium 199 for 1 hour. The medium
was discarded. Fresh medium containing the
neutralized acid extracts were then added to
the flasks and incubated for 4 hours. Extract
equivalent to 0.75, 1.50, 3.00 or 6.00 rat
hypothalami were added to each flask containing
six pituitaries. This corresponds to 0.125,
0.250, 0.500 and 1.000 hypothalamic equivalent
per incubated pituitary. Rat cerebral extract
was used as a control. The medium was then
assayed for GH by the standard tibia test of
Greenspan gt, gt, (1949).

16.

17.
Results

1. Dose response relationship between

 

hypothalamic extract and anterior pituitaty

 

(5P) GH release in vitro. Hypothalamic extracts

 

were assayed at 4 doses of 0.125, 0.250, 0.500
and 1.000 hypothalamic equivalent per incubated
pituitary. Cerebral extract was assayed

at a dose corresponding to the highest dose

of hypothalamic extract/incubated AP. It can

be seen in Table I and Fig. 1 that when
progressively greater amounts of hypothalamic
extract were incubated with male rat pituitaries,
more GH was released into the medium. Regression
analysis of the data showed these differences

to be highly significant (p40.001). Breakdown

of significance by a Duncan's Multiple range

test showed each point to be significantly
different from the next lower point (p(0.05 or
p<0.01). GH release by the pituitaries incubated
with cerebral extract was significantly less
(p(0.01) than released by the lowest dose of
hypothalamic extract (0.125 HE/incubated AP).

2. Comparison of cerebral versus hypothalamic

 

extract on AP release of GH. Cerebral and hypo-

 

thalamic extracts were assayed at two dose

levels (0.25 and 1.00 CE or HE/incubated AP).

18.

.omop HO3OH uxmc #mcwmmm UOHMQEOO .mo.ovmm
.mmOU HOBOH uxoc gunfimmm Umnmmfioo .Ho.ovmm
mo umcfimmm OOHOQEOUU

 

 

s.mwm.amm u as ooH ocnasouocnmrooosr .xoommo
m.mHm.Hom n ma mm uomnpxo Hmnnmuou «mun
wmlmwlmHz ”pumpcmum mocoummom Homuwxm OMEmamnuomms “mmm
maouucou
h.mHo.nHH w wmmmm Uxomwm o
O@.HHm.¢vm 0 mm ooo.H m
ww.mhm.hmm 0 mm oom.o w
mm.¢Hm.¢Hm 0 mm omm.o m
0.6m.ehm.vmfl e mm mma.o m
m.me.nhH 0 m0 ooo.H H
mm H acme mpmn mm poumnsocfl msouw
ADV SDUHB hmmmm \mpcwam>flsvm
Hmflnwp mmmum>< mo .02 nmo Ho mam

 

ouufl> mw.ommmamm m0 mumpflsyflm uoflnmpcm
paw pomupxm OflEmHmnpomwm cmmzpom mfi5mcoflpmamm oncommmm Omoa .H magma

TIBIAL WIDTH (U)

I .

225‘

2001-

I75-1

 

19.

 

 

* 0J2:

0.550 0.000 ' ‘ L000

LOG-DOSE HYPOTHALAMIC EXTRACT

Figure 1.

Logarithmic Dose-Response Curve Showing
Pituitary GH Release as a Response to
Hypothalamic Extract.

20.
It can be seen in Table II and Fig. 2 that
when the larger dose of cerebral extract
was incubated with male rat pituitary, GH
released into the medium was no greater than
with the lower dose. 0n the other hand the
larger dose of hypothalamic extract incubated
with rat pituitary induced release of signifi-
cantly more GH into the medium than the lower
dose. The amounts of pituitary GH release stimulated
by hypothalamic extract were significantly
greater (p(0.01) than the amounts released by
pituitary upon adding cerebral extract at
either dose level. These results compare
favorably with those obtained in Experiment 1.

3. Dose response to medium from AP incu-

 

bated with cerebral extract or medium 199

 

gtggg, Medium from AP incubated with cerebral
extract or medium 199 alone was divided into
low and high doses corresponding to 0.25

and 1.00 AP equivalents/assay rat. These

were then injected into each assay rat for 4
days. The results in Table III and Fig. 3
show that cerebral extract did not elicit a
significantly greater (p)0.05) release of GH
into the medium than medium 199 alone. The

difference in response to the low and high

.mo m5mno> mm .Ho.ovmp

 

21.

 

H.mHm.mnN n on 00H poww80poomm£momms "xomhmo
w.HHm.hmm n ms mm DOMHDXO Hmnnonoo "mun
mmlmolmHz “UHmUCmum mocoumwom uomuyxo OflEmHmsuomwn Ammo
y maonucoo
w.mHN.mMH w hmmmm Oxommm m
Uo.¢wa.¢mm 0 mm oo.H w
pH.mHm.mmm 0 mm mm.o m
¢.mHn.dom 0 m0 oo.H m
m.@HS.SmH 0 m0 mm.o H
mm H :008 mpmu poumnsocfl msouw
ADV LDUHB mommm \mpcmam>flsvm
Hmwnflv mmmuw>¢ mo.oz QmU Mommm

 

m0 m0 Ommmaom humuflspflm HOHHODG¢ co
pomupxm OHEmHmnuommm m5mu0> pomuuxm Hmunouou mo comHHmmEoo .HH magma

(U)

TIBIAL WIDTH

22.

 

 

 

290"
270--
HYPOTHALAMIC
EXTRACT
2” I.
230--
ZIO " '
J
CEREBRAL I i
EXTRACT
'90 0.25 I.0'00

toe-003E HYPOTHALAMIC EXTRACT
LOG-DOSE CEREBRAL EXTRACT '

Figure 2., Logarithmic DoseeResponse Curves Showing
Pituitary GH Release as a Response to
Hypothalamic or Cerebral Extract.

23.

dose of medium in each group was significant.

4. Dose response to medium from AP

 

incubated with cerebral or typothalamic

 

extract. Medium from AP incubated with
hypothalamic or cerebral extract was divided
into two doses as in 3 above. The results in
Table IV and Fig. 4 show that the difference
in reSponse to the low and high dose of medium
in each group was significant (p(0.05 or
p(0.01). Both doses of hypothalamic extract
(0.5 and 1.0 HE/incubated AP) elicited signifi-
cantly greater release of GH into the medium
than cerebral extract. The higher dose of HE
elicited significantly greater release of GH
than the lower dose. These results are in
agreement with those in Tables I, II and III.

Discussion

 

The amount of GH released by male rat
pituitary in a 4 hour incubation procedure was
found to be directly proportional to the
logarithm of the dose of rat hypothalamic
extract added to the medium. The log-dose
response was linear between the levels of
0.125 and 1.000 hypothalamic equivalents/
incubated AP. Graded doses of hypothalamic

extracts elicited significantly greater

24.

 

 

H.HHm.bmm u ms om HQMOHMHcmHm #oz .mo.OAmo
H.mhm.omH n m5 om UONHEouommhsmomhn "xomhmn
mmlmwlmHz ”pumpcmpm moccuomom Homupxm HmHQouoo "mun
mHonucoo
0.0Hm.wHH 0 III: mommm onmhm m
Om.¢H¢.mmH v oo.H
Om.vho.mmH 0 mm.o mo oo.H N
m.mHo.HmH 0 oo.H
m.qHH.HOH 0 mm.o mmH ESHUOZ H
mm A come upon mhmp 0 mm OmquSOcH msouw
HOV nppHB mammm \Hmn >mmmm \mpcme>HSUmmm0
HmHQHp mmmuo>¢ mo .02 \mpCOHm>HSUO
m< mo Owon
Hmpoe

 

OcoH4 mmH ESHUOZ Ho pomupxm Hmunmumo
SHHB OOHmQDOCH mm Eoum EDHOOZ op oncomwmm mmon .HHH OHQMB

(U)

TIBIAL WIDTH

25.

I85d-

I75 "

I85q
MEDIUM I99

Ififi"
CEREBRAL EXTRACT

 

ciao I63
LOG-DOSE ANTERIOR PITUITARY EQUIVALENTS

0

I45

‘Figure 3. Logarithmic Dose-Response Curves to
Medium from AP.Incubated with Cerebral
Extract or Medium 199 Alone.

Ho.ovoc

 

26.

 

mO.OVQU
m.mAs.oom n as on .ocuHEouoonsrooomr "Roommo
o.mhm.moH n on om pomnwxo Hmnnmumo “mun
mmImemHz "UanCmpm mocouomom uomnuxm OHECHmnuomws "mmm
mHonucoo
m.mHm.HmH w IIII manna Oxommm 0
oo.HA¢.me 0 oo.H
N.¢Ho.NmH w mm.o mm o.H m
0¢.mH@.HmH 0 oo.H
N.NHS.NmH 0 mm.o mm m.o N
On.mHH.mmH 0 oo.H
m.NHm.mNH 0 mm.o m0 o.H H
mm H 2008 much mhmp 0 mg OOHOQSOGH msouw
ADV SHUHB wommm \pmn kammm \muCOHm>H5Uo
HmHQHy 0mmnm>¢ mo .02 \mszHm>HSWO gnu Ho 0mm
mg no Omon
HMHOB

 

uowuuxm OHECHmsuomkm Ho
Hmunowov SDHB OOHMQSOCH mm Eoum EDHUOE 0p Omdommom omon .>H OHQCB

 

 

TIBIAL WIDTH UJ)

27.

230"

2I0--

"o" I0 HYPOTHALAMIC

EXTRACT

 

I70"

 

0.5 HYPOTHALAMIC

"so" EXTRACT

LO CEREBRAL
I30 ‘ - EXTRACT

 

 

0320 ' I50

 

LOG-DOSE ANTERIOR PITUITARY EQUIVALENTS

Figure 4. Logarithmic Dose-Response Curves to Medium

from AP Incubated with Hypothalamic or Cerebral
EXtraCt.‘

28.

release of GH into the medium in all
experiments. However, graded doses of
cerebral extract failed to elicit increased
release of GH into the medium, indicating that
this area of the brain does not contain GH-RF
activity. In all experiments, AP incubated
with the lowest dose of hypothalamic extract
resulted in significantly greater release
of GH into the medium than any dose of
cerebral extract used. Analysis of the
medium at two dose levels showed significant
log-dose responses, indicating that the
hormone assayed was GH. Some release of GH
occurred in incubations of AP with cerebral
extract. This is due to spontaneous release
of GH since it has been shown that rat pituitary
cultures release GH for up to 3 days even in
the absence of hypothalamic extract (Meites,
Hopkins and Deuben, 1962). There is no
evidence that cerebral extracts can induce GH
release by the incubated AP (Deuben and Meites,
1964; Deuben and Meites, 1965; Schally
gt, gt,, 1965; and Schally gt, gt., 1968).
Crude hypothalamic extracts can influence

the release of anterior pituitary hormones

29.

other than GH (Guillemin, 1956; Schreiber et.
gt,, 1962; McCann, 1962; Talwaker, Ratner and
Meites, 1963; Mittler and Meites, 1964). ACTH
can reduce epiphyseal cartilage width in

young rats (Evans gt, gt,, 1943; Marx gt, gt,,
1943) while TSH can increase it (Fels gt, gt.,
1955). However, intraperitoneal injections of
aqueous solutions of ACTH even at high doses
have very little effect on the tibial response
(Li gt, gt,, 1954), and injections of only
very large.doses of TSH (500 ug/day/4 days)
increase the width of the tibial cartilage
plate (Fels gt, gt,, 1955). It appears unlikely,
therefore, that either TSH or ACTH in the
medium influenced our results. If gonado-
tropins were present in the medium, they

would have reduced rather than enhanced the
tibial reSponses recorded.

Our experimental data support the concept
that a Specific GH-RF is present in the hypo-
thalamus of the rat, since only such a factor
would be expected to release pituitary GH in
a log-dose fashion. Our method has been found
to be suitable for measuring quantitative
changes in hypothalamic GH-RF content produced

by different physiological states (see next

30.

section of thesis). The sensitivity of
this tg_ytttg_method for assaying GH-RF
should be further increased with the avail-
ability of a dependable radioimmunoassay

for rat GH.

II.

EFFECTS OF STARVATION ON PLASMA GH ACTIVITY,
PITUITARY GH, AND HYPOTHALAMIC GH-RF LEVELS IN
THE RAT.

A.

Objectives

 

The purpose of these experiments was to
observe the effects of starvation on plasma
GH activity, pituitary GH and hypothalamic
GH-RF levels in the rat. An attempt was
made to correlate any Changes in pituitary and
hypothalamic content with plasma GH levels.

Procedures

 

Control animals were maintained on a
diet of Wayne Lab Blox pellets and fed gg
libitum. Food, but not water, was removed
from the experimental animals for 7 days. On
day 8, the animals were weighed, anesthetized
with ether and bled from the abdominal aorta
with a heparinized syringe. The pituitaries
and hypothalami were also removed. Blood,
pituitaries and hypothalami were prepared as
described previously and injected intra-
peritoneally into assay rats to test for GH
by the method of Greenspan gt, gt, (1949).

In a separate experiment, the starved
animals received a daily subcutaneous injection

of L—Na-thyroxine (T4) (Nutritional Biochemicals

31.

32.
Corporation, Cleveland, Ohio) at a dose of
2.5 ug/lOOg body weight/day for 5 days beginning
on day 2 of starvation. Only the plasma of the
animals was analyzed for CH activity.
Results

1. Plasma GH activity of control and

 

starved rats. Table V and Fig. 5 show the

 

effects of 7 days of starvation on plasma
GH activity. In two independent experiments
plasma GH activity in control and starved rats
was assayed at two dose levels with a fourfold
difference between doses (8 or 32 ml of
plasma equivalent per assay rat). In both
experiments, plasma from control animals
elicited a significantly greater tibial
response than plasma from starved animals at
either dose level (p(0.01).

The starved rats lost an average 87.7
and 88.6 g per animal, respectively, whereas
the gg libitum fed controls gained an average
33.8 and 40.4 g respectively. Pituitary
weights were also decreased in the starved rats.

2. Effects of starvation and T4 on plasma

 

GH activity. Starvation has been shown to

 

reduce thyroid activity in rats (Meites and

Wolterink, 1950; Yousef and Johnson, 1968)

m.cAH.ch n ma ooH

 

 

 

 

 

H.ch.cmm u on ma ooou on n> Howucoo .Ho.ovoaa
mmlmwlmHz "pumpcmym mucoummom UONHEovommmzmom>£ u xom>MH
Hwy MHoupcOO
m.mHH.mmH II 0 III IIIIIIIIII mmmmm onmmm
**¢.hhh.vom mm 0 II.
Amev .QAH cc
*«m.¢Hm.mmm m 0 0.0 m.m¢m m.mom UOMIHOHHCOU
m.¢Hw.mmm mm d
4.6Am.mmH m e m.c m.mHN H.som Away ooou 02 HH
MHoupcoo
N.@Hm.NMH II 0 III IIIIIIIIII mummm onmmm
aw are.mwo.mcm mm a .II
as Ammv .AHH on
Atm.HHm.mm~ m 0 m.m 0.0mm «.mmm COMIHOHHQOU
m.NHm.¢HN mm 0
m.mHs.NaH m e N.s m.eom c.mm~ AooHV ooou oz H
mm H cmoE Amhmp ¢\umn wmmmm mpmn hammm me o m mpmu mo .0: .mxm
ADV fiypHB \mEmmHm mo HEV m0 m0 .oz mymn HMGHm HmHuHGH w #:oEHMOHB
HmHQHu m>¢ mmoa mo #3 uanm3 moon m><
Hmuoe uHm m>¢

 

mpmm co>~mym paw Hobpcoo mo >DH>Hwog m0 mammHm .> OHQCB

34.

   

 

 

275"
250--
“ A0 LIs,I=E0 - EXP. 11 N0 FOOD - EXP. n
:1 AD LIB. FED -EXP. I.
~"22:5"-
. :I:
IS NO FOOD- EXP. I
E
200..
.J
S
g NIH-GH-SB
p.
I75" ,. '
"9 - . a at 3'2 I50

LOG - DOSE ML PLASMA
LOG - DOSE .UC NIH -CH-SB

Figure 5., Logarithmic Dose—Response Curves to Plasma
from Control or Starved Rats. Standard
Curve Extrapolated.

 

 

 

o.mHm.mm~ u as ooH
m.~H~.HmH n as mm , poem on n> Hoaucoo .Ho.ovo.«
mmlmwlmHz “OHMUCMHm oucouwmom UONHEOHOOmmsmomhn n xommmH
mHonpaoo
N.¢Hm.an III m III IIIIIIIIII mmmmm onmmm
sym.mHm.Nmm mm m I
Aomv .AAH on
an ecb.NHm.mmH m m o.m m.mmm H.nmm COMIHOHHGOU
A5
m.mHm.mmH mm m
AOHHV 03_moob
m 00H\ee ms
m.0HH.me m m 0.0 m.~m~ m.mHm m.~ + UOOM oz H
mm H cmmE Ammww «\pmu hammm myth hummm ma 0 m mumn mo .oc .mxm
ADV nuUHz. \mEmmHm mo HEV mo mo .02 mumu HMCHm HMHHHGH w acoEumone
HMHQHu m>< whom no #3 ugmHOS SOOQ m><
Hobos uHm m>¢

 

 

>DH>Hv0¢ m0 mEmMHm so 09 + COHpm>Hmum mo mvoommm

.H> OHQMB

36.

and this probably results in decreased blood
levels of thyroxine (Grossie and Turner, 1962).
Since the tibial response to GH is decreased
in the absence of thyroxine (Schooley gt, gt,,
1966), the starved animals received replace-
ment doses of tthoxine (2.5 ug/lOO g body
weight/day) during the last five days of
starvation. In this way we tried to

restore normal levels of circulating thyroxine
in the starved rats, thereby reducing the
possible effects of thyroid deficiency on GR
activity in the plasma.

As can be seen in Table VI, a significant
reduction in plasma GH activity was observed
in the thyroxine treated starved rats. Body
and pituitary weights were also reduced in
the starved animals. The reduction in plasma
GH activity, body and pituitary weights
observed in the thyroxine treated starved
rats is comparable to the reductions observed
in the previous experiments shown in Table V.

3. Effects of starvation on pituitary

 

GH content. Pituitaries were assayed at two

 

dose levels (2 or 4 mg of anterior pituitary

per assay rat). Table VII shows that starvation

 

370

 

 

 

H.mHm.~sm u so ooH
o.HAm.sm~ u a: mm coon on n> Honusoo .Ho.ovo..
mmlmemHz “Unmpcmuw monoummmm UONHEOHUOmhnmomhn n xommmH
MHOHHCOU
m.mHH.mNH I 0 III IIIIIIIII ,I hmmmm onmhm
RAH.NHo.omN 0 0 II.
.QHH pm
«Rm.mHm.nNN N v o.m N.@¢m m.mom COMIHoupcoo
N.NHm.¢vN 0 0
m.mHN.m0N N 0 N.@ m.mHN N.hom ©00m 02 H
mm H some Ammmp v\umu wumu wmmmm me m m ucmEpmmHB mxm
ADV SHUHB >0mm0\m< mo may m0 mo .02 mpmn Hmch HmeHcH
HmHQHu m>¢ mmom mo #3 pgmHOB wdon mw><

Hmuoe pHm m>¢

 

COHDMHHCOOQOU m0 mumespHm 0>Hp0Hmm co GoHpm>ngm mo mHUOmwm .HH> OHQMB

38.

significantly reduced the concentration of

GH in the pituitary (p(0.0l). These findings
are in agreement with previous reports (Meites
and Fiel, 1965; Friedman and Reichlin, 1965).
The starved rats lost an average 88.6 g and
the gg_ttg. fed control animals gained an
average 40.4 g.

4. Effects of starvation on hypothalamic

 

95:53, Hypothalamic content of GH-RF was
assayed at two dose levels (.25 or 1 hypo-
thalamic equivalent per incubated pituitary)
using the previously described l2.!l££2
incubation method. The results in Table VIII
show that starvation significantly reduced
hypothalamic content of GH-RF (p(0.01). Our
results confirm those previously obtained by
Meites and Fiel (1965) with an tg_yttg_assay
method.

Discussion

 

The data presented here indicate that
complete food removal for 7 days in the rat
significantly reduces plasma GH activity as
well as pituitary concentration of GH and
hypothalamic content of GH-RF. These results

corroborate the previous observations that

 

 

H.mHm.~sm I ma OOH soon on m> Honpsoo .Ho.ovo.a

 

 

 

 

w.HHm.NMN I vs mN . pmNHEnflommmflmomhn u xomth
mmlmemHz "Unmpcmum moconomom HOMHHxO xwpuoo HMHQOHOU u HUGH
MHOHUGOO
m.mHH.mNH IIII 0 III IIIIIIIIII mmmmm Nxomhm
m.wHw.SNH oo.H 0 III IIIIIIIIII MHOHHCOO Hmoo
www.me.H¢N oo.H v
o” .QHH Om
«3 RAN.mH¢.MHN mN.o w w.m N.@¢m m.mom OOHIHOHHGOU
©.mHH.HON oo.H v
h.mHv.HmH mN.o v N.@ m.mHN N.h0m UOOH 02 H
mm H some AhumgHspHQ mHMH mmwmm me o m pamEDMOHB .mxm
ADV SDUHB OOHMQSOCH\.MumV m0 Ho .02 mumu Hmch HMHuHcH
HMHQH#.0>¢ Omon mo #3 pan03 SUCH .m>¢
uHm m><

 

pcoucoo mmImw OHEmHm£pom>m so coHpm>H0pm Ho muvmmwm .HHH> OHQMB

40.

starvation in rats results in lower hypo-
thalamic GH-RF content (Meites and Fiel, 1965)
and decreased pituitary concentration of GH
(Meites and Fiel, 1965; Friedman and Reichlin,
1965), when compared with gg_libitum fed con-
trols, and indicate that these changes are
reflected in lower concentration of plasma GH
activity. Our observation of reduced plasma
GH activity in starved rats agrees with a
similar independent study carried out in
starved rats by Dr. A. Trenkle (Department of
Animal Science, Iowa State University, Ames,
Iowa) using a radioimmunological assay for GH
(personal communication).

Although we have used the term plasma
GH activity rather than plasma GH, we have no
evidence that the material measured in the
blood differs from that in the NIH-GH-S8.
As indicated in Fig. 5, the dose-response curves
obtained from the plasma of the control and
starved rats were parallel to those obtained
with purified NIH ovine GH. This suggests that
the tibia test is measuring GH activity.

The GH bioassay values found here in
the plasma (Fig. 5) are considerably above

those reported by radioimmunoassay in the rat

41.

(Schalch and Reichlin, 1966). However,

NIH ovine GH was used in our bioassay of

rat GH, whereas a purified rat GH was used

as the reference standard for radioimmunoassay
of rat GH (Schalch and Reichlin, 1966). The
ovine GH used in our experiments has a stated
potency of 0.79 USP units/mg, whereas a

purified rat GH prepared by Dr. S. Ellis

(Ames Research Center, Moffett Field, California,
personal communication) has a stated potency

of 2.7 to 3.9 USP units/mg. Therefore, purified
rat GH may be 3.41 to 4.94 times more potent
than ovine GH in the rat.

It has been suggested that the tibia
bioassay of plasma GH may measure ”sulfation
factor" activity. Salmon and Daughaday (see
Daughaday and Kipnis, 1966) showed that fasting
reduces sulfation factor levels in the
plasma of rats. Uptake of sulfate tg_ytttg_
by cartilage from fasted rats was also
decreased when the fast was continued for
more than 24 hours. Hypophysectomy in rats
decreased serum sulfation factor (Salmon and
Daughaday, 1957), but GH injections into

hypophysectomized rats restored values to

42.

normal levels. However, direct addition of
HGH to tg_ytttg_incubations containing
cartilage from hypophysectomized rats did
not stimulate sulfate uptake (Daughaday
and Reeder, see Daughaday and Kipnis, 1966).
The lfl.XlE£2 stimulation of sulfate uptake
was found to be a linear function of the
logarithm of the dose of normal serum
added to the incubation medium containing
cartilage from hypophysectomized rats
(Daughaday gt, gt,, 1959). Since a relation-
ship was demonstrated between the dose of GH
injected and the rise in sulfation factor
activity in the plasma of pituitary dwarfs
(Almgvist, 1960), it is believed that GH gives
rise to a component of plasma capable of
stimulating sulfate uptake by cartilage and
incorporating this sulfate into chondroitin
sulfate (Koumans and Daughaday, 1963). Serum
sulfation factor activity therefore may be an
index of GH activity in the plasma (Daughaday
gt. 9.1.. 1959).

Our observations on plasma GH levels are

in contrast to those reported on human subjects

43.

by radioimmunoassay. Roth gt. gt. (1963 b)
and Cahill gt. gt, (1966) reported that
prolonged fasting in humans resulted in
increased serum GH content. Knobil (1966)
however, found no increase in serum GH in
fasted monkeys. Machlin gt, gt. (1968)

also reported that plasma GH levels in pigs
increased during the first 48 hours of
starvation and subsequently fell to lower
levels. This may indicate that increased GH
secretion during fasting may not occur in

all species. In a study Closely related

to ours, Srebnik gt, gt, (1959) found a
significant reduction in pituitary and plasma
GH levels as measured by bioassay in rats fed
a protein-free diet for prolonged periods of
time. The possibility of a different
mechanism for control of GH secretion in

the rat as compared to humans is indicated

by the recent work of Schalch and Reichlin (1967).
They found that stimuli such as forced
exercise, moderate or severe hypoglycemia,
or cold, all known to elicit release of GH
in humans, failed to do so in rats.

The interpretation of our results may

44.

be complicated by the presence or lack of

other hormones in the plasma of starved rats,
particularly ACTH and TSH. Unpublished
observations in our laboratory (Dickerman,
Negro-Vilar, and Meites, 1967) showed that
absolute adrenal weight decreased in starved
rats. These observations confirm previous
findings by Quimby (1948). Furthermore,

Li gt. gt. (1954) showed that the tibial
response to ACTH is dependent upon the mode

of injection of ACTH. Thus, a very high dose
of d-adrenocorticotropin, of the order of 100
ug, had very little effect on the tibial response
when injected alone intraperitoneally or with
GH in an aqueous solution. Fels gt, gt, (1955)
reported that injections of a TSH
preparation into hypophysectomized rats

(200 ug/day/4 days) increased the width of the
tibial cartilage plate from l63u to l92u.
Administration of the latter dose with GH gave
no increment over the values obtained with

GH alone. Since all our assay rats were in-
jected intraperitoneally with aqueous solutions
of plasma, anterior pituitary or incubation
medium, we believe that the changes in tibial

width were due primarily to GH.

CONCLUSIONS

 

The present experiments support the concept
of a hypothalamic factor which controls anterior
pituitary secretion of GH, GH-RF. The amount of
GH released by male rat pituitary upon incubation was
directly proportional to the logarithm of the dose of
rat hypothalamic extract added. This factor is not
present in the cerebrum of rats, since cerebral
extract failed to show a log-dose response and was
also unable to increase the secretion of GH above
that released spontaneously. This method of
measuring GH-RF is sensitive enough to detect physio-
logical changes in hypothalamic content of GH-RF.
Thus, it was possible to corroborate the decrease
reported in hypothalamic GH—RF by an tg_yttg_method
observed after starvation in rats (Meites and
Fiel, 1965).

The decrease in hypothalamic content of GH—RF
leads to a decrease in synthesis and release of GH
from the pituitary and is reflected in a lower plasma
GH activity. Thus the rat appears to differ from the
primate in its response to starvation. It is also
possible that the GH measured in the serum of humans
by radioimmunoassay may measure not only biologically
active GH, but also biologically inactive GH which is

immunologically active.
45.

REFERENCES

Almgvist, s., 1960. Studies on sulfation factor (SF)
activity of human serum, effect of human growth
hormone on SF levels in pituitary dwarfism.

Acta Endocrinol. 35: 381-396.

 

Arky, R.A., and Freinkel, N., 1954. The response
of plasma human growth hormone to insulin and
ethanol-induced hypoglycemia in two patients with
"isolated adrenocorticotropic defect." Metabolism

 

3: 547-549.

Armstrong, C.N., and Durh, D.P.H., 1922. Three
cases of supra-pituitary tumors presenting
Frohlid's syndrome. Brain 45: 113-125.

Bach, L.M.N., O'Brien, C.P., and Cooper, G.P., 1964.
Some observations concerning the hypothalamic
regulation of growth and of food intake. In:
Progress in Brain Research, vol. 5: Lectures
on theIDiencephakm. W.—Bargmann and J.P. Schade
(Eds.), Elsevier, Amsterdam.

 

Bernardis, L.L., Box, B.M., and Stevenson, J.A.F.,
1963. Growth following hypothalamic lesions
in the weanling rat. Endocrinology 72: 684-692.

 

Birge, C.A., Peake, G.T., Mariz, I.K., and Daughaday,
W.H., 1967. Effects of cortisol and diethyl-
besterol on growth hormone release by rat
pituitary in vitro. Proc. Soc. Exptl. Biol. Med.
126: 342-345.

Bliss, C.I., 1967. Statistics in Biology. Vol. I,
McGraw-Hill, New YOrk.

 

Bogdanove, E.M., and Lipner, H.J., 1952. Intes-
tinal absorption of glucose in hypothalamic
obesity. Proc. Soc. Exptl. Biol. Med. 81:
410-412.

Cahane; M., and Cahane, T., 1938. Sur 1e role du

diencephale dans le developpement somatique.
Rev. Franc. d'Endocrinol. 16: 181-184.

46.

47.

Cahill, G.F., Jr., Herrera, M.G., Morgan, A.P.,
Soeldner, J.S., Steinke, J., Levy, P.L.,
Reichard, G.A., Jr., and Kipnis, D.M., 1966.
Hormone fuel interrelationships during fasting.
J. Clin. Invest. 45: 1751-1769.

 

Contopoulos, A.N., Simpson, M.E., and Koneff, A.A.,
1958. Pituitary function in the thyroidectomized
rat. Endocrinology63: 642-653.

 

Daniel, P.M., and Prichard, M.M.L., 1964. Observa-
tions on the metabolic disturbances, growth and
behaviour of young goats after transection of
the pituitary stalk. Acta Endocrinol. 45: 84-98.

 

Daughaday, W.H., Salmon, W.D., and Alexander, F.,
1959. Sulfation factor activity of sera from
patients with pituitary disorders. J. Clin.
Endocrinol. Metab. 19: 743-758.

 

Daughaday, W.H., and Kipnis,.D.M., 1966. The growth-
promoting and anti-insulin actions of somatotropin.
Recent Prog. Hormone Res. 22: 49-93.

 

Deuben, R.R., and Meites, J., 1964. Stimulation
of pituitary growth hormone release by a hypotha-
lamic extract tg_vitro. Endocrinology_74: 408—414.

 

Deuben, R., and Meites, J., 1965. tt_vitro reinitiation
of pituitary somatotropin release by an acid
extract of hypothalamus. Proc. Soc. Exptl.

Biol. Med. 118: 409-412.

 

 

Dhariwal, A.P.S., Krulich, L., Antunez-Rodrigues, J.,
and McCann, S.M., 1966. Separation of GH—RF
from CRF. Neuroendocrinology_l: 341-349.

 

Endroczi, E., Kovacs, S., and Lissak, K., 1956. Die
Wirkung der Hypothalamus-Reizung auf das endokrine
und somatische verhalten. Endokrinologie 33: 271-278.

Evans, H.M., Simpson, M.E., and Li, C.H., 1943.
Inhibiting effect of adrenocorticotrophic hormone
on8the growth of male rats. Endocrinology 33: 237-
23 .

Fels, I.G., Simpson, M.E., and Evans, H.M., 1955. Puri-
fication of the anterior hypophyseal thyrotropic
hormone. J. Biol. Chem. 213: 311-323.

 

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48.

Frantz, A.G., and Rabkin, M.T., 1964. Human growth
hormone. Clinical measurements, response to
hypoglycemia and suppression by corticosteroids.
New Engl. J. Med. 271: 1375-1381.

 

Franz, J., Haselbach, C.H., and Libert, 0., 1962.
Studies on the effect of hypothalamic extracts
on somatotrophic pituitary function. Acta
Endocrinol. 41: 336-350.

 

Friedman, R.C., and Reichlin, S., 1965. Growth hormone
content of the pituitary gland of starved rats.
Endocrinology 76: 787-788.

 

Gaarenstrom, J.H., and Levie, L.H., 1939. Disturban-
ces of growth by diethyl-stilboestrol and oestrone.
J. Endocrinol. 1:420-429.

 

Geschwind, I.I., and Li, C.H., 1955. The tibia test
for growth hormone. In Hypoptyseal Growth Hormone,
Nature and Actions, R.W. Smith, 0.H. Gaebler,
and Long, C.N.H., eds. pp. 28-58. McGraw-Hill,

New York. ,

 

 

Glick, S.M., Roth, J., Yalow, R.S., and Berson, S.A.,
1965. The regulation of growth hormone secretion.
Rec. Prog. Hormone Research 21: 241-270.

 

' Goldberg, R.C., and Knobil, E., 1957. Structure and
function of intraocular hypophyseal grafts in the
hypophysectomized male rat. Endocrinology 61:
742-754.

Green, J.D. and Harris, G.W., 1949. Observation of
the hypophysiOportal vessels of the living rat.
J. Physiol. (London) 108: 359-361.

 

Greenspan, F.S., Li, C.H., Simpson, M.E., and Evans,
H.M., 1949. Bioassay of hypophyseal growth
hormone: the tibia test. Endocrinology 45: 455-463.

 

Greep, R.0,, 1936.; Functional.pituitaryfgrafts-in..
rats. Proc. Soc. Exptl. Biol. Med. 34: 754-755.

Grossie, J., and Turner, G.W., 1962. Thyroxine
secretion rates during food restriction in rats.
Proc. Soc. Exptl. Biol. Med. 110: 631-633.

Guillemin, R., 1956. Hypothalamo-Hypophyseal
Interrelationships. Charles C. Thomas, Spring-
field, Illinois.

 

 

 

IIII lull. III

Iii.

 

 

49.

Halasz, B., Pupp, L., and Uhlarik, 8., 1962.
Hypophysiotrophic area in the hypothalamus.
J. Endocrin. 25: 147-154.

 

Halasz, B., Pupp, L., Uhlarik, S., and Thoma, L.,
1963. Growth of hypophysectomized rats bearing
pituitary transplant in the hypothalamus. Acta
Physiol. Acad. Sci. Hung. 3: 287-292.

 

 

Harris, G.W., 1950. Oestrous rhythm. Pseudopregnancy
and the pituitary stalk in the rat. J. Physiol.

Hertz, R., 1959. Growth in the hypophysectomized rat
sustained by pituitary grafts. Endocrinology 65:

926—931.

Hinton, G.G., and Stevenson, J.A.F., 1962. The
effect of hypothalamic lesions on growth. Can.
J. Biochem. Physiol. 40: 1239-1243.

 

 

Houssay, B.A., Biasotti, A., and Sammartino, R., 1935.
Modifications fonctionnelles de 1'hypophyse apres
1es lesion infundibulo-tuberiennes chez le
chapaud. Compt. Rend. Soc. Biol. 120: 725-727.

 

Hunter, W.M., and Greenwood, F.C., 1964. ~Studies on
the secretion of human pituitary growth hormone.
Brit. Med. J. 1: 804-807.

 

Jackson, C.M., 1916. Effects of inanition upon the
structure of the thyroid and parathyroid glands
of the albino rat. Amer. J. Anat. 19: 305-352.

 

Jackson, C.M., 1917. Effects of inanition and
refeeding upon the growth and structure of the
hypophysis in the albino rat. Amer. J. Anat.
21: 321-358.

Josimovich, J.B., Mintz, D.H., and Finster, J.L.,
1967. Estrogenic inhibition of growth hormone-
induced tibial epiphyseal growth in hypophysectomized
rats. Endocrinology 81: 1428-1430.

 

Katz, S.H., Dhariwal, A.P.S., and McCann, S.M., 1967.
Effect of hypoglycemia on the content of pituitary
growth hormone (GH) and hypothalamic growth
hormone-releasing factor (GHRF) in the rat.
Endocrinology_8l: 333-339.

 

50.

Knigge, H.M., 1958. Cytology and growth hormone
content of rat's pituitary gland following
thyroidectomy and stress. Anat. Record 130:

543-551.

Knobil, E., 1966. Tenth Bowditch Lecture. The
Pituitary Growth Hormone: An Adventure in
Physiology. The Physiologist 9: 25-44.

 

 

Koumans, J., and Daughaday, W.H., 1963. Amino
acid requirements for activity of partially
purified sulfation factor. Trans. Assoc.
Am. Physicians 76: 152-162.

Krulich, L, Dhariwal, A.P.S., and McCann, S.M., 1965.
Growth hormone activity of crude ovine hypo-
thalamic extracts. Proc. Soc. Exptl. Biol.

Mgg, 120: 180-184.

 

Krulich, L., Dhariwal, A.P.S., and McCann, S.M.,
1967. Stimulatory and inhibitory hypothalamic
factors regulating secretion of growth hormone.
Program 49th Meet. Endocr. Soc. p. 87.

Li, C.H., Geschwind, 1.1., Levy, A.L., Harris, J.J.,
Dixon, J.S., Pon, N.G., and Porath, J.O., 1954.
Isolation and properties of alpha-corticotropin
from sheep pituitary glands. Nature 173: 251-253.

‘ Martini, L., and de Poli, A., 1956. Neurohumoral
control of the release of adrenocorticotrophic:
hormone. J. Endocrin. 13: 229-234.

 

Marx, W., Simpson, M.E., Li, C.H., and Evans, H.M.,
1943. Antagonism of pituitary adrenocortico-
tropic hormone to growth hormone in hypophy-
sectomized rats. Endocrinology_33: 102-105.

 

McCann, S.M., 1962. A hypothalamic luteinizing
hormone-releasing factor. Am. J. Physiol. 202:

Machlin, L.J., Horino, M., Kipnis, D.M., Phillips, S.L.,
and Gordon, R.S., 1967. Stimulation of GH secre-
tion by median eminence extracts in the sheep.
Endocrinolggy 80: 205-207.

 

 

Machlin, L.J., Horino, M., Hertelendy, F., and Kipnis,
D.M., 1968. Plasma growth hormone and insulin
levels in the pig. Endocrinology 82: 369-376.

 

 

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51.

Meites, J., 1949a. Relation of food intake to growth
depressing action of natural and artificial
estrogens. Am. J. Physiol. 159: 281-286.

 

Meites, J., 1949b. Effects of starvation in rats
and mice on thyroid secretion rate as indicated
by uptake of radioactive iodine and thiouracil
action. J. Animal Science 8: 642.

 

Meites, J., and Wolterink, L.F., 1950. Uptake of
radioactive iodine by the thyroids of underfed
rats. Science 111: 175-176.

Meites, J., Hopkins, T.F., and Deuben, R., 1962.
Growth hormone production by rat pituitary tg_
vitro. Fed. Proc. 21: 196.

 

Meites, J., and Kragt, C.L., 1964. Effects of a
pituitary homotransplant and thyroxine on body
and mammary growth in immature hypophysectomized
rats. Endocrinology 75: 565-570.

 

Meites, J., and Fiel, N.J., 1965. Effect of starvation
on hypothalamic content of "somatotropin
releasing factor" and pituitary growth hormone
content. Endocrinology 77: 455-460.

 

Mittler, J.C., and Meites, J., 1964. In vitro stimula-
tion of pituitary follicle-stimulating hormone
release by hypothalamic extract. Proc. Soc. Exptl.
Biol. Med. 117: 309-313.

 

Mulinos, M.G., Pomerantz, L., Smelzer, J., and Kurzrok,
R., 1939. Estrus-inhibiting effects of inanition.
Proc. Soc. Exptl. Biol. Med. 40: 79-83.

 

Mulinos, M.G., and Pomerantz, L., 1941. The repro-
ductive organs in malnutrition. Endocrinology 29:
267-275.

Muller, E.E., and Pecile, A., 1965. Growth hormone
releasing factor of a guinea-pig hypothalamic
extract: its activity in guinea-pig and rat.
Proc. Soc. Exptl. Biol. Med. 119: 1191-1194.

 

 

Muller, E., Pecile, A., and Smirne, S., 1965.
Substances present at the hypothalamic level and
growth hormone releasing activity. Endocrinology

77: 390-392.

 

52.

Negro-Vilar, A., Dickerman, E., and Meites, J., 1968.
Effects of starvation on pituitary FSH and
hypothalamic FSH-releasing factor (FSH-RF) in
male rats. Fed. Proc. 28: 269.

 

Nikitovitch-Winer, M., and Everett, J.W., 1958.
Functional restitution of pituitary graft
re-transplanted from kidney to median eminence.
Endocrinology 63: 916—930.

 

O'Brien, C.P., Happel, L., and Bach, L.M.N., 1964.
Some hypothalamic effects of STH-influenced
growth and insulin sensitivity in kittens.
Federation Proc. 23: 205.

 

Pecile, A., Muller, E., Falconi, G., and Martini, L.,
1965. Growth hormone releasing activity of
hypothalamic extracts at different ages.
Endocrinology 77: 241-246.

 

Piacsek, B.E., and Meites, J., 1967. Reinitiation
of gonadotropin release in underfed rats by
constant light or epinephrine. Endocrinology 81:
535-541.

Popa, G.T. and Fielding, U., 1933. Hypophysio-portal
vessels and their colloid accompaniment. J. Anat.
(London) 67: 227-232.

 

Purves, H.D., and Griesbach, W.E., 1946. Observation
on the acidophil cell changes in the pituitary
in thyroxine deficiency states, acidophil
degranulation in relation to gastrogenic agents
and extrathyroidal thyroxine synthesis.

Brit. J. Exp. Pathol. 27: 170-179.

 

Quimby, F.H., 1948. Organ weights of rats receiving
' hormone supplements during recovery from chronic
starvation. Endocrinology_42: 263-272.

 

Reece, R.P., and Leonard, S.L., 1939. Further
evidence for a mammogenic factor in the rat
hypophysis. Proc. Soc. Egptl. Biol. Med. 42:
200-202.

 

Reichlin, S., 1960a. Thyroid function, body temperature
regulation and growth in rats with hypothalamic
lesions. Endocrinology_66: 340-354.

 

Reichlin, 8., 1960b. Growth and the hypothalamus.
Endocrinology 67: 760-773.

 

 

atl'.

 

53.

Reichlin, S., 1961. Growth hormone content of
pituitaries from rats with hypothalamic lesions.
Endocrinology_69: 225-230.

 

Rodger, N.W., Beck, J.C., Burgos, R., and Guillemin,
R., 1967. Variability of response in the
bioassay of ovine hypothalamic extracts for a
somatotropin releasing factor. Program 49th
Meeting Endocr. Soc. p. 88.

 

 

Roth, J., Glick, S.M., Yalow, R.S., and Berson, S.A.,
1963a. Secretion of human growth hormone:
physiologic and experimental modification.
Metabolism 12: 577-579.

 

Roth, J., Glick, S.M., Yalow, R.S., and Berson, S.A.,
1963b. Hypoglycemia: a potent stimulus to
secretion of growth hormone. Science 140: 987-988.

Rubinstein, H.S., and Solomon, M.L., 1941. The growth
stimulating effect of small doses of testosterone
prOpinate in the castrate albino rat. Endocrinology
28: 229-232.

 

Salmon, W.E., and Daughaday, W.H., 1957. A hormonally
controlled serum factor which stimulates sulfate
incorporation by cartilage in vitro. J. Lab. Clin.
Egg,, 49: 825-836.

 

Schalch, D.S., and Reichlin, S., 1966. Plasma growth
hormone concentration in the rat determined by
radioimmunoassay: influence of sex, pregnancy,
lactation, anesthesia, hypophysectomy and
extrasellar pituitary transplants. Endocrinology
79: 275-280.

Schalch, D.S., and Reichlin, S., 1967. Stress and
growth hormone release. Excerpta Medica 142: 13.

 

 

Schally, A.V., Steelman, S.L., and Bowers, C.Y., 1965.
Effects of hypothalamic extracts on release of
GH tg_vitro. Proc. Soc. Exptl. Biol. Med. 119: 208-
212.

 

Schally, A.V., Kuroshima, A., Ishida, Y., Arimura,
A., Saito, T., Bowers, C.Y., and Steelman, S.L.,
1966. Purification of GH-RF from beef hypothalamus.
Proc. Soc. Exptl. Biol. Med. 122: 821-823.

 

 

. I'lI'IIJi.I.IIlI

 

54.

Schally, A.V., Muller, E.E., and Sawano, E., 1968.
Effect of porcine growth hormone-releasing
factor on the release and synthesis of growth
hormone in vitro. Endocrinology_82: 271-276.

 

Schooley, R.A., Friedkin, S., ahd Evans, R.S., 1966.
Re-examination of the discrepancy between
acidophil numbers and growth hormone concentration
in the anterior pituitary gland following
thyroidectomy. Endocrinology 79: 1053-1057.

 

Schreiber, V., Rybak, M., Eckertova, A., Koci, J.,
Jirgl, V., Franc, Z., and Kmentova, V., 1962.
Isolation of hypothalamic peptide with TRF
(Thyreotrophin releasing factor) activity in
vitro. Experientia 18: 338-340.

 

Smith, P.E., 1961. Postponed homotransplants of
the hypophysis into the region of the median
eminence in hypophysectomized male rats.
Endocrinology 68: 130-143.

 

Solomon, J., and Greep, R.0., 1959. The effect of
alterations in thyroid function on the pituitary
growth hormone content and acidophil cytology.
Endocrinology 65: 158-164.

 

Srebnik, H.H., Nelson, M.M., and Simpson, M.E., 1959.
Reduced growth hormone content in anterior
pituitary rats on protein free diets. Proc. Soc.
Exptl. Biol. Med. 101: 97-99.

 

 

Stephens, D.J., 1940. The effect of the thyrotropic
principle of the anterior pituitary on the
thyroid of the undernourished guinea pig.
Endocrinology 26: 485-492.

 

Sullivan, L.W., and Smith, T.C., 1957. Influence
of estrogens on body growth and food intake.
Proc. Soc. Exptl. Biol. Med. 96: 60-64.

 

Swelheim, T., and Wolthius, D.L., 1962. On the
growth hormone production by pituitary trans-
plants. Acta Physiol. Pharmacol. Neerl. 11: 343-349.

 

Takahashi, Y., Kipnis, D.M., and Daughaday, W.H., 1968.
Increased growth hormone associated with initiation
of sleep. Excerpta Medica 157: 17.

 

55.

Talwaker, P.K., Ratner, A., and Meites, J., 1963.
In vitro inhibition of pituitary prolactin synthesis
and release by hypothalamic extract. Am. J.
Physiol. 205: 213-218.

Uotila, U.U., 1939. On the role of the pituitary
stalk of the anterior pituitary with special
reference with thyrotrOphic hormone.
Endocrinology 25: 605-614.

 

Westman, A. and Jacobsohn, D., 1940. Endokrinologische
Untersuchungen an Kaninchen mit durchtrenntem
Hypophysenstiel. Acta Obstet. Gynecol.20: 392-433.

 

Wislocki, G.B. and King, L.S., 1936. The permeability
of the hypophysis and the hypothalamus to
vital dyes, with a study of the hypophysial
vascular supply. Am. J. Anat. 58: 421-472.

 

Wislocki, G.B., 1937. The vascular supply of the
hypophysis cerebri of the cat. Anat. Rec. 69:
361-387.

 

Wislocki, G.B., 1938. Further observations on the
blood supply of the hypophysis of the rhesus
monkey. Anat. Rec. 72: 137-150.

 

Yousef, M.K., and Johnson, H.D., 1968. Effects of
heat and feed restriction during growth on
thyroxine secretion rate of male rats. Endocrinology

82: 353-358.

 

 

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