Paradoxical Effects of D-TRP6 -LHRH in Immature Female Rats Correlated with Changes in ACTH, Prolactin, and Corticosterone Levels

Transcription

1 BIOLOGY OF REPRODUCTION 24, (1981) Paradoxical Effects of D-TRP6 -LHRH in Immature Female Rats Correlated with Changes in ACTH, Prolactin, and Corticosterone Levels MARY V. NEKOLA,2 ESCIPION PEDROZA and ANDREW V. SCHALLY Tulane University School of Medicine and the Veterans Administration Medical Center, New Orleans, Louisiana 7146 ABSTRACT Immature female rats were treated with D-Trp -LHRH to determine if the delay in vaginal opening induced by the superactive agonist of LHRH could be correlated with changes in ACTH, prolactin, or corticosterone levels. The rats were injected daily from Day 3 to Day 4 of age with.5 or 1 g of D-Trp6 -LHRH or with saline alone. Six animals per group were sacrificed on Days 31, 35, 38, or 42 and trunk blood collected. Pituitary and adrenal weights did not vary between groups, but increased in all treatment regimens as the animals grew older. Prolactin and ACTH levels increased on Days 35 and 38 in the control animals, but were significantly reduced in both agonist-treated groups. Relative to controls, serum corticosterone was elevated (P<.5) on Day 31 in.5 Mg-treated animals and decreased on Day 38 in animals treated with 1 Mg D-Trp6 -LHRH. The addition of corticosterone (16 Mg/mI in 4% ethanol) to the drinking water reversed the inhibitory effects of 1 g of D-Trp -LHRH on vaginal opening. In animals treated with saline or.5 g D-Trp5-LHRH, corticosterone did not affect the normal vaginal opening. These results demonstrate that it is possible that D-Trp -LHRH delays vaginal opening through its effect on corticosterone levels. INTRODUCTION We have shown that the administration of 1. jig but not.5 jig D-Trp6-LHRH, a superactive agonist of LHRH, from Day 3 to Day 4 of age delays vaginal opening (VO) in rats (Vilchez-Martinez et al., 1979). Since the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, and progesterone were elevated in the plasma of agonist-treated rats, the delay in VO could not be attributed to decreased levels of these hormones. The question remains as to the mechanism for the delay in the onset of puberty induced by D-Trp6- LHRH. A complex interrelationship exists between adrenocorticotropin (ACTI-!), corticosterone Accepted November 2, 198. Received July 18, 198. I Presented in part at the 13th Annual Meeting of the Society for the Study of Reproduction, August 198. Abstr. 12. This investigation was supported by Contract HD and by the Veterans Administration. 2 Reprint requests: Dr. Mary V. Nekola, Veterans Administration Medical Center, 161 Perdido Street (151), New Orleans, LA (), and prolactin (PRL) in reproductive maturation. Adrenalectomy before but not after Day 26 delays VO in the rat (Firlit and Lawton, 1974), and replacement with physiological levels of restores the normal onset of puberty (Ramaley, 1976). It cannot be assumed, however, that the effects of adrenalectomy on VO are directly attributable to the low levels of rather than possible high levels of ACTI-I resulting from adrenalectomy. The administration of ACTH to 21-day-old animals for 2 days delayed VO in intact animals and decreased the number of ova ovulated at the first estrus in adrenalectomized animals (MacFarland and Mann, 1977). Lower PRL levels between Days 28 and 3 are also associated with adrenalectomy and, consequently, with delayed VO (Ramaley and Campbell, 1977). Importantly, Gelato et al. (1976) determined that PRL injections advanced puberty in intact animals and restored normal pubertal onset in adrenalectomized animals. Therefore, high levels of ACTH (Mac- Farland and Mann, 1977), reduced (Firlit and Lawton, 1974), and low levels of PRL (Ramaley and Campbell, 1977) all have been found to be associated with a delay in VO. It is possible that the injection of D-Trp6-55

2 56 NEKOLA ET AL. LHRH may delay puberty by affecting one or more of these hormones. To test this hypothesis, 3-day-old female rats were treated with D-Trp6-LHRH to determine whether the agonist alters the plasma levels of, PRL, or ACTH and the pituitary concentrations of PRL and ACTH. After we determined that differences occurred in serum levels of agonist-treated rats, this steroid was added to the drinking water in an attempt to reverse the effects of D-Trp6-LHRH on VO. most, but not all animals, vaginal smears were taken daily beginning with the day of VO until the first cornified smear occurred. At that time, their oviducts were excised and examined for the number of ova under a dissecting microscope. The data of VO and the number of animals ovulating at first estrus were statistically treated by an overall x analysis (Snedecor and Cochran, 1967). When this analysis was significant, individual analyses were done. The differences were considered significant at P<O.5. RESU LTS MATERIALS AND METHODS Immature female rats (CD, Charles River Breeding Labs) arrived on Day 25 of age and were housed eight to a cage under conditions of 14L:1OD (lights on 5-19 h). They were examined for VO on the day of arrival and daily during the experimental period. Only animals with closed vaginae on Day 3 of age were included in the study (only one animal was discarded). The animals were injected s.c. at 9 h from Day 3 to Day 4 with.5 or 1. g of D-Trp6- LHRH dissolved in.2 ml of.9% saline or with vehicle alone. On Days 31, 35, 38, or 42 six animals from each group were decapitated between 15 and 17 h and the adrenals and pituitaries were excised. Approximately 2 ml of trunk blood were collected into a tube containing ethylenediamine tetraacetic acid (EDTA, 2 mg, Baker) and Trasylol (2 lu, FBA Pharmaceutical Mobay Chemical Co.). The blood was stored on ice until all animals were sacrificed at which time the blood was centrifuged, and the serum divided into aliquots and frozen (-2#{176}C). The adrenals were weighed and the pituitaries were frozen on solid CO2. weighed, and then homogenized in 1. ml of.9% saline containing EDTA (1 mg/mi) and Trasylol (1 IU/ml). About.5 ml of the pituitary homogenate was transferred to another tube for ACTH determination. These tubes contained a final concentration of 5 Mg pepstatin A (Sigma) and.2 M acetic acid. ACTH was measured by radioimmunoassay (Besser et al., 1971) using Arimura antibody 4S51 and reference preparation ACTH 1-24 (Ciba-Geigy). Serum and pituitary PRL levels were determined by radioimmunoassay with materials supplied by NIAMDD including reference preparation RPL RP-1 as standard and rat PRL antibody S-4. Comp. B was measured with kits obtained from Radioassay Systems Labs., Inc., CA. The data were statistically treated with Duncan s new multiple range test after one-way analysis of variance (Steel and Torrie, 196). Differences between means were considered significant at P<.5. The ability of to restore normal VO was tested in the following manner. Comp. B was dissolved in absolute ethanol and diluted with water to a final concentration of 16 Mg/ml in 4% ethanol. Other water contained 4% ethanol alone. Animals were injected with saline,.5 g or 1. g of D-Trp - LHRH for 1 days beginning on Day 3 of age. At the same time they were exposed to either or ethanol in their drinking water ad libitum. Each day the animals were examined for VO, and every other day until Day 4 their body weights were recorded. In Experiment 1: Effect of D-Trp6-LHRH on ACTH, PRL, and Levels As in the earlier study (Vilchez-Martinez et al., 1979), VO was delayed by 1. jig of the agonist. By Day the vaginae of all animals treated with saline or.5 pg D-Trp6- LHRH were open. In contrast, the vaginae were still closed in four of six animals treated with 1. pg of the agonist and sacrificed on Day 42. D-Trp6-LHRH did not affect pituitary or adrenal weights at either.5 or 1. jig. Both the pituitaries and adrenals increased in weight between Day 31 and Day 42 in all groups. ACTH serum levels in saline-injected animals were relatively constant and did not vary significantly throughout the course of the study (Fig. 1). In the animals treated with the agonist, serum levels of ACTH were significantly lower on Days 35 and 38 in both agonist-treated groups than in comparable saline controls. Pituitary ACTH was high on Day 31 and fell on Day 35 (Fig. 2). On the other hand, pituitary ACTU was significantly lower on Day 31 in both agonist groups than in saline-treated animals. The ACTH content remained relatively unchanged through Day 42 in the agonisttreated animals. Prepubertal PRL serum levels were very low in animals injected with saline on Day 31, but rose markedly on Days 35 and 38 (Fig. 3). Treatment with either dose of agonist prevented the increase in PRL seen on Days 35 and 38 in the control animals and the levels remained low even 2 days after cessation of treatmenr (Day 42). Pituitary content of PRL was relatively low in saline-treated animals on Days 31 and 35 (Fig. 4), but it increased by Day 38 and remained elevated on Day 42. Pituitary PRL rose similarly with time in animals treated with the agonist. Serum in saline-treated animals followed a pattern similar to that of serum PRL

3 VAGINAL OPENING, D-Trp6-LHRH, AND THE ADRENAL E 2 U a DAYS 1 AGE FIG. 1. Serum ACTH levels in immature female rats treated with D-Trp -LHRH. +, Significantly different from saline controls on same day (P<.5) (Fig. 5). Serum values peaked on Days 35 and 38 in the control animals. Interestingly, treatment with.5 pg of D-Trp6-LHRH induced an elevation in over the levels found in both the saline- and 1. pg-treated animals on Day 31 but not on the other days. Comp. B remained high in the animals treated with.5 pg of the agonist. In contrast, the level of was significantly lower in the 1. pg group on Day 38 compared with the salinetreated animals. Experiment 2: Effect of on VO in Animals Treated with D-Trp6-LHRH The presence of ethanol or of in the drinking water had no appreciable effect on VO in animals treated with either saline or.5 pg D-Trp6-LHRH (Table 1). By Days 4 and 42 all but one of the animals injected with saline or.5 pg of the agonist and exposed to or to ethanol had undergone VO. Ethanol alone had no effect on VO in the 1. pg group since VO still was delayed in animals treated with 1. pg D-Trp6-LHRH and given ethanol in their water. The addition of 16 pg /mi to the drinking water reversed the effects of 1. pg of the agonist on VO. By Day 42, 9% of the animals injected with 1. pg D-Trp6-LHRH but exposed to underwent VO in contrast to only 56% of the animals treated with 1. pg DAV FIG. 2. Pituitary ACTH content in immature female rats treated with D-Trp6-LHRH., Significantly different from Day 31; +, significantly different from saline controls on same day (P<O.5). and ethanol. On Day 41, VO was significantly lower in the 1. pg/4% ethanol group than in the animals treated with 1. jig/ or saline-4% ethanol (P<.5). The body weights of all animals increased with time regardless of treatment and did not vary between groups. Although there appears to be a lengthening of time to the first estrus with agonist treatment (Table 2), the differences between D-Trp6 - LHRH and saline administration were not significant. There was no difference in the number of animals ovulating at first estrus. DISCUSSION The results indicate that D-Trp6-LI-IRI-l in doses of.5 or 1. pg alters the levels of ACTH, PRL, and in prepubertal female rats when injected from Days 3 to 4 of age; however, VO was delayed only with the larger dose. The question is then, what were the differences between the two doses which might have caused the varying effects on VO and on hormone levels? The delay in VO probably was not due to fl

4 58 NEKOLA ET AL #{149}.5 ug D-Trp6 #{149} 1. eg D-Trp8 8 #{149}.5 aq DTrp6 U 1. ig D-Trp6 12 * 1 * IOU 6 8 a a 4 z z U C 2 1 DAYS 1 AGE FIG. 3. Serum PRL levels in immature female rats treated with D-Trp -LHRH., Significantly different from Day 31; +, Significantly different from saline controls on same day (P<.5) DAYS 1 AGE FIG. 4. Pituitary PRL content in immature female rats treated with D-Trp6 -LHRH. #{176}, Significantly different from Day 31 (P<.5). changes in either ACTH or PRL alone. In the control animals, PRL rose dramatically on Days 35 and 38. Dohler and Wuttke (1974) and Meijs-Roelofs et al. 1975) reported a smaller but similar rise in PRL preceding the first ovulation in uninjected rats. Both.5 and 1. pg of D-Trp6-LHRH suppressed the prepubertal peak in PRL. At the same time that PRL was reduced on Days 35 and 38 in both agonisttreated groups, ACTH was also lower in both groups. If the reduced levels of PRL and/or ACTH delayed VO in the 1. pg groups, VO should also have been delayed in the.5 pg-treated animals. Since PRL and ACTH were lower with.5 or 1. pg of the agonist but VO was delayed only in the group treated with 1. pg, it is unlikely that the changes in PRL or ACTH were responsible for the delay in VO observed with 1. pg D-Trp6 -LHRH. The main difference between the two agonist-treated groups occurred in levels. On Day 31 was significantly higher in the animals treated with.5 pg D-Trp6-LHRH than in the other two groups, including the control group. At no point in the study were levels significantly lower in the.5 pg group than in the saline-injected ones; however, the 1. pg-treated animals had decreased concentrations. On Day 38, a time when most of the control animals had undergone VO, was lower after injection of 1. pg D-Trp6-LHRH than after saline administration. The reduced levels of are in agreement with Sandow et al. (1978) who showed that treatment of castrated male rats with a superactive analog of LHRH decreases the content of in the adrenals. They did not measure in the serum. The mechanism controlling secretion in the agonist-treated animals is unknown. Lower ACTH levels may have been responsible for the reduced concentrations of seen on Day 38 in the 1. pg group in the present study. Yet, changes in ACTH do not explain all of the differences that occurred in levels since the.5 pg animals also had reduced ACTH but levels were not reduced. The peak of in the.5 pg group on Day 31 was not accompanied by a rise in ACTH. Since PRL has been shown to stimu-

5 VAGINAL OPENING, D-Trp -LHRH, AND THE ADRENAL 59 E DAYS OF AGE FIG. 5. Serum levels in immature female rats treated with D-Trp -LHRH. o, Significantly different from the other two groups on Day 31;, Significantly different from saline controls on day 31; +, Significantly different from saline controls on same day (P<.5). late secretion from the adrenals (Lis et al., 1973), it is possible that the slight but not significant rise in PRL on Day 31 in animals treated with.5 pg D-Trp6 -LHRH was responsible for the increase in on that day. Also, Bernardo et al. (1978) determined by immunocytochemistry that LHRH receptors may be present in the adrenals of rats. Possibly, D-Trp6-LHRH directly stimulated the adrenals resulting in an increase in in the.5 pg group. Since the level of was elevated on Day 31 in the.5 pg animals over that in the 1. pg-treated ones, the effect of the agonist on the adrenal, if there is one, varies with the dose. It is interesting that the animals treated with.5 pg D-Trp6-LHRH underwent normal VO. As mentioned previously, PRL levels generally peak before the first ovulation (Dohler and Wuttke, 1974; Meijs-Roelofs et al., 1975) and alterations in PRL concentrations drastically affect the onset of puberty. For example, the administration of PRL to intact animals induces precocious puberty (Clemens et al., 1969) and restores normal puberty onset in adrenalectomized animals (Gelato et al., 1978). VO occurred in the.5 pg group even though PRL was reduced and never rose. The decrease in PRL probably was not caused by a decrease in estrogen since we have shown that estrogen is as high in the agonist-treated groups as in the saline controls (Vilchez-Martinez et al., 1979). Nor can the decrease in PRL concentrations in serum be attributed to reduced synthesis of the hormone since pituitary concentrations of PRL rose with time in all groups. Perhaps D-Trp6- LHRH acted at the level of the pituitary to decrease release of PRL. The early rise in in the.5 jigtreated animals may have compensated for the failure of PRL to increase on Days 35 and 38. Campbell and Ramaley (1977) reported that a prepubertal rise in PRL was suppressed by adrenalectomy and that PRL did not peak with TABLE 1. Vaginal opening (VO) in immature rats treated with D-Trp6-LHRH and with (16 Mg/mI) in the drinking water. D-Trp6 injection or -LHRH Drinking water Animals (n) Day of age Cumulative percentage of animals with VO 4%Ethanol Mg.5 Mg comp. B Mg 1. pg Significantly different from 1. pg- and from saline-4% ethanol (P<.5).

6 51 NEKOLA ET AL. TABLE 2. First estrus and ovulation in immature rats treated with D-Trp -LHRH and with (16 pg/ ml) in drinking water. D-Trp6 injection or -LHRH Drinking water Rats examined (n) Time of 1st estrus after VO (days ± SEM) No. of ovulating rats (%) No. of ova ± SEM No. of ovulating rats ± ±.75 6(86%) 4(1%) 1.33 ± ± pg.5 pg ± ± (67%) 2 (4%) 6. ± pg 1. pg ± ± 1.5 4(5%) 2(33%) 12. ± UTwO additional rats were cycled for 8 and 11 days, respectively, and neither one exhibited an estrous smear during that tithe. replacement although VO returned to normal. They postulated that, in some unknown fashion, compensated for the low levels of PRL. Following this argument, VO was not delayed in the present study in the.5 jig-treated animals even though PRL was low because levels remained as high as or higher than saline controls. In the animals treated with 1. pg D-Trp6-LHRH, PRL levels were reduced, and serum was also significantly lower than in controls on Day 38. Based on the levels of hormones observed, we hypothesized that the differences in production were responsible, at least in part, for the delay in VO induced by large amounts of D-Trp6-LHRH. To test this possibility we added to the drinking water of animals treated with the agonist. Indeed, the presence of 16 pg of /mi of water, a concentration that has been reported to raise serum levels in adrenalectomized animals to that of intact animals (Ramaley, 1976), restored VO in animals treated with 1. pg D-Trp6-LHRH without substantially affecting VO in animals injected with saline or.5 pg of the agonist. The fact that reverses the inhibitory effects of D-Trp6-LHRH on VO supports the conclusion that D-Trp6-LHRH possibly exerts its effects on puberty through direct or indirect action on the adrenals. If D-Trp6-LHRH does delay VO by affecting the adrenals, it is unclear why levels compared with those of the controls were reduced on only one day and not on all days of the study. REFERENCES Bernardo, L. A., Petrali, J. P., Weiss, L. P. and Sterns- berger, L. A. (1978). Adrenal luteinizing hormone releasing hormone receptors. J. Histochem. Cytochem. 26, Besser, G. M., Orth, D. N., Nicholson, W. E., Byyny, R. L., Abe, K. and Woodham, J. P. (1971). Dissociation of the disappearance of bioactive and radioimmunoreactive ACTH from plasma in man. J. Clin. Endocrinol. 32, Clemens, J. A., Minaguchi, H., Storey, R., Voogt, J. L. and Meites, J. (1969). Induction of precocious puberty in female rats by prolactin. Neuroendocrinology 4, Dohler, K. D. and Wuttke, W. (1974). Serum LH, FSH, prolactin and progesterone from birth to puberty in female and male rats. Endocrinology 94, Firlit, M. A. and Lawton, I. E. (1974). The timing of the adrenal-ovarian interaction period in the prepubertal rat. BioI. Reprod. 11, Gelato, M. C., Dibbet, J., Marshall, S., Meites, J. and Wuttke, W. (1976). Prolactin-adrenal interactions in the immature female rat. Ann. Biol. Anim. Biochem. Biophys. 16, Gelato, M. C., Meites, J. and Wuttke, W. (1978). Adrenal involvement in the timing of puberty in female rats: Interaction with serum prolactin levels. Acta Endocrinol. 89, Lis, M., Gilbardeau, C. and Chretien, M. (1973). Effect of prolactin on corticosterone production by rat adrenals. Clin. Res. 21, 127. MacFarland, L. A. and Mann, D. R. (1977). The inhibitory effects of ACTH and adrenalectomy on reproductive maturation in female rats. Biol. Reprod. 16, Meijs-Roelofs, H.M.A., Vilenbroek, J. Th. J., de Greef, W. J., Dejong, F. H. and Kramer, P. (1975). Gonadotrophin and steroid levels around the time of the first ovulation in the rat. J. Endocrinol. 67, Ramaley, J. A. (1976). The role of corticosterone rhythmicity in puberty. Biol. Reprod. 14, Ramaley, J. A. and Campbell, G. T. (1977). Serum prolactin concentrations in the adrenalectomized

7 VAGINAL OPENING, D-Trp -LHRH, AND THE ADRENAL 511 rat: Relationships to pubertal onset. Endocrinology 11, Sandow, J., Von Rechenberg, W., Jerzabek, G. and Stoll, W. (1978). Pituitary gonadotropin inhibition by a highly active analog of luteinizing hormone-releasing hormone. Fertil. Steril. 3, Snedecor, G. W. and Cochran, W. S. (1967). The variance test for homogeneity of the binomial distribution. In: Statistical Methods. (G. W. Snedecor and W. S. Cochran, eds.). The Iowa State University Press, Ames. pp Steel, R.E.D. and Torrie, J. H. (196). Data with a single criterion of classification. In: Principles and Procedures of Statistics. (R.E.D. Steel and J. H. Torrie, eds.). McGraw-Hill, New York. pp Vilchez-Martinez, J. A., Pedroza, E., Arimura, A. and Schally, A. V. (1979). Paradoxical effects of D-Trp -LH-RH on the hypothalamo-pituitarygonadal axis in immature female rats. Fertil. Steril. 31,