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Effect of Hypophysectomy on the Radioactive Phosphorus Uptake of Chick Adrenals12
P l N E A L E C T O M Y OE Q U A I L
jections in the young cockerel. Endocrinol. 51: 152-154. Shellabarger, C. J., 1953. Observations of the pineal in the White Leghorn capon and cockerel. Poultry Sci. 32: 189-197. Shellabarger, C. J., and W. R. Breneman, 1949. The effects of pinealectomy on young White Leghorn cockerels. Indiana Acad. Sci. Proc. 59: 299-302. Wilson, W. O., U. K. Abbott and H. Abplanalp, 1961. Evaluation of Coturnix (Japanese quail) as pilot animal for poultry.Poultry Sci. 40: 651-657. Wilson, W. O., H. Abplanalp and L. Arrington, 1962. Sexual development of Coturnix as affected by changes in photoperiods. Poultry Sci. 41: 17-22. Wolfson, A., 1952. The cloacal protuberance. Bird Banding, 23: 159-165. Woodard, A. E., H. Abplanalp and W. O. Wilson, 1965. Japanese quail husbandry in the laboratory (Coturnix coturnix japonica). Dept. Poultry Husbandry, Univ. of Calif., Davis. 36 pp. Wurtman, R. J., and J. Axelrod, 1965. The pineal gland. Scientific Amer. 213: 50-60.
Effect of Hypophysectomy on the Radioactive Phosphorus Uptake of Chick Adrenals1'2 DAVID B. K I N G
Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604 (Received for publication August 15, 1968)
T
HE rate of incorporation of radio- chickens would be expected to incorporate active phosphorus (32P) into the tar- less radioactivity due to lower levels of get organs of young chickens serves as an endogenous tropic hormones. Surprisingly, end-point in the bioassay procedures for investigations of the 32P uptake by these thyrotropic, gonadotropic and adrenocor- organs did not show a decreased incorticotropic hormones (Greenspan et al., poration of the isotope following hypo1956;Brenemanetal., 1965; Connell, 1965; physectomy of young cockerels (King, Bukovsan, 1967). The tropic hormones 1965). Instead, the adrenals and thyroids stimulate an increased incorporation of of hypophysectomized chickens incorporadioactivity per unit weight of their spe- rated at least twice as much radioactivity cific target organ. Since 32P uptake is per mg of tissue during a 48-hour period stimulated by tropic hormone, the pitu- following isotope administration, while the itary target organs of hypophysectomized 32P uptake by the gonads was the same or slightly greater than controls. When the 1 Part of a thesis submitted to the Zoology Deadrenals and thyroids were divided into partment of Indiana University in partial fulfillment trichloroacetic acid soluble, lipid and proof the requirements for the Ph.D. tein-nucleic acid fractions, the greater ra2 Supported in part by a pre-doctoral National dioactivity was found in each fraction at Aeronautical and Space Administration Fellowship.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 24, 2015
physiological actions of the pineal gland. Ann. Int. Med. 61: 1144-1161. Farner, D. S., 1964. The photoperiodic control of reproductive cycles in birds. Amer. Scientist, 52: 137-156. Hoffman, R. A., and R. J. Reiter, 1965a. Influence of compensatory mechanisms and of the pineal gland on dark-induced gonadal atrophy in male hamsters. Nature, 207: 658-659. Hoffman, R. A., and R. J. Reiter, 1965b. Pineal gland: Influence on gonads of male hamsters. Science, 148: 1609-1610. Kitay, J. I., and M. D. Altschule, 1954. Effects of pineal extract administration on ovary weight in rats. Endocrinol. 55: 782-784. Mather, F. B., and W. O. Wilson, 1964. Post-natal testicular development in Japanese quail (Coturnix coturnix japonica). Poultry Sci. 43: 860864. Relkin, R., 1966. The pineal gland. New England J. Med. 274: 944-950. Shellabarger, C. J., 1952. Pinealectomy vs pineal in-
459
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D.
B.
MATERIALS AND METHODS White Leghorns cockerels (State F a r m Bureau Hatchery, Indianapolis, Indiana) were hypophysectomized at about three weeks of age by a parapharyngeal method described in detail elsewhere (King, 1965). Complete removal of the adenohypophysis was confirmed at autopsy for all hypophysectomized cockerels included in the results by a careful inspection of the pituitary region under a dissecting scope. Control and hypophysectomized cockerels were housed in constant temperature animal rooms (75° F.) under continuous lighting and were fed Wayne Pullet Grower and water ad libitum. The hypophysectomized chickens were provided with extra heat for a period of at least three weeks following the operation. Hypophysectomized and control cockerels were injected with 20/uc. 3 2 P/100 g. body weight at 53 days of age (about one month after hypophysectomy) and killed three hours later. T h e 32P (Oak Ridge National Laboratories; at least 9 9 % free of non-radioactive phosphorus) was re-
ceived as phosphate in weak HC1, diluted to the desired concentration with distilled water and injected subcutaneously at a volume of 0.1 to 0.3 ml. T h e birds were killed alternately from each group b y decapitation and the adrenals removed, frozen on dry-ice and weighed to the nearest 0.2 mg. T h e tissue was homogenized in cold 10% trichloroacetic acid with a motor-driven teflon pestle (TriR Corp.) and the homogenate transferred to a centrifuge tube; the homogenization tube and pestle were washed at least three times with cold 1 0 % trichloroacetic acid. T h e homogenate was brought to a known volume with trichloroacetic acid, an aliguot plated on a disposable planchet, dried and the radioactivity measured with a Baird Atomic Multiscaler (Model 132) connected to a thin window (1.7 mg./cm. 2 ) Geiger Muller tube. T h e counts per minute (cpm) were corrected for background and decay. T h e remaining homogenate was centrifuged at 800 to l , 0 0 0 X g for 10 minutes in a refrigerated centrifuge ( 0 ° C ) . T h e pellet remaining after centrifugation was re-extracted twice with approximately 3.0 ml. of cold 1 0 % trichloroacetic acid. The supernatants from the three extractions were combined and designated the acid soluble fraction. All the tubes were kept in an ice-bath (5°C.) throughout the homogenization and acid extraction procedures. The acid soluble fraction was brought to a known volume and the radioactivity in an aliquot determined in the same manner as for the whole homogenate. The total phosphorus in the fraction was determined according to the method of Fiske and Subbarow (1925) as described by LePage (1957). Standards were prepared and analyzed simultaneously with the unknowns in order to construct a standard curve. All the samples were subjected to the entire procedure in duplicate.
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3, 12 and 24 hours after isotope administration. I n an earlier investigation involving older hypophysectomized chickens, M a and Nalbandov (1963) also observed greater 32P uptake by the whole adrenals of hypophysectomized chickens than by control adrenals. This paper reports a more detailed comparison of the 82P uptake by the acid soluble fraction of adrenals of hypophysectomized and control cockerels. Of particular importance, greater radioactivity was noted for the plasma of hypophysectomized chickens at three hours after isotope administration. Consequently, corrections were made for differences in plasma specific activity by expressing the adrenal radioactivity relative to t h a t of the plasma.
KING
ADRENAL
82
P
quot plated for counting and the remainder stored at approximately — 20° C. until phosphorus analysis. A mixture of 1.0 ml. of plasma and 1.0 ml. of cold 20% trichloroacetic acid was centrifuged at 0°C. and the pellet re-extracted twice with 1.0 ml. of cold 10% trichloroacetic acid. The combined supernatants were then brought to a known volume with water and an aliquot plated for counting. The phosphorus in a 1.0 ml. aliquot was assayed, after the addition of 0.4 ml. of 10 N H 2 S0 4 , by the same procedure used to determine the adrenal total acid soluble phosphorus. Plasma phosphorus determinations were also done in duplicate. All data were analyzed statistically with Student's "t" test (Snedecor, 1956). RESULTS AND DISCUSSION
The adrenal and plasma radioactivity of hypophysectomized and control cockerels at three hours after the administration of 32P are summarized in Tables 1 and 2. The cpm/mg. fresh adrenal were 78% higher in hypophysectomized birds than controls, while the specific activity of the
TABLE 1.—Radioactivity in the acid soluble fraction of the adrenals following hypophysectomy. All values are means+ SE
Adrenal Weight (mg.) Total Acid Soluble Phosphorus (/xg./mg. Adrenal) Inorganic Phosphorus (jig./mg. Adrenal) cpm/mg. Adrenal Relative* cpm/mg. Adrenal cpm/ug. Acid Soluble Phosphorus Relative cpm//xg. Acid Soluble Phosphorus cpm/jug. Inorganic Phosphorus Relative cpm/ug. Inorganic Phosphorus
Hypophysectomized (n=ll)
Controls (n=15)
P value
61.6+3.1
92.7 + 3.0
<.001
2.20 + 0.07
1.84 + 0.05
<.001
0.85 + 0.05 116 + 4
0.77 + 0.04 65 + 2
NS <.0Q1
53 + 3
46 + 2
NS
53 + 2
33 + 2
<.001
23 + 2
25 + 2
NS
29 + 2
15+1
<.001
13+1
12+1
NS
* cpm/mg. or cpm/ug. phosphorus divided by the specific activity of the plasma (cpm/ug. acid soluble phosphorus) X100.
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The adrenal inorganic phosphorus specific activity was determined by the method of Ernster et al. (1950). The trichloroacetic acid extracts were stored in the refrigerator and placed in an ice-bath during sampling to prevent, or at least reduce, the decomposition of labile organic compounds. A satisfactory plate for counting was obtained when small amounts of the isobutanolbenzene phase were added to a planchet and evaporated after each addition until 1.0 ml. was plated. The entire inorganic phosphorus procedure was run in duplicate. Phosphorus standards and trichloroacetic acid and water blanks were analyzed simultaneously with the unknowns. The specific activities of the whole homogenate, total acid soluble phosphorus and inorganic phosphorus were also expressed relative to the specific activity of the plasma acid soluble phosphorus. Blood was collected immediately following decapitation in heparinized centrifuge tubes and placed in an ice-bath until the end of autopsy. At this time, the plasma was collected by centrifugation (0°C), an ali-
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B.
KING
TABLE 2. Radioa-ctivity in the acid soluble fraction of the plasma following hypophysectomy. A11 values are means +SE
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the 79% higher plasma total acid soluble phosphorus specific activity in our hypophysectomized cockerels at three hours cpm/Mg. Numafter isotope administration (Table 2). Total b er Treatment „ f phosphorus X/™1' 'total ? of A similar adrenal 32P uptakef or hypophyOug./ml.) ,. . 1,/ui plasma ph. phorus birds sectomized and control cockerels, when Hypophysectomy 11 56.3±2.6 12,569+952 228+21 Controls 15 55.6±2.4 7,293+290 135+11 corrected for differences in plasma phosP value NS <.001 <.001 phorus specific activity, compared to a marked depression of 32P uptake by the adrenal total acid soluble phosphorus was adrenals of hypophysectomized rats is 6 1 % greater and the specific activity of consonant with the changes in adrenal the inorganic phosphorus 93% greater. weight and steroid production following This greater 32P uptake by the adrenals of hypophysectomy of the two species. The hypophysectomized cockerels apparently extensive atrophy of mammalian adrenals was due to the higher specific activity of following hypophysectomy is well-known, the plasma acid soluble phosphorus (Table whereas the slightly smaller adrenals of 2), since in all cases the adrenal radioac- hypophysectomized chickens may simply tivity relative to the specific activity of reflect their lesser body weight (Newthe plasma acid soluble phosphorus was comer, 1959; King, 1965). Staehelin not different for control and pituitary- et al. (1965) found that the rat adrenal less birds. Similar corrections for dif- corticosterone production was reduced to ferences in plasma specific activity were nearly nondetectable levels by one day made in studies of the phosphorus metab- after hypophysectomy and was no longer olism of the rat adrenal, but the specific increased following ACTH administration activity of the adrenal phosphorus relative at 2 or 3 days following pituitary removal. to the plasma specific activity was still Frankel et al. (1967), on the other hand, considerably less for hypophysectomized found that the corticosterone concentrarats than controls (Reidel et al., 1954 a, b). tion in the adrenal effluent plasma of At two hours after 32P administration the hypophysectomized chickens was about relative adrenal inorganic phosphorus spe- 37% of the level of intact birds at 18 to 52 cific activity of rats hypophysectomized days after removal of the adenohypophyfor 14 days was 46% less than controls, sis. Also, ACTH increased adrenal corwhile at four hours after 82P it was 58% ticosterone secretion of chickens hypophyless (Reidel et al., 1954a). Similarly, at sectomized for one month by about the two hours after isotope administration, same magnitude as intact controls (Resko the relative adrenal total acid soluble etal., 1964). phosphorus specific activity was about Apparently the lesser adrenal steroid 49% less for rats hypophysectomized 2 production of the hypophysectomized 4 days earlier than for normal controls chicken is not reflected by a decrease in (Reidel et al., 1954b). Plasma inorganic 32 P uptake like that of the more atrophic phosphorus specific activity of rats hyrat adrenal. It must be emphasized, howpophysectomized for 14 days was about ever, that more subtle changes in adrenal 86% greater than controls at two hours phosphorus metabolism following removal after isotope administration and about of the adenohypophysis may have been 167% greater at four hours after isotope missed. There was a slight increase (about (Reidel et al., 1954b). This compares with 16%) in the adrenal total acid soluble
ADRENAL
phosphorus level following hypophysectomy of cockerels but no significant change in the concentration of inorganic phosphorus (Table 1). In the rat there is a slightly lesser (about 13%) adrenal total acid soluble phosphorus concentration at 2 to 6 days following hypophysectomy and no difference in adrenal inorganic phosphorus level (Reidel et al., 1954a; Gemzell and Samuels, 1950).
REFERENCES Breneman, W. R., F. J. Zeller and R. O. Creek, 1965. Radioactive phosphorus uptake by chick testes as an end-point for gonadotropin assay. Endocrinology, 71: 790-798. Bukovsan, W., 1967. The adrenal phosphorus (32P) metabolism of the newly hatched chick. A. A radio-bioassay for adrenocorticotropin. B. The
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separation and identification of some of the perchloric acid soluble phosphate compounds. Ph.D. Thesis, Zoology Department, Indiana University. Connell, G. M., 1965. Bioassay of pregnant mare serum gonadotrophin by the chick-32P method. Acta Endocrinol. 50: 249-253. Ernster,:L., R. Zetterstrom and 0 . Lindberg, 1950. A method for the determination of tracer phosphate in biological material. Acta Chem. Scand. 4: 942947. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Frankel, A. I., J. W. Graber and A. V. Nalbandov, 1967. Adrenal function in cockerels. Endocrinology, 80:1013-1019. Gemzell, C. A., and L. T. Samuels, 1950. The effect of hypophysectomy, adrenalectomy and of ACTH administration on the phosphorus metabolism of rats. Endocrinology, 47: 48-59. Greenspan, F. S., J. D. Kriss, L. E. Moses and W. Lew, 1956. An improved bioassay method for thyrotropic hormone using thyroid uptake of radio-phosphorus. Endocrinology, 58: 767-776. King, D. B., 1965. Hypophysectomy and hormonal replacement in young cockerels. I. Effects on bone growth, body weight and certain organ weights. II. Investigation of the phosphorus metabolism of target organs. Ph.D. Thesis, Zoology Department, Indiana University. LePage, G. A., 1957. Methods for the analysis of phosphorylated intermediates, pp. 268-287. In W. W. Umbreit, R. H. Burris and J. R. Stauffer, eds., Manometric Techniques. Burgess Publishing Co., Minneapolis. Ma, R. C. S., and A. V. Nalbandov, 1963. In A. V. Nalbandov, ed., Advances in Neuroendocrinology, pp. 306-311. University of Illinois Press, Urbana. Newcomer, W. S., 1959. Effects of hypophysectomy on some functional aspects of the adrenal gland of the chicken. Endocrinology, 65: 133-135. Reidel, B. E., J. E. Logan, H. A. DeLuca and R. J. Rossiter, 1954a. Phosphorus metabolism of the adrenal gland: Effect of hypophysectomy and administration of ACTH on incorporation of P32 into inorganic phosphate. Canad. J. Biochem. Physiol. 32: 251-260. Reidel, B. E., J. E. Logan and R. J. Rossiter, 1954b. Phosphorus metabolism of the adrenal gland: Effect of hypophysectomy and administration of ACTH on incorporation of P32 in phospholipid. Endocrinology, 55: 219-229. Resko, J. A., H. W. Norton and A. V. Nalbandov,
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SUMMARY
The adrenals of cockerels hypophysectomized for about one month had incorporated considerably greater amounts of radioactivity at three hours after the administration of 32P than the adrenals of controls. The cpm/mg. fresh adrenal and the specific activities of the total adrenal acid soluble phosphorus and inorganic phosphorus were not different for hypophysectomized and control cockerels, however, when the values were expressed relative to the specific activity of the plasma acid soluble phosphorus. This same adrenal 32P incorporation, relative to plasma specific activity, for hypophysectomized and control cockerels is consonant with the known lack of adrenal weight change (relative to body weight) and continued but lesser steroid production which follows pituitary removal. In contrast, other studies show that the rat adrenal decreases considerably in weight following hypophysectomy, loses its capacity to synthesize corticosterone and incorporates less 32P (relative to plasma specific activity).
32
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D. B. KING
1964. Endocrine control of the adrenals in chickens. Endocrinology, 75: 192-200. Snedecor, G. W., 1956. Statistical Methods. 5th ed., The Iowa State College Press.
Staehelin, M., P. Barthe and P. A. Desaulles, 196S. On the mechanism of the adrenal gland response to adrenocorticotrophic hormone in hypophysectomized rats. Acta Endocrinol. 50: 55-64.
Hemoglobin Types in Chick Embryos with Different Adult Hemoglobin Genotypes
(Received for publication August 15, 1968)
I
T HAS been demonstrated that genetic variation exists in hemoglobin types of the hatched chick. The hemoglobin types have been designated as Type I (homozygous normal), Type II (homozygous abnormal) and Type III (heterozygote) by Washburn (1968a). A "major band" is present in all birds in combination with either or both of two "minor", more acidic bands. The bands differ in their electrophoretic properties; the abnormal "minor" band migrates in an electrical field at a faster rate than that of the normal "minor" hemoglobin component. The differences in electrophoretic properties of the minor hemoglobin components are due to allelic, codominant genes (Washburn, 1968a). The adult hemoglobin type can vary, depending on the genotype of the bird. This variation could explain some of the disagreement found in the literature pertaining to the normal hemoglobin types of chick embryos, when the adult type hemoglobin (Hb) appears, and which components should be considered as embryonic. D'Amelio and Salvo (1961), using electrophoretic and immunoelectrophoretic analyses, found two distinct embryonic Hb components present at 68 hours of emUniversity of Georgia College of Agriculture Experiment Stations Journal Series Paper number 323, College Station, Athens.
bryonic development. By 88 hours of incubation, they found three additional hemoglobin fractions which they stated are identical to those present in hemoglobin from adult chickens. By the eleventh day of incubation, they reported that only traces of the embryonic hemoglobin were detectable in the embryo and that the adult type hemoglobins were the dominant respiratory blood proteins. Manwell et al. (1963) found that three and four day-old embryos had a distinctly different hemoglobin from that of late embryos or adults. Prior to the fifth day of incubation, only the two embryonic hemoglobin components were visible; but by the seventh day approximately one-half of the hemoglobin was of the adult type. In agreement with D'Amelio and Salvo (1961) they observed only traces of embryonic hemoglobin remaining after eleven days of incubation. The only apparent difference between their studies and those of D'Amelio and Salvo (1961), was whether there were three rather than two hemoglobin components in the adult chicken. Manwell et al. (1963) could detect only two adult components, both of which were visible by the seventh day of embryogenesis. They also found that there was no difference in the electrophoretic behavior or distribution of the hemoglobins between the various breeds of birds tested.
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C. R. DENMARK AND K. W. WASHBURN Department of Poultry Science, University of Georgia, Athens, Georgia 30601
jections in the young cockerel. Endocrinol. 51: 152-154. Shellabarger, C. J., 1953. Observations of the pineal in the White Leghorn capon and cockerel. Poultry Sci. 32: 189-197. Shellabarger, C. J., and W. R. Breneman, 1949. The effects of pinealectomy on young White Leghorn cockerels. Indiana Acad. Sci. Proc. 59: 299-302. Wilson, W. O., U. K. Abbott and H. Abplanalp, 1961. Evaluation of Coturnix (Japanese quail) as pilot animal for poultry.Poultry Sci. 40: 651-657. Wilson, W. O., H. Abplanalp and L. Arrington, 1962. Sexual development of Coturnix as affected by changes in photoperiods. Poultry Sci. 41: 17-22. Wolfson, A., 1952. The cloacal protuberance. Bird Banding, 23: 159-165. Woodard, A. E., H. Abplanalp and W. O. Wilson, 1965. Japanese quail husbandry in the laboratory (Coturnix coturnix japonica). Dept. Poultry Husbandry, Univ. of Calif., Davis. 36 pp. Wurtman, R. J., and J. Axelrod, 1965. The pineal gland. Scientific Amer. 213: 50-60.
Effect of Hypophysectomy on the Radioactive Phosphorus Uptake of Chick Adrenals1'2 DAVID B. K I N G
Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604 (Received for publication August 15, 1968)
T
HE rate of incorporation of radio- chickens would be expected to incorporate active phosphorus (32P) into the tar- less radioactivity due to lower levels of get organs of young chickens serves as an endogenous tropic hormones. Surprisingly, end-point in the bioassay procedures for investigations of the 32P uptake by these thyrotropic, gonadotropic and adrenocor- organs did not show a decreased incorticotropic hormones (Greenspan et al., poration of the isotope following hypo1956;Brenemanetal., 1965; Connell, 1965; physectomy of young cockerels (King, Bukovsan, 1967). The tropic hormones 1965). Instead, the adrenals and thyroids stimulate an increased incorporation of of hypophysectomized chickens incorporadioactivity per unit weight of their spe- rated at least twice as much radioactivity cific target organ. Since 32P uptake is per mg of tissue during a 48-hour period stimulated by tropic hormone, the pitu- following isotope administration, while the itary target organs of hypophysectomized 32P uptake by the gonads was the same or slightly greater than controls. When the 1 Part of a thesis submitted to the Zoology Deadrenals and thyroids were divided into partment of Indiana University in partial fulfillment trichloroacetic acid soluble, lipid and proof the requirements for the Ph.D. tein-nucleic acid fractions, the greater ra2 Supported in part by a pre-doctoral National dioactivity was found in each fraction at Aeronautical and Space Administration Fellowship.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 24, 2015
physiological actions of the pineal gland. Ann. Int. Med. 61: 1144-1161. Farner, D. S., 1964. The photoperiodic control of reproductive cycles in birds. Amer. Scientist, 52: 137-156. Hoffman, R. A., and R. J. Reiter, 1965a. Influence of compensatory mechanisms and of the pineal gland on dark-induced gonadal atrophy in male hamsters. Nature, 207: 658-659. Hoffman, R. A., and R. J. Reiter, 1965b. Pineal gland: Influence on gonads of male hamsters. Science, 148: 1609-1610. Kitay, J. I., and M. D. Altschule, 1954. Effects of pineal extract administration on ovary weight in rats. Endocrinol. 55: 782-784. Mather, F. B., and W. O. Wilson, 1964. Post-natal testicular development in Japanese quail (Coturnix coturnix japonica). Poultry Sci. 43: 860864. Relkin, R., 1966. The pineal gland. New England J. Med. 274: 944-950. Shellabarger, C. J., 1952. Pinealectomy vs pineal in-
459
460
D.
B.
MATERIALS AND METHODS White Leghorns cockerels (State F a r m Bureau Hatchery, Indianapolis, Indiana) were hypophysectomized at about three weeks of age by a parapharyngeal method described in detail elsewhere (King, 1965). Complete removal of the adenohypophysis was confirmed at autopsy for all hypophysectomized cockerels included in the results by a careful inspection of the pituitary region under a dissecting scope. Control and hypophysectomized cockerels were housed in constant temperature animal rooms (75° F.) under continuous lighting and were fed Wayne Pullet Grower and water ad libitum. The hypophysectomized chickens were provided with extra heat for a period of at least three weeks following the operation. Hypophysectomized and control cockerels were injected with 20/uc. 3 2 P/100 g. body weight at 53 days of age (about one month after hypophysectomy) and killed three hours later. T h e 32P (Oak Ridge National Laboratories; at least 9 9 % free of non-radioactive phosphorus) was re-
ceived as phosphate in weak HC1, diluted to the desired concentration with distilled water and injected subcutaneously at a volume of 0.1 to 0.3 ml. T h e birds were killed alternately from each group b y decapitation and the adrenals removed, frozen on dry-ice and weighed to the nearest 0.2 mg. T h e tissue was homogenized in cold 10% trichloroacetic acid with a motor-driven teflon pestle (TriR Corp.) and the homogenate transferred to a centrifuge tube; the homogenization tube and pestle were washed at least three times with cold 1 0 % trichloroacetic acid. T h e homogenate was brought to a known volume with trichloroacetic acid, an aliguot plated on a disposable planchet, dried and the radioactivity measured with a Baird Atomic Multiscaler (Model 132) connected to a thin window (1.7 mg./cm. 2 ) Geiger Muller tube. T h e counts per minute (cpm) were corrected for background and decay. T h e remaining homogenate was centrifuged at 800 to l , 0 0 0 X g for 10 minutes in a refrigerated centrifuge ( 0 ° C ) . T h e pellet remaining after centrifugation was re-extracted twice with approximately 3.0 ml. of cold 1 0 % trichloroacetic acid. The supernatants from the three extractions were combined and designated the acid soluble fraction. All the tubes were kept in an ice-bath (5°C.) throughout the homogenization and acid extraction procedures. The acid soluble fraction was brought to a known volume and the radioactivity in an aliquot determined in the same manner as for the whole homogenate. The total phosphorus in the fraction was determined according to the method of Fiske and Subbarow (1925) as described by LePage (1957). Standards were prepared and analyzed simultaneously with the unknowns in order to construct a standard curve. All the samples were subjected to the entire procedure in duplicate.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 24, 2015
3, 12 and 24 hours after isotope administration. I n an earlier investigation involving older hypophysectomized chickens, M a and Nalbandov (1963) also observed greater 32P uptake by the whole adrenals of hypophysectomized chickens than by control adrenals. This paper reports a more detailed comparison of the 82P uptake by the acid soluble fraction of adrenals of hypophysectomized and control cockerels. Of particular importance, greater radioactivity was noted for the plasma of hypophysectomized chickens at three hours after isotope administration. Consequently, corrections were made for differences in plasma specific activity by expressing the adrenal radioactivity relative to t h a t of the plasma.
KING
ADRENAL
82
P
quot plated for counting and the remainder stored at approximately — 20° C. until phosphorus analysis. A mixture of 1.0 ml. of plasma and 1.0 ml. of cold 20% trichloroacetic acid was centrifuged at 0°C. and the pellet re-extracted twice with 1.0 ml. of cold 10% trichloroacetic acid. The combined supernatants were then brought to a known volume with water and an aliquot plated for counting. The phosphorus in a 1.0 ml. aliquot was assayed, after the addition of 0.4 ml. of 10 N H 2 S0 4 , by the same procedure used to determine the adrenal total acid soluble phosphorus. Plasma phosphorus determinations were also done in duplicate. All data were analyzed statistically with Student's "t" test (Snedecor, 1956). RESULTS AND DISCUSSION
The adrenal and plasma radioactivity of hypophysectomized and control cockerels at three hours after the administration of 32P are summarized in Tables 1 and 2. The cpm/mg. fresh adrenal were 78% higher in hypophysectomized birds than controls, while the specific activity of the
TABLE 1.—Radioactivity in the acid soluble fraction of the adrenals following hypophysectomy. All values are means+ SE
Adrenal Weight (mg.) Total Acid Soluble Phosphorus (/xg./mg. Adrenal) Inorganic Phosphorus (jig./mg. Adrenal) cpm/mg. Adrenal Relative* cpm/mg. Adrenal cpm/ug. Acid Soluble Phosphorus Relative cpm//xg. Acid Soluble Phosphorus cpm/jug. Inorganic Phosphorus Relative cpm/ug. Inorganic Phosphorus
Hypophysectomized (n=ll)
Controls (n=15)
P value
61.6+3.1
92.7 + 3.0
<.001
2.20 + 0.07
1.84 + 0.05
<.001
0.85 + 0.05 116 + 4
0.77 + 0.04 65 + 2
NS <.0Q1
53 + 3
46 + 2
NS
53 + 2
33 + 2
<.001
23 + 2
25 + 2
NS
29 + 2
15+1
<.001
13+1
12+1
NS
* cpm/mg. or cpm/ug. phosphorus divided by the specific activity of the plasma (cpm/ug. acid soluble phosphorus) X100.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 24, 2015
The adrenal inorganic phosphorus specific activity was determined by the method of Ernster et al. (1950). The trichloroacetic acid extracts were stored in the refrigerator and placed in an ice-bath during sampling to prevent, or at least reduce, the decomposition of labile organic compounds. A satisfactory plate for counting was obtained when small amounts of the isobutanolbenzene phase were added to a planchet and evaporated after each addition until 1.0 ml. was plated. The entire inorganic phosphorus procedure was run in duplicate. Phosphorus standards and trichloroacetic acid and water blanks were analyzed simultaneously with the unknowns. The specific activities of the whole homogenate, total acid soluble phosphorus and inorganic phosphorus were also expressed relative to the specific activity of the plasma acid soluble phosphorus. Blood was collected immediately following decapitation in heparinized centrifuge tubes and placed in an ice-bath until the end of autopsy. At this time, the plasma was collected by centrifugation (0°C), an ali-
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B.
KING
TABLE 2. Radioa-ctivity in the acid soluble fraction of the plasma following hypophysectomy. A11 values are means +SE
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the 79% higher plasma total acid soluble phosphorus specific activity in our hypophysectomized cockerels at three hours cpm/Mg. Numafter isotope administration (Table 2). Total b er Treatment „ f phosphorus X/™1' 'total ? of A similar adrenal 32P uptakef or hypophyOug./ml.) ,. . 1,/ui plasma ph. phorus birds sectomized and control cockerels, when Hypophysectomy 11 56.3±2.6 12,569+952 228+21 Controls 15 55.6±2.4 7,293+290 135+11 corrected for differences in plasma phosP value NS <.001 <.001 phorus specific activity, compared to a marked depression of 32P uptake by the adrenal total acid soluble phosphorus was adrenals of hypophysectomized rats is 6 1 % greater and the specific activity of consonant with the changes in adrenal the inorganic phosphorus 93% greater. weight and steroid production following This greater 32P uptake by the adrenals of hypophysectomy of the two species. The hypophysectomized cockerels apparently extensive atrophy of mammalian adrenals was due to the higher specific activity of following hypophysectomy is well-known, the plasma acid soluble phosphorus (Table whereas the slightly smaller adrenals of 2), since in all cases the adrenal radioac- hypophysectomized chickens may simply tivity relative to the specific activity of reflect their lesser body weight (Newthe plasma acid soluble phosphorus was comer, 1959; King, 1965). Staehelin not different for control and pituitary- et al. (1965) found that the rat adrenal less birds. Similar corrections for dif- corticosterone production was reduced to ferences in plasma specific activity were nearly nondetectable levels by one day made in studies of the phosphorus metab- after hypophysectomy and was no longer olism of the rat adrenal, but the specific increased following ACTH administration activity of the adrenal phosphorus relative at 2 or 3 days following pituitary removal. to the plasma specific activity was still Frankel et al. (1967), on the other hand, considerably less for hypophysectomized found that the corticosterone concentrarats than controls (Reidel et al., 1954 a, b). tion in the adrenal effluent plasma of At two hours after 32P administration the hypophysectomized chickens was about relative adrenal inorganic phosphorus spe- 37% of the level of intact birds at 18 to 52 cific activity of rats hypophysectomized days after removal of the adenohypophyfor 14 days was 46% less than controls, sis. Also, ACTH increased adrenal corwhile at four hours after 82P it was 58% ticosterone secretion of chickens hypophyless (Reidel et al., 1954a). Similarly, at sectomized for one month by about the two hours after isotope administration, same magnitude as intact controls (Resko the relative adrenal total acid soluble etal., 1964). phosphorus specific activity was about Apparently the lesser adrenal steroid 49% less for rats hypophysectomized 2 production of the hypophysectomized 4 days earlier than for normal controls chicken is not reflected by a decrease in (Reidel et al., 1954b). Plasma inorganic 32 P uptake like that of the more atrophic phosphorus specific activity of rats hyrat adrenal. It must be emphasized, howpophysectomized for 14 days was about ever, that more subtle changes in adrenal 86% greater than controls at two hours phosphorus metabolism following removal after isotope administration and about of the adenohypophysis may have been 167% greater at four hours after isotope missed. There was a slight increase (about (Reidel et al., 1954b). This compares with 16%) in the adrenal total acid soluble
ADRENAL
phosphorus level following hypophysectomy of cockerels but no significant change in the concentration of inorganic phosphorus (Table 1). In the rat there is a slightly lesser (about 13%) adrenal total acid soluble phosphorus concentration at 2 to 6 days following hypophysectomy and no difference in adrenal inorganic phosphorus level (Reidel et al., 1954a; Gemzell and Samuels, 1950).
REFERENCES Breneman, W. R., F. J. Zeller and R. O. Creek, 1965. Radioactive phosphorus uptake by chick testes as an end-point for gonadotropin assay. Endocrinology, 71: 790-798. Bukovsan, W., 1967. The adrenal phosphorus (32P) metabolism of the newly hatched chick. A. A radio-bioassay for adrenocorticotropin. B. The
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UPTAKE
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separation and identification of some of the perchloric acid soluble phosphate compounds. Ph.D. Thesis, Zoology Department, Indiana University. Connell, G. M., 1965. Bioassay of pregnant mare serum gonadotrophin by the chick-32P method. Acta Endocrinol. 50: 249-253. Ernster,:L., R. Zetterstrom and 0 . Lindberg, 1950. A method for the determination of tracer phosphate in biological material. Acta Chem. Scand. 4: 942947. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Frankel, A. I., J. W. Graber and A. V. Nalbandov, 1967. Adrenal function in cockerels. Endocrinology, 80:1013-1019. Gemzell, C. A., and L. T. Samuels, 1950. The effect of hypophysectomy, adrenalectomy and of ACTH administration on the phosphorus metabolism of rats. Endocrinology, 47: 48-59. Greenspan, F. S., J. D. Kriss, L. E. Moses and W. Lew, 1956. An improved bioassay method for thyrotropic hormone using thyroid uptake of radio-phosphorus. Endocrinology, 58: 767-776. King, D. B., 1965. Hypophysectomy and hormonal replacement in young cockerels. I. Effects on bone growth, body weight and certain organ weights. II. Investigation of the phosphorus metabolism of target organs. Ph.D. Thesis, Zoology Department, Indiana University. LePage, G. A., 1957. Methods for the analysis of phosphorylated intermediates, pp. 268-287. In W. W. Umbreit, R. H. Burris and J. R. Stauffer, eds., Manometric Techniques. Burgess Publishing Co., Minneapolis. Ma, R. C. S., and A. V. Nalbandov, 1963. In A. V. Nalbandov, ed., Advances in Neuroendocrinology, pp. 306-311. University of Illinois Press, Urbana. Newcomer, W. S., 1959. Effects of hypophysectomy on some functional aspects of the adrenal gland of the chicken. Endocrinology, 65: 133-135. Reidel, B. E., J. E. Logan, H. A. DeLuca and R. J. Rossiter, 1954a. Phosphorus metabolism of the adrenal gland: Effect of hypophysectomy and administration of ACTH on incorporation of P32 into inorganic phosphate. Canad. J. Biochem. Physiol. 32: 251-260. Reidel, B. E., J. E. Logan and R. J. Rossiter, 1954b. Phosphorus metabolism of the adrenal gland: Effect of hypophysectomy and administration of ACTH on incorporation of P32 in phospholipid. Endocrinology, 55: 219-229. Resko, J. A., H. W. Norton and A. V. Nalbandov,
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SUMMARY
The adrenals of cockerels hypophysectomized for about one month had incorporated considerably greater amounts of radioactivity at three hours after the administration of 32P than the adrenals of controls. The cpm/mg. fresh adrenal and the specific activities of the total adrenal acid soluble phosphorus and inorganic phosphorus were not different for hypophysectomized and control cockerels, however, when the values were expressed relative to the specific activity of the plasma acid soluble phosphorus. This same adrenal 32P incorporation, relative to plasma specific activity, for hypophysectomized and control cockerels is consonant with the known lack of adrenal weight change (relative to body weight) and continued but lesser steroid production which follows pituitary removal. In contrast, other studies show that the rat adrenal decreases considerably in weight following hypophysectomy, loses its capacity to synthesize corticosterone and incorporates less 32P (relative to plasma specific activity).
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1964. Endocrine control of the adrenals in chickens. Endocrinology, 75: 192-200. Snedecor, G. W., 1956. Statistical Methods. 5th ed., The Iowa State College Press.
Staehelin, M., P. Barthe and P. A. Desaulles, 196S. On the mechanism of the adrenal gland response to adrenocorticotrophic hormone in hypophysectomized rats. Acta Endocrinol. 50: 55-64.
Hemoglobin Types in Chick Embryos with Different Adult Hemoglobin Genotypes
(Received for publication August 15, 1968)
I
T HAS been demonstrated that genetic variation exists in hemoglobin types of the hatched chick. The hemoglobin types have been designated as Type I (homozygous normal), Type II (homozygous abnormal) and Type III (heterozygote) by Washburn (1968a). A "major band" is present in all birds in combination with either or both of two "minor", more acidic bands. The bands differ in their electrophoretic properties; the abnormal "minor" band migrates in an electrical field at a faster rate than that of the normal "minor" hemoglobin component. The differences in electrophoretic properties of the minor hemoglobin components are due to allelic, codominant genes (Washburn, 1968a). The adult hemoglobin type can vary, depending on the genotype of the bird. This variation could explain some of the disagreement found in the literature pertaining to the normal hemoglobin types of chick embryos, when the adult type hemoglobin (Hb) appears, and which components should be considered as embryonic. D'Amelio and Salvo (1961), using electrophoretic and immunoelectrophoretic analyses, found two distinct embryonic Hb components present at 68 hours of emUniversity of Georgia College of Agriculture Experiment Stations Journal Series Paper number 323, College Station, Athens.
bryonic development. By 88 hours of incubation, they found three additional hemoglobin fractions which they stated are identical to those present in hemoglobin from adult chickens. By the eleventh day of incubation, they reported that only traces of the embryonic hemoglobin were detectable in the embryo and that the adult type hemoglobins were the dominant respiratory blood proteins. Manwell et al. (1963) found that three and four day-old embryos had a distinctly different hemoglobin from that of late embryos or adults. Prior to the fifth day of incubation, only the two embryonic hemoglobin components were visible; but by the seventh day approximately one-half of the hemoglobin was of the adult type. In agreement with D'Amelio and Salvo (1961) they observed only traces of embryonic hemoglobin remaining after eleven days of incubation. The only apparent difference between their studies and those of D'Amelio and Salvo (1961), was whether there were three rather than two hemoglobin components in the adult chicken. Manwell et al. (1963) could detect only two adult components, both of which were visible by the seventh day of embryogenesis. They also found that there was no difference in the electrophoretic behavior or distribution of the hemoglobins between the various breeds of birds tested.
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C. R. DENMARK AND K. W. WASHBURN Department of Poultry Science, University of Georgia, Athens, Georgia 30601