BIOLOGY
OF REPRODUCTION
20, 763-772
(1979)
Dose
Effect
of a Single
Related
on Gonadotropin LHRH JAMES
Injection
in Estrogen-Primed C. McPHERSON,
Castrate III and
Department
of
Augusta,
Sensitivity Female
VIRENDRA
to
Rats’
B. MAHESH
Endocrinology,
College
Medical
of Progesterone
and Pituitary
Secretion
of Georgia,
Georgia
30901
ABSTRACT This
investigation as a single
studied the effect on gonadotropin secretion of varying doses of progesterone given injection to estrogen-primed castrated rats. Female rats were ovariectomized at 26 days of age (Day 1) and administered 0.1 Mg/kgBw estradiol-17fl for 4 days starting on the day of castration. Progesterone, in various doses, was administered at 0930 h on the fourth postcastration day and groups of animals were sacrificed between 1000 h and 1600 h. Estrogen treatment reduced but did not suppress the postcastration rise of gonadotropins. In estrogen-primed castrated rats given 0.2 or 0.8 mg/kgBW progesterone, there was a significant increase in both serum follicle stimulating hormone (FSI-I) and luteinizing hormone (LH) by 1600 h. In estrogenprimed castrated rats given 0.4 or 3.2 mg/kgBW progesterone, there was a significant suppression of both FSH and LI-I by 1600 h. By 1800 h the serum LH had returned to baseline levels in all groups. Estrogen-primed castrated rats given 0.8 or 3.2 mg/kgBW progesterone were then administered 10 ng pulses of luteinizing hormone releasing hormone (LHRH) at 1800 h 2000 h and 2200 h and bled at 0, bO, 60 and 120 after each pulse. There was a dose related effect of progesterone on the pituitary’s response to LHRH. The 0.8 mg dose was stimulatory and the 3.2 mg dose inhibitory as compared with estrogen treated controls for LFI. For FSH, there was no difference in the increment after LHRH in the progesterone treated and nonprogesterone treated estrogen-primed castrated rats. Between the 2 progesterone treated groups, however, the increment in FSH in rats treated with the 0.8 mg/kgBW dose was significantly higher than in rats given the 3.2 mg/kgBW dose. Pituitary gonadotropin content was significantly increased by the 0.8 mg/kgBW progesterone dose. In addition to the demonstration of a direct pituitary effect, this study shows that the effect of progesterone is dose dependent under conditions of uniform estrogen stimulation and time of exposure to progesterone.
INTRODUCTION Several
investigators
the effect or inhibitory ovarian istration
of
have
progesterone to gonadotropin
cycle of
depending progesterone
on
that
demonstrated
may the
be stimulatory release in the time (Everett,
of
rat
admin1948;
Zeilmaker, 1966; Goldman et a!., 1971). In a previous study (McPherson et al., 1975) we reported the dose dependent effect of progesterone on gonadotropin secretion in the estrogen-primed
immature
gonadotropin morning levels the steroids afternoon
surge.
castrated
female
rat.
the
estradiol
given for the entire length of period rather than proges-
terone
following
estradiol
current effect
investigation of a single
was injection
gonadotropin the dose
secretion dependent
during
period
the
conditions in and progesterone under
The
these
effect
pituitary studied FSH
and
in by and
of
which
the
pretreatment.
the
afternoon
the exposure was controlled.
the on
surge
the
progesterone
estrogen-primed the the
rat
pituitary pituitary
under
to estrogens Furthermore,
conditions, of
examining LH and
The
designed to study of progesterone
and to examine whether effect was manifested
controlled
dose-related
levels measured were the tonic 14 h after the last injection of rather than those during the Furthermore,
progesterone were the postcastration
direct on
the
was
also
content response
of to
LHRH.
Accepted November 28, b978. Received July 31, 1978. ‘This investigation was supported Research Grant HI) 10795.
MATERIALS by
NICHHD
Sprague-Dawley Holtzman Company
763
AND
METHODS
female rats, obtained at 23 days of age, were
from used
the in all
McPHERSON
764
of
the
studies.
environment
They (lights
and were given Ovariectomy of age. Surgery
housed in a controlled 0500 h to 1900 h, EST) and rat chow ad libitum. performed on animals at 26 days
was
was performed
under
ether
anesthesia
through bilateral dorsal incisions. After surgery, animals were divided into groups of at least 6 rats. Treatment with 0.1 tg/kg/day estradiolI 7I was begun on the day of surgery and continued for 3 additional days. The mean BW of the rats at the start of the experiment was 62.5 ± 2 g. Based on previous experience of average weight at the middle of the experimental period, the dosages of steroids were calculated on the basis of a 70 g BW with no adjustment for lesser BW at the start of the experiment and higher BW towards the end. Dosages were divided into 2 0.1 ml injections daily given s.c. in a corn oil vehicle. Additional groups of castrate and intact animals not treated with estradiol served as controls. The treatment period was terminated on the fifth day of castration (day of castration considered as Day 1) with a single injection of either: 0.2, 0.4, 0.8 or 3.2 mg/ kgBW progesterone at 0930 h to estrogen-primed castrated animals; an injection of 0.8 mg/kgBW progesterone to castrated animals not treated with estradiol; or an injection of 0.1 ml vehicle to estrogenprimed animals or to untreated castrated and intact animals. Groups of animals were then rapidly anesthetized with ether at 1000 h, 1200 h, 1400 h and 1600 h and blood was withdrawn by cardiac puncture, allowed to clot and centrifuged. The serum was
separated
and stored
at -4#{176}C until
assayed.
The experimental procedure for the next set of experiments was the same as described above. Ovariectomized immature rats were treated for 4 days with 0.1 tg/kg/day estradiol-17(3 beginning on the day of castration. Either vehicle or 0.8 or 3.2 mg/kgBW progesterone was administered at 0930 h on the fifth day of castration. Ten ng pulses of LHRH (Beckman) or 0.2 ml saline were administered i.v. in the tail vein at 1800, 2000 h and 2200 h with groups of animals sacrificed at 0, 10, 60 and 120 mm following each pulse. Blood was obtained by cardiac puncture. Pituitaries were removed and quickly frozen in 1 ml
phosphate
buffered
Individual
saline
(PBS),
pH 7.5.
serum
samples and pituitary samples were assayed for FSH and LH by double-antibody radioimmunologic methods of Eldridge et al., 1974. Pituitaries were thawed and homogenized in 3 ml PBS using a glass homogenizer. A dilution of 100 ii of the homogenate to 10 ml was made with PBS containing
1% bovine serum dards, hormones obtained Program.
albumin, pH 7.5 and assayed. for labeling and anri-rat-FSH
from the NIAMDD Anti-ovine-LH was
Gordon Niswender. the chloramine-T et
al.
gamma through in terms FSH-RP-1
(1963).
The
‘second
antibody,”
produced of goats.
in our Results
of the respective or NIAMD-LH-RP-1 serum or pg/pituitary.
ng/ml for rat serum
and
Miller and Aehnelt (1978). Statistical Duncan’s Multiple
Hormone obtained
standards (Parlow
RESULTS
Effect
of a Single
of Progesterone FSH and LH Female
To
evaluate
of
anti-rabbit
used: NIAMDet al., 1969), The use of anti-ovine-LH pituitary LH has been validated by (1977) and Greeley and Mahesh analyses were performed using Range test (Steel and Torrie, 1960).
castration at
of
and
female at 0930
and 1000
a single
FSH
FSH
1200
of
in
the
rat, progesh on the fifth
serum
h,
dose LH
h,
and
1400
LH
h
and
1600 h. Controls were castrated rats given vehicle at 0930 h, castrated rats given 0.8 mg/kgBW progesterone at 0930 h, castrated rats given 0.1 j.ig/kgBW/day of estradio! followed by vehicle at 0930 h on of
castration
at serum both in
and
0930
h.
The
intact
castrated
(P
OF REPRODUCTION
20, 763-772
(1979)
Dose
Effect
of a Single
Related
on Gonadotropin LHRH JAMES
Injection
in Estrogen-Primed C. McPHERSON,
Castrate III and
Department
of
Augusta,
Sensitivity Female
VIRENDRA
to
Rats’
B. MAHESH
Endocrinology,
College
Medical
of Progesterone
and Pituitary
Secretion
of Georgia,
Georgia
30901
ABSTRACT This
investigation as a single
studied the effect on gonadotropin secretion of varying doses of progesterone given injection to estrogen-primed castrated rats. Female rats were ovariectomized at 26 days of age (Day 1) and administered 0.1 Mg/kgBw estradiol-17fl for 4 days starting on the day of castration. Progesterone, in various doses, was administered at 0930 h on the fourth postcastration day and groups of animals were sacrificed between 1000 h and 1600 h. Estrogen treatment reduced but did not suppress the postcastration rise of gonadotropins. In estrogen-primed castrated rats given 0.2 or 0.8 mg/kgBW progesterone, there was a significant increase in both serum follicle stimulating hormone (FSI-I) and luteinizing hormone (LH) by 1600 h. In estrogenprimed castrated rats given 0.4 or 3.2 mg/kgBW progesterone, there was a significant suppression of both FSH and LI-I by 1600 h. By 1800 h the serum LH had returned to baseline levels in all groups. Estrogen-primed castrated rats given 0.8 or 3.2 mg/kgBW progesterone were then administered 10 ng pulses of luteinizing hormone releasing hormone (LHRH) at 1800 h 2000 h and 2200 h and bled at 0, bO, 60 and 120 after each pulse. There was a dose related effect of progesterone on the pituitary’s response to LHRH. The 0.8 mg dose was stimulatory and the 3.2 mg dose inhibitory as compared with estrogen treated controls for LFI. For FSH, there was no difference in the increment after LHRH in the progesterone treated and nonprogesterone treated estrogen-primed castrated rats. Between the 2 progesterone treated groups, however, the increment in FSH in rats treated with the 0.8 mg/kgBW dose was significantly higher than in rats given the 3.2 mg/kgBW dose. Pituitary gonadotropin content was significantly increased by the 0.8 mg/kgBW progesterone dose. In addition to the demonstration of a direct pituitary effect, this study shows that the effect of progesterone is dose dependent under conditions of uniform estrogen stimulation and time of exposure to progesterone.
INTRODUCTION Several
investigators
the effect or inhibitory ovarian istration
of
have
progesterone to gonadotropin
cycle of
depending progesterone
on
that
demonstrated
may the
be stimulatory release in the time (Everett,
of
rat
admin1948;
Zeilmaker, 1966; Goldman et a!., 1971). In a previous study (McPherson et al., 1975) we reported the dose dependent effect of progesterone on gonadotropin secretion in the estrogen-primed
immature
gonadotropin morning levels the steroids afternoon
surge.
castrated
female
rat.
the
estradiol
given for the entire length of period rather than proges-
terone
following
estradiol
current effect
investigation of a single
was injection
gonadotropin the dose
secretion dependent
during
period
the
conditions in and progesterone under
The
these
effect
pituitary studied FSH
and
in by and
of
which
the
pretreatment.
the
afternoon
the exposure was controlled.
the on
surge
the
progesterone
estrogen-primed the the
rat
pituitary pituitary
under
to estrogens Furthermore,
conditions, of
examining LH and
The
designed to study of progesterone
and to examine whether effect was manifested
controlled
dose-related
levels measured were the tonic 14 h after the last injection of rather than those during the Furthermore,
progesterone were the postcastration
direct on
the
was
also
content response
of to
LHRH.
Accepted November 28, b978. Received July 31, 1978. ‘This investigation was supported Research Grant HI) 10795.
MATERIALS by
NICHHD
Sprague-Dawley Holtzman Company
763
AND
METHODS
female rats, obtained at 23 days of age, were
from used
the in all
McPHERSON
764
of
the
studies.
environment
They (lights
and were given Ovariectomy of age. Surgery
housed in a controlled 0500 h to 1900 h, EST) and rat chow ad libitum. performed on animals at 26 days
was
was performed
under
ether
anesthesia
through bilateral dorsal incisions. After surgery, animals were divided into groups of at least 6 rats. Treatment with 0.1 tg/kg/day estradiolI 7I was begun on the day of surgery and continued for 3 additional days. The mean BW of the rats at the start of the experiment was 62.5 ± 2 g. Based on previous experience of average weight at the middle of the experimental period, the dosages of steroids were calculated on the basis of a 70 g BW with no adjustment for lesser BW at the start of the experiment and higher BW towards the end. Dosages were divided into 2 0.1 ml injections daily given s.c. in a corn oil vehicle. Additional groups of castrate and intact animals not treated with estradiol served as controls. The treatment period was terminated on the fifth day of castration (day of castration considered as Day 1) with a single injection of either: 0.2, 0.4, 0.8 or 3.2 mg/ kgBW progesterone at 0930 h to estrogen-primed castrated animals; an injection of 0.8 mg/kgBW progesterone to castrated animals not treated with estradiol; or an injection of 0.1 ml vehicle to estrogenprimed animals or to untreated castrated and intact animals. Groups of animals were then rapidly anesthetized with ether at 1000 h, 1200 h, 1400 h and 1600 h and blood was withdrawn by cardiac puncture, allowed to clot and centrifuged. The serum was
separated
and stored
at -4#{176}C until
assayed.
The experimental procedure for the next set of experiments was the same as described above. Ovariectomized immature rats were treated for 4 days with 0.1 tg/kg/day estradiol-17(3 beginning on the day of castration. Either vehicle or 0.8 or 3.2 mg/kgBW progesterone was administered at 0930 h on the fifth day of castration. Ten ng pulses of LHRH (Beckman) or 0.2 ml saline were administered i.v. in the tail vein at 1800, 2000 h and 2200 h with groups of animals sacrificed at 0, 10, 60 and 120 mm following each pulse. Blood was obtained by cardiac puncture. Pituitaries were removed and quickly frozen in 1 ml
phosphate
buffered
Individual
saline
(PBS),
pH 7.5.
serum
samples and pituitary samples were assayed for FSH and LH by double-antibody radioimmunologic methods of Eldridge et al., 1974. Pituitaries were thawed and homogenized in 3 ml PBS using a glass homogenizer. A dilution of 100 ii of the homogenate to 10 ml was made with PBS containing
1% bovine serum dards, hormones obtained Program.
albumin, pH 7.5 and assayed. for labeling and anri-rat-FSH
from the NIAMDD Anti-ovine-LH was
Gordon Niswender. the chloramine-T et
al.
gamma through in terms FSH-RP-1
(1963).
The
‘second
antibody,”
produced of goats.
in our Results
of the respective or NIAMD-LH-RP-1 serum or pg/pituitary.
ng/ml for rat serum
and
Miller and Aehnelt (1978). Statistical Duncan’s Multiple
Hormone obtained
standards (Parlow
RESULTS
Effect
of a Single
of Progesterone FSH and LH Female
To
evaluate
of
anti-rabbit
used: NIAMDet al., 1969), The use of anti-ovine-LH pituitary LH has been validated by (1977) and Greeley and Mahesh analyses were performed using Range test (Steel and Torrie, 1960).
castration at
of
and
female at 0930
and 1000
a single
FSH
FSH
1200
of
in
the
rat, progesh on the fifth
serum
h,
dose LH
h,
and
1400
LH
h
and
1600 h. Controls were castrated rats given vehicle at 0930 h, castrated rats given 0.8 mg/kgBW progesterone at 0930 h, castrated rats given 0.1 j.ig/kgBW/day of estradio! followed by vehicle at 0930 h on of
castration
at serum both in
and
0930
h.
The
intact
castrated
(P
