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

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1 , FERTILITY AND STERILITY Copyright" 19 The American Fertility Society Vol. 29, No.5, May 19 Printed in U.S.A. INHIBITORY EFFECT OF A LUTEINIZING HORMONE (LH)-RELEASING HORMONE AGONIST ON RAT OVARIAN LH AND FOLLICLE-STIMULATING HORMONE RECEPTOR LEVELS DURING PREGNANCY GARY S. KLEDZIK, PH.D. 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, Quebec GIV 4G2, Canada When injected at the daily dose of 1 JJg on days through 12 of pregnancy, the luteinizing hormone (LH)-releasing hormone (LHRH) agonist ID-Ala 6,des-Gly-NH21] LHRH ethylamide led to complete suppression of pregnancy and a 45% to 6% decrease in plasma progesterone concentration. The antifertility effect of the LHRH analog was accompanied by an early and almost complete inhibition of ovarian LHlhuman chorionic gonadotropin and follicle-stimulating hormone receptor levels. These data suggest that the antifertility effects of LHRH agonists are mediated by down-regulation of ovarian gonadotropin receptors and decreased luteal function. Recent reports indicate that luteinizing hormone (LH)-releasing hormone (LHRH) and its agonistic analogs have potent postcoital contraceptive activity in the rat 1 2 and rabbit.3 Other studies have shown that treatment with LH or human chorionic gonadotropin (hcg) leads to a marked loss of LHlhCG receptors in testis 4 5 and ovary.6. Receptor loss was accompanied by a reduced ability of LH to stimulate ovarian adenylate cyclase activity.6. We have recently found that treatment of adult male rats with [n-leu6,des-gly-nh21]-lhrh ethylamide led to a marked reduction of testicular LHlhCG receptor and plasma testosterone levels. 9 It thus occurred to us that the postcoital antifertility effects of LHRH agonists during pregnancy were likely mediated by endog- Received October 3, 19; revised November 1, 19; accepted November 1, 19. * Associate of the Medical Research Council of Canada. To whom reprint requests should be addressed at Groupe du Conseil de Recherches Medicales en Endocrinologie Moleculaire, Le Centre Hospitalier de l'universite Laval, 25 Boulevard Laurier, Quebec G1V 4G2, Canada. 56 enous LH-induced desensitization of the ovary. The present report describes the effect of treatment with [n-ala 6,des-Gly-NH 21]-LHRH ethyl a mide on ovarian LHlhCG and follicle-stimulating hormone (FSH) receptor levels in the pregnant rat. MATERIALS AND METHODS Animals. Virgin female Sprague-Dawley rats (Canadian Breeding Farms, St. Constant, Que.) weighing 2 to 225 gm were kept in a temperature (22 1 C)- and light (14 hours/day, lights on at 5: A.M.)-controlled environment and given Purina rat chow and tap water ad libitum. Rats showing at least three consecutive regular cycles were housed on the afternooon of proestrus with two fertile males. The presence of vaginal sperm the next morning was used as the index of day 1 of pregnancy. Hormones. [n-ala6,des-gly-nh21]-lhrh ethylamide was kindly supplied by Drs. Deghenghi and GOtz, Ayerst Research Laboratories, Montreal. Purified hcg (CR 119, 11,6 IU/mg)

2 Vol. 29, No EFFECTS OF AN LHRH AGONIST DURING PREGNANCY TABLE 1. Effects of [D-Ala 6,des-Gly-NH/O]-LHRH Ethylamide on Pregnancy and Ovarian Weight a Treatment Time of treatment Day of sacrifice Normal fetuses Resorbing fetuses days b Ovarian weight mg c c c athe LHRH analog was administered daily at : A.M., noon, 4: P.M., and : P.M. to to 1 animals per group from day of pregnancy. b [D-Ala6,des-Gly-NH2!O]-LHRH ethylamide. cp <.1 (treated animals versus controls on the same day). was generously supplied by the Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. Ovine LH (olh) (NIH-LHS19, 1.1 x NIH-LH-S1), ofsh (NIH-FSH-S12, 1.25 x NIH-FSH-S1), hfsh (LER-11-3, 419 IUlmg), and prolactin (PRL) (NIH-P-S12, 35 lui mg) were gifts of the National Pituitary Agency, National Institutes of Health. Treatment. [D-Ala 6,des-Gly-NH 2 1 J-LHRH ethyl amide (25 J1-g) or saline was injected subcutaneously four times daily for various lengths of time starting on day of pregnancy. The last injection was given 12 hours before sacrifice with the exception of groups killed on day 1, which received the last injection on day 12. Uteri were examined for fetuses and implantation sites. Organs and plasma were collected for binding FIG. 1. Effect of treatment with the LHRH agonist [D-Ala6,des-Gly-NH 2!O]-LHRH ethylamide on pregnancy in the rat. The peptide was injected daily at : A.M., noon, 4: P.M., and : P.M. from day to day 12 of pregnancy. Uteri were examined on days,,1,12, and 1.

3 562 KLEDZIK ET AL. May 19 studies and hormone measurements. Serum progesterone and LH were measured by specific double-antibody radioimmunoassays.. 9 LHlhCG, FSH, and PRL Receptorassays. Ovaries from individual animals were pooled and homogenized in appropriate volumes of Trisbovine serum albumin (BSA) buffer (.1 M Tris HCl [ph.4],.1% BSA, and 5 mm MgCI2). No difference in binding was seen between total homogenate and the 2, x g pellet. Each assay tube received 5-mg equivalents of ovarian tissue. Low concentrations of chloramine T were used to iodinate the hormones (6 to ng of chloramine T and 5 ILg of peptide). As calculated by isotope recovery, the specific activities were 25.1 (hcg), 22.3 (hfsh), and. (oprl) ILCi! ILg Hormone binding was measured by duplicate incubation for 14 to 16 hours at 23 C of 1 ILl of ovarian suspension, 2 ILl of buffer, 1 ILl of 125I-labeled hormone (a saturating quantity equivalent to approximately 1, to 15, cpm representing 3, 99, and 5 fmoles, respectively, for hcg, hfsh, and oprl), in the presence or absence of 1 ILl of unlabeled hormone ~ >. :z = = CONTROL ( D-AlA6. DES GlY-NH~O) lhrh ETHYlAMIOE._.- -. f'/..' r r r oyrl---l------~1~====~~~~~ DAY OF PREGNANCY FIG.2. Effect of treatment of pregnant rats with In-Ala",des Gly-NH.lOJ-LHRH ethylamide on ovarian 1'5I-heG binding. Animals were treated as indicated in Table 1 and Figure 1. "E >. c; E..,. :c....:: :z = o ' I r ()O O -. CONTROL (D-AlA6. DES GlY-NH1 ) lhrh ETHYlAMIOE I I I '!-----l 1 ~ F 1 DAY OF PREGNANCY FIG. 3. Effect of treatment of pregnant rats with In-Ala",des Gly-NH.lOJ-LHRH ethylamide on ovarian 125I-hFSH binding. Animals were treated as indicated in Table 1 and Figure 1. (2 ILg of olh or ofsh and 1 ILg of oprl). The reaction was stopped by the addition of 3 ml of cold Tris-BSA buffer, and the tubes were centrifuged at 2,3 x g for 15 minutes for measurement of bound radioactivity. Specific binding was expressed as femtomoles of bound hormone. Results are expressed as means standard error of the mean. RESULTS The postimplantation abortive effects of [D Ala 6,des-Gly-NH21 ]-LHRH ethylamide are illustrated in Table 1 and Figure 1. Although no apparent effect on fetuses could be seen 24 hours after the first injection of the analog, treatment from days to 9 led to visible signs of resorption of all fetuses examined on day 1. Almost complete resorption was seen on day 12, and no sign of pregnancy was found on day 1 in animals treated from days to 12. As is illustrated in Figure 2, treatment with the LHRH agonist led to an almost complete loss of ovarian LHlhCG receptors measured on days 1,

4 Vol. 29, No.5 EFFECTS OF AN LHRH AGONIST DURING PREGNANCY 563 TABLE 2. Ovarian LHlhCG and FSH Receptor Levels and Serum LH and Progesterone Concentrations in Female Rats Receiving Injections of [n-ala 6,des-Gly-NHz' J-LHRH Ethylamide from Day of Pregnancy a Treatment Time of treatment Day of sacrifice Bound heg Bound hfsh Serum LH Serum progesterone days {moies/joo mg ovary {molesll mg ovary ng/ml ng/ml " 222 ge e Ie 6.3.ge e i Ie 5..6e ge e c asame experiment as in Table 1. I> [D-Ala6,des-Gly-NH/ oj-lhrh ethylamide. ep <.1 (treated animals versus controls killed on the same day). 12, and 1. The inhibitory effect of [D-Ala6,des Gly-NH21]-LHRH ethyl amide on the level offsh receptors was already well established on day (Fig. 3). It can be seen in Table 2 that the marked loss of LH/hCG and FSH receptor levels followed the same pattern when expressed per 1 mg of ovary. It is also shown that treatment with the LHRH agonist led to a 45% to 6% reduction of plasma progesterone levels at all time intervals studied, whereas plasma LH levels were not affected at the times chosen (at least 12 hours after injection of the LHRH agonist). DISCUSSION The present data clearly demonstrate that the antifertility effects of [D-Ala6,des-Gly-NH21] LHRH ethyl amide are accompanied by a marked loss of LHlhCG and FSH receptors in ovarian tissue. The previous observation that the injection of hcg or LH caused a desensitization of ovarian adenyl ate cyclase activity which was closely paralleled by a loss of LH/hCG receptors6 strongly suggests that the contraceptive activity of the LHRH analog is due to desensitization of the ovary mediated by changes in endogenous gonadotropin release. Although LH is known to be essential for maintenance of pregnancy up to day 12 in the rat, the present findings show that increased gonadotropin secretion can have deleterious effects. The luteolytic action of treatment with [D Ala6,des-Gly-NH21 ]-LHRH ethylamide is well illustrated by the 45% to 6% decrease in plasma progesterone levels in all treated groups. A luteolytic effect of exogenous LH on established corpora lute a has been observed in pseudopregnant rats1 as well as in pregnant and pseudopregnant rabbits.1l, 12 It is of interest that the time course of the effect of the LHRH analog on the level of ovarian LHlhCG receptors is similar to that observed after a single injection of hcg in pregnant mare serum gonadotropinlhcg-treated rats,6 and of [D-Leu6,des-Gly-NH21 ]-LHRH ethylamide or hcg on testicular receptors. The low LH/hCG receptor levels measured on day 1 indicate the slow recovery of these ovarian LHlhCG receptors after desensitization. The significance of the dramatic inhibitory effects of treatment with the LHRH agonist on ovarian FSH receptors remains to be elucidated. Although the role of changes in endogenous LHRH secretion, the sensitivity of the pituitary gonadotropin response to LHRH, and the sensitivity of ovarian responsiveness to gonadotropin remains to be investigated, determination of the level of ovarian LH/hCG and FSH receptors offers a precise and sensitive parameter for study of the mechanism of the postcoital contraceptive action of LHRH. REFERENCES 1. Corbin A, Beattie CW, Yardley J, Toell TJ: Postcoital contraceptive effects of an agonistic analogue of luteinizing hormone releasing hormone. Endocr Res Commun 2:359, Humphrey RR, Windsor BL, Bousley FG, Edgren RA: Antifertility effects of an analog of luteinizing hormonereleasing hormone (LHRH) in rats. Contraception 14:625, Hilliard J, Pang C-N, Sawyer CH: Effects of luteinizing hormone-releasing hormone on fetal survival in pregnant rabbits. Fertil Steril 2:421, Hsueh AJW, Dufau ML, Catt KJ: Regulation of luteinizing hormone receptors in testicular interstitial cells by gonadotropins. BiochemBiophys Res Commun 2:1145, Scharpe RM: hcg-induced decrease in availability of rat testis receptors. Nature 264:644, 196

5 564 KLEDZIK ET AL. May Conti M, Harwood JP, Hsueh AJW, Dufau ML, Catt KJ: Gonadotropin-induced loss of hormone receptors and desensitization of adenylate cyclase in the ovary. J Bioi Chem 251:29, 196. Hunzicker-Dunn M, Birnbaumer L: Adenyl cyclase activities in ovarian tissues. IV. Gonadotropin-induced desensitization of the luteal adenylyl cyclase throughout pregnancy and pseudopregnancy in the rabbit and the rat. Endocrinology 99:211,196. Auclair C, Kelly PA, Labrie F, Coy DH, Schally AV: Inhibition of testicular luteinizing hormone receptor level by treatment with a potent luteinizing hormone-releasing hormone agonist or human chorionic gonadotropin. Biochem Biophy ResCommun 6:55, Auclair C, Kelly PA, Coy DR, Schally AV, Labrie F: Potent inhibitory activity of [n-leu,des-gly-nh.'oj. LHRH ethylamide on LHlhCG and PRL testicular receptor levels in the rat. Endocrinology 11:19, Rothchild I, Schwartz NB: The corpus luteum-hypophysis relationship; the effects of progesterone and oestrogen on the secretion of luteotrophin and luteinizing hormone in the rat. Acta Endocrinol (Kbh) 49:12, Stormshak F, Casida LF: Effects of LH and ovarian hormones on corpora lutea of pseudopregnant and pregnant rabbits. Endocrinology :33, Spies HG, Coon LL, Gier HT: Luteolytic effect of LH and hcg on the corpora lutea of pseudopregnant rabbits. Endocrinology :6, 1966