FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 3, No.3, September 1978 Printed in U S.A. 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 42, Canada A single injection of the luteinizing hormone (LH)-releasing hormone (LHRH) agonist (D-Ala 6,des-Gly-NH 2 1 )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 4 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 Steril3:348, 1978 The administration of luteinizing hormone (LH)-releasing hormone (LHRH)1 or the potent LHRH agonist [D-Ala 6,des-Gly-NH21]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/JLHRH 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 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. 348 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-Leu 6,des Gly-NH21]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 2 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 4
Vol. 3, No.3 INHIBITION OF OVARIAN LH AND FSH RECEPI'OR LEVELS 349 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-Ala 6,des-Gly-NH 2 1)LHRH ethylamide was kindly supplied by Drs. R. Deghenghi and M. Gotz, Ayerst Research Laboratories, Montreal. Purified hcg (CR119, 11,6 IU/mg) was provided by the Center for Population Research, National Institutes of Health, Bethesda, Md. hfsh (LER 181-3, 4,19 IU/mg), ofsh (NIH-FSH-S12, 1.25 x NIH-FSH-S1), olh (NIH LH-S19, 1.1 x NIH-LH-S1), and oprl (NIH P-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 1 )LHRH ethylamide (dissolved in.1% gelatin-.87% NaCl) or the vehicle alone. Groups of 8 to 1 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-Ala 6,des-Gly-NH 2 1 )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 1 A.M. and blood was collected for hormone assays. Ovaries were excised, frozen immediately on Dry Ice, and stored at -2 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 (.1 M Tris-HCl [ph 7.4), 5 mm MgC1 2, and.1% bovine serum albumin) in order to yield 5 mg of equivalent wet weight ovarian tissue/mi. The ovarian homogenate (1 JLD was incubated for 14 to 16 hours in duplicate at ambient temperature with 1 f.ll of 125 1-hCG, 125 1-hFSH, or 125 1-oPRL (a saturating quantity equivalent to approximately 1, to 15, cpm representing 83, 99, and 5 fmoles, respectively, for hcg, hfsh, and oprl) and 2 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 1 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 1 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 1JLHRH 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 1)LHRH ethylamide on testicular LH receptor levels was obtained with a single injection of only 4 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 (3%). A near-maximal inhibition (6%) of ovarian LH receptors was seen at the dose of 4 ng, the inhibitory effect remaining of similar magnitude up to a dose of25 f.lg (Fig. la). Doses of 8 to 2 ng of the LHRH agonist led to a 25% to 4% inhibition of FSH receptor levels
35 KLEDZIK ET AL. September 1978 CONTROL A... [ D-ALA6. DES- GLY-NH21] LHRH ETHYL AMIDE 4 B 2 c '15.. E - c::: '.. E... 3 2 :z: ; "' r/_/ )--/'\.. /... \! \! \ :l :...... 1 E DI on p ------._---------------- E DI on p DAY OF ESTROUS CYCLE FIG. 1. Effect of a single injection of 25 p.g of [n-ala 6,des-Gly-NH 2 1 ]LHRH ethylamide on ovarian LH-hCG (A) 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. 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 1 ]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 1 ]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 1 rats that received injections on diestrus I of 16 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-
Vol. 3, No.3 INHffiiTION OF OVARIAN LH AND FSH RECEPI'OR LEVELS 351 1 A 75 B -: -- <C : <C '.. e ' o; e.:::::. 5 <.:1 :z: "-' en Cl :z Cl :::: :z :::: 25 ""' 25... ""' OL---------------.8.4.2 5 25 o.oos.4.2 5 25 [ D-ALA6, DES-GLY-NH 2 1] LHRH ETHYL AMIDE (g) FIG. 2. Effect of treatment with increasing doses of [n-ala6,des-gly-nh2 1)LHRH ethylamide on ovarian LH/hCG (A) and FSH (B) receptor levels. Rats received injections on the morning of diestrus I of the indicated doses of the peptide and were killed 2 days later. terone concentration; and testicular, ventral prostate, and seminal vesicle weight was achieved after single injection of 4 ng of an analog of similar potency, [D-Leu6,des-Gly-NH 2 1] LHRH ethylamide. 5 However, data of Figure 3 indicate that the sensitivity to the LHRH agonist is markedly reduced during pregnancy. In fact, a single injection of 25 JLg of [D-Ala6,des-Gly NH21JLHRH ethylamide leads to a decrease of ovarian LH receptors lasting less than 2 days during pregnancy, whereas the effect is still highly significant after 3 days in cycling animals. A study of the factors responsible for this marked difference should be of great interest for a better understanding of the control of ovarian function during the estrous cycle and pregnancy. It is unlikely that the low levels oflh receptors measured reflect occupancy of binding sites by endogenous LH, since plasma levels return to normal within 12 hours after injection of [D-Ala 6,des-Gly-NH21]LHRH ethylamide.9 Moreover, we have recently found that changes in plasma LH levels following injection of the LHRH agonist lead to no more than 5% occupancy of. TABLE 1. Effect of Increasing Doses of [D-Ala 6,des-Gly-NHz' )LHRH Ethylamide on Ovarian LH/hCG, FSH, and Prolactin Receptor Levels and Ovarian Weight in Cycling Rats" Receptor level Dose of LHRH agonist LH!hCG FSH Prolactin Ovarian weight I'IJ {moles/1 mg ovary fmoles/1 mg ovary fmoles/1 mg ovary mg 195.7 ± 14.7 116.± 11.5 115.7± 8.8 94.8± 7.6.8 124. ± 11.31' 65.3 ± 5.4b 74.3 ± 6.Qb 1.9± 4.2.4 82.8± 8.1b 76.± 7.1b 95.5± 5.9 93.4± 5.4.2 69.3 ± 8.6" 83.3 ± 5.7" 78.± 9.Qb 92.± 5. 1 57.1 ± 1.1b 88.± 8. 1.± 7.7 98.7± 2.5 5 56.8± 9.6" 89.5± 13. 89.8± 6.6 12.1 ± 5.9 25 84.3 ± 14.7b 11.4 ± 1.4 112.6± 14.4 92.3 ± 7.3 a All animals were killed between 9 and 1 A.M. 2 days after a single injection of the LHRH agonist on diestrus I. b P <.1 compared with controls ( dose). c P <.5 compared with controls ( dose).
352 KLEDZIK ET AL. September 1978 TABLE 2. Effect of Increasing Doses of [D-Ala6,des-Gly-NH/ ]LHRH Ethylamide on Serum LH, FSH, and Progesterone Levels and Uterine Weight in Cycling Rats Dose of LHRH agonist Uterine weight Intrauterine fluid SerumLH Serum FSH Serum progesterone pg mg pi nglml nglml nglml 756± 15.4 163 ± 28.1 27.2 ± 4.1 99± 9.4 7.6±.6.8 735 ± 6.4 167 ± 55.3 35.7 ± 7.9 118 ± 13.9 4.7 ± 1.8".4 489± 21.& 38.7 ± 29.7C 33. ± 5.3 127 ± 18.2 5.9±.8.2 489 ± 31.6" 26.2 ± 45.5" 27.2 ± 4.1 128± 8.2 5.6±.!1' 1. 477 ± 25.1< 14.3 ± 37.6" 26.3 ± 2.7 122 ± 7.3 4.8 ±.7c 25. 395± 18.1c 1.± 26.< 22.7 ± 4.1 233± 25$ 3.4 ±.6" "All animals were killed between 9 and 1 A.M. 2 days after a single injection of the LHRH agonist on diestrus I. bp <.5 compared with controls ( dose). c P <.1 compared with controls ( dose). LH-binding sites at times of maximal plasma LH levels. 1 The low levels of LH/hCG binding sites measured up to 3 days after injection of the LHRH agonist in cycling rats suggest a slow recovery of ovarian LH receptors after desensitization in cycling animals. Whereas LH administration has been reported to reduce LH/hCG and FSH receptors in ovaries, the injection of FSH has been shown to increase the FSH receptor content and, in the presence of prolonged estrogen exposure, to increase LH receptors in granulosa cells. 11 Therefore, it would appear that, despite the ability of the LHRH analog to increase the release of both gonadotro- 4 A CONTROl...-. [ D-AlA6. DES GlY- NH21 ]LHRH ETHYLAMIDE pins, 12 LH and not FSH was largely responsible for the reduction in gonadotropin receptors seen in the present study. The relatively fast recovery of FSH-binding sites following treatment with [D-Ala6,des-Gly-NH2 1 ]LHRH ethylamide may be related to the ability of FSH to increase its own receptor content. This may also explain the biphasic and less-consistent effect of the LHRH analog on FSH than LH receptors. The mechanism(s) involved in negative regulation of ovarian receptors is not yet readily apparent. However, the present data indicate that surges of gonadotropin release, not unlike those occurring spontaneously on proestrus, can induce 5 B - <... C e 3 2 z: :: "" 1 - <... -;; e 4 3 ::c: en... -= 2 z :: = "" 1 or-------------------- 7 8 9 11 14 DAY OF PREGNANCY OY-------------------- 7 8 9 11 14 FIG. 3. Effect of treatment of pregnant rats with 25 f..lg of [n-ala 6,des-Gly-NH2 1 ]LHRH ethylamide on ovarian LH/hCG (A) and FSH (B) receptor levels. Animals received injections on the morning of day 7 of pregnancy and were..,.. killed 1, 2, 4, and 7 days later.
Vol. 3, No.3 INHffiiTION OF OVARIAN LH AND FSH RECEPI'OR LEVELS 353 marked changes in ovarian receptors and progesterone secretion. In the rat, this mechanism may play an important role in the normal regulation of the estrous cycle. REFERENCES 1. Banik UK, Givner ML: Ovulation, induction and antifertility effects of an LHRH analog (A Y2525) in cycling rats. J Reprod Fertil44:87, 1975 2. Beattie CW, Corbin A: Pre- and post-coital contraceptive activity of LHRH in the rat. Bioi Reprod 16:333, 1977 3. Kledzik GS, Cusan L, Auclair C, Kelly PA, Labrie F: Inhibitory effect of a luteinizing hormone (LH)-releasing hormone agonist on rat ovarian LH and folliclestimulatng hormone receptor levels during pregnancy. Fertil Steril 29:56, 1978 4. 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 Biophys Res Commun 76:855, 1977 5. Auclair C, Kelly PA, Coy DH, Schally AV, Labrie F: Potent inhibitory activity of [n-ala6,des-gly-nh2 ' JLHRH ethylamide on LH!hCG and PRL testicular receptor levels in the rat. Endocrinology 11:189, 1977 6. Drouin J, Labrie F: Selective effect of andogens on LH and FSH release in anterior pituitary cells in culture. Endocrinology 98:1528, 1976 7. Rodbard D, Lewarl JE: Computer analysis ofradioligand assay and radioimmunoassay data. In 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology, Edited bye Diczfalusy. Geneva, Karolinska Institutet, 197, p 79 8. Kramer CY: Extension of multiple-range tests to group means with unequal numbers of replications. Biometrics 12:37, 1956 9. Cusan L, Kledzik GS, Labrie F: Unpublished data 1. Auclair C, Labrie F: Unpublished data 11. Richard JS, Midgley AR: Protein hormone action: a key to understanding ovarian follicular and luteal cell development. Bioi Reprod 14:82, 1976 12. Coy DH, Coy EJ, Schally AV, Vilchez-Martinez J, Hirotsu Y, Arimura A: Synthesis and biological properties of [n-ala 6,des-Gly-NH2 1 ) LHRHethylamide,apeptide with greatly enhanced releasing activity. Biochem Biophys Res Commun 57:335, 1974