BIOLOGY OF REPRODUCTION 29, 25-37 (1983)

In Vivo and In Vitro Studies on the Effect of

Adrenocorticotropic Hormone or Cortisol on the Pituitary Response
to Gonadotropin Releasing Hormone

P. S. LI and W. C. WAGNER’

Department of Veterinary Biosciences

College of Veterinary Medicine
University of Illinois
Urbana, Illinois 61801

ABSTRACT

Experiment I Hyperadrenal states were induced in intact heifers (N=3) or adrenalectomized

(ADRX) heifers (N=3) by constant infusion of ACTH (20.8 pg. 1-24 ACTH/h) or hydrocortisone
succinate (HS) (30 mg/h), respectively. Control infusions consisted of the saline vehicle. All infu-
sions began on Day 2 of a normal estrous cycle. Exogenous gonadotropin releasing hormone
(GnRH) was given ass 100-pg bolus i.v. on Days 7,9, and 11 (intact) or 5,7, and 9 (ADRX)of the
cycle. In intact heifers, the cumulative luteinizing hormone (LU) response was reduced (P<0.05)
by the ACTH treatment. In ADRX heifers, the HS treatment did not alter the cumulative response
but did alter the qualitative response with a time X treatment interaction (P<0.01). The LU re-
sponse in the HS-ADRX animals had a slower onset and lower peak concentrations with a more
prolonged response.
Experiment II: Dispersed bovine pituitary cells were prepared and incubated at concentrations
of 2 X 106 viable cells in 2.0 ml per dish. Cells were exposed to cortisol at concentrations of 0.01,
0.10, 0.21 and 1.03 X iO M for time periods of 8, 14, 20 or 26 h for basal LH secretion studies
and 10, 16, 22 and 28 h for CnRH-stimulated LH secretion. Both dosage of cortisol and length of
exposure had a depressing effect on basal LI-I release. The cortisol pretreatment also decreased
(P<0.001) the LH release following addition of GnRH (8.5 X lO M) in cultures at all dosages and
exposure times of cortisol. However, there was no decrease in LH or protein content of cells. These
experiments indicate a direct action of cortisol on the pituitary gland to depress both basal and
stimulated LH release.

INTRODUCTION fined and characterized. Some indirect evi-

dence has led other investigators to suggest that
It has been demonstrated that the effect of
adrenocorticotropic hormone (ACTU) on
ACTU may directly inhibit LU secretion. For
example, administration of exogenous ACTH,
ovulation and corpus luteum development and
but not cortisol, reduced ovarian weight (Pasley
function is not an effect of ACTU itself on the
and Christian, 1972). This decrease in ovarian
pituitary or on the ovary but could be a direct
weight could be reversed by injections of pitui-
effect of adrenal hormones on either the hypo-
tary homogenates (Christian et al., 1965).
thalamus or pituitary to inhibit luteinizing hor-
ACTU, but not corticosteroids, interfered with
mone (LH) secretion (Kobayashi et al., 1966;
ovulation in the sow (Liptrap, 1970).
Hagino et al., 1969; Liptrap, 1973; Hoffmann
Uagino et al. (1969) have demonstrated that
et al., 1977; Wagner et al., 1977; Moberg and
implantation of dexamethasone into the medial
Stoebel, 1980; da Rosa and Wagner, 1981; Li
preoptic area was as effective as systemic injec-
and Wagner, 1983). However, the exact Site(s)
tions in blocking ovulation in pregnant mare’s
of action of corticoids has not been clearly de-
serum (PMS)-treated rats. Smith et al. (1971)
also implanted crystalline cortisol acetate in the
medial basal hypothalamus and were able to
block gonadal maturation in rats. There also is
Accepted February 3, 1983. evidence which indicates that corticoids have a
Received October 27, 1982.
‘Reprint requests: W. C. Wagner, Dept. of Veteri- direct depressing influence on pituitary LU re-
nary Biosciences, University of Illinois, 2001 South lease (Baldwin and Sawyer, 1974; Sakakura
Lincoln Ave., Urbana, IL 61801. et al., 1975; Luton et al., 1977; Chantarapra-

25
26 LI AND WAGNER

teep and Thibier, 1978; Fuquay and Moberg, 1979). Pre-GnRH injection data were analyzed
1980; Sowers et al., 1980; Matteri and Moberg, separately by split-plot analysis of variance (Gill,
1978).
1982).
Data for corticoids were analyzed by split-plot
The question of whether ACTU can act analysis of variance with repeat measure over time
directly on LU-secreting cells or requires the using the GLM procedure. Data prior to ACTH or
production of steroid(s) from the adrenal hydrocortisone treatment were not included in the
cortex, which then inhibits LU secretion, is analysis of variance.
The cumulative response for LH and progesterone
important. Matteri and Moberg (1982) found
was defined as the area under the curve of plasma
that ACTU treatment caused a suppression of hormone concentration for 120 mm after GnRH injec-
both the initial LU response, and the sensitiza- tion as described by Thibier and Rolland (1976). The
tion of the LH response to a second GnRU area was expressed in terms of pg/mI per 120 mm.

treatment, while cortisol treatment only

Experiment II
blocked the initial response. Uowever, the
Materials. The medium used was Eagle’s minimal
ACTU-treated animals were intact, and the
essential medium (MEM, Gibco, Grand Island, NY)
amount and type of adrenocortical secretions with Earle’s salts, supplemented with nonessential
are unknown. amino acids (10 mM, Gibco) and L-glutamine solution
The objectives of this study were 1) investi- (200 mM, Gibco). Penicillin (50 U/mI medium),
gate the depressant effect of ACTH and hydro- streptomycin solution (200 mM), and fungizone
(amphotericin B-250, 0.62 pg/mI medium) were added
cortisone on the response of LU to GnRU
to prevent microbial contamination. The pH of the
treatment in heifers and 2) determine the effect medium (7.4) and buffering capacity were adjusted by
of cortisol on basal and GnRU-stimulated LU adding NaHCO3. Growth media used for incubation of
secretion by cultured bovine pituitary cells. cells on Days 0-5 contained 10% fetal bovine serum
(FBS).
Preparation and incubation of cells. Bovine pitui-
taries (cows) were collected at a campus abattoir
MATERIALS AND METHODS
within 30 mm of killing, immediately placed in cold
Experiment I
(4#{176}C)
MEM, and transported to the laboratory. Stage
of estrous cycle was not recorded. Cells were pre-
Animals and experimental Six intact design. and 6 pared as described by Smith et al. (1974) with slight
adrenalectomized (ADRX) heifers were used for this modifications. Within 1 h of death of the animals,
study. Animals were randomly assigned to one of the anterior pituitaries were pooled, weighed, and sliced
four treatment groups: A) intact saline; B) intact with a razor blade. The pituitary slices were washed
ACTH; C) adrenalectomized (ADRX) saline; or 3X with medium and transferred into a Spinner flask.
D) ADRX hydrocortisone. Sliced pituitary pieces
dispersed were
with 0.3%
Intact heifers were infused with either saline (0.9%, collagenase (Type 1-183 U/mg tissue; Worthington,
w/v) or ACTH (20.8 pg 1-24 ACTU/h) from cycle Freehold, NJ) in MEM. The dispersion was carried out
Day 2 to Day 14. On Days 7, 9 and 11, each animal at 37#{176}Cfor two 30-mm periods with constant stirring.
received a single iv. bolus of 100 pg of GnRH. At the end of each 30-mm incubation period, the cells
Adrenalectomized animals were infused with either in the supernatant were centrifuged at 375 X g for 3
saline or hydrocortisone sodium succinate (30 mg/h) mm. The supernatant was discarded, and the cells were
from cycle Days 2 to 9. On Days 5, 7 and 9, each ani- washed 4X with MEM. The trypan blue (0.4% in
mal also received a single iv. bolus of 100 pg of saline, Gibco) dye exclusion technique (Tennant,
GnRI-I. 1964) was applied to indicate cell viability in each
Adrenalectomy was performed and animals main- step. Virtually all cells remained intact and viable
tained with deoxycorticosterone acetate (Does) and according to this procedure. The washed cells were
cortisone acetate as described by da Rosa and Wagner then resuspended in MEM containing 0.25% pan-
(1981). Under general anesthesia, two catheters were creatin (Viokase, Gibco) and incubation was con-
placed in one jugular vein for infusion (proximal) and tinued as before for 15 mm. Cells were collected and
sampling (distal). The proximal catheter is the one washed 4X in MEM. Approximately 41.19 X 10’
closest to the heart. Jugular blood samples were col-
cells were obtained per gram of anterior pituitary tis-
lected via the more distal catheter prior to and after sue. Cells were then passed through 2 layers of cheese-
GnRH treatment at 1 5-mm intervals starting 30 mm
cloth and resuspended at a concentration of about 106
pre-GnRH andfor 120 mm post-GnRH. Daily blood viable cells/mI in MEM containing 10 percent FBS.
samples were also collected at 0800, 1200 and 1600 h. The number of cells was determined with a hemocy-
Plasma was separated at once and stored at -10#{176}C tometer. Two ml of this suspension were transferred
until assayed for LH, progesterone, and corticoids by to a tissue culture dish (35 X 10 mm) and grown at
radioimmunoassay (RIA). The assay procedures have for
37#{176}C 5 days in a humidified atmosphere of 5%
been previously described (Li and Wagner, 1983). CO2 and 95% 02. The incubation schedule for dis-
Statistical analysis. The data for LU and proges- persed pituitary cells is shown in Fig. 1. During the
terone concentrations after GnRH injection were incubation period, the medium was changed at 24-h
analyzed by split-split-plot analysis of variance (Gill, intervals beginning 48 h after the cells were pre-
1978) using the General Linear Models (GLM) pro- pared. On Day 5 of culture, cells were washed twice
cedure of the Statistical Analysis System (Barr et al., with MEM (without serum) to remove high concen-
 ACTH OR CORTISOL ON PITUITARY RESPONSE TO GnRH 27

CELL INCUBATION SCHEDULE

DISPERSED MEDIUM MEDIUM MEDIUM MEDIUM MEDIUM MEDIUM

CELLS CHANGED CHANGED CHANGED REMOVED, REMOVED, REMOVED,
PREPARED CELLS 2 ml ME- CELLS
WASHED, DIUM LYSED
2 ml ME- APPROPRIATE 2 ml .5%
DIUM TREATMENT LAURYL
APPROPRIATE & GnRH SULFATE
TREATMENT ADDED
ADfr

Ih JO 24 48 12 96 12O 124 -

I. 1.
HYDRO- BASAL GnRH-STIMULATED
CORTISONE RELEASE RELEASE
TREATMENT

FIG. 1. Schematic chart showing incubation schedule for dispersed pituitary cells and design of Experiment
II. Exposure to US was begun at 6, 12, 18 or 24 h before 120 h of incubation.

trations of accumulated LH. Treatments were ap- and assayed for LH. During the subsequent 2 h, the
plied during 2 successive 2-h periods. The medium cells were incubated with 2 ml of medium contamning
was collected after each treatment period and assayed appropriate concentrations of HS and 8.5 X 10 M
for LH by radioimmunoassay. GnRH. At the end of this final 2 h of incubation, the
At the completion of the incubation, cells were media and cells were harvested separately for assay.
lysed with 2 ml of 0.5 percent sodium lauryl sulfate Data in this study were obtained from 3 preparations
(dodecyl sulfate, sodmum salt) and cell contents of LU of cells, each being prepared from 2 pituitaries. Within
and protein were determined. The lauryl sulfate may each preparation, there were 3 replicates per treat-
cause a small underestimation of the LU content using ment.
the RIA procedure. Statistical analysis. The GnRH dose-response curve
Duplicate 500 p1 aliquots of pituitary cell lysate was calculated using a multiple regression (Dunn and
were precipitated with 2 ml of 20% trichloroacetic Clark, 1974) program according to the General Linear
acid (TCA) following the procedure described pre- Models (GLM) procedure of the Statistical Analysis
viously (Moguilevsky and Christot, 1973). Briefly, the System (Barr et al., 1979). Differences in the basal
TCA-insoluble residue was washed twice with 10% release of LU were analyzed by two-way analysis of
TCA. The precipitate was separated by centrifugation variance, withconcentrations of US, duration of cell
at 4#{176}C.Five hundred p1 of 1 N NaOH was added to exposure to US, and incubations as fixed effects
the final TCA precipitate and the mixture was heated (Dunn and Clark, 1974) by using the GLM procedure.
in a 90#{176}Cwater bath for 90 mm in a stoppered tube. The 3 incubations were defined as blocks. Data ob-
Protein concentration in the aliquots of the heated tained from the cells that were not exposed to US and
mixture was measured by the method of Lowry em al. not challenged with GnRH were not included in the
(1951), using lysozyme as standards. analysis of variance for GnRU-stimulated release of
Experimental design. A single preliminary experi- LH and cell LU and protein contents. Each incubation
ment was conducted to determine whether GnRH time and its corresponding concentration of US were
increased LH release from cells into medium in a dose- considered as one treatment. The differences in
related manner. After the cells had been in culture for GnRH-stimulated LH release and cell LH and protein
5 days, the cells were incubated with 2 ml of medium contents were analyzed by one-way analysis of vari-
for 2 h in the presence of various concentrations ance of a completely randomized block design (Dunn
(6.76. 4.23, 3.38 and 1.69 X 10 M, 8.5 X 1O, and Clark, 1974) using the GLM procedure.
i0#{176}, 10b0, 10” and 1012 M) of GnRU. There were
5 replicates per dose of GnRH. The medium was col-
lected after the treatment period and assayed for LH.
RESULTS
Concentrations of hydrocortisone succinate (US)
used in this study were 0, 0.01, 0.10, 0.21 and 1.03
X 10 M (0, 5, 50, 100 and 500 ng/ml). Total ex- Experiment I
posure times of cells to US were 8, 14, 20 and 26 h
The mean plasma corticoid concentrations
for basal LII release and 2 h longer for GnRH-stimu-
Iated LU release. HS was included in the growth me- for intact and ADRX heifers are illustrated in
dium at 6, 12, 18 or 24 h preceding the start of the Figs. 2 and 3. The analysis of variance for corti-
experimental period at 120 h (Fig. 1). On Day 5, after coids in intact heifers (Table 1) showed signifi-
the preliminary incubation of cells with HS, cells were
cant differences among treatments (P<0.01),
washed and then incubated with 2 ml of medium with
appropriate concentrations of US for 2 h to determine days (P<0.05), and a treatment by day inter-
effects on basal secretion. The medium was collected action (P<0.01). The analysis also showed
 28 LI AND WAGNER

100 LA, Sahne

100
/ Hydrocortisone (nr3)
90

/ f
90 I

- 80 _ i1jJ - 80
,I \

‘1 ‘ Iv

170
E 60
60
110 50

g’50 T/ Saline (n3)

g
r
40

40 30
ACTH (n3)
20
30
10

20 A 1 2 3 4 5 6 7 8 9 10 11 12

Infusion started
10
Day of Estrous Cycle
FIG. 3. Jugular plasma corticoid concentrations in
1 234567891011121314
ADRX heifers treated with saline or hydrocortisone
Infusion started sodium succinate (30 mg per h) beginning on cycle
Day of Estrous Cycle Day 2 and continued to Day 9. Values are expressed
as the mean and SEM.
FIG. 2. Jugular plasma corticoid concentrations in
intact heifers treated with saline or ACTH (20.8 pg per
h) beginning on cycle Day 2. Values are expressed as
the mean and SEM.
treated with saline on cycle Days 7, 9 and 11,
respectively (P< 0.05).
The mean concentrations of progesterone in
significant differences among treatments the plasma prior to GnRU treatment (Fig. 5)
(P<0.001), days (P<0.001), and a treatment by differed significantly (PczO.05) between saline-
day interaction (P<0.001) for ADRX heifers. and ACTU-treated heifers. As might be ex-
The ACTU-treated group had a mean corticoid pected, the progesterone concentration had a
concentration of 80 ng/ml compared to 68 smaller increase after GnRU in the ACTU-
ng/ml in the hydrocortisone-treated ADRX treated heifers than in the saline-treated group
animals. (P<0.05).
The mean concentrations of LU in the There were no differences in the mean LU
plasma before GnRU injection did not differ concentrations before or following GnRU treat-
significantly between saline- and ACTU-treated ment or in total LU response to GnRH among
heifers (Fig. 4). The cumulative responses to saline- and hydrocortisone-treated ADRX
the GnRU treatment were smaller (P<0.001) heifers (Fig. 6). The total LU (cumulative) re-
on Days 9 and 11 than on Day 7 for both sponse to GnRH on Days 7 and 9 did not differ
ACTU- and saline-treated groups. A significant from Day 5 for either saline- or hydrocortisone-
(P<0.01) day by treatment interaction also was treated groups. LU increased sharply within 15
observed. to 30 mm after GnRH injection in ADRX hei-
Intact heifers given saline had an LU re- fers given saline. LU peaked at 17, 17 and 12
sponse to GnRU within 15 mm. Plasma LH ng/ml on cycle Days 5, 7 and 9, respectively,
concentrations peaked at 30, 22 and 17 ng/ml and then gradually decreased on all 3 days. In
within 30 to 45 mm on cycle Days 7, 9 and 11, ADRX heifers given hydrocortisone, LU first
respectively. The pituitary responsiveness to peaked at 12, 9 and 7 ng/ml within 30 mm on
GnRU in ACTU-treated animals was quite dif- cycle Days 5, 7 and 9, respectively, and then re-
ferent from that seen in the saline controls with mained relatively constant until about 105 mm
only a small amount of LU released 30 to 45 after GnRU injection (Fig. 6). Analysis of
mm after each GnRU injection. The total LU variance showed differences due to time
responses of the ACTU-treated heifers were (P<0.01) and a time by treatment interaction
24%, 30% and 23% of those observed in heifers (P< 0.01).
 ACTH OR CORTISOL ON PITUITARY RESPONSE TO GnRH 29

TABLE 1. Analysis of variance for corticoid concentrations in jugular plasma for intact (I, receiving ACTII or
saline) and adrenalectomized (II, receiving HS or saline) heifers.

Degrees Mean
Source of variation of freedom square F

Treatment 1 100673.634 643.55a

Animal (treatment) 4 156.434 - - -

Day 12 257.991 208b

Treatment X day 12 352.169 2.83
Day X animal (treatment) 48 124.307 - - -

II
Treatment 1 55514.316 182.98c
Animal (treatment) 4 303.390 - - -

Day 7 789.262 8.61c

Treatment X day 7 848.427 9.86c
Day X animal (treatment) 21 91.633 - - -

ap<OOl

bp<oo5

- Saline (n3)
- - ACTH (n3)

day 7 day 9 day 11

a
E
a
a
0.

I
-I

Time After GnRH Injection (mm)

FIG. 4. Jugular plasma LU in response to a single injection of 100 pg of GnRH (given at 0 time) on cycle
Days 7, 9 and 11 in intact heifers treated with saline or ACTU (20.8 pg per h) from cycle Day 2 to Day 14.
Values are expressed as the mean and SEM.
30 LI AND WAGNER

10 Saline (n3)
ACTH (n3)
9
day 7

E
0.
day 9 day 11
a,
#{163}5
a

iij-j--i-U-IIIj LTILf.f.JiLI
-30 0 306090 120 -30 0 30 6090120 -30 0 3060 90120

Time After GnRH Injection (mm)

FIG. 5. Jugular plasma progesterone in response to a single injection of 100 pg of GnRH (given at 0 time) on
cycle Days 7, 9 and 11 in intact heifers treated with saline or ACTH (20.8 pg per h) from cycle Day 2 to Day 14.
Values are expressed as the mean and SEM.

day 5 day 7 day 9

a Saline

E
0.

E
C

I
-j

30 Hydrocortisone
a
E
a
a
0.
20
-

#{163}
IL-.. ii
10 F
V
,\1.i V..
i-. .T. .
‘
-

I
‘iiTJ.r
I V., L-1-_.,1’
(1

-30 0 30 60 90120 -30 0 30 60 90120 -30 0 30 6090120

Time After GnRH Injection (mm)

FIG. 6. Jugular plasma LU in response to a single injection of 100 pg of GnRU (given at 0 time) on cycle
Days 5, 7 and 9 in ADRX heifers treated with saline or hydrocortisone sodium succinate (30 mg per h) from
cycle Day 2 to Day 9. Values are expressed as the mean and SEM.
 ACTH OR CORTISOL ON PITUITARY RESPONSE TO GnRH
31

- Saline (n3)
- Hydrocortisone (n=3)

day 5 day 7 day 9

8

E
-.-
a,
C
4
c
03

,,I1.tLI-I-1
-30 0 306090120 -30 0 30 60 90120 -30 0 30 60 90 120

Time After GnRH Injection (mm)

FIG. 7. Jugular plasma progesterone in response to a single injection of 100 pg of GnRH (given at 0 time) on
cycle Days 5, 7 and 9 in ADRX hemfers treated with saline or hydrocortisone sodium succinate (30 mg per h)
from cycle Day 2 to Day 9. Values are expressed as the mean and SEM.

The mean concentrations of progesterone in a significant (P<0.O01) block effect (Table 2)

the plasma prior to GnRU injection (Fig. 7) indicating wide variation between pituitary cell
differed (P<0.05) between saline- and hydro- preparations.’ That variation partially accounted
cortisone-treated ADRX heifers. As shown in for the interactions between the effects of the
Fig. 7 the post-GnRU progesterone responses of dosage of US and the incubations (P<0.05) and
the ADRX heifers given hydrocortisone were
16%, 33% and 37% of those observed in the
ADRX heifers given saline on cycle Days 5, 7
and 9, respectively (P<0.05). 100

Experiment II
- 80
a,
The effect of increasing concentrations of C T
T
GnRU on LU concentration in media is shown
60
in Fig. 8. In the absence of GnRU, LU concen-
tration in the medium averaged 12 ng/ml. LU
release increased linearly (P<0.001, r=0.94) 40
with increasing log concentration of GnRH over
the range of 8.5 x 10_12 M to 1.69 x 10 M. 20

n1
Since the 8.5 x 108 M concentration of GnRU
provided a response at 85% of the maximum,
3 U) U) U) U) U) 0) W (‘J (

this concentration was used in the subsequent n

experiments in order to avoid the possibility of 0 c.,
exceeding the maximum response area.
The effects of various concentrations of US
GnRH (Molar Cone.. X10.-8)
on the basal release of LU by bovine pituitary
cells following 8, 14, 20 and 26 h of exposure FIG. 8. The effect of various doses of synthetic
GnRI-l upon the release of LH by cultured bovine
are illustrated in Fig. 9. As shown in Fig. 9, the
pituitary cells (2 X 10’). The incubation time was 2 h.
very first column is the average of pooled data
Each column represents the mean amount of LH re-
resulting from no US exposure, indicating a leased into the medium of 5 replications. Vertical bars
mean LU concentration of 12 ng/ml. There was indicate SEM of the responses.
32 LI AND WAGNER

Hydrocortisone
a o.o
E .O1x1O6M

.ioxio-6rvi
.21x1OM

0 fl 1.O3x1OM

0
ilO
C
N
C

8h 14h 20h 26h

Duration of Hydrocortisone Exposure
FIG. 9. LU concentration in the medium following 8, 14, 20 and 26 h of incubation of bovine pituitary cells
(2 X 106) with hydrocortisone. Values are expressed as the mean and SEM.

between the length of hydrocortisone exposure lease of LU in the medium was decreased
and the incubations (P<0.001). There was also (P<0.001) linearly when pituitary cells were
a significant effect of hydrocortisone dosage exposed to 0.01, 0.10, 0.21 or 1.03 x 10_6 M
(P<O.001) and length of hydrocortisone ex- cortisol.
posure (P<0.001), but no interaction between The effects of various doses of US on GnRU-
exposure time and dosage of US. The basal re- induced release of LU following 10, 16, 22 and

TABLE 2. Analysis of variance for basal release of LH in the medium of cultured bovine pituitary cells.

Degrees Mean
Source of variation of freedom square F

Block 2 918.814 247.05a

Exposure time 4 41.914 11.27a
GnRH control 1 17.215 463b
Exposure 3 50.153 13.48k
Hydrocortisone 4 51.084 13.74a
Lmnear 1 193.292 51.97a
Quadratic 1 0.057 0.02
Residual 2 5.493 1.48
Hydrocortmsone X block 8 11.055 2.97c
Exposure time X block 8 25.685 6.91a
Exposure X hydrocortisone 12 5.595 1.50
Exposure time X block X hydrocortisone 24 5.017 1.35
Residual 126 3.719 - - -

aP<0.0o1.

bp<005

cp<OOl
 ACTH OR CORTISOL ON PITUITARY RESPONSE TO GnRH 33

100

Hydrocortisone

180 .01x106M

.10x106M

.21x106M

D1.03x10_6M

0
lOh 16h 22h 28h
Duration of Hydrocortisone Exposure
FIG. 10. LH concentration in media of cultured bovine pituitary cells (2 X 106) incubated with various con-
centrations of hydrocortisone for 10, 16, 22 and 28 h and exposed to 8.5 X i0 M GnRU during the final 2 h
of incubation. Values are expressed as the mean and SEM.

28 h of incubation are illustrated in Fig. 10. for cells treated with HS for 16 h. All other
The analyses of variance for GnRH-induced LU treatment periods were comparable to the un-
release in the medium and pituitary cell LU and treated control value.
protein contents are given in Table 3. The There was a tendency for a greater (P<0.10)
analysis showed a block effect (P<O.001) on protein content in cells incubated with US at all
GnRU-induced LH release in the medium and exposure times than in those incubated with
on pituitary cell LU and protein contents, indi- GnRU alone and no US exposure. There was a
cating wide variation between pituitary cell linear effect of hydrocortisone dosage
preparations. In the absence of US and GnRU, (P<0.001) and length of hydrocortisone ex-
a mean LU concentration of 27 ng/ml was ob- posure (P<0.001) on cell protein content
tained. When pituitary cells were incubated (Fig. 11).
with GnRU alone and had no US exposure
during the final 2 h of incubation, the mean LU
DISCUSSION
concentration in the medium was 78 ng/ml,
which was significantly (P<0.001) greater than Infusion of intact heifers with ACTH and
that of cells exposed to all concentrations of ADRX heifers with cortisol produced a similar
hydrocortisone at all exposure times. There was plasma corticoid concentration (ACTU, 80
a linear effect of hydrocortisone dosage ng/ml; hydrocortisone, 68 ng/ml) throughout
(P<0.001) but no linear effect of length of the experimental period. This amount of corti-
hydrocortisone exposure. coid was higher than that reported by Stoebel
LU content of pituitary cells incubated with and Moberg (1979) and Moberg and Stoebel
GnRH alone and no HS was not different from (1980) for heifers after ACTU (200 lU/day)
that after exposure to various doses of US for injection for 5.5 days and cortisol (30 mg/h)
10, 16, 22 and 28 h (Table 3). The linear effect infusion for 141 h.
of length of hydrocortisone exposure (P<0.05) Our data presented in this study showed that
related primarily to an unexplained higher value continuous infusion of ACTH reduced the LU
 34 LI AND WAGNER

response to GnRU. This finding is in agreement completely block the pituitary responsiveness
with the observation from previous studies that to GnRH under our experimental conditions.
administration of ACTU to rams inhibited Matteri and Moberg (1982) also found that
LURU-induced LU release (Fuquay and ACTU treatment was more effective than corti-
Moberg, 1980) and that ACTU or cortisol treat- sol in suppressing LU secretion.
ment similarly reduced the LU response to The ADRX animals treated with hydrocorti-
GnRU in dairy heifers (Matteri and Moberg, sone exhibited an LU response that was qualita-
1982). The inhibitory effect of ACTU was not tively different from that observed in ADRX
due to increased plasma progesterone concen- saline controls. The onset of the LU increase
trations originating from the adrenal cortex. If was delayed with the maximum LU concentra-
there was a progesterone block on LU release tion somewhat reduced. Whether or not these
by GnRU, then the marked decrease in plasma qualitative differences would affect the ovula-
progesterone following ACTU treatment in tory response is not known because these ani-
these heifers would be expected to constitute mals were all in the luteal phase when chal-
removal of this block. It has also been observed lenged with GnRU. Uowever, our data suggest
that serum progesterone had no influence on that hydrocortisone may reduce or alter the
GnRU-induced pituitary LU release in heifers sensitivity of the pituitary to respond to GnRU.
(Zolman et al., 1974) and ewes (Cumming et The difference in degree of suppression of LU
al., 1972). The small amount of LU released in release between ACTU-treated intact heifers
response to GnRU in ACTU-treated heifers and US-treated ADRX heifers was rather sur-
would suggest that ACTU treatment does not prising since plasma concentrations of gluco-

TABLE 3. Analyses of variance for LH concentration in the medium (I) and pituitary cell LU (II) and protein
(III) contents after in vitro GnRH treatment.

Degrees Mean
Source of variation of freedom square F

Block 2 10237.612 126.35

Treatment 16 1262.592 15.58a
GnRH control vs. rest 1 17551.690 216.62a
Hydrocortisone vs. linear 1 1898.321 23.43a
Exposure time vs. linear 1 0.748 0.01
Residual 134 80.421 - - -

II
Block 2 448349.020 195.70a
Treatment 16 56310.353 246b
GnRH control vs. rest 1 7105.353 0.31
Hydrocortisone vs. linear 1 12582.302 0.55
Exposure time vs. linear 1 152674.998 6.66c
Residual 134 22934.733 - - -

II!
Block 2 18479.515 24.85a
Treatment 16 2386.482 3.21a
GnRH control vs. rest 1 2091.574 281d
Hydrocortisone vs. linear 1 14574.780 l9.6O
Exposure time vs. linear 1 14621.069 19.66a
Residual 134 734.680 - - -

aP<0.ool.
bp<0o1
Cp<O.OS.
dp<ol 0.
 ACTH OR CORTISOL ON PITUITARY RESPONSE TO GnRH 35

Hydrocortisone
OJ3

D .O1x1O6M
2 .ioxio-6ivi
C
0 : .21x1O6M
C.)
0 1.O3x1O6M

C
a,
0a,
(‘C-)
Co
ao
‘-I-

C-)

lOh 16h 22h 28h

Duration of Hydrocortisone Exposure
FIG. 11. Protein content in cultured bovine pituitary cells (2 X jQ6) after incubation with various concen-
trations of hydrocortisone for 10, 16, 22 and 28 h and exposed to 8.5 X 1o M GnRU during the final 2 h of
incubation. Values are expressed as the mean and SEM.

corticoids were similar in the two groups. Other results or there might be a difference between
steroids such as estrogens and androgens were plasma and tissue concentrations of these hor-
not measured however. Since da Rosa and mones. Uowever, in the present experiment, the
Wagner (1981) have shown that ACTU adminis- incubation medium was essentially free of
tration caused increased estrogen concentra- endogenous hormones, except for the fact that
tions, the role of such steroids produced by the the system was supplemented with 10% fetal
adrenal cortex under ACTU stimulation cannot bovine serum. The concentration of corticoid
be ignored. Exposure of bovine pituitary cells was undetectable in medium containing 10%
to hydrocortisone at concentrations of 0.01 x fetal bovine serum.
10_6 M or higher for periods of 8 h or longer Uydrocortisone, at the concentrations used,
decreased basal LU release. The range in con- significantly suppressed the G nRU-stimulated
centrations of hydrocortisone used in this study LU release when added to dispersed pituitary
resemble the plasma corticoid concentrations of cell cultures for exposure periods of 10 h or
adrenalectomized heifers given saline (5 ng/ml longer prior to addition of the GnRU. This
plasma) and hydrocortisone (68 ng/ml plasma), finding is in agreement with the observations
and intact heifers given ACTU (53 and 80 from several studies that glucocorticoids, either
ng/ml plasma) as reported by Li and Wagner natural or synthetic, can affect the responsive-
(1983). It appears that the in vitro system was ness of the pituitary to GnRU in the rat (Bald-
more sensitive to the depressing effect of win and Sawyer, 1974), human (Boccuzzi et
hydrocortisone on LU secretion than is true in al., 1975; Sakakura et al., 1975; Luton et al.,
the animal itself. The inconsistency could be 1977; Sowers et al., 1980), ram (Fuquay and
due to the fact that in the whole animal, the Moberg, 1980) and cattle (Chantaraprateep and
endogenous hormone levels might influence the Thibier, 1978; Matteri and Moberg, 1982). The
36 LI AND WAGNER

authors of these studies interpreted their results ACKNOWLEDGMENTS

as indicating that glucocorticoids suppress LU
The authors are grateful to Dr. J. J. Chart (Ciba),
release at the anterior pituitary by diminishing Dr. J. Lauderdale (The Upjohn Co.) and Dr. Myron
its responsiveness to GnRU. By contrast, the Brown (Abbott Labs.) for their generous gifts of
study by Hagino et al. (1969) showed that ovu- ACTH, hydrocortisone and GnRU, respectively. We
also wish to thank Dr. Roger Shanks for statistical
lation was blocked when dexamethasone was
consultation. Special thanks are also given to Dr.
implanted into the medial preoptic area of Claudio Pimentel and Mr. Cliff Smith for their assist-
PMS-treated immature rats. Smith et al. (1971) ance with pituitary collections.
showed that cortisol implants in the medial This study was supported in part by USPHS,
basal hypothalamus in immature rats delayed
NICHD Grant No. R01-HD08698.
sexual maturation. They suggested that the
probable site of the action of glucocorticoids
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