and Pituitary Sensitivity to

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