Developmental Changes in Pituitary Responsiveness to Luteinizing Hormone-Releasing Hormone (LHRH) in the Female Rat: Ovarian-Adrenal Influence During the Infantile Period1 S. R. OJEDA, H. E. JAMESON, AND S. M. M C C A N N Department of Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 but not with estradiol benzoate (EB) or testosterone propionate (TP) suppressed the post-Ovx-Adrx rise in plasma LH and FSH. Progesterone (P) potentiated the effect of DHT. Restoration of basal plasma LH and FSH levels (by DHT and/or P) restored FSH responsiveness to exogenous LHRH. EB and TP were ineffective. The LH response was slightly depressed by EB + DHT. It is concluded that the elevated plasma FSH levels in the infantile female rat may be due at least in part to a high degree of pituitary responsiveness to LHRH and/or FSH-RF brought about by steroidal signals of ovarian origin. DHT and P appear to be the steroids responsible for such a stimulatory action. (Endocrinology 100: 440, 1977)
ABSTRACT. Pituitary LH and FSH responses to synthetic LHRH as estimated by increases in plasma FSH and LH 15 and 45 min following its iv injection were enhanced during the first 2 weeks of life, reaching a maximum around day 10-15 and declining thereafter.
No A.M.-P.M. variations in pituitary
responsiveness were observed at any age studied. The increased pituitary response found in infantile rats did not appear to be caused by a slower rate of disappearance of LHRH in blood of the younger animals. Ovariectomy-adrenalectomy (OvxAdrx) or Ovx at day 10, but not Adrx alone, resulted in elevated LH and FSH levels 5 days later and almost complete obliteration of the FSH response to LHRH. The LH response was not altered. Treatment with 5a-dihydrotestosterone (DHT)
P
LASMA FSH and, to a lesser extent, LH are elevated in early life in the female rat and the values decline beginning at day 15, to reach quite low values just prior to the first preovulatory LH surge ( 1 4). The present study was undertaken to determine if changes in pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH) might in part account for this pattern of plasma hormone titers. Since changes in responsiveness to LHRH during post-natal development were observed, a study was carried out on the effects of steroid replacement therapy on responsiveness to the neurohormone following ovariectomy and ovariectomy-adrenalectomy. Received December 1, 1975. 1 Supported by grants from NIH (AM 10073 and HD 05151), the Ford Foundation, and the Regents Appropriation for Organized Research, University of Texas. The following abbreviations are used: 5a-dihydrotestosterone (DHT): 17/3-hydroxy-5a-androstan-3-one; 3a-androstanediol: 5a-androstane-3a,17/3-diol.
The results reported herein suggest that high levels of circulating FSH during the first two weeks of life are related to an enhanced pituitary responsiveness to LHRH which appears to be brought about by a direct stimulatory action exerted on the gland by steroids of gonadal (and perhaps adrenal) origin. A preliminary report of these results has been presented (5). Materials and Methods Female rats of the Holtzman strain (Holtzman Co., Madison, Wisconsin) were used. They were housed under controlled conditions of lighting (lights on 0500-1900 h) and temperature (24-26 C). Tap water and Purina laboratory chow were available ad libitum. Up to day 21 animals were housed with their mothers (7 pups/dam). Beyond this age they were housed in group cages (5-6 rats/cage). Experimental procedures a) LHRH injections. Synthetic LHRH (Beckman, lot #04702) was injected iv dissolved in 0.9%
440
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RESPONSIVENESS TO LHRH IN IMMATURE RATS NaCl in a volume of 0.2 ml/100 g BW. Controls were injected with an equivalent volume of saline solution. In all cases the injections were made while the animals were lightly anesthetized with ether. Doses and time intervals used are indicated in Results. In one experiment, LHRH was injected iv and its disappearance rate from plasma of 10 and 28 day old rats was determined. The half-life of LHRH in plasma was calculated as reported by Keye et al. (6). b) Surgical procedures. Ovariectomy and adrenalectomy were performed through bilateral dorsal incisions in ether-anesthetized animals at day 10 or day 13 (see Results). Following the removal of the organs, both the muscular layer and the skin were sutured separately. Animals were returned to their mothers 4-6 h after the operation. Since adrenalectomy was performed only in nursing animals, no NaCl replacement therapy was provided. c) Steroid replacement therapy. Animals were ovariectomized and adrenalectomized at day 13. Starting the same day, they were treated with different doses of steroids or combinations of steroids for two days. Injections were administered twice a day (0830 and 1600 h) starting in the afternoon of the day of the operation. All steroids were dissolved in corn oil and injected in a volume of 0.2 ml/100 g BW. Controls were injected with an equivalent volume of corn oil. The effect of the different steroid treatments on pituitary responsiveness to LHRH was determined by injecting LHRH (25 ng/100 g BW) at 1300 h on the day of the last steroid injection (day 15). The steroids and doses injected were: estradiol benzoate (EB, Sigma), 0.02 /i,g/100 g BW; 5a-dihydrotestosterone (DHT, Sigma), 20 /u,g/ 100 g BW; progesterone (P, Schering), 50 //.g/100 g BW; and testosterone propionate (TP, Schering), 20 /Ltg/100 g BW. d) Blood samples. A single blood sample from each animal was drawn into heparinized syringes by cardiac puncture while the animals were anesthetized with ether. Radioimmunoassays Plasma LH2 was measured by the method of Niswenderet al. (7) using the RP-1 rat pituitary
441
LH reference preparation and is expressed in terms of the NIH LH-S-1 standard. Plasma FSH was measured as recommended in the directions supplied with the NIAMDD kit3 with minor modifications and is expressed in terms of the FSH RP-1 reference preparation. All assays were run using the anti-FSH serum S-6 at a dilution of 1:2250. LHRH2 was measured according to the procedure described by Nett et al. (8) with minor modifications (9) and is expressed in terms of the synthetic LHRH (Beckman, lot #04702) used as reference preparation. Statistics Differences between plasma gonadotropin levels induced by each dose of LHRH at different ages were analyzed by means of a one way analysis of variance and the Schaeffe's comparison test for unequal replications (10). Student's t test was used to compare plasma hormone levels attained after different doses of LHRH at a particular age with plasma hormone levels of age-matched controls injected with saline. The statistical significance of the differences between slopes of the disappearance curves of LHRH in plasma was calculated according to Zar (11) using a DEC-system 10 computer program.
Results
Effect of various doses of LHRH on plasma LH and FSH concentrations at different ages LHRH injected iv at four different dose levels induced a dose-related increase in plasma LH 15 min following its injection at all ages studied (Fig. 1). The LH response to each dose of LHRH increased significantly from day 5 to day 10 (P
ABSTRACT. Pituitary LH and FSH responses to synthetic LHRH as estimated by increases in plasma FSH and LH 15 and 45 min following its iv injection were enhanced during the first 2 weeks of life, reaching a maximum around day 10-15 and declining thereafter.
No A.M.-P.M. variations in pituitary
responsiveness were observed at any age studied. The increased pituitary response found in infantile rats did not appear to be caused by a slower rate of disappearance of LHRH in blood of the younger animals. Ovariectomy-adrenalectomy (OvxAdrx) or Ovx at day 10, but not Adrx alone, resulted in elevated LH and FSH levels 5 days later and almost complete obliteration of the FSH response to LHRH. The LH response was not altered. Treatment with 5a-dihydrotestosterone (DHT)
P
LASMA FSH and, to a lesser extent, LH are elevated in early life in the female rat and the values decline beginning at day 15, to reach quite low values just prior to the first preovulatory LH surge ( 1 4). The present study was undertaken to determine if changes in pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH) might in part account for this pattern of plasma hormone titers. Since changes in responsiveness to LHRH during post-natal development were observed, a study was carried out on the effects of steroid replacement therapy on responsiveness to the neurohormone following ovariectomy and ovariectomy-adrenalectomy. Received December 1, 1975. 1 Supported by grants from NIH (AM 10073 and HD 05151), the Ford Foundation, and the Regents Appropriation for Organized Research, University of Texas. The following abbreviations are used: 5a-dihydrotestosterone (DHT): 17/3-hydroxy-5a-androstan-3-one; 3a-androstanediol: 5a-androstane-3a,17/3-diol.
The results reported herein suggest that high levels of circulating FSH during the first two weeks of life are related to an enhanced pituitary responsiveness to LHRH which appears to be brought about by a direct stimulatory action exerted on the gland by steroids of gonadal (and perhaps adrenal) origin. A preliminary report of these results has been presented (5). Materials and Methods Female rats of the Holtzman strain (Holtzman Co., Madison, Wisconsin) were used. They were housed under controlled conditions of lighting (lights on 0500-1900 h) and temperature (24-26 C). Tap water and Purina laboratory chow were available ad libitum. Up to day 21 animals were housed with their mothers (7 pups/dam). Beyond this age they were housed in group cages (5-6 rats/cage). Experimental procedures a) LHRH injections. Synthetic LHRH (Beckman, lot #04702) was injected iv dissolved in 0.9%
440
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 February 2016. at 17:06 For personal use only. No other uses without permission. . All rights reserved.
RESPONSIVENESS TO LHRH IN IMMATURE RATS NaCl in a volume of 0.2 ml/100 g BW. Controls were injected with an equivalent volume of saline solution. In all cases the injections were made while the animals were lightly anesthetized with ether. Doses and time intervals used are indicated in Results. In one experiment, LHRH was injected iv and its disappearance rate from plasma of 10 and 28 day old rats was determined. The half-life of LHRH in plasma was calculated as reported by Keye et al. (6). b) Surgical procedures. Ovariectomy and adrenalectomy were performed through bilateral dorsal incisions in ether-anesthetized animals at day 10 or day 13 (see Results). Following the removal of the organs, both the muscular layer and the skin were sutured separately. Animals were returned to their mothers 4-6 h after the operation. Since adrenalectomy was performed only in nursing animals, no NaCl replacement therapy was provided. c) Steroid replacement therapy. Animals were ovariectomized and adrenalectomized at day 13. Starting the same day, they were treated with different doses of steroids or combinations of steroids for two days. Injections were administered twice a day (0830 and 1600 h) starting in the afternoon of the day of the operation. All steroids were dissolved in corn oil and injected in a volume of 0.2 ml/100 g BW. Controls were injected with an equivalent volume of corn oil. The effect of the different steroid treatments on pituitary responsiveness to LHRH was determined by injecting LHRH (25 ng/100 g BW) at 1300 h on the day of the last steroid injection (day 15). The steroids and doses injected were: estradiol benzoate (EB, Sigma), 0.02 /i,g/100 g BW; 5a-dihydrotestosterone (DHT, Sigma), 20 /u,g/ 100 g BW; progesterone (P, Schering), 50 //.g/100 g BW; and testosterone propionate (TP, Schering), 20 /Ltg/100 g BW. d) Blood samples. A single blood sample from each animal was drawn into heparinized syringes by cardiac puncture while the animals were anesthetized with ether. Radioimmunoassays Plasma LH2 was measured by the method of Niswenderet al. (7) using the RP-1 rat pituitary
441
LH reference preparation and is expressed in terms of the NIH LH-S-1 standard. Plasma FSH was measured as recommended in the directions supplied with the NIAMDD kit3 with minor modifications and is expressed in terms of the FSH RP-1 reference preparation. All assays were run using the anti-FSH serum S-6 at a dilution of 1:2250. LHRH2 was measured according to the procedure described by Nett et al. (8) with minor modifications (9) and is expressed in terms of the synthetic LHRH (Beckman, lot #04702) used as reference preparation. Statistics Differences between plasma gonadotropin levels induced by each dose of LHRH at different ages were analyzed by means of a one way analysis of variance and the Schaeffe's comparison test for unequal replications (10). Student's t test was used to compare plasma hormone levels attained after different doses of LHRH at a particular age with plasma hormone levels of age-matched controls injected with saline. The statistical significance of the differences between slopes of the disappearance curves of LHRH in plasma was calculated according to Zar (11) using a DEC-system 10 computer program.
Results
Effect of various doses of LHRH on plasma LH and FSH concentrations at different ages LHRH injected iv at four different dose levels induced a dose-related increase in plasma LH 15 min following its injection at all ages studied (Fig. 1). The LH response to each dose of LHRH increased significantly from day 5 to day 10 (P
