Zoological Institute, Faculty of Science, University of Tokyo, Tokyo

Transcription

1 EFFECTS OF GONADECTOMY AND SEX STEROIDS ON THE ACID PHOSPHATASE ACTIVITY OF THE HYPOTHALAMO- HYPOPHYSIAL SYSTEM IN THE BIRD, EMBERIZA RUSTICA LA TIFASCIA HARUKO UEMURA Zoological Institute, Faculty of Science, University of Tokyo, Tokyo Since the pioneer experiments of Rowan(1925 and 1926)and Bissonnette(1930 and 1931), there have been many investigations of photoperiodic stimulation on avain gonads. The extensive literature on the subject has been reviewed recently by Benoit and Assenmacher(1959), Benoit(1961 and 1962), Farner(1959 and 1962), Farner and Oksche(1962), Wolfson(1959 a, b and 1960)and Wolfson and Kobayashi(1962). Despite the many studies of photoperiodic stimulation of the avian gonad, the mechanisms involved in gonadal involution following its full development have not been sufficiently studied. Bailey(1950)has suggested that prolactin, which might be secreted following gonadotropin secretion, may be responsible, directly or indirectly, for the regression of the gonad, since the injection of prolactin inhibits light-induced gonadal development in Zonotrichia leucophrys pugetensis. Lofts and Marshall(1956)also have shown that prolactin causes testicular involution in Passer domesticus, Chloris chloris and Fringilla coelebs. On the other hand, Laws and Farner(1960)were unable to find an inhibitory action of prolactin on gonadal development of the White-crowned Sparrow subjected to prolonged daily photoperiods. Thus, it is still uncertain whether or not prolactin is involved in the natural regression of the avian gonads at the end of the breeding season. Apart from the prolactin theory, a gonadal negative feedback mechanism, which has been fully investigated in laboratory mammals(see Harris, 1955), should be considered in relation to gonad involution in birds. Several attempts to obtain experimental evidence for a feedback mechanism have been relatively unsuccessful in birds thus far(burger, 1949;Farner and Wilson, 1959; Wolfson and Harris, 1959). In these attempts, administration of androgen did not completely prevent the gonadal development induced by prolonged daily photoperiods. However, Kobayashi(1954)described inhibitory effects of testosterone and estradiol on Received for publication May 28, 1964.

2 light-induced gonadal development of Zosterops palpebrosa japonica. In his experiments the sex steroids were administered just prior to the light treatment. Thus, there has not been agreement concerning the effects of sex steroids on avian gonads. Recently Lofts(1962)has clearly demonstrated that the apparent contradictions among the investigators of this phenomenon is due to the differences in the developmental stages of the testis at the time when androgen is administered; androgen stimulates the testis during the regenerating phase, while it has a depressive effect on the testis during the early stages of the recrudescence. It is assumed that the stimulating effect of androgen on the testis at its early regenerating phase may be a direct effect; several investigators have demonstrated that androgen is able to restore spermatogenesis of hypophysectomized rats(see Boccabella, 1963). Considering this direct effect, we cannot exclude a possibility that androgen inhibits, directly or indirectly, gonadotropic function of the adenohypophysis; that is, it forms part of a negative feedback mechanism. If there is a negative gonadal feedback mechanism in birds, it is likely that it is involved in natural gonadal involution of birds at the end of the breeding season. In the present experiments an attempt was made to learn whether or not a negative gonadal feedback mechanism operates during the breeding cycle of birds. If so, how does such a gonadal feedback mechanism operate in those birds whose breeding season lasts a month or more?castration and implantation of pellets of sex steroids were done on migratory temperate-zone birds, Emberiza rustica latifascia, subjected to prolonged daily photoperiods; the functional activities of their adenohypophysial and the hypothalamic tissues were assessed by measuring their weight and/or acid phosphatase content. The prolactin theory was not critically tested in this study. In addition to the questions mentioned above, attention was also paid to the effects of castration and the sex steroids on body weight change due to vernal premigratory fat deposition(king and Farner, 1959; Wolfson, 1959 c; King, 1961 a and b). MATERIALS AND METHODS Specimens of the migratory temperate-zone species, Emberiza rustica latifascia Portenko were caught at Asama, Nagano Prefecture, and transferred to the laboratory in Tokyo during October and November, They were kept under controlled 8-hour daily photoperiods(short days) for about 5 months in indoor cages until the beginning of the experiments. The birds were fed a commercial mixture of dry grains consisting of millet, canary seed, rape seed, and radish seed (2:6:1:1). In the first experiment(experiment 1), intact(long-day control, 20, Š17)and gonadectomized birds( 31, Š4)were subjected to 20-hour daily photoperiods(long days). Those birds( 4, Š2)retained under 8-hour daily photoperios served as short-day controls. The castration was done 3 to 15 days before the short- to long-day change was made. At particular

3 intervals following the exposure to long days, groups consisting of 5 to 7 birds were killed and the weights of the pars distalis of the adenohypophysis, gonad and body were recorded. Acid phosphatase activity was also measured in the median eminence region, the supraoptic region, the small tissue of ventral portion of the lateral forebrain bundle, and the pars distalis of the adenohypophysis of the individual sacrificed birds(fig.1). All the birds were killed during the period Fig.1. Schematic diagram of sagittal section of the hypothalamo-hypophysial system of Emberiza, showing the samples of the median eminence and supraoptic regions used in the estimation of acid phosphatase activity. The sample is enclosed by the heavy straight black lines. III, third ventricle; ca, caudal lobe of pars distalis; ce, cephalic lobe of pars distalis; ME, median eminence region containing the deposit of neurosecretory material; OC, optic chiasma; OR, optic recess; PD, pars distalis of adenohypophysis; PN, pars nervosa; PT, pars tuberalis; PVN, paraventricular nucleus; SOR, supraoptic region including the neurosecretory cells; TSH, supraoptico-hypophysial tract. March through April, It is known already that the acid phosphatase activity in these tissues changes with gonadal development in Zonotrichia leucophrys gambelii (Kobayashi and Farner, 1960)and Zonotrichia albicollis(kobayashi et al., 1960; Wolfson and Kobayashi, 1962.) In the second experiment(experiment 2),intact birds(long-day control, )and birds implanted intraperitoneally with pellets of testosterone( 19, Š5)or estradiol( 16, Š6)were subjected to 20-hour daily photoperiods. The ranges of weights of the pellets of testosterone and estradiol were 2 to 4.5 mg and 1.1 to 3.0mg,respectively. There was no short-day control group in Experiment 2. The implantation of the pellets has been done 1 day before the birds were subjected to long days, except that the birds killed on 0 day of the experiment had received the pellets 1 week before the sacrifice. The weights of the pars distalis of the adeno-

4 hypophysis, gonad and body were measured for the birds killed at stated intervals following the beginning of exposure to long days. Acid phosphatase activity was measured in the same way as in Experiment 1. These birds were killed during May and June, The pellets were recovered from all the implanted birds at the time of sacrifice. In both experiments, the difference between the body weights at the beginning of the experiment and at the time of death was calculated for individual birds, except for the birds kept on 8-hour daily photoperiods(short-day control)in Experiment 1. Particular hypothalamic regions(median eminence region,supraoptic region)and the ventral portion of the lateral forebrain bundle were removed for phosphatase assay according to the method of Kobayashi and Farner(1960). The masses of tissue thus removed weighed 0.4 to 0.6mg. The samples from the median eminence region contained small amount of pars tuberalis. The phosphatase activity of the tiny tissue of pars tuberalis was considered to be negligible, as in the case of the White-crowned Sparrow(Kobayashi and Farner, 1960). Phosphatase activity was estimated by a procedure essentially the same as their method. The tissues were homogenized in 0.5ml of chilled deionized water.the reaction mixture consisted of 0.3ml of 0.2M acetate-buffer solution(ph 5.2,see below), 0.3ml of 1% sodium para-nitrophenylphosphate and 0.4ml of the homogenate.the enzymatic action was initiated by addition of the substrate to the homogenate-buffer mixture and it was terminated by the addition of 0.2ml of 20% trichloracetic acid after 50mins'.incubation. After centrifugation a 1ml aliquot was drawn from the deproteinated supernatant and added to 2.0ml of saturated sodium carbonate. The quantity of para-nitrophenol in the aliquot was measured from the optical density at 415mƒÊ using a Hitachi Spectrophotometer.Phosphatase activity is expressed as ug of para-nitrophenol liberated in 1 hr. at 37.2 Ž.All the determinations were controlled with blanks containing all constituents of the reaction mixture except the tissue homogenate. Under the conditions mentioned above, the optimal ph for acid phosphatase in each region was determined between ph 4.5 to 6.0(at 0.2 intervals)using 0.25 to 0.4mg tissue of each region. The optimum for the enzymatic activity in all tissues was about ph 5.2.At ph 5.2 enzymatic activity was a linear function of the enzyme concentrations 0.2,0.4 and 0.8 mg used for each tissue. In both experiments the incubation time was 50mins. There was also a linear relation between the amount of para-nitrophenol released and the time of incubation of the reaction mixture for periods of 20,35 and 50mins. RESULTS Experiment 1 Effect of gonadectomy on the pars distalis weight of birds subjected to long days As shown in Table1, a significant increase in the weight of pars distalis of adenohypophysis as observed on day 15(compared to initial control value)and on day 22(compared to final value of short-day control)in the long-day control group, and thereafter the weight decreased to, and was maintained at, the initial level.in the gonadectomized group, the weight was increased on day 15 and this increased weight was maintained until the end of the experiment(48 days of long-day treatment).the small pieces of the testicular regenerates, which were observed in 4 of 6 birds killed on day 36(Table 8), did not suppress the weight increase of the pars distalis(table 1). In the birds maintained on 8-hour photoperiods(short-day control), the pars distails weight did not show any increase at

5 AVIAN GONADO-HYPOPHYSIAL FEEDBACK Table 1. Effect of gonadectomy on the weight of the pars distalis of birds sublected to long days the end of the experiment. Effect of gonadectomy on the acid phosphatase activity of the pars distalis of birds subjected to long days The acid phosphatase activity of the pars distalis of the adenohypophysis expressed on the basis of activity per whole gland increased significantly on day 15 and day 48 in the long-day control group(table 2). In the gonadectomized group, enzymatic activity was significantly elevated by the treatment with long Table 2. Effect of gonadectomy on acid phosphatase of the pars distalis, expressed as activity per entire gland in birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per gland liberated in 1 hr.

6 days. The increase was first observed on day 15 and the increased activity continued to the end of the experiment. The activity of the pars distalis of shortday control birds at the end of the experiment did not differ from the initial activity(0 day value on the long-day control). When the acid phosphatase activity was expressed in terms of activity of per mg tissue, only one case of increase was observed on day 48 in the long-day control group (Table 3). However, this increase was not significant when compared to the final activity of the short-day control group. In the other groups, there was no significant change during the experimental period. Table 3. Effect of gonadectomy on acid phosphatase of the pars distalis, expressed as activity per mg tissue in birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1 hr. Effect of gonadectomy on the acid phosphatase activity of the hypothalamic regions and ventral portion of the lateral forebrain bundle of birds subjected to long days Significant increases of the acid phosphatase activity of the median eminence were observed on day 15 and day 48 in the gonadectomized group (Table 4). In the other groups, the enzymatic activity of the median eminence did not show any significant change during the experimental period. An increased enzymatic activity of the supraoptic region was observed only on day 48 in the gonadectomized group (Table 5). In the other groups no significant change was observed in the acid phosphatase activity of the supraoptic region. The acid phosphatase activity of the ventral portion of the lateral forebrain bundle did not show any change either in the experimental or control groups (Table 6).

7 AVIAN GONADO-HYPOPHYSIAL FEEDBACK Table 4. Effect of gonadectomy on acid phosphatase activity of the median eminence region of birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1hr. Table 5. Effect of gonadectomy on acid phosphatase activity of the supraoptic region of birds subjected to long days Phosphatase activity in expressed is Đg p-nitrophenol per mg tissue liberated in 1hr. Table 6. Effect of gonadectomy on acid phosphatase activity of the ventral portion of the lateral forebrain bundle of birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1hr.

8 Weight of the gonad As shown in Table 7, the gonads of long-day control birds increased in weight, while those of short-day control birds showed no significant change at the end of the experiment. Effect of gonadectomy on the body weight of birds subjected to long days In the long-day control group, the body weight increased significantly 27, 36 and 48 days after the exposure to long days in both males and females (Table 8). In gonadectomized birds, no significant change in body weight was observed following the same long-day treatment, excepting the birds which had small pieces of the regenerated testicular tissue. These exceptional birds were found among the animals killed on day 36, and they showed increases in their body weights. In the short-day control group, the average body weight of 6 birds was }0.46g and }0.59g, at the beginning and at the end of the experiment, respectively. It seems likely that during the experimental period no change in body weight took place in the short-day control group. Experiment 2 Effects of testosterone and estradiol on the pars distalis weight of birds subjected to long days The pars distalis weight showed a tendency toward an increase on day 8 and day 16 in the long-day control, whereas there was no such a tendency observed in the birds implanted with the pellets of testosterone or estradiol (Table 9). Effects of testosterone and estradiol on the acid phosphatase activity of the pars distalis of birds subjected to long days The enzymatic activity of the pars distalis was higher in the birds of Experiment 2 than in the birds of Experiment 1. In the long-day control, there were significant increases in acid phosphatase activity expressed per whole gland on day 8 and day 16 (Table 10). In the other groups, no such increase in enzymatic activity was observed. The enzymatic activity on day 0 of the birds in the estradiol group was higher than that of control animals. This is due to the fact that one bird had an exceptionally large pars distalis. The activity measured on day 8 was lower in the estradiol group than in the long-day control. In the testosterone group the enzymatic activities on day 8 and day 16, respectively, were lower than those of the long-day control. On the basis of enzymatic activity per mg tissue, significant increases were observed on day 8 and day 16 in the long-day control (Table 11). In the est-

9 AVIAN GONADO-HYPOPHYSIAL FEEDBACK Table 7. Growth of gonads of birds subjected to long days Table 8. Effect of gonadectomy on the body weight of birds subjected to long days Table 9. Effects of estradiol and testosterone on the pars distalis weight of birds subjected to long days

10 UEMURA Table 10. Effects of estradiol and testosterone on acid phosphatase of the pars distalis, expressed as activity per entire gland in birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per gland liberated in 1hr. Table 11. Effects of estradiol and testosterone on acid phosphatase of the pars distalis, expressed as activity per mg tissue in birds subjected to long days Phosphatase activity is expressed in pg p-nitrophenol per mg tissue liberated in 1hr. radiol group significant decreases were observed on day 8 and day 16.There was no change in enzymatic activity after the long-day treatment in the testosterone group. However, the birds of the estradiol and testosterone groups had lower

11 AVIAN GONADO-HYPOPHYSIAL FEEDBACK activities on day 8 and day 16 than the birds of the long-day control measured on the same days Effects of testosterone and estradiol on the acid phosphatase activity of the hypothalamic regions of birds subjected to long days The enzymatic activities on the hypothalamic regions were also higher in the birds of Experiment 2 than in the birds of Experiment 1. The acid phosphatase activity of the median eminence was increased on day 16 in the long-day control (Table 12). However, in the other groups the phosphatase activities of the median eminences did not differ from the respective initial activities. The enzymatic activity of the median eminence on day 16 was significantly lower in the testosterone group than in the long-day control. In the supraoptic region (Table 13) and the ventral portion of the lateral forebrain bundle (Table 14), the acid phosphatase activities were not significantly different from the respective initial activities in all the groups, excepting the ventral portion of the lateral forebrain bundle of day 16 in the long-day control group. However, the phosphatase activities of the supraoptic region and the ventral portion of the lateral forebrain bundle, measured on day 16, were lower in both estradiol and testosterone groups than in the long-day control. Effects of testosterone and estradiol on the weight of gonads of birds subjected to long days As shown in Table 15, the gonads of the birds implanted with testosterone or estradiol pellets did not develop, whereas those of the birds of the long-day control group showed a marked development. Effects of testosterone and estradiol on the body weight of birds subjected to long days The body weights of control birds killed on day 8 were significantly decreased, but the weights of other birds of this group, killed thereafter, did not differ significantly from the initial values (Table 16). In the estradiol group, the body weight was significantly decreased by implantation of estradiol already at the beginning of the experiment, and decreases were also observed on day 16 and 24 after the subjection to long days. In the testosterone group, the birds showed a significant decrease in their body weights on 0 day of the experiment. As mentioned before, the steroid pellets were implanted one day before the exposure to long days, excepting the birds killed on 0 day of the experiment, in which the pellets were implanted one week before the sacrifice.

12 Table 12. Effects of estradiol and testosterone on acid phosphatase activity of the median eminence region of birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1hr. Table 13. Effects of estradiol and testosterone on acid phosphatase activity of the supraoptic region of birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1hr. Table 14. Effects of estradiol and testosterone on acid phosphatase activity of the ventral portion of the lateral forebrain bundle of birds subjected to long days Phosphatase activity is expressed in Đg p-nitrophenol per mg tissue liberated in 1hr.

13 AVIAN GONADO-HYPOPHYSIAL FEEDBACK Table 15. Effects of estradiol and testosterone on gonadal growth of birds subjected to long days Table 16. Effects of estradiol and testosterone on the body weight of birds subjected to long days

14 DISCUSSION In the long-day control group, the weight of pars distalis was significantly increased on day 15 and day 22 in Experiment 1 and also showed a tendency toward an increase on day 8 and day 16 in Experiment 2; thereafter the weight decreased to an initial weight level. These phenomena may be explained by assuming an inhibitory effect of gonadal hormones on adenohypophysial function. In other words, insufficient gonadal hormones are secreted into the circulation from lightstimulated gonads to prevent the weight increase of the pars distalis of adenohypophysis until about 15 to 22 days after the exposure to long days. However, thereafter the circulating gonadal hormones increase to an amount sufficient to reduce the weight of the pars distalis. This conclusion is supported by the observations that in gonadectomized birds the pars distalis weights increased significantly on day 15 and the increased level was maintained until the end of the experiment. The maintenace of increased pars distalis weight in gonadectomized birds appears to be due to the lack of inhibition by the gonadal hormones. Small pieces of testicular regenerates were unable to prevent the weight increase of the pars distalis. The findings, in sum, suggest that a negative gonadal feedback mechanism operates in these birds. Furthermore, the influence of a feedback mechanism is suggested by the results of administration of testosterone or estradiol. These hormones inhibited completely the increase of pars distalis weight and also the gonadal development of the birds subjected to long-days. The acid phosphatase activities of the tissues from the birds of Experiment I were always lower than those of Experiment 2. The reason is not known at the moment, but one of possible explanations is that the difference in the enzymatic activities may be due to the difference in the season when two experiments were done. In the first, the phosphatase activity was measured in March and April, and in the second it was done in May and June. The period between the end of Experiment 1 and the beginning of Experiment 2 was about 20 days. The basal metabolic rate in the regions measured which may reflect acid phosphatase activity might be higher in early summer than in late winter, even under the same day-light condition. The experimental variations in acid phosphatase activity of the pars distalis also suggest the existence of a negative gonadal feedback mechanism in Emberiza rustica latifascia. In Experiment 1, the acid phosphatase activity of the pars distalis of adenohypophysis expressed on the basis of activity per entire gland increased only on day 15 in the long-day control. In gonadectomized birds, the activity

15 AVIAN GONADO-HYPOPHYSIAL FEEDBACK also increased on day 15, and the increased level continued until the end of the experiment. In Experiment 2, there were increases in acid phosphatase activity of the pars distalis on day 8 and day 16, whereas no increase was observed in either the testosterone or the estradiol group. Furthermore, the enzymatic activities were lower on day 8 and day 16 in the testosterone and estradiol groups than in the long-day controls. The sex steroids apparently inhibited the lightinduced gonadal development. Thus, it appears probable that endogenous sex steroids do not inhibit the increase of acid phosphatase activity until about 16 days after the beginning of exposure to long days. After this, the titer of endogenous steroids seemingly increases and eventually it becomes effective in preventing the activity of the pars distalis. These data also may be interpreted in support of a gonadal feedback mechanism in birds. The increase of the phosphatase activity per entire gland observed on day 48 is not explicable at the moment. Although after day 15 the weight and acid phosphatase activity of the pars distalis were at the starting level in the long-day control (Experiment 1), the gonads were still showing development. This show that a lower level of gonadotropin continues to be secreted by the adenohypophysis even under the inhibitory effect of the gonadal hormones, and in turn, the gonads continue to develop gradually even after day 15. It is assumed that the methods for measuring pars distalis weight and acid phosphatase activity are not sensitive enough to reveal the increased (compared to initial activity) gonadotropic activity which occurred after day 15. Such an interaction between adenohypophysis and gonads may be one of the reasons for the long-lasting breeding season in birds. It appears likely that the endogenous sex steroids secreted from the developing gonads may finally cause the complete inhibition of the gonadotropic activity of the pars distalis of adenohypophysis, thus resulting in the natural regression of the gonad. The more complete inhibitory effect of the exogenous sex steroids on gonadal development in Experiment 2 supports this postulation. After the complete involution of the gonad, they do not respond any more to long days for a certain period (refractory period, see Kobayashi, 1957; Wolfson, 1959 c; Farner, 1959). This mechanism, which is probably different from a negative gonadal feedback, is not understood at the moment and further studies are needed to clarify it. It has already been demonstrated that the supraoptic and median eminence regions of Zonotrichia leucophrys gambelii and Zonotrichia albicollis showed increases in the acid phosphatase activity, as the testis developed following exposure to long days (Kobayashi and Farner, 1960; Kobayashi et al., 1960; Wolfson and Kobayashi, 1962). In Emberiza rustica latifascia used in the present experiment, the change in phosphatase activity of the median eminence was detected on day 15 and day 48

16 in gonadectomized birds, but not in the long-day control (Experiment 1). In Experiment 2, an increase in the enzymatic activity of the median eminence was observed in the long-day group, but not in the birds implanted with the steroids. These results suggest that the negative gonadal feedback mechanism is expressed at least through the median eminence. The participation of hypothalamus in avian reproduction has been -claimed by many investigators (see Wingstrand, 1951; Legait, 1959; Ralph, 1959; Ralph and Fraps, 1959a and b; Benoit and Assenmacher, 1959; Benoit, 1962; Farner and Oksche, 1962; Egge and Chiasson, 1963; Kobayashi, 1963). However, in the supraoptic region, we found no change after exfiposure to long days; furthermore, gonadectomy and sex steroids did not exert a definite effect on the phosphatase activity. Therefore, in the present experiments, it is not possible to decide whether or not the supraoptic region is involved in the negative gonadal feedback mechanism. In this species of bird, however, a more sensitive method may perhaps reveal an activity change in this region following exposure to long days. With respect to the degree of change of acid phosphatase activity in the hypothalamic regions and adenohypophysis following long-day treatment, a species difference already has been found between Zonotrichia leucophrys gambelii (Kobayashi and Farner, 1960) and Zonotrichia albicollis (Kobayashi et al., 1960; Wolfson and Kobayashi, 1962). In the ventral portion of the lateral forebrain bundle, enzymatic activity increased on day 16 in the long-day control group in Experiment 2 but not in Experiment 1. This region of the brain may not be involved in gonadal development since Kobayashi et al. (1962) did not observe any change in proteinase activity here, although it was demonstrable in the median eminence, at a time when the gonad showed development in the White-crowned Sparrow. In several migratory temperate-zone avian species, it is well established that the increase of body weight due to fat deposition is induced by subjecting the birds to long days, and that this is brought by hyperphagia (King and Farner, 1959; King, 1961a and b). In Experiment 1, the birds of the long-day control, both males and females, significantly gained body weight, whereas the gonadectomized birds did not. This shows clearly that the gonad is necessary for fat deposition following long days. This conclusion is supported by the observation of gain in body weight in gonadectomized birds bearing regenerates of testicular tissue. According to Morton and Mewaldt (1962), the castrated male Zonotrichia atricapilla deposits fat but not as much as intact birds during the premigratory period. They have suggested that the mechanism controlling photoperiod-induced fat deposition in male birds in the spring has at least two components. One factor is dependent upon an increased production of testosterone, and the other is not.

17 AVIAN GONADO-HYPOPHYSIAL FEEDBACK the castrated male birds experienced no weight increase. Therefore, in Emberiza rustica latifascia, light-induced fat deposition depends entirely on gonadal hormones, directly or indirectly, unlike Zonotrichia atricapilla. As mentioned before, King (1961a and b) has demonstrated that premigratory fat deposition is dependent upon a photoperiodically induced hyperphagia. On the other hand, in mammals it is known that there is a feeding center as well as a regulatory center in the hypothalamus. Bilateral lesions in the lateral hypothalamic area cause aphagia and those in the ventromedial hypothalamic nucleus induce hyperphagia. Electrical stimulation of the former induces hyperphagia, and stimulation of the latter suppresses it (see Oomura et al., 1964). Therefore, it appears that gonadal hormones, either androgen or estrogen, have stimulatory or inhibitory effect on either the hypothalamic feeding center or the regulatory center, thus inducing marked fat deposition through hyperphagia. However, in the second experiment, the implantation of sex steroids did not elicit any fat deposition. Unfortunately, these birds were killed within 25 days after the exposure to long days. During that period, even in normal long-day birds, no increase in body weights occurs (Experiment 1). It is assumed, then, that they were killed before they might have shown increase in body weight. An alternative explanation for the failure of fat deposition in the birds of the estrogen and testosterone groups is that the doses of the sex steroids ( mg pellets) were toxic and unphysiological. Wolfson and Harris (1959) have also shown that 75 mg pellets of testosterone inhibit gonadal regression of birds subjected to 8-hour photoperiods after treatment with 16-hour photoperiods for 1 month, but they could not prevent the loss of fat deposits. This amount of testosterone must have been too great and toxic, since most Emberiza rustica latifascia implanted with testosterone pellets weighing about 70 mg died, even though the testes were stimulated by the hormone (Uemura, unpublished data). The body weight decreases observed on 0 day of the experiment in the testosterone and estradiol groups may have been caused by trauma from the actual pellet implantation, which was done 1 week prior to the sacrifice. SUMMARY 1. The pars distalis weight of the adenohypophysis of intact birds, Emberiza rustica latifascia, increased significantly after 15 days of exposure to long days; thereafter the weight decreased and returned to an initial level. In gonadectomized birds the weight of the pars distalis increased significantly after 15 days of exposure to long days, and this increase was maintained until the end of the experi-

18 UEMURA ment (48 days after beginning of exposure to long days). 2. The acid phosphatase activity of the pars distalis of intact birds increased after 15 days of exposure to long days; thereafter it decreased to an initial level. The elevated phosphatase activity of the pars distalis of gonadectomized birds observed after 15 days of exposure to long days was maintained until the end of the experiment (48 days after beginning of exposure to long days). 3. Birds implanted with pellets of testosterone or estradiol exibited no increase in the weight and acid phosphatase activity of the pars distalis after the exposure to long days. 4. Testosterone and estradiol inhibited gonadal development in birds subjected to long days. 5. The acid phosphatase activity of the median eminence increased in intact birds (Experiment 2) and gonadectomized birds subjected to long days, but not in birds implanted with the sex steroid pellets. 6. From these findings it is concluded that a negative gonadal feedback mechanism operates at least through the median eminence, even in the birds which have a long-lasting breeding season. A possibility is discussed that the negative gonadal feedback mechanism is involved in the natural gonadal involution at the end of the breeding season of the bird. 7. Intact birds, whether female Or male, experienced an increase of body weight after 27 days of exposure to long days and the increase was maintained until the end of the experiment (48 days after beginning of exposure to long days). Gonadectomized birds did not show such an increase during the experimental period. ACKNOWLEDGEMENT The author wishes to acknowledge her indebtedness to Dr. H. Kobayashi for his valuable guidance in the course of this study and also to Dr. A. Gorbman for reading the manuscript. She also acknowledges helpful suggestions of Dr. S. Ishii, and the invaluable assistance of Mr. T. Matsui. The investigation was partly supported by a grant from Ministry of Education of Japan and partly by United States Public Health Research Grant, No. A-3678, to Dr. H. Kobayashi from the National Institute of Arthritis and Metabolic Diseases.

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