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Gonadotropin Content in the Cephalic and the Caudal Lobe of the Anterior Pituitary in Laying, Non-Laying and Broody Hen
739
GLYCARBYLAMIDE AND REPRODUCTION
ACKNOWLEDGMENT
The radioactive studies using glycarbylamide-C" were a collaborated effort of the senior author with Dr. C. Rosenblum and Mr. H. Meriwether of Merck Sharp &
Dohme Research Laboratories. Appreciation is expressed for the statistical advice of Mr. A. G. Itkin, Merck Sharp & Dohme Research Laboratories, and to Miss M. Loukides for technical assistance. REFERENCES Cuckler, A. C , L. R. Chapin, C. M. Malanga, E. F. Rogers, H. J. Becker, R. L. Clark, W. J. Leanza, A. A. Pessolano, T. Y. Shen and L. H. Sarett, 1958. Antiparasitic drugs. II. Anticoccidial activity of 4,5-imidazoledicarboxamide and related compounds. Proc. Soc. Exp. Biol. Med. 98: 167-170. Kosin, I. L., 1944. Macro- and microscopic methods of detecting fertility in unincubated hen's eggs. Poultry Sci. 23: 266-269. Snedecor, G. W., 1946. Statistical Methods, 4th Edition. Iowa State College Press, Ames, Iowa. Warren, D. C , and R. M. Conrad, 1939. Growth of the hen's ovum. J. Agr. Res. 58: 875-893.
Gonadotropin Content in the Cephalic and the Caudal Lobe of the Anterior Pituitary in Laying, Non-Laying and Broody Hen SEIICHI NAKAJO AND KIYOSHI IMAI Laboratory
of Animal Breeding, Faculty of Agriculture, Nagoya University, Anzyo,
Japan
(Received for publication August 2, 1960)
"PHILLIPS (1942) used a change in the principle elicited the increase of testes •*• histology of rat vaginal epithelium as weight of newly hatched White Leghorn a measure of gonadotropic activity and es- cockerels. In the previous study, we demontimated gonadotropic potency of the an- strated (Nakajo and Imai, 1956) that the terior pituitaries of fowls in relation to age testes weight response of one-day-old chicks and egg laying. Riley and Fraps (1942), was adequate to use as quantitative assay employing an increase of uterine weight in for fowl pituitary gonadotropins. immature mouse, assayed gonadotropic acThe anterior pituitary of fowl could be tivity of avian pituitary in various repro- classified into two cytologically different ductive stages, and found that the anterior portions which were designated as cephalic pituitaries from hens with ovarian follicles "lobe" and caudal "lobe" (Rahn and in regressive or quiescent phases showed a Painter, 1941). Yasuda (1953) observed higher gonadotropic effect than those from that beginning and advance of broody period hens in full reproductive condition. in female fowl were accompanied by cytoBreneman (1945) reported that a rela- logical changes respectively in these two tively small amount of anterior pituitary lobes. Moreover, Nakajo and Tanaka
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 29, 2015
again at initial levels. This effect appears to be correlated with the amount of glycarbylamide found in albumen. Furthermore, the embryo mortality during incubation occurs in increasing amounts at earlier stages. Roosters fed glycarbylamide at levels which cause the decrease in fertile eggs maintain a high fertilizing capacity. Hens fed high levels of the drug lay a high percentage of eggs which are apparently infertile even though artificially inseminated with semen from non-medicated roosters.
740
S. NAKAJO AND K. IMAI TABLE 1.—Fresh weight of the anterior piluilaries from hens in various reproductive stages Group of hen
Breed
Reproductive stage
No. of birds
Anterior pituitary (mg.)
Cephalic lobe (mg.)
NA. NA. NA. NA.
Non-laying Laying Early broody Late broody
17 26 14 19
6.9+0.57* 7.8 + 0.36 8.1+0.66 8.9 + 0.71
4.4 + 4.4 + 5.5 + 5.9 +
0.36* 0.26 0.57 0.32
W.L. W.L.
Non-laying Laying
21 17
8.4 + 0.68 9.0±0.59
5.3 + 0.48 5.4±0.50
Caudal lobe (mg.) 2.4 + 2.9 + 2.2 + 2.9 +
0.27* 0.27 0.28 0.34
2.8 + 0.32 3.1+0.39
(1956) reported that expression and cessation of broodiness were associated with the changes in prolactin content in the anterior pituitary and these changes occurred mainly in the caudal lobe. The purpose of this study was to determine the relationship between the gonadotropin content in each lobe of the anterior pituitary and the reproductive stage of the hen, employing the chick assay method. MATERIALS AND METHODS
Anterior pituitaries were obtained from hens in the following six groups; a) nonlaying hens of Nagoya breed (NA.), 12 to 24 month-old, b) laying hens of NA., 12 to 18 month-old, c) early stage in broody period of NA., 12 to 16 month-old, d) late stage in broody period of NA., 12 to 16 month-old, e) non-laying hens of White Leghorn (W.L.), 12 to 24 month-old, f) laying hens of W.L., 12 to 18 month-old. The non-laying hens produced no eggs for several weeks and their ovaries were in late regressive or quiescent phase at the time of autopsy. Average ovarian weights in these groups from N.A. and W.L. were 4.0 gm. and 3.8 gm. respectively. The egg production of the laying hens was confirmed in individual cage. Their ovaries were in full active phase with normal sequence of developing follicles. The early broody hens were sacrificed on 5th day in nesting and
their ovaries were in quiescent phase (average ovarian weight was 3.8 gm.). The late broody hens were killed on 15th day in nesting and average ovarian weight in this group was 2.9 gm. The anterior pituitaries of all birds were separated into the two lobes under the dissecting microscope. After storing each lobe in acetone for 60 hours, they were kept in a desiccator at room temperature for a week. Then, each dried lobe was ground with an agate-mortar and stored in the desiccator until ready for use. One mg. of the dried lobe powder from each group was suspended in 1.0 ml. of distilled water immediately before the first injection. Data on the fresh weights of the anterior pituitaries from the hens are summarized in Table 1. The fact that in most cases the sum of cephalic and caudal lobes is less than the anterior pituitary weight is due to the weighing procedure in which the anterior pituitary was weighed and then separated into the two lobes. The chick assay was performed according to the method described previously (Nakajo and Imai, 1956). One-day-old male chicks of W.L. were injected subcutaneously with the suspended preparations into the dorsal neck region at 24, 36, 48, 60 and 72 hours after hatching. They were killed 24 hours after the final injection and the testes were excised and weighed.
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Abbreviations: NA.—Nagoya. W.L.—White Leghorn. Note: *Mean + Fiducial limit (Degree of confidence = 0.95).
741
GONADOTROPIN AND PRODUCTION
The injected chicks and the distilled water injected controls received no food and water during the test period. The response of chick testes weight was expressed as an increase-rate which was calculated by the following formula; exp. wt. — control wt. control wt.
- X 100 = increase-rate (as percent)
The estimation of the gonadotropin content of the pituitary was made in terms of "chick unit." When the increase rate showed 30%, the difference of the testes weights between the injected chicks and the controls was statistically significant if 5 or more birds were used in the assay. Therefore, considering a slight additional allowance for error, 35% increase was adopted to constitute a "chick unit" (C.U.). RESULTS
Gonadotropic potency per mg. of the dried powders of each lobe from the hens of the six groups is shown in Table 2. Since one control group was provided for each treated group, the determination of the "chick unit" was made as an increment over the control for each experiment. In the group of NA., the gonadotropic potency per mg. of the powder ranged from
TABLE 2.—Gonadotropic potency per mg. of acetone-dried powder of the cephalic and the caudal lobe from hens in various reproductive stages Assay in chicks
Breed
Reproductive stage
(No.)
(mg.)
(No.)
(mg.)
(%)
Gonadotropic potency/ mg. (chick unit)
NA.
Non-laying
Cephalic Caudal
(11) ( 7)
7.8 + 0.62* 9.9±1.09
( 8) (8)
4.0+0.28* 4.7±0.29
95.0 109.8
2.7 3.1
NA.
Laying
Cephalic Caudal
( 8) ( 5)
6.6±1.14 7.5+1.25
( 7) (9)
3.8+0.30 3.8+0.28
73.7 98.2
2.1 2.8
NA.
Early broody
Cephalic Caudal
( 9) (5)
8.0+0.42 10.8±1.16
( 8) ( 8)
5.7 + 0.48 5.7+0.48
40.0 89.6
1.1 2.5
NA.
Late broody
Cephalic Caudal
(12) ( 6)
5.6+0.44 5.7 + 0.92
(10) (10)
4.5±0.61 4.5±0.61
24.4 26.7
0.7 0.8
W.L.
Non-laying
Cephalic Caudal
( 7) ( 5)
7.7±0.81 7.5 ± 1 . 2 0
( 7) ( 7)
3.9+0.32 3.9+0.32
98.5 92.3
2.8 2.6
W.L.
Laying
Cephalic Caudal
(13) ( 7)
7.1 + 0.43 7.5 + 0.79
(10) (10)
4 . 5 + 0.32 4 . 5 + 0.32
58.2 66.7
1.7 1.9
Group of donors Testes weight of chick Treated
Control
Lobe
Notes: *Mean ± Fiducial limit (Degree of confidence =0.95). (No.)—No. of assay chicks.
Increaserate
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0.7 C.U. of late broody hen to 2.7 C.U. of non-layer in the cephalic lobe and from 0.8 C.U. of late broody hen to 3.1 C.U. of nonlayer in the caudal lobe. The cephalic lobe powder from the non-layer was more active than that from the layer, and the cephalic lobe from the broody hen had a considerably lower gonadotropic activity than that from the layer. The lowest activity was found in the cephalic lobe powder from the late broody hen. The relation of the gonadotropic potency of the cephalic lobe to the reproductive stage (non-layer > layer > early broody > late broody) was applicable for the caudal lobe. In W.L., the potencies per mg. of the cephalic and the caudal lobe powders from the non-layer were higher than those from the layer. These findings in W.L. were similar to those observed in NA. described above. The potency per mg. of the caudal lobe was higher than that of the cephalic lobe in NA., while the potencies of the two lobes in W.L. were approximately equal. The powders of each lobe from NA. were slightly more active than those from W.L. in the same reproductive stage, but these differences were not significant. On the basis of the weight of the powder
742
S. NAKAJO AND K. IMAI TABLE 3.—Gonadotropin content of the cephalic lobe, the caudal lobe and the entire gland of the anterior pituitary from hens in various reproductive stt Gonadotropin content (chick unit)
Group of hen
Per anterior pituitary
Reproductive stage
NA.
Non-laying
Cephalic Caudal
2.1 1.1
3.2
NA.
Laying
Cephalic Caudal
1.5 1.0
2.6
NA.
Early broody
Cephalic Caudal
0.9 0.9
1.9
NA.
Late broody
Cephalic Caudal
0.7 0.3
1.0
W.L.
Non-laying
Cephalic Caudal
2.5 1.1
3.5
W.L.
Laying
Cephalic Caudal
1.5 1.0
2.5
Lobe
obtained and the potency per mg. of each lobe powder, approximate values of the gonadotropin content per lobe and per entire gland of the anterior pituitary were calculated. These results are shown in Table 3. No significant differences of the content of each lobe and the entire gland were observed between NA. and W.L. in the same stage. The gonadotropin content per anterior pituitary ranged from 1.0 to 3.2 C.U. in NA. hens. The pituitary from the nonlayer was definitely more abundant in the hormone than that from the other groups, and the hormone content of the gland from the late broody hen was the least. In the light of the gonadotropin content of each lobe, the cephalic lobe had more content than the caudal lobe with the exception of the early broody hen, in which the hormone contents of the two lobes were almost equal. As it can be clearly seen in Tables 2 and 3, there were marked differences of the total gonadotropin contents of the anterior pituitaries among the groups of non-laying, laying, early broody and late broody hen. Moreover, these changes were closely as-
Per lobe
sociated with those in the content of the cephalic lobe, whereas the contents of the caudal lobe were not greatly affected by the changes in these reproductive stages with the exception of the late stage in broody period. In the late broody hen, remarkable reduction of the gonadotropin content was found in both the cephalic and the caudal lobe. DISCUSSION
The anterior pituitary from the nonlayer showed the highest gonadotropic potency among the three reproductive stages, laying, non-laying and broody. Moreover, Bailey and Phillips (1952) and our recent studies (Imai and Nakajo, 1958, 1959) indicated that the gonadotropic activity of blood serum from the non-layer was also higher than' that from the layer. Under these hormonal conditions, the fact that their ovaries were in late regressive or quiescent phase (lack of follicular growth) might result either from (a) a lowering of sensitivity of their ovary to circulating gonadotropins, or (b) a failure in process of production, mobilization and/or deposition of the yolk materials, or (c) an
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Breed
743
GONADOTROPIN AND PRODUCTION
time, i.e. the content ranged from 0.6 C.U. immediately before ovulation to 3.1 C.U. at the time of 3 to 4 hours before expected ovulation. In the present study, the pituitaries from the layer were removed at the time from 11:30 a.m. to 2:00 p.m., when the gonadotropin content in their pituitaries was considered to be relatively lowered. The marked differences of the gonadotropin content of the cephalic lobe existed among the three reproductive stages of laying, non-laying and broody. The content of the caudal lobe remained comparatively unchanged among the five groups, while the hormone content of the late broody hen reduced markedly in both the cephalic and the caudal lobe. Yasuda (1953) found a marked reduction of the relative percentage of the basophiles in the cephalic lobe at the beginning of the broody period, and the lowest percentage at the middle (11th to 20th days) of the broody period. In the caudal lobe, the relative percentage of the basophiles of the broody hen was approximately equal to that of the laying hen. These findings are consistent with the present results on gonadotropin assay of the two lobes. In view of these results, it is concluded that the changes of the reproductive stage in the hen are closely associated with the increase or the decrease of the gonadotropin content in the anterior pituitary and these changes may be mainly due to the changes of the gonadotropin level in the cephalic lobe. SUMMARY
Acetone-dried powders of the cephalic and the caudal lobes of the anterior pituitary Nagoya and White Leghorn hens of six groups in various reproductive stages were assayed for their gonadotropic activity by means of chick testes method. Estimation of the gonadotropin content was made in terms of "chick unit." The potency per mg. powder of the two
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unbalance of FSH and ICSH factors in circulating gonadotropin. The lowered ovarian function caused by one or combinations of these possibilities might contribute to the high level of gonadotropin in the pituitary and the blood of the non-layer as seen in gonadectomized animal (Evans and Simpson, 1929; Cozens and Nelson, 1958; and others). The lowest gonadotropin content was found in the pituitary from the broody hen among the three reproductive stages. A marked drop of the gonadotropic potency of the pituitary occurred in the early broody period followed by a further reduction as the broody period advances. Since the blood serum from the early broody hen was found to have a considerable lowered gonadotropic activity than those from the layer and the non-layer (Imai and Nakajo, 1959), the circulating gonadotropin in the broody hen might be insufficient to cause normal development of the ovarian follicles. It was found previously that the pituitary of the broody hen contained more prolactin than that of the laying hen (Burrows and Byerly, 1936; and others). Moreover, a remarkably lowered level of the prolactin content was found in the pituitary from the non-laying hen (Imai and Nakajo, 1959). It is suggested that the gonadotropin level in the pituitary may tend to be inversely proportional to the prolactin level. The result of the gonadotropic potency of the anterior pituitary from the layer being lower than that from the non-layer agreed with the findings of Riley and Fraps (1942) and Phillips (1942). It was reported that in regularly laying hens the ovulating hormone was released from the pituitary at the time of several hours before ovulation (Rothchild and Fraps, 1949 a,b). Recently, we demonstrated (Imai and NTakajo, 1960) that the gonadotropin content of the pituitary from the laying hen fluctuated diurnally in relation to ovulating
744
S. NAKAJO AND K. IMAI
ACKNOWLEDGMENT
This work was carried out through a grant from the Research Fund of Ministry of Education of Japan. REFERENCES Bailey, R. L., and R. E. Phillips, 1952. Gonadotropins: potency of avian blood serum. Poultry Sci. 3 1 : 68-71. Breneman, W. R., 1945. Response of chicks to pituitary gonadotropins and pregnant mare serum. Proc. Indiana Acad. Sci. 54: 207-213. Burrows, W. H., and T. C. Byerly, 1936. Studies of prolactin in the fowl pituitary. I. Broody hens compared with laying hens and males. Proc. Soc. Exp. Biol. Med. 34: 841-844. Cozens, D. A., and M. M. Nelson, 1958. Increased
follicle-stimulating activity in the plasma of ovariectomized rats. Proc. Soc. Exp. Biol. Med. 98:617-620. Evans, H. M., and M. E. Simpson, 1929. A comparison of anterior hypophyseal implants from normal and gonadectomized animals with reference to their capacity to stimulate the immature ovary. Am. J. Physiol. 89: 371-374. Imai, K., and S. Nakajo, 1958. Studies on gonadotropin of the anterior pituitary in domestic fowl. III. Fractionation and potency of gonadotropic preparation from blood plasma. Jap. J. Anim. Reprod. 3 : 135-138. Imai, K., and S. Nakajo, 1959. Unpublished data. Imai, K., and S. Nakajo, 1960. Studies on gonadotropin of the anterior pituitary in domestic fowl. V. Diurnal change on the pituitary content in the laying hen. Jap. J. Anim. Reprod. In press. Nakajo, S., and K. Imai, 1956. Investigation on chick assay method for avian gonadotropins. Endocrinol. Japan, 3 : 197-202. Nakajo, S., and K. Tanaka, 1956. Prolactin potency of the cephalic and the caudal lobe of the anterior pituitary in relation to broodiness in the domestic fowl. Poultry Sci. 35: 990-994. Phillips, R. E., 1942. Comparative gonadotropic potency of unfractionated extracts of poultry pituitaries. Poultry Sci. 21: 161-172. Rahn, H., and B. T. Painter, 1941. A comparative histology of the birds pituitary. Anat. Rec. 79: 297-308. Riley, G. M., and R. M. Fraps, 1942. Relationship of gonad-stimulating activity of female domestic fowl anterior pituitaries to reproductive condition. Endocrinol. 30: 537-541. Rothchild, I., and R. M. Fraps, 1949a. The interval between normal release of ovulating hormone and ovulation in the domestic hen. Endocrinol. 44: 134-140. Rothchild, L, and R. M. Fraps, 1949b. The induction of ovulating hormone release from the pituitary of the domestic hen by means of progesterone. Endocrinol. 44: 141-149. Yasuda, M., 1953. Cytological studies of the anterior pituitary in the broody fowl. Proc. Japan Acad. 29: 586-594.
AUGUST 8-11. POULTRY SCIENCE ASSOCIATION, ANNUAL MEETING, PENNSYLVANIA STATE UNIVERSITY, UNIVERSITY PARK, PA. AUGUST 21-24. COMBINED CANADIAN TURKEY AND HATCHERY FEDERATION CONVENTION, BANFF SPRINGS HOTEL, BANFF, ALBERTA.
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lobes was highest in the non-layer, followed by the layer, the early broody and the late broody hen in the order of their magnitude. The estimated total gonadotropin content of the anterior pituitary of the non-layer was more than that of the layer. A marked drop of the gonadotropin content of the pituitary occurred in the early broody period followed by a further reduction as the broody period advanced. The marked differences of the gonadotropin content of the cephalic lobe existed among the non-laying, the laying, the early broody and the late broody hen, whereas the content of the caudel lobe remained comparatively unchanged with the exception of the late broody hen with a remarkable reduction. It was concluded that the changes of the reproductive stage in the hen were accompanied by the changes of the gonadotropin content in the anterior pituitary and these changes were mainly due to those in the cephalic lobe.
GLYCARBYLAMIDE AND REPRODUCTION
ACKNOWLEDGMENT
The radioactive studies using glycarbylamide-C" were a collaborated effort of the senior author with Dr. C. Rosenblum and Mr. H. Meriwether of Merck Sharp &
Dohme Research Laboratories. Appreciation is expressed for the statistical advice of Mr. A. G. Itkin, Merck Sharp & Dohme Research Laboratories, and to Miss M. Loukides for technical assistance. REFERENCES Cuckler, A. C , L. R. Chapin, C. M. Malanga, E. F. Rogers, H. J. Becker, R. L. Clark, W. J. Leanza, A. A. Pessolano, T. Y. Shen and L. H. Sarett, 1958. Antiparasitic drugs. II. Anticoccidial activity of 4,5-imidazoledicarboxamide and related compounds. Proc. Soc. Exp. Biol. Med. 98: 167-170. Kosin, I. L., 1944. Macro- and microscopic methods of detecting fertility in unincubated hen's eggs. Poultry Sci. 23: 266-269. Snedecor, G. W., 1946. Statistical Methods, 4th Edition. Iowa State College Press, Ames, Iowa. Warren, D. C , and R. M. Conrad, 1939. Growth of the hen's ovum. J. Agr. Res. 58: 875-893.
Gonadotropin Content in the Cephalic and the Caudal Lobe of the Anterior Pituitary in Laying, Non-Laying and Broody Hen SEIICHI NAKAJO AND KIYOSHI IMAI Laboratory
of Animal Breeding, Faculty of Agriculture, Nagoya University, Anzyo,
Japan
(Received for publication August 2, 1960)
"PHILLIPS (1942) used a change in the principle elicited the increase of testes •*• histology of rat vaginal epithelium as weight of newly hatched White Leghorn a measure of gonadotropic activity and es- cockerels. In the previous study, we demontimated gonadotropic potency of the an- strated (Nakajo and Imai, 1956) that the terior pituitaries of fowls in relation to age testes weight response of one-day-old chicks and egg laying. Riley and Fraps (1942), was adequate to use as quantitative assay employing an increase of uterine weight in for fowl pituitary gonadotropins. immature mouse, assayed gonadotropic acThe anterior pituitary of fowl could be tivity of avian pituitary in various repro- classified into two cytologically different ductive stages, and found that the anterior portions which were designated as cephalic pituitaries from hens with ovarian follicles "lobe" and caudal "lobe" (Rahn and in regressive or quiescent phases showed a Painter, 1941). Yasuda (1953) observed higher gonadotropic effect than those from that beginning and advance of broody period hens in full reproductive condition. in female fowl were accompanied by cytoBreneman (1945) reported that a rela- logical changes respectively in these two tively small amount of anterior pituitary lobes. Moreover, Nakajo and Tanaka
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 29, 2015
again at initial levels. This effect appears to be correlated with the amount of glycarbylamide found in albumen. Furthermore, the embryo mortality during incubation occurs in increasing amounts at earlier stages. Roosters fed glycarbylamide at levels which cause the decrease in fertile eggs maintain a high fertilizing capacity. Hens fed high levels of the drug lay a high percentage of eggs which are apparently infertile even though artificially inseminated with semen from non-medicated roosters.
740
S. NAKAJO AND K. IMAI TABLE 1.—Fresh weight of the anterior piluilaries from hens in various reproductive stages Group of hen
Breed
Reproductive stage
No. of birds
Anterior pituitary (mg.)
Cephalic lobe (mg.)
NA. NA. NA. NA.
Non-laying Laying Early broody Late broody
17 26 14 19
6.9+0.57* 7.8 + 0.36 8.1+0.66 8.9 + 0.71
4.4 + 4.4 + 5.5 + 5.9 +
0.36* 0.26 0.57 0.32
W.L. W.L.
Non-laying Laying
21 17
8.4 + 0.68 9.0±0.59
5.3 + 0.48 5.4±0.50
Caudal lobe (mg.) 2.4 + 2.9 + 2.2 + 2.9 +
0.27* 0.27 0.28 0.34
2.8 + 0.32 3.1+0.39
(1956) reported that expression and cessation of broodiness were associated with the changes in prolactin content in the anterior pituitary and these changes occurred mainly in the caudal lobe. The purpose of this study was to determine the relationship between the gonadotropin content in each lobe of the anterior pituitary and the reproductive stage of the hen, employing the chick assay method. MATERIALS AND METHODS
Anterior pituitaries were obtained from hens in the following six groups; a) nonlaying hens of Nagoya breed (NA.), 12 to 24 month-old, b) laying hens of NA., 12 to 18 month-old, c) early stage in broody period of NA., 12 to 16 month-old, d) late stage in broody period of NA., 12 to 16 month-old, e) non-laying hens of White Leghorn (W.L.), 12 to 24 month-old, f) laying hens of W.L., 12 to 18 month-old. The non-laying hens produced no eggs for several weeks and their ovaries were in late regressive or quiescent phase at the time of autopsy. Average ovarian weights in these groups from N.A. and W.L. were 4.0 gm. and 3.8 gm. respectively. The egg production of the laying hens was confirmed in individual cage. Their ovaries were in full active phase with normal sequence of developing follicles. The early broody hens were sacrificed on 5th day in nesting and
their ovaries were in quiescent phase (average ovarian weight was 3.8 gm.). The late broody hens were killed on 15th day in nesting and average ovarian weight in this group was 2.9 gm. The anterior pituitaries of all birds were separated into the two lobes under the dissecting microscope. After storing each lobe in acetone for 60 hours, they were kept in a desiccator at room temperature for a week. Then, each dried lobe was ground with an agate-mortar and stored in the desiccator until ready for use. One mg. of the dried lobe powder from each group was suspended in 1.0 ml. of distilled water immediately before the first injection. Data on the fresh weights of the anterior pituitaries from the hens are summarized in Table 1. The fact that in most cases the sum of cephalic and caudal lobes is less than the anterior pituitary weight is due to the weighing procedure in which the anterior pituitary was weighed and then separated into the two lobes. The chick assay was performed according to the method described previously (Nakajo and Imai, 1956). One-day-old male chicks of W.L. were injected subcutaneously with the suspended preparations into the dorsal neck region at 24, 36, 48, 60 and 72 hours after hatching. They were killed 24 hours after the final injection and the testes were excised and weighed.
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Abbreviations: NA.—Nagoya. W.L.—White Leghorn. Note: *Mean + Fiducial limit (Degree of confidence = 0.95).
741
GONADOTROPIN AND PRODUCTION
The injected chicks and the distilled water injected controls received no food and water during the test period. The response of chick testes weight was expressed as an increase-rate which was calculated by the following formula; exp. wt. — control wt. control wt.
- X 100 = increase-rate (as percent)
The estimation of the gonadotropin content of the pituitary was made in terms of "chick unit." When the increase rate showed 30%, the difference of the testes weights between the injected chicks and the controls was statistically significant if 5 or more birds were used in the assay. Therefore, considering a slight additional allowance for error, 35% increase was adopted to constitute a "chick unit" (C.U.). RESULTS
Gonadotropic potency per mg. of the dried powders of each lobe from the hens of the six groups is shown in Table 2. Since one control group was provided for each treated group, the determination of the "chick unit" was made as an increment over the control for each experiment. In the group of NA., the gonadotropic potency per mg. of the powder ranged from
TABLE 2.—Gonadotropic potency per mg. of acetone-dried powder of the cephalic and the caudal lobe from hens in various reproductive stages Assay in chicks
Breed
Reproductive stage
(No.)
(mg.)
(No.)
(mg.)
(%)
Gonadotropic potency/ mg. (chick unit)
NA.
Non-laying
Cephalic Caudal
(11) ( 7)
7.8 + 0.62* 9.9±1.09
( 8) (8)
4.0+0.28* 4.7±0.29
95.0 109.8
2.7 3.1
NA.
Laying
Cephalic Caudal
( 8) ( 5)
6.6±1.14 7.5+1.25
( 7) (9)
3.8+0.30 3.8+0.28
73.7 98.2
2.1 2.8
NA.
Early broody
Cephalic Caudal
( 9) (5)
8.0+0.42 10.8±1.16
( 8) ( 8)
5.7 + 0.48 5.7+0.48
40.0 89.6
1.1 2.5
NA.
Late broody
Cephalic Caudal
(12) ( 6)
5.6+0.44 5.7 + 0.92
(10) (10)
4.5±0.61 4.5±0.61
24.4 26.7
0.7 0.8
W.L.
Non-laying
Cephalic Caudal
( 7) ( 5)
7.7±0.81 7.5 ± 1 . 2 0
( 7) ( 7)
3.9+0.32 3.9+0.32
98.5 92.3
2.8 2.6
W.L.
Laying
Cephalic Caudal
(13) ( 7)
7.1 + 0.43 7.5 + 0.79
(10) (10)
4 . 5 + 0.32 4 . 5 + 0.32
58.2 66.7
1.7 1.9
Group of donors Testes weight of chick Treated
Control
Lobe
Notes: *Mean ± Fiducial limit (Degree of confidence =0.95). (No.)—No. of assay chicks.
Increaserate
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0.7 C.U. of late broody hen to 2.7 C.U. of non-layer in the cephalic lobe and from 0.8 C.U. of late broody hen to 3.1 C.U. of nonlayer in the caudal lobe. The cephalic lobe powder from the non-layer was more active than that from the layer, and the cephalic lobe from the broody hen had a considerably lower gonadotropic activity than that from the layer. The lowest activity was found in the cephalic lobe powder from the late broody hen. The relation of the gonadotropic potency of the cephalic lobe to the reproductive stage (non-layer > layer > early broody > late broody) was applicable for the caudal lobe. In W.L., the potencies per mg. of the cephalic and the caudal lobe powders from the non-layer were higher than those from the layer. These findings in W.L. were similar to those observed in NA. described above. The potency per mg. of the caudal lobe was higher than that of the cephalic lobe in NA., while the potencies of the two lobes in W.L. were approximately equal. The powders of each lobe from NA. were slightly more active than those from W.L. in the same reproductive stage, but these differences were not significant. On the basis of the weight of the powder
742
S. NAKAJO AND K. IMAI TABLE 3.—Gonadotropin content of the cephalic lobe, the caudal lobe and the entire gland of the anterior pituitary from hens in various reproductive stt Gonadotropin content (chick unit)
Group of hen
Per anterior pituitary
Reproductive stage
NA.
Non-laying
Cephalic Caudal
2.1 1.1
3.2
NA.
Laying
Cephalic Caudal
1.5 1.0
2.6
NA.
Early broody
Cephalic Caudal
0.9 0.9
1.9
NA.
Late broody
Cephalic Caudal
0.7 0.3
1.0
W.L.
Non-laying
Cephalic Caudal
2.5 1.1
3.5
W.L.
Laying
Cephalic Caudal
1.5 1.0
2.5
Lobe
obtained and the potency per mg. of each lobe powder, approximate values of the gonadotropin content per lobe and per entire gland of the anterior pituitary were calculated. These results are shown in Table 3. No significant differences of the content of each lobe and the entire gland were observed between NA. and W.L. in the same stage. The gonadotropin content per anterior pituitary ranged from 1.0 to 3.2 C.U. in NA. hens. The pituitary from the nonlayer was definitely more abundant in the hormone than that from the other groups, and the hormone content of the gland from the late broody hen was the least. In the light of the gonadotropin content of each lobe, the cephalic lobe had more content than the caudal lobe with the exception of the early broody hen, in which the hormone contents of the two lobes were almost equal. As it can be clearly seen in Tables 2 and 3, there were marked differences of the total gonadotropin contents of the anterior pituitaries among the groups of non-laying, laying, early broody and late broody hen. Moreover, these changes were closely as-
Per lobe
sociated with those in the content of the cephalic lobe, whereas the contents of the caudal lobe were not greatly affected by the changes in these reproductive stages with the exception of the late stage in broody period. In the late broody hen, remarkable reduction of the gonadotropin content was found in both the cephalic and the caudal lobe. DISCUSSION
The anterior pituitary from the nonlayer showed the highest gonadotropic potency among the three reproductive stages, laying, non-laying and broody. Moreover, Bailey and Phillips (1952) and our recent studies (Imai and Nakajo, 1958, 1959) indicated that the gonadotropic activity of blood serum from the non-layer was also higher than' that from the layer. Under these hormonal conditions, the fact that their ovaries were in late regressive or quiescent phase (lack of follicular growth) might result either from (a) a lowering of sensitivity of their ovary to circulating gonadotropins, or (b) a failure in process of production, mobilization and/or deposition of the yolk materials, or (c) an
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Breed
743
GONADOTROPIN AND PRODUCTION
time, i.e. the content ranged from 0.6 C.U. immediately before ovulation to 3.1 C.U. at the time of 3 to 4 hours before expected ovulation. In the present study, the pituitaries from the layer were removed at the time from 11:30 a.m. to 2:00 p.m., when the gonadotropin content in their pituitaries was considered to be relatively lowered. The marked differences of the gonadotropin content of the cephalic lobe existed among the three reproductive stages of laying, non-laying and broody. The content of the caudal lobe remained comparatively unchanged among the five groups, while the hormone content of the late broody hen reduced markedly in both the cephalic and the caudal lobe. Yasuda (1953) found a marked reduction of the relative percentage of the basophiles in the cephalic lobe at the beginning of the broody period, and the lowest percentage at the middle (11th to 20th days) of the broody period. In the caudal lobe, the relative percentage of the basophiles of the broody hen was approximately equal to that of the laying hen. These findings are consistent with the present results on gonadotropin assay of the two lobes. In view of these results, it is concluded that the changes of the reproductive stage in the hen are closely associated with the increase or the decrease of the gonadotropin content in the anterior pituitary and these changes may be mainly due to the changes of the gonadotropin level in the cephalic lobe. SUMMARY
Acetone-dried powders of the cephalic and the caudal lobes of the anterior pituitary Nagoya and White Leghorn hens of six groups in various reproductive stages were assayed for their gonadotropic activity by means of chick testes method. Estimation of the gonadotropin content was made in terms of "chick unit." The potency per mg. powder of the two
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unbalance of FSH and ICSH factors in circulating gonadotropin. The lowered ovarian function caused by one or combinations of these possibilities might contribute to the high level of gonadotropin in the pituitary and the blood of the non-layer as seen in gonadectomized animal (Evans and Simpson, 1929; Cozens and Nelson, 1958; and others). The lowest gonadotropin content was found in the pituitary from the broody hen among the three reproductive stages. A marked drop of the gonadotropic potency of the pituitary occurred in the early broody period followed by a further reduction as the broody period advances. Since the blood serum from the early broody hen was found to have a considerable lowered gonadotropic activity than those from the layer and the non-layer (Imai and Nakajo, 1959), the circulating gonadotropin in the broody hen might be insufficient to cause normal development of the ovarian follicles. It was found previously that the pituitary of the broody hen contained more prolactin than that of the laying hen (Burrows and Byerly, 1936; and others). Moreover, a remarkably lowered level of the prolactin content was found in the pituitary from the non-laying hen (Imai and Nakajo, 1959). It is suggested that the gonadotropin level in the pituitary may tend to be inversely proportional to the prolactin level. The result of the gonadotropic potency of the anterior pituitary from the layer being lower than that from the non-layer agreed with the findings of Riley and Fraps (1942) and Phillips (1942). It was reported that in regularly laying hens the ovulating hormone was released from the pituitary at the time of several hours before ovulation (Rothchild and Fraps, 1949 a,b). Recently, we demonstrated (Imai and NTakajo, 1960) that the gonadotropin content of the pituitary from the laying hen fluctuated diurnally in relation to ovulating
744
S. NAKAJO AND K. IMAI
ACKNOWLEDGMENT
This work was carried out through a grant from the Research Fund of Ministry of Education of Japan. REFERENCES Bailey, R. L., and R. E. Phillips, 1952. Gonadotropins: potency of avian blood serum. Poultry Sci. 3 1 : 68-71. Breneman, W. R., 1945. Response of chicks to pituitary gonadotropins and pregnant mare serum. Proc. Indiana Acad. Sci. 54: 207-213. Burrows, W. H., and T. C. Byerly, 1936. Studies of prolactin in the fowl pituitary. I. Broody hens compared with laying hens and males. Proc. Soc. Exp. Biol. Med. 34: 841-844. Cozens, D. A., and M. M. Nelson, 1958. Increased
follicle-stimulating activity in the plasma of ovariectomized rats. Proc. Soc. Exp. Biol. Med. 98:617-620. Evans, H. M., and M. E. Simpson, 1929. A comparison of anterior hypophyseal implants from normal and gonadectomized animals with reference to their capacity to stimulate the immature ovary. Am. J. Physiol. 89: 371-374. Imai, K., and S. Nakajo, 1958. Studies on gonadotropin of the anterior pituitary in domestic fowl. III. Fractionation and potency of gonadotropic preparation from blood plasma. Jap. J. Anim. Reprod. 3 : 135-138. Imai, K., and S. Nakajo, 1959. Unpublished data. Imai, K., and S. Nakajo, 1960. Studies on gonadotropin of the anterior pituitary in domestic fowl. V. Diurnal change on the pituitary content in the laying hen. Jap. J. Anim. Reprod. In press. Nakajo, S., and K. Imai, 1956. Investigation on chick assay method for avian gonadotropins. Endocrinol. Japan, 3 : 197-202. Nakajo, S., and K. Tanaka, 1956. Prolactin potency of the cephalic and the caudal lobe of the anterior pituitary in relation to broodiness in the domestic fowl. Poultry Sci. 35: 990-994. Phillips, R. E., 1942. Comparative gonadotropic potency of unfractionated extracts of poultry pituitaries. Poultry Sci. 21: 161-172. Rahn, H., and B. T. Painter, 1941. A comparative histology of the birds pituitary. Anat. Rec. 79: 297-308. Riley, G. M., and R. M. Fraps, 1942. Relationship of gonad-stimulating activity of female domestic fowl anterior pituitaries to reproductive condition. Endocrinol. 30: 537-541. Rothchild, I., and R. M. Fraps, 1949a. The interval between normal release of ovulating hormone and ovulation in the domestic hen. Endocrinol. 44: 134-140. Rothchild, L, and R. M. Fraps, 1949b. The induction of ovulating hormone release from the pituitary of the domestic hen by means of progesterone. Endocrinol. 44: 141-149. Yasuda, M., 1953. Cytological studies of the anterior pituitary in the broody fowl. Proc. Japan Acad. 29: 586-594.
AUGUST 8-11. POULTRY SCIENCE ASSOCIATION, ANNUAL MEETING, PENNSYLVANIA STATE UNIVERSITY, UNIVERSITY PARK, PA. AUGUST 21-24. COMBINED CANADIAN TURKEY AND HATCHERY FEDERATION CONVENTION, BANFF SPRINGS HOTEL, BANFF, ALBERTA.
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lobes was highest in the non-layer, followed by the layer, the early broody and the late broody hen in the order of their magnitude. The estimated total gonadotropin content of the anterior pituitary of the non-layer was more than that of the layer. A marked drop of the gonadotropin content of the pituitary occurred in the early broody period followed by a further reduction as the broody period advanced. The marked differences of the gonadotropin content of the cephalic lobe existed among the non-laying, the laying, the early broody and the late broody hen, whereas the content of the caudel lobe remained comparatively unchanged with the exception of the late broody hen with a remarkable reduction. It was concluded that the changes of the reproductive stage in the hen were accompanied by the changes of the gonadotropin content in the anterior pituitary and these changes were mainly due to those in the cephalic lobe.