Gonadotropin Secretion in Ovariectomized Ewes: Effect of Passive ...

BIOLOGY

OF REPRODUCTION

42, 273-280

(1990)

Gonadotropin Secretion in Ovariectomized Ewes: Effect of Passive Immunization against Gonadotropin-Releasing Hormone (GnRH) and Infusion of a GnRH Agonist and Estradiol1 and T. E. ADAMS2

M. E. HERMAN Department

of Animal

University Davis,

Science

of California California

95616

ABSTRACT Gonadotropin secretion was examined in ovariectomized sheep after passive immunization against gonadotropin-releasing hormone (GnRH). Infusion of ovine anti-GnRR serum, but not control antiserum, rapidly depressed serum concentrations of luteinizing hormone (LII). The atui-GnRI-I-induced reduction in serum LII was reversed by circhora! (hourly) administration of a GnRH agonist that did not cross-react with the an:i-GnRH serum. In contrast, passive immunization against GnRH led to only a modest reduction in serum concentrations offolliclestimulating hormone (FSH). Pulsatile delivery of the GnRH agonist did not influence serum concentrations of FSH. Continuous infusion of estradiol inhibited and then stimulated gonadotropin secretion in animals passively immunized against GnRH, with gonadotrope function driven by GnRH agonist. However, the magnitude of the positive feedback response was only 10% of the response noted in controls. These data indicate that the estradiol-induced surge of LH secretion in ovariectomized sheep is the product of estrogenic action at both hypothalamic and pituitary loci. Replacement of the endogenous GnRH pulse generator with an exogenous generator of GnRH-like pulses that were invariant infrequency and amplitude could not fully reestablish the preovulatory-like surge of LH induced by estradiol.

GnRH

from the hypothalamus (Clarke and Cummins, Moenter et al., 1989). Increases in GnRH pulse amplitude during the estrogen-induced LH surge in the ovariectomized rhesus macaque have also been reported (Levine et al., 1985). Estrogen may also act directly at adenohypophyseal sites. Administration of GnRI-I cannot reverse the negative feedback effect of estradiol in a number of species (Kesner et al., 1981; Clarke and Cummins, 1984; Kesner et al., 1987; Condon et al., 1988). Similarly, estradiol increases the concentration of GnRH receptors in pituitary tissue even when the tissue is disconnected from the hypothalamus by physical (Gregg and Nett, 1989) or immunological (Adams and Adams, 1988) methods. Moreover, as Schillo et al. (1985) reported, rates of hypothalamic secretion of GnRH are unaltered during the estradiol-induced LH surge in ovariectomized sheep. In addition, both the negative and positive feedback effects of estradiol are expressed in vitro (Turgeon and Waring, 1981; Dumesic et al., 1987). In this study, the magnitude of the negative and positive feedback responses induced by estradiol was examined in ovariectomized sheep in which endogenous GnRH had been neutralized by passive immuniza-

INTRODUCTION

1985;

The high level of secretion of estradiol from mature Graafian follicles leads to the preovulatory surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the estrous cycle of sheep (Webb et al., 1981). Exogenous estradiol induces a similar augmentation of LH and FSH secretion in ovariectomized sheep (Adams and Adams, 1986). The increase in LH and FSH secretion induced by esiradiol may reflect estrogenic actions at hypothalamic and/or adenohypophyseal loci. Steroid action at the hypothalamic level is suggested by the observation that portal concentrations of gonadotropin-releasing hormone (GnRH) and concentrations of LH in the peripheral circulation are reduced in parallel during estradiol administration (Karsch et al., 1987). Similarly, the estradiol-induced augmentation of LH secretion in ovariectomized sheep is associated with a marked increase in secretion of

Accepted October 24, 1989. Received August 8, 1989. tSupported by USDA Grant tural Experiment Station. 2Reprint requests.

CRCR-1-1870

and the California

Agricul-

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HERMAN

tion. The primary objective of this study was mine whether episodic delivery of GnRH-like an unvarying, circhoral pattern could support like secretion of LH and FSH induced by

to deterstimuli in the surgeestradiol.

MATERIALS

AND

METHODS

Animals

Thirteen mature whiteface sheep were used. All animals were ovariectomized at least 3 weeks before the experiment. Animals were penned together in an opensided barn under natural lighting. Animals were afforded free access to water. Alfalfa pellets supplemented with grain, and vitamin and mineral premix were made available in amounts sufficient to maintain animal weight and condition.

AND

ADAMS

of an equal volume of saline was begun at the same time in animals in Groups 2 and 3. All animals received 10% ethanol-saline as a continuous infusion for a 90-h period that began at the start of antiserum delivery. Continuous delivery of estradiol (5 J.tg/ml in 10% ethanol-saline) was initiated in all animals 90 h after the start of antiserum delivery and continued for 70 h thereafter. Antibody titer in recipient ewes was determined by incubation of 0.1 ml of a 1:1500 dilution of serum with 8 fmoi 1251-GnRH in a final volume of 0.3 ml. A 1: 1500 dilution of serum collected 4 h after initiation of passive immunization bound 38.2 ± 5.1% of the labeled GnRH added. Anti-GnRH titer in recipient sheep was 21.6 ± 4.3% and 15.5 ± 5.9% three and eight days after passive immunization, respectively. Cannulation

Im,nunization

against

GnRH

Ovariectomized sheep that served as donors of antisera were actively immunized against a GnRH-keyhole limpet hemocyanin (KLH) conjugate or KLH alone. Synthetic GnRH (US Biochemical, Cleveland, OH) was made immunogenic by covalent linkage to KLH (Calbiochem, San Diego, CA), using conjugation and immunization procedures described previously (Adams and Adams, 1986). Blood from donor ewes was collected in evacuated 250-mi serum bottles. Serum containing GnRH or KLH antibodies was collected by centrifugation. Antibody titer and specificity were determined as described previously (Adams and Adams, 1986). The pool of serum used for passive immunization against GaRH had an anti-GnRH titer of 1:60,000 (0.1 ml diluted serum bound 40% of ‘I-GnRH added in fmal volume of 0.3 ml) and displayed only limited cross-reaction (O.OS) depressed by passive immunization until 76 h after infusion of anti-GnRH sera began. By 90 h after immunization against GnRH, serum concentrations of FSH were reduced by 27%, while serum concentrations of LH were reduced by 95%. In contrast to the suppression of LH and FSH secretion noted after passive immunization against GnRH,

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FiG. I. Serum concentrations of LII (least squares mean standard error = in ovarteciomized sheep after passive immunization against GnRH ( - #{149} - - .n=5)or KLH( - - A - - .n=3),orpassive immunizationagainstGnRH and concurrent circhoral administration of GnRH agonist (---, n = 5). 0.92)

HERMAN

276

AND

concentration in Group 3 reflect a temporal mean more than an estimate of basal concentration. In contrast to the GnRH agonist-induced stimulation of LH secretion, the gradual suppression of FSH secretion induced by passive immunization against GnRH was not reversed by episodic administration of the GnRH agonist (Fig. 2). Serum concentrations of FSH declined in parallel in Groups 1 and 2, and at no time were significant differences between groups noted.

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FIG. 2. Relative changes in serum concentrations of FSH (least squares mean standard error = 5.43) in ovariectomized sheep after passive immunizationagainstGnRH(----,n=5)orKLH(--A--,n=3)orpassive immunization against GnRH and concurrent circhoral administration of GnRH agonist (--, n = 5)The relative change in serum FSH for each animal was determined by using the serum concentration of FSH pnor to passive immunization as a reference.

passive immunization against KLH (Group 3) did not significantly (p>O.OS) affect serum concentrations of either gonadotropin (Figs. 1 and 2). Circhoral

ADAMS

Administration

of

a GnRH

Agonist

Pulsatile administration of a GnRH agonist that did not cross-react with the anti-GnRIi serum increased secretion of LH in animals passively immunized against GnRH (Fig. 1). Serum concentrations of LH were significantly (p