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Homologous radioimmunoassay for plasma and pituitary prolactin in the toad, Bufo japonicus
GENERAL AND COMPARATIVEENDOCRINOLOGY 74, 373-376 (1989)
Homologous Radioimmunoassay for Plasma and Pituitary Prolactin in the Toad, Bufo japonicus K A Z U T O S H I YAMAMOTO, SAKAE K I K U Y A M A , 1 AND S U S U M U ISHII
Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169, Japan Accepted October 6, 1988 A specific and sensitive homologous radioimmunoassay (RIA) was developed for the measurement of toad (Bufo japonicus) prolactin (PRL). PRL isolated from toad pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and a homogenate of the anterior lobe of the pituitary gland of toads yielded dose-response curves which paralleled the standard. Plasma from hypophysectomized toads showed the least amount of cross-reaction. Bovine PRL, ovine PRL, ovine growth hormone, and a homogenate of toad neurointermediate lobes showed no inhibition of binding even at relatively high doses in this RIA. A preliminary application of homologous RIA for toad PRL was performed by determining plasma and pituitary PRL levels in adult toads of both sexes captured in spring and late summer. Plasma PRL levels of male toads remaining in the pond for breeding were significantly higher than those of the males feeding in the bush or hibernating under the ground. The pituitary PRL content in spring toads of both sexes was higher than those in summer toads. The pituitary PRL content in females was invariably lower than those in simultaneously captured males. 9 1989AcademicPress, Inc.
Amphibian prolactin (PRL) was first pudried from bullfrog (Rana catesbeiana) pituitary glands and its physicochemical characteristics were determined in our laboratory (Yamamoto and Kikuyama, 1981). Recently, another amphibian PRL was obtained from the pituitary gland of the toad, Bufo japonicus, by monitoring purification using heterologous radioimmunoassay (RIA) (Yamamoto et al., 1986a). The present paper deals with the development of homologous RIA for toad PRL and its application to the measurement of plasma and pituitary PRL of adult Japanese toads captured during the breeding season (spring) and the nonbreeding season (summer). MATERIALS AND METHODS Toad PRL. PRL for the production of antiserum, t To whom all correspondence and requests for reprints should be addressed.
radioiodination, and use as a reference standard was purified from the anterior pituitary glands of adult toads (B. japonicus) by extraction of acetone-dried powder with acid acetone and chromatography on DEAE--cellulose and Sephadex G-100 (Yamamoto et al., 1986a), according to the method used to isolate bullfrog PRL (Yamamoto and Kikuyama, 1981). Production of antiserum. Antiserum was raised in a mature female albino rabbit by the multiple-site injection technique (Vaitukaitis et al., 1971). For immunization, I nag of highly purified toad PRL was dissolved in 1.5 ml of saline, emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI), and injected subcutaneously into 10-20 sites over the dorsal surface of the rabbit. The rabbit received three sets of injections in a similar fashion at 2-week intervals. Blood was collected from the marginal ear vein after the second set of injections and the antibody titer was checked. One week after the last set of injections, the rabbit was bled from the carotid arteries. The serum was separated by centrifugation and stored at - 7 0 ~ Radioimmunoassay. Radioiodination of toad PRL with Na125I (carrier free; The Radiochemical Centre Amersham, England) was carried out at room temperature according to the modified lactoperoxidase method (Sakai et al., 1975). The specific radioactivity of the radioiodinated PRL was about 40-50 ~Ci/ixg. The antiserum to toad PRL exhibited an ability to bind
373 0016-6480/89 $1.50 Copyright9 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
374
YAMAMOTO, KIKUYAMA, AND ISHII
the radioligand when 100 I~1 of the diluted antiserum and approximately 0.2 ng 9f the labeled PRL (about 20,000 cpm) in 100 ~1 of diluent were added to each incubation tube containing 300 I~1 of assay buffer (1% BSA-PBS, pH 7.5). A 30% specific binding of the added radioligand was obtained in the absence of any unlabeled PRL when the antiserum was used at a final dilution of 1:20,000. The details are similar to those described for bullfrog PRL RIA (Yamamoto and Kikuyama, 1982, 1986b). Statistics for linearity, parallelism, precision, and potency estimation in the parallel line assay were computed according to the method of Bliss (1952). In order to evaluate the specificity of the RIA, serum samples from toads hypophysectomized for 7 days were tested. Ovine PRL (NIAMDD oPRL-18), bovine PRL (NIAMDD bPRL6), ovine GH (NIAMDD oGH-14), toad neurointermediate lobe homogenate, and bullfrog anterior lobe homogenate were also tested. Pituitary and plasma samples for RIA. Blood and pituitary samples were collected between 18:00 and 20:00 hr from adult toads immediately after capture in the suburbs of Tomobe City, Ibaraki Prefecture, during the spring and summer of 1983. Animals collected on March 18, March 30, April 1, and August 22 were those staying still under the ground, moving toward or around a pond, mating or swimming in the pond, and feeding in bushes near the pond, respectively. On each occasion, seven females and seven males were captured. Pituitaries were individually homogenized in 1 ml of distilled water (DW), since there was no difference in the amount of immunoassayable PRL between pituitary samples homogenized in DW and samples extracted with 2.5 M urea, which secures the complete extraction of rat pituitary PRL (Haggi and Aoki, 1981). Aliquots of the pituitary homogenates were used for protein determination. Blood samples were individually collected into heparinized tubes by cardiac puncture. Plasma was separated by centrifugation. Both pituitary and plasma samples were stored at - 20~until assayed. Statistical analysis. Analysis of variance and Duncan's multiple range test were used to analyze the results. In some cases, the logarithmic transformation of data was employed in order to satisfy the requirement of homosedasticity. A P value less than 0.05 was considered significant.
RESULTS The standard PRL gave a log-dose inhibition of the binding of 125I-labeled PRL to the antiserum (Fig. 1). Sensitivity of the RIA, defined as 2 SD below the 100% bound point, averaged 0.12 -+ 0.01 ng of standard toad PRL/100 ixl assay buffer in 10
101 8(
.-~,~
?~(~~i~oGH NIlobes OPRL
=
b/PRL
~
0.01 0.1
1
10 100 1000 10000
ng p r o t e i n / t u b e
FIG. l. Displacement of ]zsI-toad PRL with homogenates of toad anterior lobes, toad neurointermediate lobes (NI), and bullfrog anterior lobes, and three mammalian hormones at varying concentrations. The antiserum to toad PRL was used at a final dilution of 1:20,000. All points are the averages of duplicated determinations.
assays. Precision of the assay was determined by assaying the toad PRL standard. The interassay coefficient of variation was 9.2% when the estimated dose at 50% inhibition in I0 assays was employed. The intraassay coefficient of variation of 8.0% was obtained by repeated determinations of 1 ng standard PRL. The slope of the inhibition curve produced by the toad anterior pituitary homogenate was parallel to that of the standard. The pituitary from the bullfrog showed significant cross-reaction in the RIA, but the displacement curve obtained was not parallel to that of the standard. Ovine PRL, bPRL, and oGH showed no cross-reactivity in this RIA. The neurointermediate lobe homogenate also did not cross-react to the antiserum. Plasma and pituitary homogenates of adult toads produce displacement curves parallel to that of purified toad PRL (Fig. 2), and that plasma of toads which were hypophysectomized for 1 week had no detectable immunoreactive PRL. The PRL concentration was estimated from the linear portion of the displacement curve. Plasma and pituitary PRL levels of spring and summer toads are shown in Fig. 3. The analysis of variance revealed that the overall difference between males and females was significant in pituitary PRL levels but
375
RIA F O R TOAD PROLACTIN Dilution
100
~
content in spring females was invariably lower than those in simultaneously captured males. In both sexes, the pituitary PRL contents in summer toads were lower than those in spring toads.
L4 ? !
4G
DISCUSSION 0.1
1 10 100 1000 ng protein/tube FIG. 2. Representative data from the RIA of toad PRL. Purified toad PRL standard is compared with p l a s m a from intact and h y p o p h y s e c t o m i z e d (HX) toads.
not in plasma PRL levels. Plasma PRL levels of male toads remaining in the water were significantly higher than those of males either hibernating under the ground or feeding in the bush. The average plasma PRL value of nonhibernating females captured in the spring was higher than that of hibernating females or females captured in the summer, but the difference was not statistically significant. The pituitary PRL
1~176~
A,5_.~.s c_..~o ~
_c
D
Male Female FIG. 3. Plasma and pituitary PRL concentration in Japanese toads. (A) hibernating (captured on March 18); (B) moving toward or around a pond (captured on March 30); (C) swimming or mating in a pond (captured on April 1); and (D) feeding in bushes (captured on August 22). Each column and vertical bar represents the mean and SEM, respectively. Significantly different by Duncan's multiple range test: a vs c, b vs d, c vs d, i vs 1,j vs 1, k vs 1, m vs n , m vs o , m vs p, n vs p, and o vs p.
As demonstrated in the present experiment, the RIA developed for the measurement of toad PRL is specific and sensitive enough to measure plasma and pituitary PRL levels in the toad. The fact that neither plasma samples from hypophysectomized animals nor neurointermediate lobe homogenate showed a significant cross-reactivity in the RIA indicates that the cross-reacting material was of pars distalis origin. Employing this RIA system, plasma and pituitary P R L levels of adult Japanese toads captured in spring and summer were determined. The toads of this species lead a terrestial life, except for a brief period during the breeding season in spring when both sexes migrate to the water. It has been known that, in several amphibian species, PRL plays an osmoregulatory role similar to that found in teleosts, when animals are exposed to a hypotonic environment (see Ensor, 1978). Therefore, it is reasonable to expect that PRL levels in toads are elevated during the breeding season. In fact, the average plasma PRL value was higher in animals remaining in the water than in hibernating toads or summer toads, although in the case of females, the difference was not significant. The results coincide with those previously reported by us on the basis of heterologous RIA data (Yoneyama et al., 1984; Ishii et al., 1989. It should be pointed out, however, that among the animals remaining in the water or moving toward or around the water, there were several in which plasma PRL values were as low as those of summer toads. The possible explanation for this fluctuation may be that during the breeding
376
YAMAMOTO, KIKUYAMA, AND ISHII
season a pulsatile release of PRL occurs and/or that elevation of PRL levels is not directly related to migration toward the water but to some other events associated with breeding such as ovulation and clasping. Judging from the relatively low values of plasma and pituitary PRL concentrations in summer toads, PRL synthesis may decline after the breeding season. It is noteworthy that sex differences in the pituitary PRL content exist especially in the spring toads. PRL concentrations in the pituitary gland were invariably higher in the males than in the females. Considering that plasma PRL levels were not higher in the female than in the male, PRL synthesis may be higher in the male than in the female. This is just opposite to the case in mammals where prolactin content is higher in the female than in the male due to the estrogen-induced increase in the cellular content of PRL messenger RNA (Gorski, 1981).
ACKNOWLEDGMENTS The authors express their sincere thanks to Professor C. S. Nicoll, University of California, for his valuable suggestion. We are also grateful to Dr. T. Seki of Juntendo University; Dr. K. Kawamura, Mr. K. Koiwai, and Mr. H. Inaco of Waseda University for their help during the course of the experiment; and to the Pituitary Hormones and Antisera Center and the National Pituitary Agency for supplying oPRL, oGH, and bPRL. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a research grant from Waseda University to S.K and S.I.
REFERENCES Bliss, C. I. (1952). "The Statistics of Bioassay." Academic Press, New York. Ensor, D. M. (1978). Prolactin in amphibians. In "Comparative Endocrinology of Prolactin," pp. 45-72. Chapman and Hall, London. Gorski, J. (1981). Prolactin biosynthesis and its regulation by estrogens. In "Prolactin" (L. V. Aviol, Ed.), pp. 57-83. Elsevier, New York. Haggi, E., and Aoki, A. (1981). Prolactin content in rat pituitary gland: RIA of prolactin after different extraction procedures. Acta Endocrinol. 97, 338-342. Ishii, S., Yoneyama, H., Inoue, M., Yamamoto, K., and Kikuyama, S. (1989). Changes of plasma level and pituitary content of prolactin in the toad, Bufo japonicus, by month and during the breeding season with special reference to the breeding migration. Gen. Comp. Endocrinol. 73, in press. Sakai, S., Kohmoto, K., and Johke, T. (1975). A receptor site for prolactin in lactating mouse mammary tissues. Endocrinol. Japon. 22, 379--387. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, T. (1971). A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. 33, 988-991. Yamamoto, K., and Kikuyama, S. (1981). Purification and properties of bullfrog prolactin. Endocrinol. Japon. 28, 59---64. Yamamoto, K., and Kikuyama, S. (1982). Radioimmunoassay of prolactin in plasma of bullfrog tadpoles. Endocrinol. Japon. 29, 159-167. Yamamoto, K., Kobayashi, T., and Kikuyama, S. (1986a). Purification and characterization of toad prolactin. Gen. Comp. Endocrinol. 63, 11)4-109. Yamamoto, K., Niinuma, K., and Kikuyama, S. (1986b). Synthesis and storage of prolactin in the pituitary gland of bullfrog tadpoles during metamorphosis. Gen. Comp. Endocrinol. 62, 247-253. Yoneyama, H., Ishii, S., Yamamoto, K., and Kikuyama, S. (1984). Plasma prolactin levels of Bufo japonicus before, during and after breeding in the pond. Zool. Sci. 1, 969.
Homologous Radioimmunoassay for Plasma and Pituitary Prolactin in the Toad, Bufo japonicus K A Z U T O S H I YAMAMOTO, SAKAE K I K U Y A M A , 1 AND S U S U M U ISHII
Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169, Japan Accepted October 6, 1988 A specific and sensitive homologous radioimmunoassay (RIA) was developed for the measurement of toad (Bufo japonicus) prolactin (PRL). PRL isolated from toad pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and a homogenate of the anterior lobe of the pituitary gland of toads yielded dose-response curves which paralleled the standard. Plasma from hypophysectomized toads showed the least amount of cross-reaction. Bovine PRL, ovine PRL, ovine growth hormone, and a homogenate of toad neurointermediate lobes showed no inhibition of binding even at relatively high doses in this RIA. A preliminary application of homologous RIA for toad PRL was performed by determining plasma and pituitary PRL levels in adult toads of both sexes captured in spring and late summer. Plasma PRL levels of male toads remaining in the pond for breeding were significantly higher than those of the males feeding in the bush or hibernating under the ground. The pituitary PRL content in spring toads of both sexes was higher than those in summer toads. The pituitary PRL content in females was invariably lower than those in simultaneously captured males. 9 1989AcademicPress, Inc.
Amphibian prolactin (PRL) was first pudried from bullfrog (Rana catesbeiana) pituitary glands and its physicochemical characteristics were determined in our laboratory (Yamamoto and Kikuyama, 1981). Recently, another amphibian PRL was obtained from the pituitary gland of the toad, Bufo japonicus, by monitoring purification using heterologous radioimmunoassay (RIA) (Yamamoto et al., 1986a). The present paper deals with the development of homologous RIA for toad PRL and its application to the measurement of plasma and pituitary PRL of adult Japanese toads captured during the breeding season (spring) and the nonbreeding season (summer). MATERIALS AND METHODS Toad PRL. PRL for the production of antiserum, t To whom all correspondence and requests for reprints should be addressed.
radioiodination, and use as a reference standard was purified from the anterior pituitary glands of adult toads (B. japonicus) by extraction of acetone-dried powder with acid acetone and chromatography on DEAE--cellulose and Sephadex G-100 (Yamamoto et al., 1986a), according to the method used to isolate bullfrog PRL (Yamamoto and Kikuyama, 1981). Production of antiserum. Antiserum was raised in a mature female albino rabbit by the multiple-site injection technique (Vaitukaitis et al., 1971). For immunization, I nag of highly purified toad PRL was dissolved in 1.5 ml of saline, emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI), and injected subcutaneously into 10-20 sites over the dorsal surface of the rabbit. The rabbit received three sets of injections in a similar fashion at 2-week intervals. Blood was collected from the marginal ear vein after the second set of injections and the antibody titer was checked. One week after the last set of injections, the rabbit was bled from the carotid arteries. The serum was separated by centrifugation and stored at - 7 0 ~ Radioimmunoassay. Radioiodination of toad PRL with Na125I (carrier free; The Radiochemical Centre Amersham, England) was carried out at room temperature according to the modified lactoperoxidase method (Sakai et al., 1975). The specific radioactivity of the radioiodinated PRL was about 40-50 ~Ci/ixg. The antiserum to toad PRL exhibited an ability to bind
373 0016-6480/89 $1.50 Copyright9 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
374
YAMAMOTO, KIKUYAMA, AND ISHII
the radioligand when 100 I~1 of the diluted antiserum and approximately 0.2 ng 9f the labeled PRL (about 20,000 cpm) in 100 ~1 of diluent were added to each incubation tube containing 300 I~1 of assay buffer (1% BSA-PBS, pH 7.5). A 30% specific binding of the added radioligand was obtained in the absence of any unlabeled PRL when the antiserum was used at a final dilution of 1:20,000. The details are similar to those described for bullfrog PRL RIA (Yamamoto and Kikuyama, 1982, 1986b). Statistics for linearity, parallelism, precision, and potency estimation in the parallel line assay were computed according to the method of Bliss (1952). In order to evaluate the specificity of the RIA, serum samples from toads hypophysectomized for 7 days were tested. Ovine PRL (NIAMDD oPRL-18), bovine PRL (NIAMDD bPRL6), ovine GH (NIAMDD oGH-14), toad neurointermediate lobe homogenate, and bullfrog anterior lobe homogenate were also tested. Pituitary and plasma samples for RIA. Blood and pituitary samples were collected between 18:00 and 20:00 hr from adult toads immediately after capture in the suburbs of Tomobe City, Ibaraki Prefecture, during the spring and summer of 1983. Animals collected on March 18, March 30, April 1, and August 22 were those staying still under the ground, moving toward or around a pond, mating or swimming in the pond, and feeding in bushes near the pond, respectively. On each occasion, seven females and seven males were captured. Pituitaries were individually homogenized in 1 ml of distilled water (DW), since there was no difference in the amount of immunoassayable PRL between pituitary samples homogenized in DW and samples extracted with 2.5 M urea, which secures the complete extraction of rat pituitary PRL (Haggi and Aoki, 1981). Aliquots of the pituitary homogenates were used for protein determination. Blood samples were individually collected into heparinized tubes by cardiac puncture. Plasma was separated by centrifugation. Both pituitary and plasma samples were stored at - 20~until assayed. Statistical analysis. Analysis of variance and Duncan's multiple range test were used to analyze the results. In some cases, the logarithmic transformation of data was employed in order to satisfy the requirement of homosedasticity. A P value less than 0.05 was considered significant.
RESULTS The standard PRL gave a log-dose inhibition of the binding of 125I-labeled PRL to the antiserum (Fig. 1). Sensitivity of the RIA, defined as 2 SD below the 100% bound point, averaged 0.12 -+ 0.01 ng of standard toad PRL/100 ixl assay buffer in 10
101 8(
.-~,~
?~(~~i~oGH NIlobes OPRL
=
b/PRL
~
0.01 0.1
1
10 100 1000 10000
ng p r o t e i n / t u b e
FIG. l. Displacement of ]zsI-toad PRL with homogenates of toad anterior lobes, toad neurointermediate lobes (NI), and bullfrog anterior lobes, and three mammalian hormones at varying concentrations. The antiserum to toad PRL was used at a final dilution of 1:20,000. All points are the averages of duplicated determinations.
assays. Precision of the assay was determined by assaying the toad PRL standard. The interassay coefficient of variation was 9.2% when the estimated dose at 50% inhibition in I0 assays was employed. The intraassay coefficient of variation of 8.0% was obtained by repeated determinations of 1 ng standard PRL. The slope of the inhibition curve produced by the toad anterior pituitary homogenate was parallel to that of the standard. The pituitary from the bullfrog showed significant cross-reaction in the RIA, but the displacement curve obtained was not parallel to that of the standard. Ovine PRL, bPRL, and oGH showed no cross-reactivity in this RIA. The neurointermediate lobe homogenate also did not cross-react to the antiserum. Plasma and pituitary homogenates of adult toads produce displacement curves parallel to that of purified toad PRL (Fig. 2), and that plasma of toads which were hypophysectomized for 1 week had no detectable immunoreactive PRL. The PRL concentration was estimated from the linear portion of the displacement curve. Plasma and pituitary PRL levels of spring and summer toads are shown in Fig. 3. The analysis of variance revealed that the overall difference between males and females was significant in pituitary PRL levels but
375
RIA F O R TOAD PROLACTIN Dilution
100
~
content in spring females was invariably lower than those in simultaneously captured males. In both sexes, the pituitary PRL contents in summer toads were lower than those in spring toads.
L4 ? !
4G
DISCUSSION 0.1
1 10 100 1000 ng protein/tube FIG. 2. Representative data from the RIA of toad PRL. Purified toad PRL standard is compared with p l a s m a from intact and h y p o p h y s e c t o m i z e d (HX) toads.
not in plasma PRL levels. Plasma PRL levels of male toads remaining in the water were significantly higher than those of males either hibernating under the ground or feeding in the bush. The average plasma PRL value of nonhibernating females captured in the spring was higher than that of hibernating females or females captured in the summer, but the difference was not statistically significant. The pituitary PRL
1~176~
A,5_.~.s c_..~o ~
_c
D
Male Female FIG. 3. Plasma and pituitary PRL concentration in Japanese toads. (A) hibernating (captured on March 18); (B) moving toward or around a pond (captured on March 30); (C) swimming or mating in a pond (captured on April 1); and (D) feeding in bushes (captured on August 22). Each column and vertical bar represents the mean and SEM, respectively. Significantly different by Duncan's multiple range test: a vs c, b vs d, c vs d, i vs 1,j vs 1, k vs 1, m vs n , m vs o , m vs p, n vs p, and o vs p.
As demonstrated in the present experiment, the RIA developed for the measurement of toad PRL is specific and sensitive enough to measure plasma and pituitary PRL levels in the toad. The fact that neither plasma samples from hypophysectomized animals nor neurointermediate lobe homogenate showed a significant cross-reactivity in the RIA indicates that the cross-reacting material was of pars distalis origin. Employing this RIA system, plasma and pituitary P R L levels of adult Japanese toads captured in spring and summer were determined. The toads of this species lead a terrestial life, except for a brief period during the breeding season in spring when both sexes migrate to the water. It has been known that, in several amphibian species, PRL plays an osmoregulatory role similar to that found in teleosts, when animals are exposed to a hypotonic environment (see Ensor, 1978). Therefore, it is reasonable to expect that PRL levels in toads are elevated during the breeding season. In fact, the average plasma PRL value was higher in animals remaining in the water than in hibernating toads or summer toads, although in the case of females, the difference was not significant. The results coincide with those previously reported by us on the basis of heterologous RIA data (Yoneyama et al., 1984; Ishii et al., 1989. It should be pointed out, however, that among the animals remaining in the water or moving toward or around the water, there were several in which plasma PRL values were as low as those of summer toads. The possible explanation for this fluctuation may be that during the breeding
376
YAMAMOTO, KIKUYAMA, AND ISHII
season a pulsatile release of PRL occurs and/or that elevation of PRL levels is not directly related to migration toward the water but to some other events associated with breeding such as ovulation and clasping. Judging from the relatively low values of plasma and pituitary PRL concentrations in summer toads, PRL synthesis may decline after the breeding season. It is noteworthy that sex differences in the pituitary PRL content exist especially in the spring toads. PRL concentrations in the pituitary gland were invariably higher in the males than in the females. Considering that plasma PRL levels were not higher in the female than in the male, PRL synthesis may be higher in the male than in the female. This is just opposite to the case in mammals where prolactin content is higher in the female than in the male due to the estrogen-induced increase in the cellular content of PRL messenger RNA (Gorski, 1981).
ACKNOWLEDGMENTS The authors express their sincere thanks to Professor C. S. Nicoll, University of California, for his valuable suggestion. We are also grateful to Dr. T. Seki of Juntendo University; Dr. K. Kawamura, Mr. K. Koiwai, and Mr. H. Inaco of Waseda University for their help during the course of the experiment; and to the Pituitary Hormones and Antisera Center and the National Pituitary Agency for supplying oPRL, oGH, and bPRL. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a research grant from Waseda University to S.K and S.I.
REFERENCES Bliss, C. I. (1952). "The Statistics of Bioassay." Academic Press, New York. Ensor, D. M. (1978). Prolactin in amphibians. In "Comparative Endocrinology of Prolactin," pp. 45-72. Chapman and Hall, London. Gorski, J. (1981). Prolactin biosynthesis and its regulation by estrogens. In "Prolactin" (L. V. Aviol, Ed.), pp. 57-83. Elsevier, New York. Haggi, E., and Aoki, A. (1981). Prolactin content in rat pituitary gland: RIA of prolactin after different extraction procedures. Acta Endocrinol. 97, 338-342. Ishii, S., Yoneyama, H., Inoue, M., Yamamoto, K., and Kikuyama, S. (1989). Changes of plasma level and pituitary content of prolactin in the toad, Bufo japonicus, by month and during the breeding season with special reference to the breeding migration. Gen. Comp. Endocrinol. 73, in press. Sakai, S., Kohmoto, K., and Johke, T. (1975). A receptor site for prolactin in lactating mouse mammary tissues. Endocrinol. Japon. 22, 379--387. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, T. (1971). A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. 33, 988-991. Yamamoto, K., and Kikuyama, S. (1981). Purification and properties of bullfrog prolactin. Endocrinol. Japon. 28, 59---64. Yamamoto, K., and Kikuyama, S. (1982). Radioimmunoassay of prolactin in plasma of bullfrog tadpoles. Endocrinol. Japon. 29, 159-167. Yamamoto, K., Kobayashi, T., and Kikuyama, S. (1986a). Purification and characterization of toad prolactin. Gen. Comp. Endocrinol. 63, 11)4-109. Yamamoto, K., Niinuma, K., and Kikuyama, S. (1986b). Synthesis and storage of prolactin in the pituitary gland of bullfrog tadpoles during metamorphosis. Gen. Comp. Endocrinol. 62, 247-253. Yoneyama, H., Ishii, S., Yamamoto, K., and Kikuyama, S. (1984). Plasma prolactin levels of Bufo japonicus before, during and after breeding in the pond. Zool. Sci. 1, 969.