AGE-, STRAIN-, AND SEX-DIFFERENCES IN THE ANTERIOR PITUITARY GROWTH HORMONE CONTENT OF MICE

Transcription

1 AGE-, STRAIN-, AND SEX-DIFFERENCES IN THE ANTERIOR PITUITARY GROWTH HORMONE CONTENT OF MICE REIKO YANAI AND HIROSHI NAGASAWA Pharmacology Division, National. Cancer Center Research Institute, Tokyo SYNOPSIS The growth, the anterior pituitary weight, and the anterior pituitary growth hormone (GH) content of C3H/He and C57BL/6 strains of mice were investigated during days of age in the male and days of age in the female. The pituitary GH content was measured by disc electrophoresis. The pattern of growth and the increasing rate of body weight were similar in either strain, whereas the body weight of C3H/He was always greater than that of C57BL/6 in both sexes. The whole pituitary weight increased with age until days and became plateau thereafter in the male of both strains and the C57BL/6 female. On the other hand, the pituitary weight of the C3H/He female continued to increase until the 90th day. Although the pituitary weights were greater in C3H/He than in C57BL/6 until the 60th and 90th days of age in both sexes the pattern of its change was little different according to the strains. The variation of the relative pituitary weight per 10g body was not so remarkable with age in both strains and sexes. In both strains, the total GH content and the relative GH content per 10g body changed in a similar pattern with age, which increased until days of age and became plateau or inclined to decrease a little after that in the male, while they continued to increase until the 90th day in the female. The total GH content in C3H/He was significantly higher than that in C57BL/6 in either sex. The relative GH content per 10g body showed no difference between strains in the male, but it was higher in C3H/He than in C57BL/6 in the female. In C3H/He, the female excelled the male not only in the total GH content but also in the GH content per observed in these areas in C57BL/6. The GH content per mg pituitary showed no noticeable variations with age, strain and sex. The significance of these findings was discussed. There is experimental evidence to show that growth hormone (OH) is essential for growth process, general metabolism, mammary gland development and lactation on which the mechanisms of GH action are not always completely clear. Further, the accerelating effect of GH on transplantable mammary tumor Received for publication January 16, growth of mice has been reported (Speiser et al., 1966). The authors demonstrated that the C3H/He strain (a high mammary tumor strain) was superior to the C57BL/6 strain (a low mammary tumor strain) in growth, mammary gland development and lactational performance (Nagasawa et al., 1967a, b, c), and it was thought worthwhile to study the difference

2 YANAI AND NAGASAWA Fig. 1. Growth curves of male and female mice of the two strains Vertical bars indicate standard error of the mean. between these two strains in GH secretory capacity of the anterior pituitary and in its mode of action. As the first step, in the present experiment, the age-, strain-, and sex-differences in the anterior pituitary GH content were investigated using C3H/He and C57BL/6 strains of mice by the method of disc electrophoresis which was applied for semiquantitative measurement of anterior pituitary GH content of mice (Yanai et al., 1968). Animals The mice of the C3H/He and C57BL/6 strains used in the present experiment came from the parents which had been inbred for at least 75 and 35 generations of brother ~sister matings respectively..they. were weaned at days of age, kept in a group of 5-6 each in a teflon cage (18 ~ 30 ~12cm), provided commercial diet and water ad libitum, and maintained at a temperature and relative humidity controlled (24 Ž, and 70 }1%) and artificially illuminated (12 hr. light from 8:00 a.m. to 8:00p.m.) room throughout the experiment. The male mice at the ages of 12, 20, 30, 40, 50, 60, 90 and 120 days and the female mice at the ages of 30, 60 and 90 days were killed by decapi- MATERIALS AND METHODS tation. The pituitary gland was immediately removed, the posterior lobe discarded and the anterior lobe weighed and stored at-20 Ž until its usage. Preparation of the anterior pituitary and disc electrophoresis for determination of GH content One to seven anterior pituitaries were pooled as

3 Male Table 1. Increasing rate of body weight* in the two strains of mice Female * one sample according to their weights. The number of samples in each age group was The pituitary for disc electrophoresis was prepared in the same method as described in the previous report (Yanai et al., 1968). Disc electrophoress was performed by the method of Davis (1964). The concentration of acrylamide employed for separating gel was 7.5%. A current of 4 milliamperes per column was applied at ph 9.5. The optical density of each GH band was measured with microdensitometer (Canalco, Model E), and the content of GH was expressed in terms of bovine GH standard (NIH- GH-B12), ƒÊg of which had the linear relation with their optical densities (Yanai et al., 1968). RESULTS Body weight The changes in body weight are shown in Figure 1. Although the body weight was always significantly greater in C3H/He than in C57BL/6 not only in the male but also in the female, the patterns of growth were similar in either strain and the differences in increasing rates of body weight during days in the male and days in the female were negligible between strains (Table 1). These results were in good agreement with the authors' previous ones (Nagasawa et al., 1967a). Anterior pituitary weight The changes in weight of the whole anterior pituitary are shown in Figure 2-A. The whole pituitary weight increased with age until days and became plateau thereafter in the males of both strains and in the C57BL/6 female. On the other hand, the pituitary weight of the C3H/He female continued to increase until the 90th day. The pituitary weight of C3H/He was significantly greater than that of C57BL/6 until the 60th and 90th days in both sexes. The sex-differences in pituitary weight were little at any age within strain except the weight on the 90th day of C3H/He which was significantly greater in the female. As shown in Figure 2-B, the relative pituitary weight per 10g body remained fairly constant until the 90th day and inclined to decrease after that in the male of either strain. The similar tendency was observed in the C57BL/6 female. However, the relative pituitary weight tended to increase with age in the C3H/He female. The strain-differences in the relative pituitary weight were not so remarkable irrespective of age, especially in the male. While no sex-differences in the relative pituitary weight were found in C57BL/6, the relative weight of the female was greater with

4 (A) Whole pituitary weight (B) Pituitary weight per 10g body weight Fig. 2. Changes in the anterior pituitary weight in male and female mice of the two strains. Vertical bars indicate standard error of the mean. age than that of the male in C3H/He. GH content Total GH content per whole pituitary In Figure 3-A are presented the changes in the total GH content. In the male, the total GH content increased with age in the similar pattern until the 60th day and became plateau or a small decline thereafter in both strains. On the other hand, it had the tendency to increase in the female until the 90th day and the increasing rate was more remarkable in C3H/He than in C57BL/6. The total GEE content was larger in C3H/ He than in C57BL/6 at all ages in both sexes the differences were much more remarkable in the female than in the male. The total GH content was significantly larger in the female than in the male of C3H/ He on the 60th and 90th days, but only slight sex-differences were noted. GH content per mg pituitary The GH content per mg pituitary increased only slightly until the 60th day and little change was found after that in the male of both strains as seen in Figure 3-B. No clear tendencies were observed in the female which

5 Fig. 3. Changes in the anterior pituitary growth hormone (GH) content in male and female mice of the two strains. Vertical bars indicate standard error of the mean. was probably due to only three stages of age groups of both sexes. sampling. The sex-differences of the GH content per There existed no strain-differences of the mg pituitary were not always distinct at any age of either strain.

6 YANAI AND NAGASAWA Endocrinol. Dec Japon. GH content per 10g body found that there was less GH content per 10g Figure 3-C shows the changes in the GH body as the male rat aged. Recently Birge et al. content per 1Og body. The GH content per (1967) studied the variations of pituitary GH content as to age and sex of the rat by radioimmunoassay. total GH content; it increased with age until the 60th-90th days and became almost plateau thereafter regardless of strain and sex. The strain-differences were hardly seen in the male but were significant on the 60th and 90th days in the female, C3H/He being larger. The GH content per 10g body of the female was significantly larger than that of the male in both strains on the 30th, 60th and 90th days except on the 60th day in C57BL/6. They demonstrated that the total GH content and GH concentration of the rat pituitary were greater in the male than in the female after puberty, and that castration of the male rat lowered the levels to those of the female and the testosterone administration to the female rat resulted in accumulation of GH in the pituitary, while the estrogen administration to the male decreased the pituitary GH content and concentration. Similar results were obtained by Jones et al. (1965). The DISCUSSION The relationship between age and the anterior pituitary GH content has been extensively studied in several species and reviewed by Kirk (1962), Verzar (1966) and others. It has been generally accepted that the total GH content in the pituitary increased with age and it was in agreement with the present results. However, the change in the relative GH concentration (content per unit weight of pituitary or body) has not always showed the consistent tendencies. Baker et al. (1956) described that the pituitaries of pigs under 20 days of age contained a distinctly lower GH concentration than the glands of older animals and that the GH concentration became constant after 20 days of age. A significant tendency toward a decrease in the GH level of the gland with age was demonstrated in heifers (Armstrong and Hansel, 1956). Furthermore, in humans, no correlation existed between age and sex of the individuals and the GH content of the gland (Gemzell and Heijkenskjold, 1956), and it was found there was a higher GH concentration in the pituitary of an adult than in the gland of a child (Gershberg, 1957). No significant variation with age in the GH concentration in the female rat was recorded bysolomon and Greep (1958), while Bowman (1961) present results in mice are not always in accordance with those obtained by these authors. Namely, in mice, the GH content per 10g body increased gradually with age until days and inclined to be plateau thereafter, while the GH content per mg pituitary was rather constant irrespective of age, strain and sex. And the GH content per 10g body was larger in the female than in the male in both strains. Moreover, the similar differences were observed in the total GH content of C3H/He. These discrepancies in the relation between GH concentration and age or sex may be partly due to the differences in the assay method and in the time and interval of sampling as well as the difference in the species of animals examined. The studies on the mechanisms involved in the regulation of GH secretion are difficult to make, inasmuch as GH, unlike the other pituitary tropic hormones, does not elicit specific activation in a single target peripheral gland (Pecile and Muller, 1966). But fine controls of GH secretion in general metabolic adjustments have been demonstrated (Korner, 1965; Glick et al., 1965; Pecile and Muller, 1966; Reichlin, 1966; Van Der Werff ten- Bosch, 1966). Among these metabolic factors, hypoglycemia is one of the most potent stimuli of GH (Krulich and McCann, 1966c). It

7 PITUITARY GH CONTENT OF MICE Produced in normal human subjects an abrupt and sustained rise in the level of GH (Glick et al., 1965; Roth et al., 1963). Insulin-induced hypoglycemia has caused the rapid and consistent stimuli of GH depletion from the anterior pituitary of rats (Katz et al., 1967; Miiller and Pecile, 1966) and the decrease of hypothalamic GH releasing factor (Katz et al., 1967). Ravinowitz and Zierler (1963) stated the hypothesis that metabolism is dominated alternatively by the action of insulin or of GH or by combined effects of the two in the three phase circle determined by the intake of food. Furthermore the intravenous injection of several amino acids has been observed to influence the plasma GH level (Knoff et al., 1965). The hormonal influences such as thyroxine, insulin, gonadal hormones (Pecile and Muller, 1966; Reichlin, 1966; Schooley et al., 1966) and GH (Krulich and McCann, 1966a ; Muller et al., 1967a) are involved as the factors regulating GH secretion. Starvation (Roth et al., 1963), a variety of non-specific stimuli (Krulich and McCann, 1966b; Muller et al., 1967b, c) and moderate exercise (Hunter et al., 1965) also altered GH secretion. Thus GH secretion is supposed to be regulated very delicately mostly via hypothalamus (Guillemin, 1967; Muller et al., 1967d ; Schally et al., 1968) in order to maintain the homeostasis of body, and synthesis and release of GH may be varied from moment to moment by the several metabolic and other factors. Since it is generally accepted that GH, as other hormones, is synthetized and released simultaneously from the anterior pituitary in response to demand, the results of GH content in the anterior pituitary obtained in normal physiological conditions, such as in the present experiment would suggest the pituitary secretory capacity of GH to be fairly consistent, although the interpretation of changes in GH content may be speculative to some extent. In the present experiment, there is no straindifferences between the males of C3H/He and C57BL/6 in the GH contents both per mg pituitary and per log body, while the total GH content was higher in the former than in the latter, and neither the pattern nor the increasing rate of body weight was different in either strain. These results would indicate that in the male, the secretory capacity of GH in the anterior pituitary itself was little different between strains and that the larger quantity of total GH in C3H/He was ascribed only to the larger pituitary in this strain. Meanwhile, the strain-difference of the female in the GH content was not similar to that of the male; the C3H/He female was higher than the C57- BL/6 female not only in the total GH content but also in the GH content per 10g body. This infers the larger quantity of GH acting on each peripheral tissue unit in the former than in the latter, and may account for the part of the superiority of C3H/He to C57BL/6 in mammary gland development and lactational performance (Nagasawa et al., 1967a,b), because GH. plays an important role in mammary gland development and lactation (Meites, 1966) as well as body growth and general metabolism. The present observation of the whole mount preparation of the right third thoracic gland also revealed that the degree of mammary gland development of C3H/He female was much more conspicuous than that of C57BL/6 female at 90 days of age; the gland of the former showed highly branched thin ducts with many scattered alveoli, whereas the gland of the latter was restricted only to the ductal development. ACKNOWLEDGEMENTS The authors' appreciation is expressed to Dr. W. Nakahara (the Director of this Institute) and Dr. K. Kuretani (the Chief of this Division) for their continued interest and encouragement in this work.

8 YANAI AND NAGASAWA Endocrinol. Dec Japon. They are also indebted to Associate Prof. K. Usui and Dr. A. Takeuchi (Department of Animal Surgery, Faculty of Agriculture, University of Tokyo) for their valuable advice and suggestion. REFERENCES Armstrong, D. T. and W. Hansel (1956). J. Anim. Sci. 15, 640. Baker, B. Jr., R. Hollandbeck, H. W. Norton and A. V. Nalbandov (1956). Ibid. 15,407. Birge, A. C., G. T. Peake, I. K. Mariz and W. H. Daughaday (1967). Endocrinology 81, 195. Bowman, R. H. (1961). Nature 192, 976. Davis, B. J. (1964). Annals of the New York Academy of Science 121, article 2, 404. Gemzell, C. A. and F. Heijkenskjold (1956). Endocrinology 59, 681. Gersh-berg, H. (1957). Ibid. 61, 160. Glick, S. M., J. Roth, R. S. Yalow and S. A. Berson (1965). Recent Progr. Hormone Res. 21, 241. Guillemin, R. (1967). Annual Review of Physiology 29, 313. Hunter, W. M., C. C. Fonseka and R. Passmore (1965). Quat. J. Exp. Physiol. 50, 406. Jones, A. E., J. N. Fisher, U. J. Lewis and W. P. Vanderlaan (1965). Endocrinology 76, 578. Katz, S. H., A. P. S. Dhariwal and S. M. Mc- Cann (1967). Ibid. 81, 333. Kirk, J. E. (1962). Vitamins and Hormones 20, 93. Knoff, R. F., J. W. Conn, S. S. Fajans, J. C. Floyd, E. Gastache and J. Rull (1965). J. Clin. Endocr. 25, Korner, A. (1965). Recent Progr. Hormone Res. 21, 205. Krulich, L. and S. M. McCann (1966a). Proc. Soc. Exp. Biol. Med. 121, Krulich, L. and S. M. McCann (1966b). Ibid. 122, 612. Krulich, L. and S. M. McCann (1966c) Endocrinology 78, 759. Meites, J. Neuroendocrinology, Vol. I. Academic Press, New York and London, PP (1966). Muller, E. E. and A. Pecile (1966). Proc. Soc. Exp. Biol. Med. 122, Muller, E. E., S. Sawano, A. Arimura and A. V. Schally (1967a). Acta Endocrinol. 56, 499. Muller, E. E., A. Arimura, S. Sawano, T. Saito and A. V. Schally (1967b). Proc. Soc. Exp. Biol. Med. 123, 874. Muller, E. E., T. Saito, A. Arimura and A. V. Schally (1967c). Endocrinology 80, 109. Muller, E. E., A. Arimura, S. Sawano, T. Saito and A. V. Schally (1967d). Proc. Soc. Exp. Biol. Med. 125, 874. Nagasawa, H., M. Fujimoto, H. Iwahashi and K. Kuretani (1967a). Gann 58, 115. Nagasawa, H., H. Iwahashi, H. Kanzawa and K. Kuretani (1967b). Ibid. 58, 49. Nagasawa, H., F. Kanzawa and K. Kuretani (1967c). Ibid. 58, 331. Pecile, A. and E. E. Muller Neuroendocrinology, Vol. II. Academic Press, New York and London, pp (1966). Ravinowitz, D. and K. L. Zierler (1963). Nature 199, 913. Reichlin, S. The Pituitary Gland, Vol. II. Butterworth, London, pp (1966). Roth, J., S. M. Glick, R. S. Yalow and S. A. Berson (1963). Science 140, 987. Schally, A. V., E. E. Muller and S. Sawano (1968). Endocrinology 82, 271. Schooley, R. A., S. Friedkin and E. S. Evans (1966). Ibid. 79, Solomon, J. and R. O. Greep (1958). Proc. Soc. Exp. Biol. Med. 99, 725. Speiser, Z., S. Gitter and Z. Laron (1966). Experientia 22, 523. Van Der Werff tenbosch, J. J. The Pituitary Gland, Vol. II. Butterworth, London, pp (1966). Verzar, F. Ibid. pp (1966). Yanai, R., H. Nagasawa and K. Kuretani (1968). Endocrinol. Japon. 15, 365.