M. W. Morrison, S. L. Davis and L. J. Spicer 3. University of Idaho 4, Moscow 83843

Transcription

1 AGE-ASSOCIATED CHANGES IN SECRETORY PATTERNS OF GROWTH HORMONE, PROLACTIN AND THYROTROPIN AND THE HORMONAL RESPONSES TO THYROTROPIN-RELEASING HORMONE IN RAMS1, 2 M. W. Morrison, S. L. Davis and L. J. Spicer 3 University of Idaho 4, Moscow Summary The objectives were to determine the relationship between advancing age in rams and (1) secretory patterns of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH); (2) clearance rate of thyrotropin-releasing hormone (TRH) from blood plasma; (3) ability of the pituitary to respond to exogenous TRH stimulation, and (4) weights of kidneys and various endocrine glands. Random episodic secretion of GH, PRL and TSH was observed in rams of all three ages (2, 3 and 5 years; n = 6, 6 and 4 respectively). Significant increases in mean overall and mean baseline GH concentrations were found with increasing age. Frequency and amplitude of GH secretory spikes were not significantly influenced by age. PRL secretion was highly variable among rams. Although not statistically significant, mean overall and baseline concentrations appeared to increase with advancing age. Amplitude and frequency of PRL secretory spikes were significantly lower in the older animals. In contrast to GH, secretory patterns of TSH were unaffected by age. The mean tl h of TRH did not differ among age groups. Furthermore, the metabolic clearance rate (MCR) and secretion rate (SR) of TRH were found to be similar at different ages. Injection of TRH significantly increased PRL and TSH concentration in rams of all ages. The degree of the PRL and TSH responses to TRH did not differ among ages. Body weights of these rams, as well as the absolute and relative weights of the kidneys, adrenal glands, anterior pituitaries, posterior pituitaries, thyroids and testicles, did not change significantly with age. Based on the results of this research, the following conclusions are suggested: (1) advancing age in mature sheep is associated with an increase in the overall and baseline concentrations of GH, while spike amplitude and frequency do not change significantly; (2) the amplitude of PRL secretory spikes is decreased in 5-year-old rams; (3) advancing age has no effect on the secretory pattern of TSH in rams; (4) the clearance and secretion rates of TRH are not influenced by age, and (5) body, kidney and endocrine gland weights do not change appreciably in rams up to 5 years old. These observations disagree with the hypothesis that advancing age is associated with a reduction in secretion of GH, PRL or TSH in rams. (Key Words: Growth Hormone, Prolactin, Thyrotropin, Aging, Rams.) Introduction The endocrine system has long been thought Univ. of Idaho Age. Exp. Sta. No This research was conducted in partial fulfillment to be involved in the aging process (Ascheim, of the requirements for the M.S. degree of M. Wayne 1976; Dilman, 1976; Everitt, 1976; Mills and Morrison and was funded principally by the Idaho Mahesh, 1978), because of the key role of hor- Age. Exp. Sta. mones in growth, sexual development, metaboathe authors thank D. L. Ohlson and Ms. D. Blann and Dr. J. Klindt for technical assistance and advice: lism and homeostasis. Most hypotheses con- Dr. J. G. Pierce for purified bovine TSII; Dr. L. E. cerning the role of the endocrine system in Reichert, Jr., for purified ovine PRL, and Dr. A. E. aging suggest endocrine failure with advancing Wilhelmi for purified GH used for radioiodination. We age (Ingbar, 1978; Mills and Mahesh, 1978). are also indebted to the National Pituitary Agency, NIH, for providing ovine hormone preparations used Endocrine failure may represent either a reducas reference standards. tion in hormone secretion or a reduction in the 4 Dept. of Anita. Sci. response of target cells to hormonal stimula- 160 JOURNAL OF ANIMAL SCIENCE, Vol. 53, No. 1, 1981

2 GH, PRL AND TSH IN RAMS OF DIFFERENT AGES 161 tion. While numerous studies have examined the relationship between aging and the secretion of metabolically active hormones - such as growth hormone (GH), thyroid hormones and prolactin (PRL) - in rats and humans, there is a virtual absence of such information for domestic ruminants. The present study was conducted, therefore, to test the hypothesis that aging in rams is associated with a reduction in hormone secretion. We tested this hypothesis by defining the following: (1) patterns of GH, PRL and thyrotropin (TSH) secretion; (2) TSH and PRL responsiveness to thyrotropin-releasing hormone (TRH) stimulation; (3) metabolic clearance rate of TRH, and (4) body weight and weight of selected organs and endocrine glands. Materials and Methods Exp. 1. Secretory Patterns of GH, PRL and TSH in Relation to Age. Sixteen Suffolk rams (free of noticeable disorders such as pneumonia and epididymitis) were segregated into three groups according to age. The age groups were 1-year-olds (n = 6), 2-year-olds (n = 6) and 5- year-olds (n = 4). According to Brody (1945) 28 months of age in sheep is chronologically equivalent to 24 years of age in humans. Brody's (1945) graph does not extend beyond this point. However, if we consider 1 year of age in sheep to be equivalent to 10.3 years in human age, the oldest rams used in this study (5 years) would have been equivalent to 51 human years. All rams were raised under the same environmental conditions (i.e., the same sheep flocks). Two weeks before the start of the experiment, the rams were placed in metabolism crates in an unheated building under natural spring (May) photoperiod. During the 2-week adaptation period, the animals were fed twice daily. At 0900 hr, they were given 2.5 kg of a complete pelleted diet containing alfalfa, barley, oats and dried beet pulp. At 1800 hr, each ram received 2 kg of coarse alfalfa hay. Water was provided ad libitum. All rams were fasted the evening before and the day of the experiment. Twenty-four hours before the experiment, the rams were anesthetized with sodium thiopental (10 mg/kg body weight), and jugular cannulas were inserted. At 0800 hr the following day, blood sample collection began and continued at 15-min intervals through 1600 hours. Ten-milliliter samples were placed into 17 x 100 mm centrifuge tubes containing 100 IU sodium heparin. Blood samples were chilled on ice, stored overnight at 4 C and then centrifuged for separation of the plasma. Plasma samples were stored at -20 C until assayed for GH, PRL and TSH by various radioimmunoassay methods (Davis et al., 1971; Davis, 1972; Borger and Davis, 1974). Reference standard preparations used for the respective hormone assays were Wilhelmi ovine GH 0743B, NIH-P- S10 and NIH-TSH-S6. Secretory patterns of GH, PRL and TSH were defined by measurement of the following variables: overall plasma concentration, baseline concentration, secretory spike amplitude and secretory spike frequency. These variables were determined with the computer program of Christian et al (1978), with one modification used for the calculation of mean spike amplitude. By definition (Christian et al., 1978), mean spike amplitude is the mean of the maximum values of the secretory spikes in an individual experimental unit (animal). In previous studies (Davis et al, 1978; Ohlson et al., 1978), when secretory spikes were not detected in some animals, it had been assumed that the baseline concentration could be used in place of the missing spike amplitude value. This practice, however, appeared to be inflationary, since, in effect, a value was included in the calculation of the mean spike amplitude that did not represent real secretory spike activity. The result was a larger mean spike amplitude than actually existed. To avoid this situation, we used a variable termed "adjusted amplitude" to compare spike activity in animals across treatment groups. While the amplitude derived by the computer analysis was inclusive of the baseline concentration, as well as the spike concentration, the adjusted amplitude was the hormone concentration above baseline only (i.e., adjusted amplitude equaled amplitude minus baseline concentration). In the case of those animals without spikes, the overall concentration was substituted for amplitude, as before, but when baseline was subtracted (baseline and overall are the same when spikes are not present), the result was zero. Since a zero amplitude (no spike) may be as biologically significant as any other amplitude value, the zero was then included in the calculation of the group mean for that variable. Thus, the mean adjusted amplitude should more accurately represent the spike activity occurring in a group of animals.

3 162 MORRISON ET AL. Exp. 2. Relationsbip of Age to TRH Halflife, Metabolic Clearance Rate and Secretion Rate and to Exogenous TRH Stimulation of PRL and TSH in Rams. All rams from the first experiment were also used in this study, except for one that was removed because of a malfunctioning cannula. Immediately after collection of the 1600 hr sample in Exp. 1, each ram was given a single injection (.5 #g/kg body weight) of synthetic TRH s in saline. Samples (5 ml) for TRH assay were collected at 5-min intervals from 1605 through 1630 hr and placed immediately into heparinized tubes containing 3.75 mg BAL (2,3 dimercaptopropanol 6) according to the method of Klindt et at. (1979). Additional 10-ml samples were taken at 15-min intervals from 1615 through 1800 hr for analysis of TSH and PRL. The halflife of disappearance of TRH (tl/2) was determined by the method described by Klindt et al. (1979). These measures were further used for estimating the metabolic clearance rate (MCR) of TRH for a single injection of hormone by the method of Trenkle (1976). Effect of Age on Glandular Tissue Weigbt. One week after the blood collections were made, the rams were sacrificed and the following glands were removed and weighed: kidneys, adrenals, anterior and posterior pituitaries, testicles and thyroids. Body weights were also recorded at this time so that relative tissue weights of the three age groups could be compared. Statistical Analysis of Data. Age effects on the secretory patterns of GH, PRL and TSH were analyzed by a one-way analysis of variance, and then by a least-squares predicted difference (Barr et al., 1976) for those comparisons with a significant F test. Before the analysis of variance, the values were transformed into logarithms because of the correlation between the mean and the variance (Steel and Torrie, 1960). The same procedure was used in the analysis of o/2, MCR, secretion rate (SR) of TRH and gland weights as a function of body weight, absolute gland weight and body weight. The age effect on PRL and TSH responses to the TRH injection was analyzed by a split-plot analysis of variance (Gill and Hafs, 1971). In this analysis, a nested effect (rams within age) SCalbiochem, lot , San Diego, CA. 6 Sigma Chemical Co., St. Louis, MO. was used to test age differences in the response to TRH, and an interaction term (age time) was used to test for different responses among age groups at each of the 5-min intervals. Results Exp. 1. Secretory Patterns of GH, PRL and TSH in Relation to Age Figures 1, 2 and 3 depict temporal plasma GH, PRL and TSH secretory profiles (nanograms/milliliter) over an 8-hr period for two representative rams from each group (1, 2 and 5 years old). These figures show that GH, PRL and TSH were secreted in an episodic manner at all three ages. Timing between spikes apparently occurred randomly, without noticeable temporal synchronization for any of the hormones (table 1). Age-related differences in the secretory patterns of GH, PRL and TSH are not apparent in figures 1 through 3. However, computer analysis of these patterns demonstrated that advancing age was associated with altered secretory patterns of GH and PRL, but not TSH, as shown in table 1 and discussed in the sections below. GH Patterns. Mean overall concentration of GH increased from ng/ml in yearling rams to and ng/ml in 2- and 5- year-old rams, respectively (table 2). These values represent increases of 36 (P<.02) and 68% (P<.001) for the 2- and 5-year-olds, respectively. Mean overall concentration was 23% higher for the 5-year-olds than the 2-year-olds, but this difference was not significant. Similarly, mean baseline GH concentrations increased significantly with age, from ng/ml at 1 year to (41%, P<.02) and ng/ml (78%, P<.001) at 2 and 5 years, respectively. There was no significant difference between the values for 2- and 5-year-old rams. There were no age differences (P>.05) in mean spike amplitude or frequency of GH spikes. PRL Patterns. A nonsignificant, age-related trend was also observed in the overall mean plasma PRL concentrations (table 2). Absolute values increased with age from ng/ml in the yearlings to and ng/ml in the 2- and 5-year-olds, respectively. While these increases in plasma PRL levels were substantial, they were not statistically significant. Unlike overall and baseline PRL concentra-

4 . GH, PRL AND TSH IN RAMS OF DIFFERENT AGES 163 tion, amplitude and frequency of PRL spikes decreased significantly with advancing age (table 2). While there was virtually no difference in mean amplitude between 1- and 2-year- olds ( and ng/ml, respectively), a 75% decrease (P<.05) had occurred by 5 years of age ( ng/ml). Mean frequency of PRL secretory spikes in 2- and year ! I I I I I I! I i- I I I I T l S I i t~t m~ '6 t,2] 2 years I I I! I! I I I ' I I I I I I I I 5 years 8-4- ol...,,.d 0e~ ' 1(~:)0 ' 12'o0 ' ' ', f I hour of day Io'oo ' 12'oo ' 1400 ' ' 1600 ' Figure 1. Temporal plasma growth hormone concentrations in two rams from each age group. Blood was sampled at 15-rain intervals from 0800 to 1600 hours.

5 164 MORRISON ET AL. year-old rams decreased to 65 (P<.05) and 91% (P<.05) to that observed in the 1-year-old rams. 7S1t Patterns. Unlike with GH and PRL secretion, there were no significant age-related changes in any of the measures of TSH secretion (table 2). Although frequency of secretory spikes appeared to be greater in 5-year-old rams, this difference was not significant. 1 year I i I I!!! I I! I I I I!! I I 2 years ~ 40-! I I I!! W k i I I I I I I I l 5 years o~ w I000 ' ' 1 14'00 w ~ 0800 r ' T~(X] w t i,,:'~o w 1200 I 1200,~o hour of day Figure 2. Temporal plasma prolactin concentrations in two rams from each age group. Blood was sampled at 15-rain intervals from 0800 to 1600 hours.

6 GH, PRL AND TSH IN RAMS OF DIFFERENT AGES year 4- O- I i I 1 I i I 1 i i I I I i! I I '1 2 years 4. O" I I I I 1 I "'1 I ~ I 1 ] I I I I 16-5 years , O- I I i I I 1 I ~ ( 1 0 I I000 ' 12~)0! 1400 ' I I&O0 I hour of day Figure 3. Temporal plasma thyrotropin concentrations in two rams from each age group. Blood was sampled at 15-min intervals from 0800 to 1600 hours.

7 166 MORRISON ET AL. TABLE 1. AVERAGE WITHIN-ANIMAL CORRELATION COEFFICIENTS OVER ALL AGES FOR TEMPORAL SECRETORY PATTERNS OF GH, PRL AND TSHa Hormone GH PRL TSH GH PRL TSH 1.00 acalculated with the SAS program (Barr et al, 1976). Exp. 2. Relationship of Age to TRH t89 MCR and SR and to Endogenous TRH Stimulation of PRL and TStt Plasma TRH concentrations increased within 5 rain after TRH injection in rams of all three ages and remained above basal levels throughout the 30-rain sampling period (figure 4). Estimates of tv~, MCR and SR of TRH are presented in table 3. There were no significant changes in these variables with age. Injection of TRH stimulated marked in YEAR 2 YEARS 5 YEARS ~ T T "1" n- 40" 30"3 I' t b- 0 ~ _ ~21 I', ,..,......, '0 60 9'0 1:20 t" '1' 'I, TRH TRH TRH MIN POST-TRH Figure 4. Plasma concentrations of thyrotropin-releasing hormone (upper panel), thyrotropin (middle panel) and prolactin (lower panel) in rams 1, 2 and 5 years of age (left to right) after the injection of TRH.

8 GH, PRL AND TSH IN RAMS OF DIFFERENT AGES 167 TABLE 2. ANALYSIS OF GROWTH HORMONE, PROLACTIN AND THYROTROPIN SECRETORY PATTERNS IN SUFFOLK RAMS 1, 2 AND 5 YEARS OF AGEa Frequency, spike Age Overall, Baseline, Amplitude d, number/ group No. ng/ml ng/ml ng/ml 8 hr GH 1 year (6) b b years (6) c c years (4) c c PRL 1 year (6) b b 2 years (6) b c 5 years (4) c c TSH 1year (6) years (6) years (4) avalues are means SEM. b'cmeans that are different (P<.05) are denoted by different superscripts. Means within a column without letter superscripts do not (P>.05) differ. damplitude indicated here is the mean adjusted amplitude derived from amplitude minus baseline. creases (P<.01) in plasma levels of PRL and TSH over basal levels in all rams (figure 4). There were no significant age-related differences in the mean peak PRL or TSH in response to TRH stimulation. Furthermore, there were no significant age x time interactions, indicating that the ability of the pituitary to maintain secretion of PRL and TSH over time (at least 2 hr), in response to TRH, was not altered in the age groups tested in this experiment. Effect of Age on Body Weight and Glandular Tissue Weight Table 4 lists the body weights (kilograms) and relative tissue weights (units/kilogram) or various glands from 15 rams used in the two experiments. Weights of paired organs (adrenals, kidneys and testes) were recorded as mean weights of the two organs. Mean body weights were not significantly affected by age, although the 2-year-olds had somewhat lower body weights than the animals in the other age groups. The relative weights of the adrenals, anterior pituitaries and thyroids appeared to increase with age, while those of the other tissues did not change noticeably. Nevertheless, neither the relative nor absolute weights of any of these organs differed significantly among age groups. TABLE 3. ESTIMATES OF METABOLIC CLEARANCE RATE, HALF-LIFE OF DISAPPEARANCE AND SECRETION RATE OF THYROTROPIN-RELEASING HORMONE AS RELATED TO AGE IN RAMS MCR, SR, Age n tv/, rain liters/hr ng/hr 1 year a years years amean SEM.

9 168 MORR1SON ET AL. z~ o ~o Z -,<,=( o~ 4,,..1.< <E e l +I +~ +I +) +t +~ +I +I +I +L +~ +I +~ +I +I 9 -,i ip, I ~a'~,j c- O II ~e q. e~ o 9.-. A" Li, "~ ~~ g2 Discussion The present study attempted to define the basic relationship of ram age to GH, PRL and TSH secretory patterns, as well as to the clearance of TRH and its stimulation of the anterior pituitary. Previous studies, particularly with GH, have suggested that a decrease in hormone secretion occurs with age. For example, Carlson et al. (1972) and Finkelstein et al. (1972) reported a decrease in GH secretory spike activity with advancing age in humans. Similar results were obtained with humans (20 to 80 years old) in studies in which less frequent blood sampling schedules were used (Dudl et al., 1973; Vidalon et al., 1973). However, mice aged 12 and 28 months showed no significant differences in basal GH concentrations. Our results on GH do not agree with those reported above. In the male sheep, a clearcut rise in baseline plasma GH concentrations was observed, while GH secretory spike amplitude remained essentially unchanged. It could be argued that the difference between our results and those reported previously is due to the more rigorous blood sampling schedule used in the present study, which allowed a more accurate estimation of plasma GH concentrations. However, Finkelstein et al. (1972) also used a multiple sequential blood sampling schedule9 Therefore, the reasons for the discrepancy (whether they be related to species, sexes, inadequate consideration of diurnal or circannual rhythms) are not obvious. Certainly, the present results do not agree with the hypothesis that advancing age in the adult ram is associated with a decrease in GH secretion. No obvious age-related changes in serum PRL have been reported in previous studies with humans (Yamaji et al., 1976), mice (Finch et al., 1977) or sheep (Ravault, 1976)9 In contrast, the present study suggested that baseline PRL concentrations, like baseline GH concentrations, might increase with age, although the trend was not statistically significant9 A significant decrease, however, was observed in both the amplitude of PRL secretory spikes and the frequency of their occurrence9 This decrease might, in fact, have been an artifact of the method of determination of secretory spikes. Under this procedure (Christian et al., 1978), an increase in variation in the baseline population (one might call it "noise") decreases the probability of detecting a secretory spike. From table 2, it is obvious that variability in basal PRL concentrations was increased con-

10 GII, PRL AND TSH IN RAMS OF DIFFERENT AGES 169 siderably in the 2- and 5-year-old groups. That is probably one of the reasons why the change in baseline PRL concentrations from 58.6 ng/ml in yearlings to 95.7 ng/ml in 5-year-old rams was not significant. Nevertheless, the resuits of this study do not agree with the hypothesis that PRL secretion is reduced with advancing age. Previous reports on changes in TSH concentrations in relation to age are conflicting. Valueva and Verzhikovskaya (1977) found that plasma TSH levels in adult rats increased significantly with age until 28 months. Similarly, Cuttelod et al. (1974) reported that senescent humans had twice the basal plasma TSIt concentration of young adults. In contrast, Wagner et al. (1974) reported that basal TSH concentrations were not correlated with increasing age in humans. These conflicting reports typify studies that have been conducted to determine whether a relationship exists between thyroid function and the aging process. As lngbar (1978) summarizes, "it has been neither possible to rationalize the age-related deviations found into an integrated, internally consistent pattern of function, nor possible to conclude whether a 'hypothyroid state' is an invariable accompaniment or contributor to the process of aging." Certainly, the thyroid function tests in the present study would argue against that hypothesis (at least for rams up to 5 years old), since none of the results (TRH kinetics, TSH response to TRH stimulation and measures of temporal TSH secretion) differed between ages. Assuming that the MCR of GH is not reduced with advancing age, the present results suggest that, in contrast to the hypothesis, GH secretion is increased with advancing age. Although the evidence is less conclusive, the same appears to be true of PRL secretion. Therefore, if these hormones are involved in a functional manner in the aging process, it appears that there may be a reduction in tissue responsiveness to hormonal stimulation with age. Perhaps such a reduction would result in a compensatory increase in GH and PRL secretion. Literature Cited Ascheim, P Aging in the hypothalamic-hypophyseal-ovarian axis in the rat. P In A. V. Everitt and J. A. Burgess (Ed.) Hypothalamus, Pituitary and Aging. Vol. I. Charles C Thomas, Springfield, IL. Barr, A. J., J. H. Goodnight, J. P. Sail and J. T. Helwig A User's Guide to SAS 76. SAS lnsti- tute, Inc., Raleigh, NC. Borger, M. L. and S. L. Davis Development of a specific ovine thyrotropin (TSH) radioimmunoassay from non-specific antisera. J. Anim. Sci. 39:768. Brody, S Bioenergetics and Growth. Reinhold Publishing Co., New York, p Carlson, H. E., J. C. Gillin, P. Gorden and F. Snyder Absence of sleep related growth hormone peaks in aged normal subjects and in acromegaly. J. Clin. Endocrinol. Metab. 34:1102. Christian, L. E., D. O. Everson and S. L. Davis A statistical method for detection of hormone secretory spikes. J. Anim. Sci. 46:699. Cuttelod, S., T. Lemarchand-B~raud, P. Magnenat, C. Perret, S. Poll and A. Vannotti Effect of age and role of kidneys and liver on thyrotropin turnover in man. Metabolism 23:101. Davis, S. L Plasma levels of prolactin, growth hormone and insulin in sheep following the infusion of arginine, leucine and phenylalanine. Endocrinology 91:549. Davis, S. L., D. L. Ohlson, J. Klindt and M. S. Anfinson Episodic patterns of prolactin and thyrotropin secretion in rams and wethers: Influence of testosterone and diethylstilbestrol. J. Anim. Sci. 46:1724. Davis, S. L., L. E. Reichert, Jr. and G. D. Niswender Serum levels of prolactin in sheep as measured by radioimmunoa~ay. Biol. Reprod. 4: 145. Dilman, V. M The hypothalamic control of aging and age associated pathology: The elevation mechanism of aging. P In A. V. Everitt and J. A. Burgess (Ed.) Hypothalamus, Pituitary and Aging. Vol. I. Charles C Thomas, Springfield, IL. Dudl, R. J., J. W. Ensinck, H. E. Palmer and R. H. Williams Effect of age on growth hormone secretion in man. J. Clin. Endocrinol. Metab. 37: 11.' Everitt, A. V The nature and measurement of aging. P In A. V. Everitt and J. A. Burgess (Ed.) Hypothalamus, Pituitary and Aging. Vol. I. Charles C Thomas, Springfield, IL. Finch, C. E., V. Jonec, J. R. Wisner, Jr., Y. N. Sinha, J. S. devellis and R. S. Swerdloff Hormone production by the pituitary and testes of male C57BL/J6 mice during aging. Endocrinology 101:1310. Finkelstein, J. W., H. P. Roffwarg, R. M. Boyar, J. Kream and L. Hellman Age-related change in the twenty-four hour spontaneous secretion of growth hormone. J. Clin. Endocrinol. Metab. 35: 665. Gill, J. L. and H. D. Hafs Analysis of repeated 9 measurements of animals. J. Anita. Sci. 33:313. Ingbar, S. H Influence of aging on the human thyroid hormone economy. P In R. B. Greenblatt (Ed.) Geriatric Endocrinology. Vol. V. Raven Press, New York. KIindt, J., S. L. Davis and D. L. Ohlson Estimation of half-life and metabolic clearance rate of thyrotropin-releasing hormone in sheep using a double antibody radioimmunoassay. J. Anita. Sci. 48:1165. Mills, T. M. and V. B. Mahesh Pituitary func-

11 170 MORRISON ET AL. tion in the aged. P In R. B. Greenblatt (Ed.) Geriatric Endocrinology. Vol. V. Raven Press, New York. Ohlson, D. L., S. L. Davis, M. S. Anfinson and J. Klindt Thyrotropin and prolactin secretory patterns during 24-hour infusion of thyrotropin-releasing hormone in calves. Neuroendocrinology 26:163. Ravault, J. P Prolactin secretion in the ram: Seasonal variations in the concentrations of blood plasma from birth until three years old. Acta Endocrinol. 83:720. Steel, R.G.D. and J. H. Torrie Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, p Trenkle, A Estimates of the kinetic parameters of growth hormone metabolism in fed and fasted calves and sheep. J. Anita. Sci. 43:1035. Valueva, G. V. and N. V. Verzhikovskaya Thyrotropic activity of hypophysis during aging. Exp. Gerontol. 12:97. Vidalon, C., R. C. Khurana, S. Chae, C. G. Gegick, T. Stephan, S. Nolan and T. S. Danowski Age-related changes in growth hormone in nondiabetic women. J. Amer. Geriatr. Soc. 21:253. Wagner, H., H. Vosberg, K. B~Sckel, M. Hrubesch, G. Grote and W. H. Hauss Influence of age on response of TSH to thyrotropin releasing hormone in normal subjects. Acta Endocrinol. (Kbh.) Suppl. 184, Abstr. #119. Yamaji, T., K. Shimamoto, M. Ishibashi, K. Kosaka and M. Orimo Effect of age and sex on circulating and pituitary prolactin levels in human. Acta Endocrinol. (Kbh.) 83:711.