STEROID PRIMING OF THE LUTEINIZING HORMONE RESPONSE TO LUTEINIZING HORMONE RELEASING HORMONE L. V. BECK, M. BAY, A. F. SMITH, D. KING AND R. LONG Section ofPharmacology, Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana 47401, U.S.A.

(Received 5 August 1977) SUMMARY

experiments were performed to study the stimulatory effects of luteinizing horreleasing hormone (LH-RH) on the release of LH from anterior pituitary tissue. Exposure of pituitary tissue from normal male rats to LH-RH (5 ng/ml for 5 min) induced a small release of LH; in tissue from ovariectomized rats receiving no pretreatment, the release was more than three times greater and in tissue from gonadectomized male or female rats pretreated with oestradiol benzoate and progesterone, the release was six times greater than that observed in normal rats. Further exposure of pituitary tissue from gonadectomized steroid-pretreated male and female rats to LH-RH (5 ng/ml) induced an increase in the level of LH even greater than that seen after the initial exposure (priming action of LH-RH); in tissue from ovariectomized rats receiving no pretreatment, less LH was Perifusion mone

released than after the first exposure to LH-RH and in tissue from normal male rats the response

was

unchanged. INTRODUCTION

Luteinizing hormone releasing hormone (LH-RH) has been reported to act on anterior pituitary tissue to make it more reactive to the stimulatory effect of LH-RH on the release of luteinizing hormone (LH). This action, referred to as priming, has been reported to occur in intact female rats, especially those in pro-oestrus (Aiyer, Chiappa & Fink, 1974; Castro-Vasquez & McCann, 1975), and in perifusion experiments performed with pituitary tissue from rats in dioestrus (Edwardson & Gilbert, 1976). In contrast, neither Dowd, Barofsky, Chaudhuri, Lloyd & Weisz (1975) nor Kao & Weisz (1975) observed priming, as distinct from the direct stimulatory effect on the release of LH, in experiments where anterior pituitary tissue from normal male rats was treated with extract of hypothalamic median eminence (it is generally assumed that the stimulatory effect of median eminence extract on the release of LH and follicle-stimulating hormone (FSH) is due mainly to LH-RH in the extract). In perifusion experiments performed in this laboratory (Beck, Roberts, Basu «fe Bay, 1976) to estimate how rapidly LH-RH acts to induce the release of LH and FSH from anterior pituitary tissue, we observed that when tissue from ovariectomized rats pretreated with oestradiol benzoate plus progesterone (ovariectomized primed rats; see Ramirez «fe McCann, 1963) was used, LH-RH had both direct and priming stimulatory effects on the release of LH. This paper describes the results of experiments suggested by this observation. It should be noted that the release of LH by anterior pituitary tissue from ovariectomized primed rats is known to be highly sensitive to the stimulatory effects of both crude hypo¬ thalamic extracts (Ramirez & McCann, 1963) and substances therein with LH-RH-like activity (Schally & Bowers, 1964).

MATERIALS AND METHODS

Anterior

Dawley

pituitary tissue

was

obtained from male

(400 g)

and female

(300 g) Spraguepretreated

rats. The animals were divided into four groups: ovariectomized rats

with oestradiol benzoate and progesterone (ovariectomized primed rats; see Ramirez «fe McCann, 1963); ovariectomized rats given no pretreatment (ovariectomized unprimed rats); orchidectomized rats pretreated with oestradiol benzoate and progesterone (orchidectomized primed rats) ; normal intact male rats. Ovariectomy or orchidectomy was performed 3 weeks before removal of the pituitary gland. In some experiments each excised anterior pituitary gland was halved; one half was placed in chamber A, the other in chamber B. This pro¬ cedure was repeated until each chamber held the desired number of halves (two to six, some left, some right). Each chamber consisted of a Millipore Co. filter holder, XX30 012 00. Throughout each experiment, perifusion fluid was pumped at a constant rate (about 1 ml/min) simultaneously through both chambers and samples of effluent were collected at 5 min intervals. Some samples were pooled before the concentration of LH was determined. Stock perifusion fluid (Ml99), pH 7-4 after aeration with 95% 02: 5% C02, was prepared as described by GIBCO using their Medium 199 powder and NaHC03 as required. Solutions of LH-RH were prepared by dissolving synthetic LH-RH (Beckman or Parke-Davis Co.) in Medium 199. Dead space and mixing problems inevitably associated with the use of pulses of reagent were minimized by controlling the beginning and end of each pulse with a fourway stopcock placed in front of each tissue chamber and using two channels of fluid per chamber as described by Beck et al. (1976). The use of two chambers per experiment permitted the stimulatory effects of LH-RH on the release of LH to be compared in two sets of anterior pituitary halves treated differently. It also allowed two identical experiments to be carried out simultaneously. The concentrations of LH in samples of effluent were estimated by a modification of the radioimmunoassay described by Rommler & Saxena (1973). In this method 'ethanolammonium acetate' is used to precipitate antibody-bound, but not free, labelled LH. This reagent was prepared by dissolving 46 g ammonium acetate in 60 ml H20 in a 500 ml volumetric flask and adding 95% ethanol to make the volume up to 500 ml. Phosphatebuffered saline (PBS, pH7-4; 0-05 M-sodium phosphate buffer containing 0-875% NaCl and 0-1% merthiolate) was used for the preparation of gel-PBS (PBS containing 0-1% gelatin brought into solution by warming) and EDTA-PBS (0-05 M-EDTA in PBS, pH 7-4, obtained by addition of EDTA disodium salt and 10 M-NaOH to PBS). Normal rabbit serum (NRS) was used diluted 1 :400 (v/v) with EDTA-PBS (NRS-EDTA-PBS) and NIAMDD anti-rat LH-I-3 was used diluted 1:16 000 (v/v) with NRS-EDTA-PBS. Doubling concentrations of NIAMDD rat LH-I-3 standard between 2-5 and 160 ng/ml were obtained by diluting the standard with perifusion fluid (Medium 199). The labelling of NIAMDD rat LH-I-3 with 125I (Amersham-Searle), the subsequent passage of the labelled hormone through cellulose and its final elution with 1 : 1 (v/v) gel-PBS : horse serum were performed essentially as described by Jeffcoate (1971). The 125I-labelled LH was then diluted just before use with a 3 : 1 (v/v) mixture of gel-PBS and horse serum to give a full count of 200 000 counts min-1 ml-1 (about 500 000 disintegrations min-1 ml-1). Mixtures for incubation and subsequent radioimmunoassay were prepared in 12 mm 75 mm disposable glass tubes and reagents were added using Hamilton syringes (Chaney or automatically controlled). Control tubes, tubes containing no antibody and tubes containing the full number of counts were prepared in groups of ten ; each standard and unknown was prepared in triplicate. All reagents were cooled to about 0 °C before use. To each tube were added in turn 005 ml LH standard or unknown, 0-1 ml 125I-labelled LH-I-3 and 0-05 ml anti-rat LH diluted 1 : 16 000 (or NRS-EDTA-PBS for tubes containing no antibody). The tubes were covered with Parafilm, vortex-mixed and kept at 4 °C for 2 or 3 days. Ethanol-ammonium acetate (1 ml) was then added to each tube and the Parafilm

replaced. The tubes were vigorously vortex-mixed and centrifuged for 30 min at about a Model PR-2 International refrigerated centrifuge. The supernatant fractions were carefully decanted and the tubes allowed to drain over absorbent paper for at least 20 min. The precipitates were counted in a Nuclear Chicago (Serial 28386) automatic cover

1700 g in

gamma spectrometer. For a given set of radioimmunoassays, the percentages of bound hormone and logit values used to prepare the line from which the concentrations of LH in the unknown samples were read off were derived from the count rates for samples of standard as described by Rodbard, Bridson «fe Rayford (1969; Method 1). Mean count rates were calculated for precipitates obtained for each dilution of LH-I-3 standard (St), for each unknown sample (X), for the tubes containing no LH (Z) and for tubes containing no antibody (NoAB). The percentage of 125I-labelled LH-I-3 bound by antibody (B)= 100% [(St-NoAB)/ (Z-NoAB)] or 100% [(X-NoAB)/(Z-NoAB)]. Where the percentage of free (i.e. not bound to antibody) 125I-labelled LH-I-3 (F)= 100% -B, logit In (B : F). The read-off line was drawn on percentage versus logit-three cycle log graph paper obtained from TEAM (Box 25, Tamworth, New Hampshire 03886, U.S.A.). The slope (S) is given by =

logit for Sta-logit for Stb log Sta-logStè

'

In these experiments, the ratio Sta : Stb was set at 0-5, S was negative and 5-10% steeper when the concentration of LH-I-3 was between 2-5 and 10 ng/ml than when it was between 40 and 160 ng/ml. In the mid-range, 5* varied from curve to curve between —2-7 and 3-0. Curves generated by suitably diluted samples of effluent containing high concen¬ trations of immunoreactive LH were, allowing for errors arising during counting, parallel to those generated by the corresponding LH-I-3 standards. The 95% confidence levels (ng rat LH-I-3/ml) were at about ± 7% when the ratio antibody bound : free LH (B : F)= 1 and at about 14% when : F— A or 0-25. For a given sample of effluent —

, LH release t

__

=

LH-I-3/ml)(ml effluent/min) (ng —;-:—:-:-—-:——. (mg pituitary tissue/chamber) RESULTS

Table 1 shows data obtained from experiments performed to investigate whether a 5 min pulse of 5 ng LH-RH/ml would alter the LH release response of rat pituitary tissue to a second such pulse of LH-RH, applied 60 min later. It was found necessary to perifuse rat pituitary tissue with control fluid, e.g. Medium 199, for at least 30 min before the release of LH reached a low and almost constant level (initial control rate). The equilibration period used was 95 min, so that LH-RH pulsing was done initially at 95-100 min and again at 155-160 min after the initiation of each experiment. In each of the experiments in which tissue came from an ovariectomized or orchidectomized steroid-primed rat, the second pulse of LH-RH induced a numerically greater increase in the release of LH, compared with the initial control rate, than did the first pulse of LH-RH. By paired i-test, the mean increase in the release of LH induced by the second pulse of LH-RH was significantly greater than the mean increase in the release of LH induced by the first pulse of LH-RH, for tissue from both ovariectomized and orchidectomized steroid-primed rats (see column (d)-(b) of Table 1). Exactly the opposite result was obtained in experiments with tissue from ovariectomized rats not pretreated with steroid hormones. In each of these experiments, the second pulse of LH-RH induced an increase in the release of LH numerically smaller than that induced by the first pulse of LH-RH ; for the entire set of experiments the mean increase in the

Table 1. Comparative effects of two pulses ofluteinizing hormone releasing hormone (LH-RH, 5 ng/mlfor 5 min), one at 95 min and one 60 min later, on the release ofLH in perifused anterior pituitary tissue from gonadectomized male and female rats pretreated with oestradiol benzoate (50 \Lg/rat, s.c.) and progesterone (25 mg/rat, s.c.) for 3 days before death and from ovari¬ ectomized (sesame oil only) and intact male (no treatment) rats (means ± s.e.m., number of experiments given in parentheses; for ovariectomized steroid-primed rats values for each particular experiment are also given) Release of LH First LH-RH

pulse

Ovariectomized

steroid-primed rats (6)

Orchidectomized

Second LH-RH pulse Control

Control Donor of pituitary tissue (ri)

(pg rat LH-I-3 min-1 mg-1)

rate

Increase

rate

Increase

(a)

(b)

(c)

325 ±40 287 249 399 192 446 375 327 ±23

280 ±24* 277 221 251 316 378 234 285 ±28*

374 ±40 384 226 439 292 492 410 407 ±23

(d) 520 ±38*

179+16

166±22*

193 + 7

47±12*

488 407 661 449 589 523

211 186 410 133 211 289

463±31*

178±26f

172+15

98±13*

-68±24f

197± 14

48±9*

steroid-primed rats (6) Ovariectomized rats

(8)

Intact normal male rats (10)

(d)-(b) (criterion of LH-RH priming action) 240±40f

1 ± 14 (NS)

* Significant (P

Steroid priming of the luteinizing hormone response to luteinizing hormone releasing hormone.

STEROID PRIMING OF THE LUTEINIZING HORMONE RESPONSE TO LUTEINIZING HORMONE RELEASING HORMONE L. V. BECK, M. BAY, A. F. SMITH, D. KING AND R. LONG Sect...
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