Vol. 30, No.3, September 1978 Printed in U S.A.

FERTILITY AND STERILITY Copyright © 1978 The American Fertility Society

INHIBITION OF OVARIAN LUTEINIZING HORMONE (LH) AND FOLLICLE-STIMULATING HORMONE RECEPTOR LEVELS WITH AN LH-RELEASING HORMONE AGONIST DURING THE ESTROUS CYCLE IN THE RAT

GARY S. KLEDZIK LIONEL CUSAN CLAUDE AUCLAIR, PH.D. PAUL A. KELLY, PH.D. FERNAND LABRIE, M.D., PH.D.*

Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de l'Universite Laval, Laval, Quebec GlV 402, Canada

A single injection of the luteinizing hormone (LH)-releasing hormone (LHRH) agonist (D-Ala 6,des-Gly-NH2 10 )LHRH ethylamide to female rats on diestrus I produced a marked reduction in ovarian LH!human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) receptor levels, uterine weight, and plasma progesterone levels measured 2 days later on expected proestrus. A maximal inhibitory effect was seen after a dose of only 40 ng of the peptide. No consistent effect of the LHRH analog was seen on ovarian prolactin receptors. When the analog was injected on day 7 of pregnancy, the inhibition of ovarian LH/hCG receptors was of shorter duration and was much less sensitive than that in nonpregnant animals. These data indicate that ovarian LH and FSH receptor levels are highly sensitive to changes in endogenous gonadotropin secretion and suggest that the gonadotropin surge occurring spontaneously on proestrus may play an important role in the regulation of ovarian gonadotropin receptors during the estrous cycle. Fertil Steril30:348, 1978

The administration of luteinizing hormone (LH)-releasing hormone (LHRH) 1 or the potent LHRH agonist [D-Ala6 ,des-Gly-NH210 ]LHRH ethylamide2 on diestrus I or II in cycling rats has been shown to delay· the expected day of mating and vaginal cornification. Since we have recently found that the postcoital contraceptive activity of [D-Ala 6 ,des-Gly-NH/ 0JLHRH ethylamide was associated with a marked inhibition of ovarian LH/human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) receptor levels, the present study describes the effect of treatment with the same LHRH agonist on ovarian LH/hCG, FSH, and prolactin (PRL) receptor levels in cycling animals and compares the sensitivity of the response during the estrous

cycle with that observed in pregnant animals. The interest of this study is strengthened by the recent observation that the administration of an LHRH agonist of similar potency ( [D-Leu6 ,desGly-NH210]LHRH ethylamide) to intact adult male rats can lead to a marked reduction of testicular LH/hCG and prolactin receptor levels; lower plasma testosterone concentrations; decreased seminal vesicle, ventral prostate, and testicular weight; as well as inhibition of spermatogenesis. 4 • 5 MATERIALS AND METHODS

Animals. Virgin female Sprague-Dawley rats (Canadian Breeding Farms, St. Constant, Que.), weighing 200 to 225 gm, were housed in a light (14 hours/day)- and temperature (22 ± 1° C)controlled environment and fed a diet of Purina rat chow and tap water ad libitum. Only rats showing at least three consecutive and regular

Received December 7, 1977; revised April3, 1978; accepted April 21, 1978. *Associate of the Medical Research Council of Canada. To whom reprint requests should be addressed.

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INHIBITION OF OVARIAN LH AND FSH RECEPI'OR LEVELS

4-day cycles were used. In some experiments, rats were housed on the afternoon of proestrus with two fertile males and the presence of vaginal sperm the next morning was recorded as day 1 of pregnancy. Hormones. [D-Ala6 ,des-Gly-NH2 10)LHRH ethylamide was kindly supplied by Drs. R. Deghenghi and M. Gotz, Ayerst Research Laboratories, Montreal. Purified hCG (CR119, 11,600 IU/mg) was provided by the Center for Population Research, National Institutes of Health, Bethesda, Md. hFSH (LER 1801-3, 4,019 IU/mg), oFSH (NIH-FSH-S12, 1.25 x NIH-FSH-S1), oLH (NIHLH-S19, 1.01 x NIH-LH-S1), and oPRL (NIHP-S-12, 35 IU/mg) were gifts from the National Pituitary Agency, National Institutes of Health. Treatment. In the first series of experiments, at 9 A.M. on diestus I cycling rats received subcutaneous injections of either 25 f.Lg of [D-Ala6 , des-Gly-NH2 10)LHRH ethylamide (dissolved in 0.1% gelatin-0.87% NaCl) or the vehicle alone. Groups of 8 to 10 rats each were killed 1, 2, and 3 days later. In a second series of experiments, increasing doses of the LHRH analog were injected at 9 A.M. on diestrus I, and all rats were killed 2 days later. In a last series of experiments, 25 f.Lg of [D-Ala6 ,des-Gly-NH 2 10 )LHRH ethylamide or the vehicle alone were injected at 8 A.M. on day 7 of pregnancy. These rats were killed 1 to 7 days later. In all studies, animals were killed by decapitation between 9 A.M. and 10 A.M. and blood was collected for hormone assays. Ovaries were excised, frozen immediately on Dry Ice, and stored at -20° C until assayed for gonadotropin receptors. Hormone Assays. Serum LH, FSH, and progesterone were measured by specific double-antibody radioimmunoassays. 4- 6 Materials for LH and FSH radioimmunoassay were provided by Dr. A. F. Parlow of the Rat Pituitary Hormone Program, National Institute of Arthritis, Metabolism and Digestive Diseases; the progesterone radioimmunoassay was developed in our laboratory. 3 Radioimmunoassay data were analyzed by using a program based on model II of Rodbard and Lewald. 7 Statistical significance has been measured according to the multiple-range test of Duncan-Kramer. 8 Data are expressed as means ± standard error of the mean. Receptor Assays. Hormones were radioiodinated by a chloramine T method previously described. 3 - 5 Ovaries from each animal were pooled and homogenized in Tris buffer (0.1 M Tris-HCl [pH 7.4), 5 mM MgC1 2 , and 0.1% bovine serum albumin) in order to yield 50 mg of equiva-

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lent wet weight ovarian tissue/mi. The ovarian homogenate (100 JLD was incubated for 14 to 16 hours in duplicate at ambient temperature with 100 f.Ll of 125 1-hCG, 1251-hFSH, or 125 1-oPRL (a saturating quantity equivalent to approximately 100,000 to 150,000 cpm representing 83, 99, and 50 fmoles, respectively, for hCG, hFSH, and oPRL) and 200 JLl of Tris buffer in the presence or absence of an excess of unlabeled hormone (2 f.Lg of oLH or oFSH and 1 f.Lg of oPRL) in 100 f.Ll. The incubations were terminated by the addition of 3 ml of Tris-bovine serum albumin buffer, and the bound and free hormones were separated by centrifugation at 1000 x g for 15 minutes at 4° C. Radioactivity contained in the pellets was counted in an LKB Autogamma spectrometer. Specific binding, expressed as femtomoles bound, is the difference between counts per minute bound in the presence and absence of excess unlabeled hormone. In selected instances, we determined by Scatchard plot analysis that changes in LH binding corresponding to parallel changes in numbers of binding sites rather than affinity. RESULTS

As is illustrated in Figure 1, LH and FSh ovarian receptors are at their lowest levels on estrus in intact 4-day cycling rats and increase progressively to a maximum on proestrus. A single injection of 25 f.Lg of [D-Ala 6 ,des-Gly-NG 2 10 JLHRH ethylamide on the morning of diestrus I led to a marked reduction of LH and FSH receptor levels as measured on diestrus II and proestrus. While the amount of LH receptors was still slightly below that of controls on the expected morning of estrus, the level of FSH receptors had reached values higher than control at that time. Since we recently observed that a maximal inhibitory effect of [D-Leu6 ,des-Gly-NH2 10 )LHRH ethylamide on testicular LH receptor levels was obtained with a single injection of only 40 ng of the peptide in the intact male rat, 5 we next examined the effect of increasing doses of the LHRH agonist on ovarian LH and FSH receptor levels measured on the morning of expected proestrus. As is illustrated in Figure 2 and Table 1, a single injection of as little as 8 ng of the LHRH agonist on diestrus I led to a significant reduction of ovarian LH receptors (30%). A near-maximal inhibition (60%) of ovarian LH receptors was seen at the dose of 40 ng, the inhibitory effect remaining of similar magnitude up to a dose of25 f.Lg (Fig. lA). Doses of 8 to 200 ng of the LHRH agonist led to a 25% to 40% inhibition of FSH receptor levels

September 1978

KLEDZIK ET AL.

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DAY OF ESTROUS CYCLE FIG. 1. Effect of a single injection of 25 p.g of [n-Ala6 ,des-Gly-NH 2 10]LHRH ethylamide on ovarian LH-hCG and FSH (}3) receptor levels. Cycling female rats received injections on the morning of diestrus I and were killed 1, 2, or 3 days later.

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while higher doses were without significant effect (Fig. 2B). No consistent effect on prolactin receptors was observed (Table 1). It can be seen in Table 2 that the decrease of ovarian LH receptors was accompanied by a marked reduction of uterine wet weight, intrauterine fluid, and plasma progesterone concentration as measured on the morning of expected proestrus; serum LH and FSH levels remained unchanged (except for plasma FSH at the 25-ILg dose of the analog). Since we had previously observed a marked reduction of ovarian LH, FSH, and prolactin receptors after repeated injection of 25 ILg of [D-Ala 6 ,des-Gly-NH 2 10]LHRH ethylamide in the pregnant rat, we studied the time-course of the effect of a single injection of peptide given on day 7 of pregnancy. It can be seen in Figure 3 that a significant decrease in ovarian LH and FSH receptors was observed 24 hours after injection of the peptide, with a return to or near control levels on day 9 of pregnancy. It should be mentioned that ovarian LH receptor levels were higher than those of controls on days 11 and 14 of pregnancy.

DISCUSSION

The present data demonstrate that the single injection of relatively low doses of the LHRH agonist [D-Ala6 ,des-Gly-NH2 10]LHRH ethylamide on the morning of diestrus I in 4-day cycling rats leads to a marked loss of ovarian LH receptors. This receptor loss is accompanied by a decreased plasma progesterone concentration and uterine weight as measured on the morning of expected proestrus. Of the 10 rats that received injections on diestrus I of 160 ng of the same peptide, only 7 ovulated; it thus appears that the maximal inhibitory effect on ovarian LH receptors occurs at a subovulatory dose of the LHRH analog. This observation might have relevance for a potential clinical application of the antifertility effects of LHRH and its agonists. Data of Figure 2 show that the sensitivity of the ovarian LH receptors to the inhibitory effect of treatment with the LHRH agonists is very similar to that of the testicular LH receptors. In fact, we have observed that a maximal inhibitory effect on testicular LH receptors; plasma testos-

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