Mechanisms of Ageing and Development, 8 (1978) 63-68
63
@Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
IMPAIRMENT OF THE HYPOTHALAMO-PITUITARY-OVARIAN OF THE ATHYMIC "NUDE" MOUSE
AXIS
Y. WEINSTEIN Department of Hormone Research, The lCeizmann Institute of Science, Rehovot (Israel)
(Received October 19, 1977)
SUMMARY Pituitary and serum levels of LH and FSH and hypothalamic GnRH content were measured in acyclic, congenitally athymic (nu/nu) female mice. No significant differences were found between athymic and normal dioestrous mice of the same age (3 months). The serum LH level of the athymic mouse failed to increase 6 days after ovariectomy, but increased in response to injection of GnRH. The results suggest that the athymic nude mice suffer from impairment of hypothalamic control of the pituitary.
INTRODUCTION The function of the thymus in the development of the immunological system is well established. It is now believed that the thymus produces hormone(s) which influence(s) the maturation of prothymocytes to T lymphocytes [1 ]. In recent years it was found that the thymus may influence the maturation and the function of the female reproductive system. Studies of females from the congenitally athymic mutant strain of "nude" mice show severe deficiencies in reproductive function. The first ovulation is delayed until the age of 2.5 months [2]. By 4 months there is an absence of non-atretic large follicles and newly formed corpora lutea [3]. The ovaries and the uterus of the 3-4 month old animals are abnormally small; the oestrous cycle is abnormal and the females are usually sterile [4]. These abnormalities can be prevented by grafting thymic tissue at birth. Similar abnormalities result from neonatal thymectomy of normal female mice, and these can be prevented by thymus grafting before the 7th day of age [5]. The atrophic ovaries of the athymic mice can be restored to normal morphology by injection of gonadotrophin [6]. This suggests that the retardation of follicular growth in nude mice is the result of gonadotrophin insufficiency. Therefore the primary endocrine defect may be the pituitary gland or the hypothalamic neural centers which control hypophysical gonadotrophin secretion. In order to understand the interrelationship between the thymus and the endocrine system, it is important to locate the endocrine disorder of the female reproductive system
64 and analyze its pathogenesis. We measured serum and pituitary levels of gonadotrophins, the hypothalamic gonadotrophin releasing hormone (GnRH), the pituitary response to ovariectomy and to administration of exogenous GnRH in the athymic mouse.
MATERIALS AND METHODS Mice Athymic C57BL/6J (nu/nu) and phenotypically normal heterozygous C57BL/6J (nu/+) were derived from stock supplied by G. L. Bomholtgaard, Denmark. The mice were bred and grown in an air-conditioned room (25 -+ 1 °(7) under controlled illumination of 14 h/clay. Three to four-month-old animals were used. Vaginal smears were examined daily. Athymic mice were used only after they had shown a constant dioestrous smear for at least 2 weeks (70% of the colony). The control haired littermates had normal oestrous cycles. Hormonal treatments (1). Pregnant mare serum gonadotrophin (PMSG, Organon, Holland; 4 I.U./mouse) was injected intraperitoneally at 12.00 a.m. followed by human chorionic gonadotrophin (Pregnyl, Organon, Holland, 2 I.U./mouse) 50 h later. The morning after the hCG injection, the animals were sacrificed. The ovaries were weighed and the Fallopian tubes were examined for presence of ova. (2). GnRH (a gift from Dr. Y. Koch; 50 ng/mouse) was iniected intraperitoneally in 0.5 ml phosphate buffered saline and 30 rain later blood samples were drawn from the tail vein. Radioimmunoassay Blood samples collected at 10-12 a.m., hypophysis were homogenized in water. The samples were kept at - 2 0 °C until required for assay. Blood and tissue samples from normal control mice were always collected on the day of dioestrus. Serum and pituitary LH and FSH levels were determined by radioimmunoassay as described by Daane and Parlow [7] using reagents supplied by The National Institute of Arthritis and Metabolic Diseases (NIAMD) through the courtesy of Dr. A. F, Parlow. The results are expressed in terms of the reference preparations NIAMD-Rat LH-RP-1, NIAMD Rat-FSH-RP-1. Hypothalamic GnRH was assayed by the method described by Koch et al. [8], using reagents kindly supplied by Koch. Protein determination was done by the method of Lowry et al. [9]. Thymus transplantation Neonate athymic female mice 24 h after birth were used. One thymus from a heterozygous littermate of the same age and sex was inserted under the skin on the back of the mouse with the aid of a trocar. At the age of 3 months, spleen cells from these mice responded to T-cell mitogens, viz. phytohaemagglutinin and concanavalin A.
65 RESULTS Ovarian weight was 45% smaller in athymic than in control mice (P < 0.001 by Student's t test; Table I). The body weight o f athymic mice was 25% less than that o f control mice, but ovarian weight in the athymic mice was significantly less than that o f control even in relation to body weight (P < 0.001). Ninety hours after administration o f PMSG followed by injection o f hCG, the athymic mice ovaries attained normal weight (Table I); formed follicles were fluid filled, and evidence of ovulation was obtained in all the mice tested in both groups (9.2 + 1.4 ova/control mouse -+ SEM and 6.0 -+ 2.2 ova[ athymic mouse, P > 0.2). The hypothalamic GnRH levels o f the athymic mice were slightly higher than that o f control (0.05 < P < 0.1, Table II). The pituitary protein content o f the athymic mouse was 25% lower per gland than that o f control, but similar to that o f control when related to body weight (Table II). The content o f FSH was similar in both groups of mice, LH
TABLE I OVARIAN WEIGHT OF ATHYMIC AND CONTROL MICE
Mice
Control (nu/+) mg
Ovarian weight Ovarian weight after PMSG, hCG treatment
A thymic (nu/nu) mg/10 g body weight
mg
mg/10 g body weight
8.4 ± 0.3
3.3 -+ 0.06
4.7 ± 0.3
2.4 ± 0.1
10.5 ± 0.5
4.4 ± 0.2
8.2 ± 0.4
4.5 ± 0.3
3-month-old mice were used. The weight of a pair of ovaries is given (mean +- SEM, n = 6-10). The athymic and control mice weight (mean ± SEM) was 19.0 +- 0.5 and 25.3 -+ 0.6 respectively. Ovulation was detected in all groups of mice treated with PMSG and hCG.
TABLE II LEVELS OF HYPOTHALAMIC GnRH, PITUITARY AND SERUM LH AND FSH IN THE ATHYMIC MOUSE
Hormone
Control {nu/+]
A thymic (nu/nu)
Hypothalamic GnRH (ng) Pituitary FSH (ug) Pituitary LH (tzg) Serum FSH (gg/ml) Serum LH (#g/ml)
2.3 4.6 20.1 0.53 0.024
3.0 4.8 10.9 0.58 0.024
± 0.2 ± 0.5 (11.2) ± 3.8 (49.1) ± 0.04 ± 0.007
± 0.3 ± 0.5 (15.3) ± 2.3 (34.8) ± 0.06 ± 0.04
Serum level and the total amount of hormone(s) found in the hypothalamus and the pituitary are shown (mean ± S.E., n = 6-10). The pituitary protein content was 0.409 ± 37 mg and 0.313 +- 25 for control and athymic mice respectively. The hypophysial hormone concentration (/~g hormone/mg protein) are given in parentheses. The control mice were bled in the morning of the dioestrus.
66 was 46% lower per gland (0.05 < P "< 0.1) and 30% lower after correction for pituitary protein content. The serum LH and FSH levels were similar in nude and control groups. Three-month-old control athymic mice and athymic equipped with thymus graft were ovariectomized. The serum LH levels were measured 3 and 6 days after the operation (Table III). Removal of the ovaries did not cause an increase in the serum LH levels in athymic mice, the normal control mice and athymic mice implanted with thymus at birth showed the expected rise in serum LH levels. On the seventh day after ovariectomy, GnRH (50 ng) was injected intraperitoneally into all mice and serum LH levels were determined 30 min later. The ovariectomized athymic mice responded to exogenous GnRH with elevated serum levels of LH (6 fold above basal level). TABLE III SERUM LH LEVELS IN OVARIECTOM1ZED AND GnRH TREATED ATHYMIC MICE Mice
Untreated LH (ng/ml~
Ovariectomized LH (ng/ml) day 3
day 6
24.0 ± 4.1
day 7 30 min after injection o f 50 ng GnRH
Athymic (nu/nu)
24.0 ± 4.5
19.0 ± 2.4
145.0 ± 54.0
Athymic implanted with thymus at birth
20.0 ÷ 3.0
N.T.
250.1 ± 42.3
N.T.
Control (nu/+)
24.0 ± 7.1
89.1 ± 26.0
225.0 ± 25.0
476.0 ± 80.0
Six to eight 4-month-old mice were used in each group, serum LH levels (mean ± SEM) a_re shown. N.T. not tested.
DISCUSSION The congenitally athymic mouse is a useful model for the analysis o f the role o f the thymus in the formation of the endocrine system. Lintern-Moore and Pantelouris [6] demonstrated the presence of specific structural and functional abnormalities in prepuberal and adult ovaries from athymic mice. Normal structure could be restored by injection of gonadotrophin. We found that injection of gonadotrophins to adult athymic mice caused ovaluation. These results led to the concept that the athymic mice ovaries are normal and their malfunction resulted from insufficient gonadotrophins secretion from the pituitary. However, direct measurement of the gonadotrophin content of the pituitary or of serum gonadotrophin concentration were not performed adequately. The measurement o f GnRH, LH and FSH in athymic and control mice revealed that hypothalamic GnRH levels of athymic mouse were slightly significantly higher than those o f
67 normal control, and that the contents of pituitary FSH and serum FSH and LH concentration in the peripheral blood were not significantly different in the two groups of mice. However, the gonadotrophin levels in the control animals were determined only during the leukocyclic phase of the cycle and not during the proestrous gonadotrophin surge. The pituitary content of LH was 46% lower per gland in the athymic mice. This may be a result of the acyclic state. Ovariectomy in normal adult mice and rats results in greatly increased secretion of LH and FSH [10, and present control data]. However, 6 days after ovariectomy, serum LH levels in the athymic mice were unchanged. Injection of exogenous GnRH stimulates LH secretion by the athymic mouse pituitary. It therefore appears that the lack of cyclic ovulatory gonadotrophin release, and the lack of feedback response to ovariectomy are due to defective hypothalamic control of LH secretion rather than an inhibition of the pituitary to respond to GnRH. It should be pointed out however, that we have no evidence that the ovaries of the athymic mice at time of ovariectomy secrete significant amounts of oestrogen; but if ovarian steroid secretion was inadequate plasma gonadotrophin levels should have been elevated, given normal hypothalamic function. The factors responsible for the hypothalamic defect remained to be identified. It was suggested [11, 17] that a perinatal androgenic influence might change the normal programming of the neural centers in the brain of the nude mice because the morphology of the atrophic follicles in the ovaries of the athymic mouse are similar to those of mice neonatally treated with testosterone propionate [12], which also blocks the cyclic release of gonadotrophins at maturity [13]. Injection of thymocytes protected neonate rats from androgenization caused by treatment with testosterone [14], and neonatal thymectomy prolonged the period in which the rat is susceptible to androgenization [15]. Recently we have found progesterone-metabolizing enzymes in the thymus of neonate mice [16]. Therefore the possibility exists that one of the ways by which the thymus may influence the endocrine system is through steroid metabolism in the neonatal period of life, when the brain is most sensitive to steroid effects.
ACKNOWLEDGEMENTS This work was supported by grants from the Israel Commission for Basic Research (to Y. W.), and the Ford Foundation and the Population Council, N.Y. (to H. R. Lindner). REFERENCES 1 J. Rygaard, Thymus and self. Immunobiology of the mouse mutant "nude". F.A.D.L., Copenhagen, 1973. 2 H. O. Besedovsky and E. Sorkin, Thymus involvement in sexual maturation, Nature, 249 (1974) 356-358. 3 S. Lintern-Moore and E. M. Pantelouris, Ovarian development in the athymic nude mice. I. The size and composition of the follicle population, Mech. Ageing Dev., 4 (1975) 385-390.
68 4 J. G. M. Shire and E. M. Pantelouris, Comparison of endocrine function in normal and genetically athymic mice, Cornp. Biochem. Physiol., 47A (1974) 93-100. 5 Y. Nishisuka and T. Sakakura, Thymus and reproduction; sex-linked dysgenesia of the gonad after neonatal thymectomy in mice, Science, 66 (1969) 753-755. 6 S. Lintern-Moore and E. M. Pantelouris, Ovarian development in athymic nude mice. III. The effect of PMSG and oestradiol upon the size and composition of the ovarian follicle population, Mech. AgeingDev., 5 (1976) 33-38. 7 T. A. Daane and A. F. Parlow, Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle: effect of pentobarbital, Endocrinology, 88 (1971) 653-663. 8 Y. Koch, M. Wilchek, M. Fridkin, P. Chobsieng, U. Zor and H. R. Lindner, Production and characterization of an antiserum to synthetic gonadotropbin-releasing hormone, Biochem. Biophys. Res. Commun., 55 (1973) 616-622. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 N. B. Schwartz and C. E. McCormack, Reproduction: gonadal function and its regulation, A. Rev. Physiol., 34 (1972) 425-472. 11 E. M. Pantelouris, Effects of athymia on development, in M. Balls and A. E. Wilds (eds.), The Early Development of Mammals, Second Symp. of the Brit. Soc. Developmental BioL, Cambridge Univ. Press, London, 1975, pp. 373-388. 12 H. Peters, I. N. Sorensen, A. G. Byskov, T. Pedersen and T. Krarup, The development of the mouse ovary after testosterone propionate on day 5, in W. R. Butt, A. C. Crooke and M. Ryle (eds.), Gonadotrophins and Ovarian Development, Churchill-Livingstone, Edinburgh, 1970, pp. 351-361. 13 R. A. Gorski, Mechanism of androgen induced masculine differentiation of the rat brain, in K. Lissak (ed.), Hormone and Brain Function, Plenum, New York, 1973, pp. 27-46. 14 F. A. Kind, A. Oriol, A. Folch Pi and M. Maqueo, Prevention of steroid induced sterility in neonatal rats with thymic cell suspension, Proc. Soc. Exp. Biol. Meal., 120 (1965) 252-255. 15 N. Zabuzkova and F. A. Kinci, The influence of thymectomy and steroid hormones in neonatal rats, Proc. Soc. Exp. Biol. Meal., 135 (1970) 874-877. 16 Y. Weinstein, H. R. Lindner and B. Eckstein, Thymus metabolises progesterone - possible enzymatic marker for T lymphocytes, Nature, 266 (1977) 632-633. 17 W. Pierpaoli and H. O. Bsedovsky, Role of the thymus in programming of neuroendocrine functions, Clin. Exp. Immunol., 20 (1975) 323-338.
63
@Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
IMPAIRMENT OF THE HYPOTHALAMO-PITUITARY-OVARIAN OF THE ATHYMIC "NUDE" MOUSE
AXIS
Y. WEINSTEIN Department of Hormone Research, The lCeizmann Institute of Science, Rehovot (Israel)
(Received October 19, 1977)
SUMMARY Pituitary and serum levels of LH and FSH and hypothalamic GnRH content were measured in acyclic, congenitally athymic (nu/nu) female mice. No significant differences were found between athymic and normal dioestrous mice of the same age (3 months). The serum LH level of the athymic mouse failed to increase 6 days after ovariectomy, but increased in response to injection of GnRH. The results suggest that the athymic nude mice suffer from impairment of hypothalamic control of the pituitary.
INTRODUCTION The function of the thymus in the development of the immunological system is well established. It is now believed that the thymus produces hormone(s) which influence(s) the maturation of prothymocytes to T lymphocytes [1 ]. In recent years it was found that the thymus may influence the maturation and the function of the female reproductive system. Studies of females from the congenitally athymic mutant strain of "nude" mice show severe deficiencies in reproductive function. The first ovulation is delayed until the age of 2.5 months [2]. By 4 months there is an absence of non-atretic large follicles and newly formed corpora lutea [3]. The ovaries and the uterus of the 3-4 month old animals are abnormally small; the oestrous cycle is abnormal and the females are usually sterile [4]. These abnormalities can be prevented by grafting thymic tissue at birth. Similar abnormalities result from neonatal thymectomy of normal female mice, and these can be prevented by thymus grafting before the 7th day of age [5]. The atrophic ovaries of the athymic mice can be restored to normal morphology by injection of gonadotrophin [6]. This suggests that the retardation of follicular growth in nude mice is the result of gonadotrophin insufficiency. Therefore the primary endocrine defect may be the pituitary gland or the hypothalamic neural centers which control hypophysical gonadotrophin secretion. In order to understand the interrelationship between the thymus and the endocrine system, it is important to locate the endocrine disorder of the female reproductive system
64 and analyze its pathogenesis. We measured serum and pituitary levels of gonadotrophins, the hypothalamic gonadotrophin releasing hormone (GnRH), the pituitary response to ovariectomy and to administration of exogenous GnRH in the athymic mouse.
MATERIALS AND METHODS Mice Athymic C57BL/6J (nu/nu) and phenotypically normal heterozygous C57BL/6J (nu/+) were derived from stock supplied by G. L. Bomholtgaard, Denmark. The mice were bred and grown in an air-conditioned room (25 -+ 1 °(7) under controlled illumination of 14 h/clay. Three to four-month-old animals were used. Vaginal smears were examined daily. Athymic mice were used only after they had shown a constant dioestrous smear for at least 2 weeks (70% of the colony). The control haired littermates had normal oestrous cycles. Hormonal treatments (1). Pregnant mare serum gonadotrophin (PMSG, Organon, Holland; 4 I.U./mouse) was injected intraperitoneally at 12.00 a.m. followed by human chorionic gonadotrophin (Pregnyl, Organon, Holland, 2 I.U./mouse) 50 h later. The morning after the hCG injection, the animals were sacrificed. The ovaries were weighed and the Fallopian tubes were examined for presence of ova. (2). GnRH (a gift from Dr. Y. Koch; 50 ng/mouse) was iniected intraperitoneally in 0.5 ml phosphate buffered saline and 30 rain later blood samples were drawn from the tail vein. Radioimmunoassay Blood samples collected at 10-12 a.m., hypophysis were homogenized in water. The samples were kept at - 2 0 °C until required for assay. Blood and tissue samples from normal control mice were always collected on the day of dioestrus. Serum and pituitary LH and FSH levels were determined by radioimmunoassay as described by Daane and Parlow [7] using reagents supplied by The National Institute of Arthritis and Metabolic Diseases (NIAMD) through the courtesy of Dr. A. F, Parlow. The results are expressed in terms of the reference preparations NIAMD-Rat LH-RP-1, NIAMD Rat-FSH-RP-1. Hypothalamic GnRH was assayed by the method described by Koch et al. [8], using reagents kindly supplied by Koch. Protein determination was done by the method of Lowry et al. [9]. Thymus transplantation Neonate athymic female mice 24 h after birth were used. One thymus from a heterozygous littermate of the same age and sex was inserted under the skin on the back of the mouse with the aid of a trocar. At the age of 3 months, spleen cells from these mice responded to T-cell mitogens, viz. phytohaemagglutinin and concanavalin A.
65 RESULTS Ovarian weight was 45% smaller in athymic than in control mice (P < 0.001 by Student's t test; Table I). The body weight o f athymic mice was 25% less than that o f control mice, but ovarian weight in the athymic mice was significantly less than that o f control even in relation to body weight (P < 0.001). Ninety hours after administration o f PMSG followed by injection o f hCG, the athymic mice ovaries attained normal weight (Table I); formed follicles were fluid filled, and evidence of ovulation was obtained in all the mice tested in both groups (9.2 + 1.4 ova/control mouse -+ SEM and 6.0 -+ 2.2 ova[ athymic mouse, P > 0.2). The hypothalamic GnRH levels o f the athymic mice were slightly higher than that o f control (0.05 < P < 0.1, Table II). The pituitary protein content o f the athymic mouse was 25% lower per gland than that o f control, but similar to that o f control when related to body weight (Table II). The content o f FSH was similar in both groups of mice, LH
TABLE I OVARIAN WEIGHT OF ATHYMIC AND CONTROL MICE
Mice
Control (nu/+) mg
Ovarian weight Ovarian weight after PMSG, hCG treatment
A thymic (nu/nu) mg/10 g body weight
mg
mg/10 g body weight
8.4 ± 0.3
3.3 -+ 0.06
4.7 ± 0.3
2.4 ± 0.1
10.5 ± 0.5
4.4 ± 0.2
8.2 ± 0.4
4.5 ± 0.3
3-month-old mice were used. The weight of a pair of ovaries is given (mean +- SEM, n = 6-10). The athymic and control mice weight (mean ± SEM) was 19.0 +- 0.5 and 25.3 -+ 0.6 respectively. Ovulation was detected in all groups of mice treated with PMSG and hCG.
TABLE II LEVELS OF HYPOTHALAMIC GnRH, PITUITARY AND SERUM LH AND FSH IN THE ATHYMIC MOUSE
Hormone
Control {nu/+]
A thymic (nu/nu)
Hypothalamic GnRH (ng) Pituitary FSH (ug) Pituitary LH (tzg) Serum FSH (gg/ml) Serum LH (#g/ml)
2.3 4.6 20.1 0.53 0.024
3.0 4.8 10.9 0.58 0.024
± 0.2 ± 0.5 (11.2) ± 3.8 (49.1) ± 0.04 ± 0.007
± 0.3 ± 0.5 (15.3) ± 2.3 (34.8) ± 0.06 ± 0.04
Serum level and the total amount of hormone(s) found in the hypothalamus and the pituitary are shown (mean ± S.E., n = 6-10). The pituitary protein content was 0.409 ± 37 mg and 0.313 +- 25 for control and athymic mice respectively. The hypophysial hormone concentration (/~g hormone/mg protein) are given in parentheses. The control mice were bled in the morning of the dioestrus.
66 was 46% lower per gland (0.05 < P "< 0.1) and 30% lower after correction for pituitary protein content. The serum LH and FSH levels were similar in nude and control groups. Three-month-old control athymic mice and athymic equipped with thymus graft were ovariectomized. The serum LH levels were measured 3 and 6 days after the operation (Table III). Removal of the ovaries did not cause an increase in the serum LH levels in athymic mice, the normal control mice and athymic mice implanted with thymus at birth showed the expected rise in serum LH levels. On the seventh day after ovariectomy, GnRH (50 ng) was injected intraperitoneally into all mice and serum LH levels were determined 30 min later. The ovariectomized athymic mice responded to exogenous GnRH with elevated serum levels of LH (6 fold above basal level). TABLE III SERUM LH LEVELS IN OVARIECTOM1ZED AND GnRH TREATED ATHYMIC MICE Mice
Untreated LH (ng/ml~
Ovariectomized LH (ng/ml) day 3
day 6
24.0 ± 4.1
day 7 30 min after injection o f 50 ng GnRH
Athymic (nu/nu)
24.0 ± 4.5
19.0 ± 2.4
145.0 ± 54.0
Athymic implanted with thymus at birth
20.0 ÷ 3.0
N.T.
250.1 ± 42.3
N.T.
Control (nu/+)
24.0 ± 7.1
89.1 ± 26.0
225.0 ± 25.0
476.0 ± 80.0
Six to eight 4-month-old mice were used in each group, serum LH levels (mean ± SEM) a_re shown. N.T. not tested.
DISCUSSION The congenitally athymic mouse is a useful model for the analysis o f the role o f the thymus in the formation of the endocrine system. Lintern-Moore and Pantelouris [6] demonstrated the presence of specific structural and functional abnormalities in prepuberal and adult ovaries from athymic mice. Normal structure could be restored by injection of gonadotrophin. We found that injection of gonadotrophins to adult athymic mice caused ovaluation. These results led to the concept that the athymic mice ovaries are normal and their malfunction resulted from insufficient gonadotrophins secretion from the pituitary. However, direct measurement of the gonadotrophin content of the pituitary or of serum gonadotrophin concentration were not performed adequately. The measurement o f GnRH, LH and FSH in athymic and control mice revealed that hypothalamic GnRH levels of athymic mouse were slightly significantly higher than those o f
67 normal control, and that the contents of pituitary FSH and serum FSH and LH concentration in the peripheral blood were not significantly different in the two groups of mice. However, the gonadotrophin levels in the control animals were determined only during the leukocyclic phase of the cycle and not during the proestrous gonadotrophin surge. The pituitary content of LH was 46% lower per gland in the athymic mice. This may be a result of the acyclic state. Ovariectomy in normal adult mice and rats results in greatly increased secretion of LH and FSH [10, and present control data]. However, 6 days after ovariectomy, serum LH levels in the athymic mice were unchanged. Injection of exogenous GnRH stimulates LH secretion by the athymic mouse pituitary. It therefore appears that the lack of cyclic ovulatory gonadotrophin release, and the lack of feedback response to ovariectomy are due to defective hypothalamic control of LH secretion rather than an inhibition of the pituitary to respond to GnRH. It should be pointed out however, that we have no evidence that the ovaries of the athymic mice at time of ovariectomy secrete significant amounts of oestrogen; but if ovarian steroid secretion was inadequate plasma gonadotrophin levels should have been elevated, given normal hypothalamic function. The factors responsible for the hypothalamic defect remained to be identified. It was suggested [11, 17] that a perinatal androgenic influence might change the normal programming of the neural centers in the brain of the nude mice because the morphology of the atrophic follicles in the ovaries of the athymic mouse are similar to those of mice neonatally treated with testosterone propionate [12], which also blocks the cyclic release of gonadotrophins at maturity [13]. Injection of thymocytes protected neonate rats from androgenization caused by treatment with testosterone [14], and neonatal thymectomy prolonged the period in which the rat is susceptible to androgenization [15]. Recently we have found progesterone-metabolizing enzymes in the thymus of neonate mice [16]. Therefore the possibility exists that one of the ways by which the thymus may influence the endocrine system is through steroid metabolism in the neonatal period of life, when the brain is most sensitive to steroid effects.
ACKNOWLEDGEMENTS This work was supported by grants from the Israel Commission for Basic Research (to Y. W.), and the Ford Foundation and the Population Council, N.Y. (to H. R. Lindner). REFERENCES 1 J. Rygaard, Thymus and self. Immunobiology of the mouse mutant "nude". F.A.D.L., Copenhagen, 1973. 2 H. O. Besedovsky and E. Sorkin, Thymus involvement in sexual maturation, Nature, 249 (1974) 356-358. 3 S. Lintern-Moore and E. M. Pantelouris, Ovarian development in the athymic nude mice. I. The size and composition of the follicle population, Mech. Ageing Dev., 4 (1975) 385-390.
68 4 J. G. M. Shire and E. M. Pantelouris, Comparison of endocrine function in normal and genetically athymic mice, Cornp. Biochem. Physiol., 47A (1974) 93-100. 5 Y. Nishisuka and T. Sakakura, Thymus and reproduction; sex-linked dysgenesia of the gonad after neonatal thymectomy in mice, Science, 66 (1969) 753-755. 6 S. Lintern-Moore and E. M. Pantelouris, Ovarian development in athymic nude mice. III. The effect of PMSG and oestradiol upon the size and composition of the ovarian follicle population, Mech. AgeingDev., 5 (1976) 33-38. 7 T. A. Daane and A. F. Parlow, Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle: effect of pentobarbital, Endocrinology, 88 (1971) 653-663. 8 Y. Koch, M. Wilchek, M. Fridkin, P. Chobsieng, U. Zor and H. R. Lindner, Production and characterization of an antiserum to synthetic gonadotropbin-releasing hormone, Biochem. Biophys. Res. Commun., 55 (1973) 616-622. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 N. B. Schwartz and C. E. McCormack, Reproduction: gonadal function and its regulation, A. Rev. Physiol., 34 (1972) 425-472. 11 E. M. Pantelouris, Effects of athymia on development, in M. Balls and A. E. Wilds (eds.), The Early Development of Mammals, Second Symp. of the Brit. Soc. Developmental BioL, Cambridge Univ. Press, London, 1975, pp. 373-388. 12 H. Peters, I. N. Sorensen, A. G. Byskov, T. Pedersen and T. Krarup, The development of the mouse ovary after testosterone propionate on day 5, in W. R. Butt, A. C. Crooke and M. Ryle (eds.), Gonadotrophins and Ovarian Development, Churchill-Livingstone, Edinburgh, 1970, pp. 351-361. 13 R. A. Gorski, Mechanism of androgen induced masculine differentiation of the rat brain, in K. Lissak (ed.), Hormone and Brain Function, Plenum, New York, 1973, pp. 27-46. 14 F. A. Kind, A. Oriol, A. Folch Pi and M. Maqueo, Prevention of steroid induced sterility in neonatal rats with thymic cell suspension, Proc. Soc. Exp. Biol. Meal., 120 (1965) 252-255. 15 N. Zabuzkova and F. A. Kinci, The influence of thymectomy and steroid hormones in neonatal rats, Proc. Soc. Exp. Biol. Meal., 135 (1970) 874-877. 16 Y. Weinstein, H. R. Lindner and B. Eckstein, Thymus metabolises progesterone - possible enzymatic marker for T lymphocytes, Nature, 266 (1977) 632-633. 17 W. Pierpaoli and H. O. Bsedovsky, Role of the thymus in programming of neuroendocrine functions, Clin. Exp. Immunol., 20 (1975) 323-338.
