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Pattern of Recovery of Pituitary Gonadotropins in Intact Male Rats Following Long-Term Treatment with Estrogen
Pattern of Recovery of Pituitary Gonadotropins in Intact Male Rats Following Long-T ernt Treatment 'With Estrogen S. P. KALRA, l'vl. R. N. PRASAD, and N. K. UBEROI
IT
IS GENERALLY AGREED that estrogens act by modifying or suppressing the secretion of the adenohypophysis, 13 · 35 · 36 and their inhibitory effects are believed to be mediated either through the hypothalamo-hypophysial axis or due to a direct effect on the pituitary gland. 4 • 11 • 20· 25 • 29 These studies indicate that the ability of estrogens to deplete the gonadotropin content of the pituitary depends upon the dose, duration of treatment, sex, age, and the physiologic state of the animal. Acute treatment with estrogen lowers pituitary as well as serum FSH and LH levels in intact and castrate male rats; 12 • 16•27 • 28 however, these workers used nonspecific qualitative assay methods whose specificity for FSH and LH have been questioned.5· 21 · 31 • 36 Small doses of estrogen inhibit postcastration rise in plasma LH and pituitary FSH and LH; however, much higher doses are needed to suppress postcastration rise of pituitary FSH.31 Recently Ryan and Philpott reported marked decrease in LH and FSH content of the pituitary fol1owing chronic treatment of castrated male rats with estrone; in no case was a complete depletion of pituitary gonadotropin obtained following acute treatment with estrogens. Very few studies have been done to demonstrate the pattern of pituitary gonadotropin secretion following the cessation of chronic treatment with gonadal hormones. The occurrence of a "rebound effect" following gonadal hormone therapy for treatment of infertility in human males and females is wel1 documented in the literature.10· 15· 17 Increase in ovulation rate occurs following cessation of prolonged treatment with norethynodrel. 19 Although these studies indicate increased gonadotropic secretion after
Supported by a grant from the Ford Foundation. The authors wish to thank Dr. B. R. Seshachar for his interest in this study. The gift of NIH-FSH-83 and NIH-LH-811 from the Endocrinology study section of N.I.H. is gratefully acknowledged.
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gonadal hormone treatment, specific bioassays for FSH or LH were not done to demonstrate such an effect. The following investigations were undertaken to study the effects of acute treatment with estrogen on pituitary FSH and LH activity in adult male rats, with special emphasis on the time sequence of the pattern of secretion of gonadotropins after the cessation of treatment. MATERIAL AND METHODS
Colony-bred adult rats ( 3-3}~ months of age, average body weight 235 gm.) of the Holtzman strain were used for these experiments. They were maintained in an air-conditioned room (78 -+- 2° F.) and provided with 14 hr. of light and 10 hr. of darkness. They were fed a standard diet and given water ad libitum. Estradiol 17-fJ, 12.5 JLg.jday in 0.25 ml. of olive oil, was injected subcutaneously for 60 days; control rats were given injections of 0.25 ml. of olive oil only. One group of 6 rats treated with estrogen was autopsied 24 hr. after the last injection and is referred to as the Day 0 group in the text. In order to study the gonadotropic activity of the pituitary after the cessation of treatment, groups of 6 rats each were autopsied at weekly intervals thereafter. The control group was autopsied along with the estrogen-treated rats at the end of 28 days of recovery. Pituitary glands were dissected free of the posterior lobe and weighed on a torsion balance to the nearest 0.2 mg. They were preserved in cold acetone and dried in a vacuum. The acetone-dried pituitaries were pooled by groups and stored in the cold ( -4 o C.). At the time of the assay they were homogenized in 0.9% saline, diluted and divided for FSH and LH assay. Assay of Follicle Stimulating Hormone (FSH)
The FSH content of the pituitary was determined by the method of Steelman and Pohley using 24-day-old female rats of the Holtzman strain. The pituitaries of rats autopsied at the end of estrogen treatment (Day 0) were tested at 1 dose level, and pituitary homogenate equivalent to 1 pituitary was injected into each of the 4 assay rats. Pituitary homogenate from all the other groups was assayed at 2 dose levels of one-quarter and three-quarter equivalents of pituitary per assay animal. Four rats were used for each dose level. Each assay included 2 doses of reference standard, namely, 50 JLg. and 150 JLg. of NIH-FSH-S3, run simultaneously with the unknown. Assay of Interstitial Cell Stimulating Hormone ( ICSH or LH)
The LH content of the pituitary was estimated according to the method of Parlow.30 Twenty-five-day-old Holtzman rats were treated with 50 I.U.
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of PMS; 65 hr. later, each rat was given an injection of 30 I.U. of HCG. The assay material was injected via the tail vein on the sixth day after HCG injection. Four hours later, the ovaries were removed and homogenized in 2.5% metaphosphoric acid. The ascorbic acid content was determined by the method of Mindlin and Butler. The LH activity in the Day 0 group was tested at the 1-dose level; pituitary homogenate equivalent to one-half pituitary was injected into each of the 4 assay rats. The LH activity of pituitaries of the other groups was tested at 2 dose levels of one-sixteenth and one-fourth pituitary per assay animal. Five animals were used for each dose. The reference standard, NIH-LH-Sll, was tested at dose levels of 0.4 and 1.6 f.Lg. The potency of LH and FSH of anterior pituitary was calculated by standard statistical methods. 3 RESULTS Weights of the Pituitary
The weights of the pituitaries at the end of the treatment and during recovery are shown in Table 1. There is an increase of pituitary weights ( 14.6 mg.) of rats autopsied at the end of estrogen treatment. Thereafter, the pituitary weights decrease to 9.6 mg. at the end of 7 days of recovery. The weights of the pituitaries of the rats autopsied at the end of 14 and 21 days of recovery show slight increase and by 28 days they are heavier than those of the control group. Pituitary FSH Levels
There is a marked reduction in the content as well as concentration of FSH in the pituitaries of rats at the end of chronic treatment with estrogen TABLE 1. Pattern of Recovery of Pituitary FSH Activity Following Long-Term Treatment with Estrogen Duration of recovery (days)
Control
ot 7 14 21 28
Anterior pituitary weight (mg.)
FSH*
FSHt
content of ant. pituit.
concentration (pg/mg.)
95% limits
Design
Lambda
10.1 ± 0.74 14.6 ± 1.6§ 9.6 ± 0.68 11.9 ± 0.67 11.1 ± 0.01 13.2 ± 0.68§
166.0 9.6 57.5 288.4 256.4 166.3
16.43 0.65 5.98 24.23 23.99 12.59
159.1-173.0 6.6- 13.1 46.o- 69.2 253.2-323.8 242.2-270.9 148.2-184.6
2+2 2+1 2+2 2+2 2+2 2+2
0.164 0.223 0.212 0.212 0.243 0.210
*Expressed as 1-1g. equivalents of NIH-FSH-..'la/anterior pituitary. t Expressed as 1-1g. equivalents of NIH-FSH-8 3 /mg. of fresh tissue of anterior pituitary. t Adult male rats treated with 12..5 1-1g/day of estradiol 17-/3 for 60 days and autopsied at weekly intervals following ressation of treatment. Controls were treated with olive oil only. § p <0.05.
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(Table 1). Within 7 days of withdrawal of treatment, there is a nearly sixfold increase in the pituitary FSH content and concentration, but these levels are still very much below those of the control group. A significant elevation in the level of FSH of the pituitary occurs in 14 days, an increase of about 50% above that of the controls. This elevated level is maintained at 21 days of recovery. Thereafter, by 28 days, the content of FSHjpituitary falls to the control level, while the concentration of FSH is less than that of the control. Pituitary LH Levels
The pituitary LH activity is reduced to about one-fourth of the control level after chronic treatment with estrogen (Table 2). There is a gradual increase in LH concentration at 7 days, reaching a level above that of the controls by 14 days of the recovery period. This level is maintained till 21 days and is followed by a slight reduction at 28 days. The content and concentration of LH in the pituitary at the end of 28 days of recovery are much higher than those of the control group. DISCUSSION
Daily treatment with estrogen at doses of 12.5 f.Lg.jday for 60 days caused an increase in the pituitary weights and is in accord with the earlier observation of Clifton and Meyer and of Creep. This increase is associated with the hypertrophy of acidophils and chromophobes. 1 • 2 • 33 A significant reduction in the pituitary weight which occurs 7 days after the withdrawal of estrogen treatment is followed by a gradual increase in weight till 28 days of the recovery period. Mandl found that the pituitaries of immature TABLE 2. Pattern of Recovery of Pituitary LH Activity Following Long-Tenn Treabnent with Estrogen Dura.tion of rec,overy (days)
Control
Ot 7 14 21 28
LH* content LHt concentraUon of ant. (p.g./mg.) pituit.
7.0 2.4 5.6 28.3 28,3 25.4
0.69 0.16 0.58 2.37 2.55 1. 91
95% limits
Design
4.8- 9.4 1.7-3.2 3.6-7.8 22.2-34.5 22.3-33.9 20.1-30.9
2+2 2+ 1 2+2 2+2 2+2 2+2
Ratio Lambda (FSH:LH)
0.307 0.157 0.241 0.332 0.214 0.232
23.7 4.06 10.31 10.23 9.40 6.54
• Expressed as p.g. equivalents of NIH-LH-811 /anterior pituitary. t Expressed as p.g. equivalents of NIH-LH-811 /mg. of fresh tissue of anterior pituitary. t Adult male rats treated with 12.5 p.g./day of estradiol17-{J for 60 days and autopsied a.t weekly intervals following cessation of treatment. Controls were trea.ted with olive oil only.
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female rats treated with estrogen were heavier than those of the controls about 2 months after the cessation of treatment. The FSH and LH content in the pituitaries of untreated adult male rats reported in this study are comparable to the earlier reports of Parlow32 and Steinberger and Duckett. Small doses of estrogen inhibit pituitary FSH, 7 • 27 • 33 whereas higher doses markedly reduce pituitary27 • 34 as well as serum FSH levels; 27 • 31 however, a complete abolition of FSH activity was never obtained. Our results show that estradiol 17-{3, at doses of 12.5 p.g.jday for 60 days, almost completely depleted the content of pituitary FSH. Estrogens have a direct feedback on LH secretion, and this axis is more sensitive than that of FSH to the circulating levels of estrogen. 31 Using the ventral prostate assay for LH, Paesi and de Jongh 28 and Cans and Van Rees reported depression of both pituitary and serum LH content after chronic treatment with estrogen. McCann and Taleisnik showed that a single massive dose of estrogen (50 p.g. ) reduced the serum LH levels in ovariectomized rats, an effect which lasted for 3 days. Later, Parlow31 showed that LH concentration in serum and pituitary decreased after treatment with estrogen; small physiological doses of estrogen ( 0.025 p.g. f day for 16 days) which stimulated the uterus caused lowering of pituitaryJ LH levels, whereas larger doses ( 0.1 p.g.jday) depressed pituitary and serum LH concentration. He concluded, therefore, that estrogen inhibited the synthesis of LH. Ryan and Philpott reported the depletion of LH activity in castrated adult rats treated with estrone for 30 days. Our results confirm these observations that long-term treatment of adult intact male rats with high doses of estrogens markedly depresses pituitary LH activity. In the present study serum FSH and LH levels were not assayed. However, testicular histology and the secretory activity (fructose and citric acid) of the accessory glands of reproduction (reported in detail elsewhere) were used as sensitive indexes of activity of circulating gonadotropins. 23 Treatment with estrogen for 60 days resulted in a marked reduction in the weights of the testis and accessory glands. Spermatogenesis was arrested at the primary spermatocyte stage with occasional degenerating spermatids present in some tubules similar to the condition following hypophysectomy. 8 Fructose and citric acid in the accessory glands of estrogen-treated rats were undetectable. These results indicate that longterm treatment with estrogen results in marked lowering of circulating FSH and LH in addition to the depletion of both gonadotropins in the pituitary. It is likely that synthesis and release of FSH and LH are nearly completely inhibited by prolonged estrogen treatment.
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Since estrogen treatment effectively abolished FSH and LH activity in the pituitary, it was of interest to study the gonadotropic content of the pituitary after the withdrawal of h·eatment. This study was done to elucidate ( 1) the time required for re-initiation of synthesis of FSH and LH and recovery to the control level and ( 2) the time sequence of secretory pattern of FSH and LH during the recovery period. The FSH and LH content as well as concentration increased by 7 days following cessation of estrogen treatment, but the levels were still less than those of the controls. During this period the testes showed significant increase in weight and advance of spermatogenesis to the spermatid stage, while the accessory glands were still nonsecretory ( unpub1ished observations). The slow recovery of the pihtitary gonadotropins immediately following the discontinuation of estrogen treatment may be due to the slow clearance of circulating estrogen which possibly still exerted its inhibitory influence on the pituitary. The gonadotropin content ( FSH and LH) of the pituitary increased sharply by 14 days much above the control levels and remained high till 21 days. While the LH remained at the same level till 28 days, FSH was reduced to the control level by the end of same period. There was a progressive increase in the weight of the testis and advance of spermatogenesis leading to the formation of spermatozoa by 21 days of the recovery period. However, the pattern of recovery of secretory activity of the accessory glands showed a lag which reached the normal level only by 28 days following cessation of treatment. These results indicate that release of FSH and LH was apparently normal. The marked increase in the gonadotropin content ( FSH and LH) of pituitary during the period 14-28 days following the withdrawal of treatment raises the question of differential rate of synthesis and release of these hormones. The fact that the testis and accessory glands showed normal recovery, while at the same time pituitary gonadotropin content increased markedly, indicates that there was possibly an enhanced rate of synthesis accompanied by a lower rate of release of gonadotropins during this period. This needs to be substantiated by analysis of serum levels of gonadotropins. The shift in the ratio of FSHjLH in the pituitary following different treahnents is another interesting problem. 5 • 6 The pituitary of an adult male rat has more FSH than LH. 14 • 32 The FSHjLH ratio of untreated control males in our study was 23.7 (Table 2). It was reduced to 4.0 following estrogen treatment in the Day 0 group. The ratio ranged between 9.0 and 10.0 during the recovery period of 7-21 days when there was a marked increase in content and concentration of FSH as well as LH. The reduction in FSHjLH ratio under these circumstances would indicate an increase in LH. .Whether this is reflected in the different hormone levels of circulating
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gonadotropins is not clear from our study. Kar et al., who studied "the sequence of recovery of the testis and accessory glands in monkeys during the recovery following long-term estrogen treatment, raised an interesting question whether estrogen promotes a preferential synthesis of ICSH by the pituitary for a substantial part of the post-treatment period. Our results indicate that this possibly may be true. Long-term treatment with gonadal hormones causes a rebound effect on the gonads, reflecting ·increased secretion of gonadotropins. 10 • 1 ~· 17 • 19 Igarashi et al. recorded increase in urinary gonadotropins in human ·females during recovery following estrogen treatment. However, no quantitative data on FSH and LH levels of the pituitary in such rebound studies are available. Our results show that there is a marked increase in pituitary FSH and LH during the recovery period following long-term estrogen treatment. The present report is the first quantitative study in support of the rebound phenomenon in intact male rats.
SUMMARY Long-term treatment with estrogen markedly depressed FSH and LH activity of the pituitary. The concentration of both FSH and LH in the pituitary increased within 7 days of cessation of treatment, but their levels were still less than those of the controls. The concentration of pituitary gonadotropins increased sharply above the control levels by 14 days and remained high till 21 days of recovery. Although the LH concentration remained at the same elevated level at the end of 28 days of recovery, the FSH activity of pituitary was reduced to the control level at the end of the same period. The significance of high gonadotropin levels and the differential secretion of FSH and LH during the period between 14-28 days foilowing withdrawal of estrogen treatment is discussed.
REFERENCES
Department of Zoology University of Delhi Delhi 7, India
1. BAKER, B. L., and EvERETT, N. B. The effect of small doses of diethylstilbesterol on the anterior hypophysis of the immature rat. Endocrinology 34:254, 1944. 2. BAKER, B. L., and EvERETT, N. B. The effect of DES on the anterior hypophysis of thyroidectomized rats. Endocrinology 41:144, 1947. 3. Buss, C. I. The Statistics of Bioassay. Acad. Press, New York, 1952. 4. BoGDANOVE, E. M. Direct gonad-pituitary feedback. An analysis of effects of intracranial estrogenic depots of gonadotrophin secretion. Endocrinology 73:696. 196.3. . 5. BOGDANOVE, E. M. The role of the brain in the regulation of pituitary gonadotrophin secretion. Vitamins Hormones 22:206, 1964. 6. BoGDANOVE, E. M., and GAY, V. C. Effects of testosterone propionate treatment
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on FSH and LH secretion and pituitary structure of intact and spayed female rats. Anat Rec 157:216, 1967. BYRNES, W. W., and MEYER, R. K. Effect of physiological amounts of estrogen on the secretion of follicle stimulating and luteinizing hormone. Endocrinology 49: 449, 1951. CLERMONT, Y., and MoRGENTALER, H. Quantitative study of spermatogenesis in the hypophysectomized rat. Endocrinology 57:369, 1955. CLIFTON, K. H., and MEYER, R. K. Mechanism of anterior pituitary tumor induction by estrogen. Anat Rec 125:65, 1956. DrcZFALUSY, E. Probable mode of action of oral contraceptives. Brit Med ] 2: 1394, 1965. EvERETT, J. W. Central neural control of reproductive functions of the adenohypophysis. Physiol Rev 44:313, 1964. CANS, E., and VAN REES, G. P. Effect of small doses of oestradiol benzoate on pituitary production and release of ICSH in gonadectomized male and female rats. Acta Endocr (Kobenhavn) 39:245, 1962. CREEP, R. 0., and JoNES, I. C. Steroid control of pituitary function. Recent Progr Hormone Res 5:191, 1950. CREEP, R. 0. "Physiology of the Anterior Hypophysis in Relation to Reproduction." In Sex and Internal Secretions, Young, W. C., Ed. Williams & Wilkins, Baltimore, 1961, p. 240. HELLER, c. G., NELSON, \V. 0., HILL, I. B., HENDERSON, E., MADDOCK, w. 0., JuNGCK, E. C., PAULSEN, C. A., and MoRTIMORE, G. E. Improvement of spermatogenesis following depression of the human testis with testosterone. Fertil Steril 1:415, 1950. HoocsTRA, M. J., and PAESI, F. J. A. The FSH content of the hypophysis of the rat as influenced by androgen. Acta Endocr (Kobenhavn) 24:353, 1957. IGARASHI, M., MATSUMOTO, S., and HosAK.o\, H. Further studies on the rebound phenomenon of ovarian function. Fertil Steril16:251, 1965. KAR, A. B., CHOWDHURY, S. R., CHOWDHURY, A. R., KAMBOJ, V. P., and CHANDRA, H. Changes in the biochemical composition of the rhesus monkey pituitary in relation to suppression of gonadotrophic activity by estrogen. Steroids 5:519, 1965. LAKSHMAN, A. B., and NELSON, W. 0. "Rebound effect" of ovulation inhibiting steroid in rat. Nature 199:608, 1963. LisK, R. D. Estrogen-sensitive centers in the hypothalamus of the rat. ] Exp Zool 145:191, 1960. LORAINE, J. A. The Clinical Application of Hormone Assay. Livingstone, Edinburgh. MANDL, A. M., as quoted by HoLMES, R. L., and MANDL, A. M. Lancet 1:1114, 1962. MANN, T. Biochemistry of Semen and of the Male Reproductive Tract, (ed. 2). Methuen, London. McCANN, S. M., and TALEISNJK, S. The effect of estrogen on plasma luteinizing hormone activity in the rat. Endocrinology 69:909, 1961. McCANN, S. M., and RAMIREZ, V. D. The neuroendocrine regulation of hypophyseal luteinizing hormone secretion. Recent Progr Hormone Res 20:131, 1964. MINDLIN, R. L., and BUTLER, A. M. Detection of ascorbic acid in plasma, a macromethod and micromethod. ] Biol Chern 122:613, 1938. PAESI, F. J. A., DE JoNCH, S. E., HoocsTRA, M. J., and ENCELBRECT, A. The follicle stimulating hormone content of hypophysis of the rat as influenced by gonadectomy and estrogen treabnent. Acta Endocr (Kobenhavn) 19:49, 1955. PAESI, F. J. A., and DE JoNCH, S. E. The effect of castration and/or estrogen treat-
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ment on the interstitial cell stimulating hormone content of hypophysis of male rats. Acta Physiol Pharmacol Neerl 7:217, 1958. 29. PALKA, Y. S., RAMIREZ, V. D., and SAWYER, C. H. Distribution of biological effects of tritiated estradiol implanted in the hypothalamo-hypophysial region of female rats. Endocrinology 78:481, 1967. 30. PARLOW, A. F. In Human Pituitary Gonadotrophins, Albert, A., Ed. Thomas, Springfield, Ill., 1961, p. 300. 31. PARLOW, A. F. Differential action of small doses of estradiol on gonadotrophins in the rat. Endocrinology 75:1, 1964. 32. PARLOW, A. F. Importance of differential, quantitative bio-assays of pituitary gonadotrophins in the rat. Endocrinology 74:138, 1964. 33. PuRvEs, H. D. "Morphology of the Hypophysis Related to its Function." In Sex and Internal Secretions, Young, W. C., Ed. Williams & Wilkins, Baltimore, 1961, p. 161. 34. RYAN, R. J., and PHILPOTT, J. E. Effects of androgens on concentrations of LH and FSH in the male rat pituitary. Proc Soc Exp Biol Med 124:240, 1967. 35. SAUNDERS, F. J. Effects of steroids on pituitary gonadotrophin and fertility. Recent Progr Hormone Res 20:395, 1964. 36. SHIPLEY, E. G. "Antigonadotropic Steroids, Inhibition of Ovulation and Mating." In Methods in Hormone Research, Dorfman, R. 1., Ed. Acad. Press, New York, 1962, p. 179. 37. STEELMAN, S. L., and PoHLEY, F. M. Assay of follicle-stimulating 'hormone based on the augmentation with human chorionic gonadotrophins. Endocrinology 53: 604, 1953. 38. STEINBERGER, E., and DucKETT, G. E. Pituitary "total" gonadotrophins, FSH and LH in orchiectomized or cryptorchid rats. Endocrinology 79:912, 1966.
IT
IS GENERALLY AGREED that estrogens act by modifying or suppressing the secretion of the adenohypophysis, 13 · 35 · 36 and their inhibitory effects are believed to be mediated either through the hypothalamo-hypophysial axis or due to a direct effect on the pituitary gland. 4 • 11 • 20· 25 • 29 These studies indicate that the ability of estrogens to deplete the gonadotropin content of the pituitary depends upon the dose, duration of treatment, sex, age, and the physiologic state of the animal. Acute treatment with estrogen lowers pituitary as well as serum FSH and LH levels in intact and castrate male rats; 12 • 16•27 • 28 however, these workers used nonspecific qualitative assay methods whose specificity for FSH and LH have been questioned.5· 21 · 31 • 36 Small doses of estrogen inhibit postcastration rise in plasma LH and pituitary FSH and LH; however, much higher doses are needed to suppress postcastration rise of pituitary FSH.31 Recently Ryan and Philpott reported marked decrease in LH and FSH content of the pituitary fol1owing chronic treatment of castrated male rats with estrone; in no case was a complete depletion of pituitary gonadotropin obtained following acute treatment with estrogens. Very few studies have been done to demonstrate the pattern of pituitary gonadotropin secretion following the cessation of chronic treatment with gonadal hormones. The occurrence of a "rebound effect" following gonadal hormone therapy for treatment of infertility in human males and females is wel1 documented in the literature.10· 15· 17 Increase in ovulation rate occurs following cessation of prolonged treatment with norethynodrel. 19 Although these studies indicate increased gonadotropic secretion after
Supported by a grant from the Ford Foundation. The authors wish to thank Dr. B. R. Seshachar for his interest in this study. The gift of NIH-FSH-83 and NIH-LH-811 from the Endocrinology study section of N.I.H. is gratefully acknowledged.
258
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gonadal hormone treatment, specific bioassays for FSH or LH were not done to demonstrate such an effect. The following investigations were undertaken to study the effects of acute treatment with estrogen on pituitary FSH and LH activity in adult male rats, with special emphasis on the time sequence of the pattern of secretion of gonadotropins after the cessation of treatment. MATERIAL AND METHODS
Colony-bred adult rats ( 3-3}~ months of age, average body weight 235 gm.) of the Holtzman strain were used for these experiments. They were maintained in an air-conditioned room (78 -+- 2° F.) and provided with 14 hr. of light and 10 hr. of darkness. They were fed a standard diet and given water ad libitum. Estradiol 17-fJ, 12.5 JLg.jday in 0.25 ml. of olive oil, was injected subcutaneously for 60 days; control rats were given injections of 0.25 ml. of olive oil only. One group of 6 rats treated with estrogen was autopsied 24 hr. after the last injection and is referred to as the Day 0 group in the text. In order to study the gonadotropic activity of the pituitary after the cessation of treatment, groups of 6 rats each were autopsied at weekly intervals thereafter. The control group was autopsied along with the estrogen-treated rats at the end of 28 days of recovery. Pituitary glands were dissected free of the posterior lobe and weighed on a torsion balance to the nearest 0.2 mg. They were preserved in cold acetone and dried in a vacuum. The acetone-dried pituitaries were pooled by groups and stored in the cold ( -4 o C.). At the time of the assay they were homogenized in 0.9% saline, diluted and divided for FSH and LH assay. Assay of Follicle Stimulating Hormone (FSH)
The FSH content of the pituitary was determined by the method of Steelman and Pohley using 24-day-old female rats of the Holtzman strain. The pituitaries of rats autopsied at the end of estrogen treatment (Day 0) were tested at 1 dose level, and pituitary homogenate equivalent to 1 pituitary was injected into each of the 4 assay rats. Pituitary homogenate from all the other groups was assayed at 2 dose levels of one-quarter and three-quarter equivalents of pituitary per assay animal. Four rats were used for each dose level. Each assay included 2 doses of reference standard, namely, 50 JLg. and 150 JLg. of NIH-FSH-S3, run simultaneously with the unknown. Assay of Interstitial Cell Stimulating Hormone ( ICSH or LH)
The LH content of the pituitary was estimated according to the method of Parlow.30 Twenty-five-day-old Holtzman rats were treated with 50 I.U.
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of PMS; 65 hr. later, each rat was given an injection of 30 I.U. of HCG. The assay material was injected via the tail vein on the sixth day after HCG injection. Four hours later, the ovaries were removed and homogenized in 2.5% metaphosphoric acid. The ascorbic acid content was determined by the method of Mindlin and Butler. The LH activity in the Day 0 group was tested at the 1-dose level; pituitary homogenate equivalent to one-half pituitary was injected into each of the 4 assay rats. The LH activity of pituitaries of the other groups was tested at 2 dose levels of one-sixteenth and one-fourth pituitary per assay animal. Five animals were used for each dose. The reference standard, NIH-LH-Sll, was tested at dose levels of 0.4 and 1.6 f.Lg. The potency of LH and FSH of anterior pituitary was calculated by standard statistical methods. 3 RESULTS Weights of the Pituitary
The weights of the pituitaries at the end of the treatment and during recovery are shown in Table 1. There is an increase of pituitary weights ( 14.6 mg.) of rats autopsied at the end of estrogen treatment. Thereafter, the pituitary weights decrease to 9.6 mg. at the end of 7 days of recovery. The weights of the pituitaries of the rats autopsied at the end of 14 and 21 days of recovery show slight increase and by 28 days they are heavier than those of the control group. Pituitary FSH Levels
There is a marked reduction in the content as well as concentration of FSH in the pituitaries of rats at the end of chronic treatment with estrogen TABLE 1. Pattern of Recovery of Pituitary FSH Activity Following Long-Term Treatment with Estrogen Duration of recovery (days)
Control
ot 7 14 21 28
Anterior pituitary weight (mg.)
FSH*
FSHt
content of ant. pituit.
concentration (pg/mg.)
95% limits
Design
Lambda
10.1 ± 0.74 14.6 ± 1.6§ 9.6 ± 0.68 11.9 ± 0.67 11.1 ± 0.01 13.2 ± 0.68§
166.0 9.6 57.5 288.4 256.4 166.3
16.43 0.65 5.98 24.23 23.99 12.59
159.1-173.0 6.6- 13.1 46.o- 69.2 253.2-323.8 242.2-270.9 148.2-184.6
2+2 2+1 2+2 2+2 2+2 2+2
0.164 0.223 0.212 0.212 0.243 0.210
*Expressed as 1-1g. equivalents of NIH-FSH-..'la/anterior pituitary. t Expressed as 1-1g. equivalents of NIH-FSH-8 3 /mg. of fresh tissue of anterior pituitary. t Adult male rats treated with 12..5 1-1g/day of estradiol 17-/3 for 60 days and autopsied at weekly intervals following ressation of treatment. Controls were treated with olive oil only. § p <0.05.
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(Table 1). Within 7 days of withdrawal of treatment, there is a nearly sixfold increase in the pituitary FSH content and concentration, but these levels are still very much below those of the control group. A significant elevation in the level of FSH of the pituitary occurs in 14 days, an increase of about 50% above that of the controls. This elevated level is maintained at 21 days of recovery. Thereafter, by 28 days, the content of FSHjpituitary falls to the control level, while the concentration of FSH is less than that of the control. Pituitary LH Levels
The pituitary LH activity is reduced to about one-fourth of the control level after chronic treatment with estrogen (Table 2). There is a gradual increase in LH concentration at 7 days, reaching a level above that of the controls by 14 days of the recovery period. This level is maintained till 21 days and is followed by a slight reduction at 28 days. The content and concentration of LH in the pituitary at the end of 28 days of recovery are much higher than those of the control group. DISCUSSION
Daily treatment with estrogen at doses of 12.5 f.Lg.jday for 60 days caused an increase in the pituitary weights and is in accord with the earlier observation of Clifton and Meyer and of Creep. This increase is associated with the hypertrophy of acidophils and chromophobes. 1 • 2 • 33 A significant reduction in the pituitary weight which occurs 7 days after the withdrawal of estrogen treatment is followed by a gradual increase in weight till 28 days of the recovery period. Mandl found that the pituitaries of immature TABLE 2. Pattern of Recovery of Pituitary LH Activity Following Long-Tenn Treabnent with Estrogen Dura.tion of rec,overy (days)
Control
Ot 7 14 21 28
LH* content LHt concentraUon of ant. (p.g./mg.) pituit.
7.0 2.4 5.6 28.3 28,3 25.4
0.69 0.16 0.58 2.37 2.55 1. 91
95% limits
Design
4.8- 9.4 1.7-3.2 3.6-7.8 22.2-34.5 22.3-33.9 20.1-30.9
2+2 2+ 1 2+2 2+2 2+2 2+2
Ratio Lambda (FSH:LH)
0.307 0.157 0.241 0.332 0.214 0.232
23.7 4.06 10.31 10.23 9.40 6.54
• Expressed as p.g. equivalents of NIH-LH-811 /anterior pituitary. t Expressed as p.g. equivalents of NIH-LH-811 /mg. of fresh tissue of anterior pituitary. t Adult male rats treated with 12.5 p.g./day of estradiol17-{J for 60 days and autopsied a.t weekly intervals following cessation of treatment. Controls were trea.ted with olive oil only.
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female rats treated with estrogen were heavier than those of the controls about 2 months after the cessation of treatment. The FSH and LH content in the pituitaries of untreated adult male rats reported in this study are comparable to the earlier reports of Parlow32 and Steinberger and Duckett. Small doses of estrogen inhibit pituitary FSH, 7 • 27 • 33 whereas higher doses markedly reduce pituitary27 • 34 as well as serum FSH levels; 27 • 31 however, a complete abolition of FSH activity was never obtained. Our results show that estradiol 17-{3, at doses of 12.5 p.g.jday for 60 days, almost completely depleted the content of pituitary FSH. Estrogens have a direct feedback on LH secretion, and this axis is more sensitive than that of FSH to the circulating levels of estrogen. 31 Using the ventral prostate assay for LH, Paesi and de Jongh 28 and Cans and Van Rees reported depression of both pituitary and serum LH content after chronic treatment with estrogen. McCann and Taleisnik showed that a single massive dose of estrogen (50 p.g. ) reduced the serum LH levels in ovariectomized rats, an effect which lasted for 3 days. Later, Parlow31 showed that LH concentration in serum and pituitary decreased after treatment with estrogen; small physiological doses of estrogen ( 0.025 p.g. f day for 16 days) which stimulated the uterus caused lowering of pituitaryJ LH levels, whereas larger doses ( 0.1 p.g.jday) depressed pituitary and serum LH concentration. He concluded, therefore, that estrogen inhibited the synthesis of LH. Ryan and Philpott reported the depletion of LH activity in castrated adult rats treated with estrone for 30 days. Our results confirm these observations that long-term treatment of adult intact male rats with high doses of estrogens markedly depresses pituitary LH activity. In the present study serum FSH and LH levels were not assayed. However, testicular histology and the secretory activity (fructose and citric acid) of the accessory glands of reproduction (reported in detail elsewhere) were used as sensitive indexes of activity of circulating gonadotropins. 23 Treatment with estrogen for 60 days resulted in a marked reduction in the weights of the testis and accessory glands. Spermatogenesis was arrested at the primary spermatocyte stage with occasional degenerating spermatids present in some tubules similar to the condition following hypophysectomy. 8 Fructose and citric acid in the accessory glands of estrogen-treated rats were undetectable. These results indicate that longterm treatment with estrogen results in marked lowering of circulating FSH and LH in addition to the depletion of both gonadotropins in the pituitary. It is likely that synthesis and release of FSH and LH are nearly completely inhibited by prolonged estrogen treatment.
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Since estrogen treatment effectively abolished FSH and LH activity in the pituitary, it was of interest to study the gonadotropic content of the pituitary after the withdrawal of h·eatment. This study was done to elucidate ( 1) the time required for re-initiation of synthesis of FSH and LH and recovery to the control level and ( 2) the time sequence of secretory pattern of FSH and LH during the recovery period. The FSH and LH content as well as concentration increased by 7 days following cessation of estrogen treatment, but the levels were still less than those of the controls. During this period the testes showed significant increase in weight and advance of spermatogenesis to the spermatid stage, while the accessory glands were still nonsecretory ( unpub1ished observations). The slow recovery of the pihtitary gonadotropins immediately following the discontinuation of estrogen treatment may be due to the slow clearance of circulating estrogen which possibly still exerted its inhibitory influence on the pituitary. The gonadotropin content ( FSH and LH) of the pituitary increased sharply by 14 days much above the control levels and remained high till 21 days. While the LH remained at the same level till 28 days, FSH was reduced to the control level by the end of same period. There was a progressive increase in the weight of the testis and advance of spermatogenesis leading to the formation of spermatozoa by 21 days of the recovery period. However, the pattern of recovery of secretory activity of the accessory glands showed a lag which reached the normal level only by 28 days following cessation of treatment. These results indicate that release of FSH and LH was apparently normal. The marked increase in the gonadotropin content ( FSH and LH) of pituitary during the period 14-28 days following the withdrawal of treatment raises the question of differential rate of synthesis and release of these hormones. The fact that the testis and accessory glands showed normal recovery, while at the same time pituitary gonadotropin content increased markedly, indicates that there was possibly an enhanced rate of synthesis accompanied by a lower rate of release of gonadotropins during this period. This needs to be substantiated by analysis of serum levels of gonadotropins. The shift in the ratio of FSHjLH in the pituitary following different treahnents is another interesting problem. 5 • 6 The pituitary of an adult male rat has more FSH than LH. 14 • 32 The FSHjLH ratio of untreated control males in our study was 23.7 (Table 2). It was reduced to 4.0 following estrogen treatment in the Day 0 group. The ratio ranged between 9.0 and 10.0 during the recovery period of 7-21 days when there was a marked increase in content and concentration of FSH as well as LH. The reduction in FSHjLH ratio under these circumstances would indicate an increase in LH. .Whether this is reflected in the different hormone levels of circulating
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gonadotropins is not clear from our study. Kar et al., who studied "the sequence of recovery of the testis and accessory glands in monkeys during the recovery following long-term estrogen treatment, raised an interesting question whether estrogen promotes a preferential synthesis of ICSH by the pituitary for a substantial part of the post-treatment period. Our results indicate that this possibly may be true. Long-term treatment with gonadal hormones causes a rebound effect on the gonads, reflecting ·increased secretion of gonadotropins. 10 • 1 ~· 17 • 19 Igarashi et al. recorded increase in urinary gonadotropins in human ·females during recovery following estrogen treatment. However, no quantitative data on FSH and LH levels of the pituitary in such rebound studies are available. Our results show that there is a marked increase in pituitary FSH and LH during the recovery period following long-term estrogen treatment. The present report is the first quantitative study in support of the rebound phenomenon in intact male rats.
SUMMARY Long-term treatment with estrogen markedly depressed FSH and LH activity of the pituitary. The concentration of both FSH and LH in the pituitary increased within 7 days of cessation of treatment, but their levels were still less than those of the controls. The concentration of pituitary gonadotropins increased sharply above the control levels by 14 days and remained high till 21 days of recovery. Although the LH concentration remained at the same elevated level at the end of 28 days of recovery, the FSH activity of pituitary was reduced to the control level at the end of the same period. The significance of high gonadotropin levels and the differential secretion of FSH and LH during the period between 14-28 days foilowing withdrawal of estrogen treatment is discussed.
REFERENCES
Department of Zoology University of Delhi Delhi 7, India
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