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Isolation and identification of testosterone from the serum and testes of the American lobster (Homarus americanus)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
54, 429-432 (1984)
Isolation and Identification of Testosterone from the Serum and Testes of the American Lobster (Homarus americanus) B. G. BURNS, G. B. SANGALANG, H. C. FREEMAN, AND M. MCMENEMY Fisheries
and Environmental P.O.
Sciences, Department Box 550 Halifax, Nova
of Fisheries and Oceans, Scotia B3J 2S7, Canada
Halifax
Laboratory,
Accepted May 6, 1983 Testosterone was isolated and quantified from male lobster serum and testes by solvent extraction, sequential thin-layer chromatography, and high-performance liquid chromatography. The identity of the isolated steroid was established by its isopolarity and isomorphicity with authentic radiolabeled testosterone and its acetate derivative. The concentrations of testosterone were determined by high-performance liquid chromatography, by a doubleisotope derivative assay, and by radioimmunoassay. The concentrations of testosterone as determined by the three methods were the same and were 0.3 rig/ml and 14.3 rig/g in lobster serum and testes, respectively.
The occurrence of vertebrate-type steroid hormones in marine invertebrates is well documented (Donahue, 1948, 1952, 1957; Hagerman et al., 1957; Lisk, 1961; Schwerdtfeger, 1932; Steidle, 1930; Gottfried et al., 1967; Teshima and Kanazawa, 1970; Nikitina et al., 1977). The identification of steroids, with the exception of the ecdysteroid molting hormones (Gagosian and Bourbonniere, 1976), in the American lobster Homarus americanus has received little attention. Previous investigations have shown that the lobster contains some of the enzyme systems necessary for steroidogenesis. Gilgan and Idler (1967) reported that lobster testes and androgenic glands were able to transform 4-[‘4C]androstenedione-3, 17dione to testosterone in vitro but did not establish the presence of testosterone in the animal. Burns et al. (1982) demonstrated the ability of the lobster testes to transform the steroid hormone precursors pregnenolone and progesterone to 20 a-dihydro-derivatives in vitro. Kanazawa and Teshima (1971) reported the in vivo conversion of cholesterol to vertebrate steroid hormones in the spiny lobster Panulirus japonica.
In the present study testosterone was isolated and identified in both male lobster serum and testes. MATERIALS AND METHODS Collection oflobsters. Adult male lobsters were collected from the Miramichi River estuary (New Brunswick, Canada) during late spring of 1981. The animals were transported to the aquarium at the Halifax laboratory where they were held in tanks of continuously flowing filtered seawater for a maximum of 2 weeks before sampling. Lobster hemolymph was collected from 50 male lobsters (mean weight ca. 300 g) by syringe from the abdominal sinus. The pooled sample was centrifuged (8000 rpm for 15 min) at O-4” and the serum was removed and stored at - 30” until used. The testes (6 g from 12 lobsters) were carefully excised from the lobsters, minced with scissors, pooled, and extracted foI steroids immediately. Extraction
of tesfosterone
from
serum
and testes.
The serum and minced lobster testes were each adjusted to pH 9.0 with 1 N NaOH and were extracted once with 10 vol of methylene chloride. Each of the extracts was washed with 0.1 N sodium bicarbonate, 0.1 N acetic acid, and distilled water as described previously (Idler et al., 1971). The methylene chloride was removed by flash evaporation at 40” and the residue was partitioned between 80% methanol and hexane as described by Idler et al. (1971). Thin-layer chromatography (TLC). TLC was carried out on glass plates coated with silica gel HFZS4-366 (Merck, Darmstadt, West Germany). The solvent sys-
429 0016~6480/84 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved
BURNS ET AL.
430
terns used (by volume) were (I) hexane:ethyl acetate (4: 1); (II) chloroform:methanol:water (188:12:1); (III) t-butanol:hexane (25:75); (IV) methylene chloride:acetone (95:5); (V) cyclohexane:ethyl acetate (30:70); (VI) isopropanol:cyclohexane (30:70). High-performance
liquid
chromatography
(HPLC).
HPLC was performed using a Beckman Model 1lOA pump, a Waters Model U6K septumless injector, a Vydac 201TP (S-brn particle size, 4.6 mm x 25 cm) column, and a Schoeffel Model SF770 variable-wavelength detector. The ultraviolet absorbance of the testosterone in the effluent was measured at 241 nm. The solvent systems used were (I) 45% acetonitrile (isocratic); (II) 55% acetonitrile in water (isocratic); (III) a linear gradient of 30 to 70% acetonitrile in water (30 min) and then held at 70% acetonitrile in water. The flow rates of all systems was 1.0 ml/min. The linear gradient was obtained by pumping acetonitrile, at a constant rate under controlled conditions, into a mixing cylinder containing acetonitrile and water, which was the solvent reservoir for the Beckman pump. Radioactive chemicals. [14C]- and [3H]testosterone and r3H]acetic anhydride were purchased from New England Nuclear Corporation, Boston, Massachusetts. The radioactive steroids were purified by chromatography before use. Radioactive measurements. Radioactivity was measured using a Packard Tri-Garb, Model 3225, liquid scintillation spectrometer equipped with automatic external standardization. Isolation of steroid fractions. One-tenth volume of each of the extracts (serum and testes) was applied to a TLC plate and was developed twice in system I and once in system II. Steroids were detected by ultraviolet (uv) absorption (240 nm) and sulfuric acid:ethanol (1: 1) spray followed by heating at 105” for a few minutes for color development. The remaining nine-tenths of each extract was run similarly but steroids were located only by (uv) absorption. Three major areas were eluted: one isopolar with testosterone, one less polar (RT = 1.19), and one more-polar (RT = 0.77). Each of the above eluted areas were divided into two parts (one-tenth and nine-tenths). Each part was rechromatographed by TLC III, the steroids were located, and the area isopolar with testosterone was eluted as in the first chromatography. The testosterone area was further purified and quantified using HPLC (system I). One-third of the testosterone purified by HPLC was rechromatographed by HPLC with a trace of standard [3H]testosterone (4000 dpm). The remaining two-thirds was acetylated with [3H]acetic anhydride as described by Idler et al. (1972). Radioimmunoassay. The radioimmunoassay procedure for testosterone was as described by Sangalang and Freeman (1977). Double-isotope derivative assay (DZDA). The DIDA
procedure was similar to that reported by Idler et al. (1972). A trace quantity of [14C]testosterone (5000 dpm) was added to quantify 3H-acetylation as well as correct for losses during the procedure. The chromatographic systems used and sequences were TLC system IV, TLC system V, HPLC system II, TLC system VI, and HPLC system III. Approximately 20 pg of authentic nonradioactive testosterone acetate was added as carrier to the [14C]testosterone[3H]acetate before chromatography. Purification of the acetate was discontinued when ‘H/14C isotope ratios were constant for three consecutive chromatographic procedures. The identity of the isolated testosterone as testosterone acetate was then confirmed by recrystallization with nonradioactive standard testosterone acetate to constant 3H/‘4C ratios as described by AXelrod et al. (1965).
RESULTS AND DISCUSSi6N
Testosterone was isolated from lobster serum and its concentration was 0.3 r&ml by HPLC. Serum testosterone was homogeneous with standard testosterone in HPLC system I and its [3H]acetate derivative was isopolar with standard testosterone acetate in TLC system IV and HPLC system I, indicating that the isolited steroid was testosterone. The isolated testosterone reacted positively and specifically with testosterone antiserum when quantified by RIA. Its concentration by RIA was also 0.3 @ml. The testosterone in lobster serum was isolated and also quantified by DIDA and its concentration was again confirmed at 0.3 rig/ml. The final 3H/14C ratios of 3H-acetylated serum sample plus [‘4C]testosterone tracer and of [14C]testosterone tracer only were 2.83 and 0.79, respectively (Table 1). The specific activity of the [3H]acetic anhydride was 25.8 &i/pmol. Testosterone was also similarly isolated and quantified from the lobster testes. The concentration of testosterone in the lobster testes was 14.3 rig/g. Previous investigations have shown that lobster testes possess many of the enzyme systems necessary for steroidogenesis (Burns et al., 1982; Gilgan and Idler, 1967; Kanazawa and Teshma, 1971); however, to
TESTOSTERONE TABLE ‘H/i4C RATIOS OF 3H-A~~~~~~~~~
431
IN LOBSTER 1
ISOLATED TESTOSTERONE PLUS AUTHENTIC [‘4C]T~~~~~~~~~~~ ONLY
[L4C]T~~~~~~~~~~
AND
3H/‘4C ratio System TLC System IV TLC System V LC System II TLC System VI LC System III Recrystallization Ia Recrystallization IIc
Isolated + authentic testosterone acetate
Authentic testosterone acetate
261 24.5 2.81 2.81 2.83 2.83(2.83)b (2.83)(2.82)b
173 20.9 0.80 0.78 0.79 (0.79)(0.79)b (0.79)(0.79)b
a Acetonelhexane. b Ratios of mother liquor. c Acetonelpentane.
the authors’ knowledge, no steroids, other than the molting hormones, have been positively isolated and identified from the hemolymph of the American lobster. The level of testosterone in the serum of the lobster is low compared to the concentrations reported in the plasma of most fishes, amphibians, and birds where the values are approximately 100 times higher (Ozon, 1972). The plasma levels of testosterone in 90 vivaparous lizards during one annual cycle, however, ranged approximately two to six times higher than the level in lobster serum (Courty and Dufaure, 1982). The testosterone levels in the testes of the lizards ranged from 14.0 to 1472.9 rig/g during one annual cycle (Courty and Dufaure, 1982). The lowest concentration of testosterone in the lizard testes is close to the value of 14.3 rig/g found in the testes of the lobster in this study. The levels of testosterone in both lobster testes and serum may also have seasonal fluctuations as in the lizard, and the sampling of the lobsters in the present investigation could have been done at a time when testosterone levels were low. The occurrence of testosterone in the testes of the American lobster indicates that this steroid may function as an androgen as it does in
vertebrates. The role of testosterone in the reproductive physiology of the lobster deserves investigation. REFERENCES Axelrod, L. R., Matthijssen, C., Goldzieher, J. W., and Pulliam, J. E. (1965). Definitive identification of microquantities of radioactive steroids by recrystallization to constant specific alterity. Acta Endocrinol. Copenhngen 49 (Suppl. 99). Burns, B. G., Sangalang, G. B., Freeman, H. C., and McMenemy, M. (1982). In vitro bioconversion of steroids by the testes of the American lobster Homarus americanus. In preparation. Courty, II., and Dufaure, J. P. (1982). Circannual testosterone, dihydrotestosterone and androstanediols in plasma and testes of Lacerta vivipara, a seasonally breeding vivaparous lizard. Steroids 39, 517-529. Donahue, J. K. (1948). Fluorometric and biological determination of estrogens in the eggs of the American lobster (Homarus americanus). Proc. Sot. Exp. Biol. Med. 69, 179-181. Donahue, J. K. (1952). “Studies on Ecdysis in the American Lobster (Homarus americanus). I. The Lobster Egg as a Source of Estrogenic Hormone.” State of Maine, Department of Sea and Shore Fisheries Res. Bull. No. 8. Donahue, J. K. (1957). -“Chromatographic Identification of Lobster Egg Estrogen. State of Maine, Department of Sea and Shore Fisheries Res. Bull. No. 28. Idler, D. R., Horne, D. A., and Sangalang, G. B. (1971). Identification and quantification of the major androgens in testicular and peripheral
432
BURNS ET AL.
plasma of Atlantic salmon (Salmo salar) during sexual maturation. Gen. Comp. Endocrinol. 16, 251-267. Idler, D. R., Sangalang, G. B., and Truscott, B. (1972). Corticosteroids in the South American lungfish. Gen. Comp. Endocrinol. Suppl. 3, 238244. Kanazawa, A., and Teshima, S. (1971). In vivo conversion of cholesterol to steroid hormones in the spiny lobster, Panulirus japonica. Bull. Japan. Sot. Sci. Fish. 37, 891-898. Lisk, R. D. (1961). Estradiol-17 in the eggs of the American lobster, Homarus americanus. Canad. J. Biochem.
Physiol.
39, 659-662.
Nikitina, S. M., Savchenko, 0. N., Kogan, M. E., and Ezhkova, N. S., (1977). Preparative isolation of progesterone, testosterone, and estrogens from tissues of marine invertebrates. Zh. Evol. Biokhim. Fiziol. 13, 443-447.
Ozon, R. (1972). In “Steroids in Nonmammalian Vertebrates” (D. R. Idler, ed.), pp. 328-389. Academic Press, New York. Sangalang, G. B., and Freeman, H. C. (1977). A new sensitive and precise method for determining testosterone and 1I-ketotestosterone in fish plasma by radioimmunoassay. Gen. Comp. Endocrinol. 32, 432-439. Schwerdtfeger, H. (1932). Beitrlge zum Vorkomen und zur Wirkung der weiblichen Sexual-hormone. Arch. Exp. Pathol. Pharmakol. 163, 487-492. Steidle, H. (1930). Uber die Verbreitung des weiblichen sexual Hormones. Arch. Exp. Pathol. Pharmakol. 157, 89. Teshima, S., and Kanazawa, A. (1970). Production of 1 I-ketotestosterone and other steroids by the sliced ovaries of the crab Portunus trituberculatus.
Bull.
Japan.
Sot.
Sci. Fish.
36, 246-249.
AND
COMPARATIVE
ENDOCRINOLOGY
54, 429-432 (1984)
Isolation and Identification of Testosterone from the Serum and Testes of the American Lobster (Homarus americanus) B. G. BURNS, G. B. SANGALANG, H. C. FREEMAN, AND M. MCMENEMY Fisheries
and Environmental P.O.
Sciences, Department Box 550 Halifax, Nova
of Fisheries and Oceans, Scotia B3J 2S7, Canada
Halifax
Laboratory,
Accepted May 6, 1983 Testosterone was isolated and quantified from male lobster serum and testes by solvent extraction, sequential thin-layer chromatography, and high-performance liquid chromatography. The identity of the isolated steroid was established by its isopolarity and isomorphicity with authentic radiolabeled testosterone and its acetate derivative. The concentrations of testosterone were determined by high-performance liquid chromatography, by a doubleisotope derivative assay, and by radioimmunoassay. The concentrations of testosterone as determined by the three methods were the same and were 0.3 rig/ml and 14.3 rig/g in lobster serum and testes, respectively.
The occurrence of vertebrate-type steroid hormones in marine invertebrates is well documented (Donahue, 1948, 1952, 1957; Hagerman et al., 1957; Lisk, 1961; Schwerdtfeger, 1932; Steidle, 1930; Gottfried et al., 1967; Teshima and Kanazawa, 1970; Nikitina et al., 1977). The identification of steroids, with the exception of the ecdysteroid molting hormones (Gagosian and Bourbonniere, 1976), in the American lobster Homarus americanus has received little attention. Previous investigations have shown that the lobster contains some of the enzyme systems necessary for steroidogenesis. Gilgan and Idler (1967) reported that lobster testes and androgenic glands were able to transform 4-[‘4C]androstenedione-3, 17dione to testosterone in vitro but did not establish the presence of testosterone in the animal. Burns et al. (1982) demonstrated the ability of the lobster testes to transform the steroid hormone precursors pregnenolone and progesterone to 20 a-dihydro-derivatives in vitro. Kanazawa and Teshima (1971) reported the in vivo conversion of cholesterol to vertebrate steroid hormones in the spiny lobster Panulirus japonica.
In the present study testosterone was isolated and identified in both male lobster serum and testes. MATERIALS AND METHODS Collection oflobsters. Adult male lobsters were collected from the Miramichi River estuary (New Brunswick, Canada) during late spring of 1981. The animals were transported to the aquarium at the Halifax laboratory where they were held in tanks of continuously flowing filtered seawater for a maximum of 2 weeks before sampling. Lobster hemolymph was collected from 50 male lobsters (mean weight ca. 300 g) by syringe from the abdominal sinus. The pooled sample was centrifuged (8000 rpm for 15 min) at O-4” and the serum was removed and stored at - 30” until used. The testes (6 g from 12 lobsters) were carefully excised from the lobsters, minced with scissors, pooled, and extracted foI steroids immediately. Extraction
of tesfosterone
from
serum
and testes.
The serum and minced lobster testes were each adjusted to pH 9.0 with 1 N NaOH and were extracted once with 10 vol of methylene chloride. Each of the extracts was washed with 0.1 N sodium bicarbonate, 0.1 N acetic acid, and distilled water as described previously (Idler et al., 1971). The methylene chloride was removed by flash evaporation at 40” and the residue was partitioned between 80% methanol and hexane as described by Idler et al. (1971). Thin-layer chromatography (TLC). TLC was carried out on glass plates coated with silica gel HFZS4-366 (Merck, Darmstadt, West Germany). The solvent sys-
429 0016~6480/84 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved
BURNS ET AL.
430
terns used (by volume) were (I) hexane:ethyl acetate (4: 1); (II) chloroform:methanol:water (188:12:1); (III) t-butanol:hexane (25:75); (IV) methylene chloride:acetone (95:5); (V) cyclohexane:ethyl acetate (30:70); (VI) isopropanol:cyclohexane (30:70). High-performance
liquid
chromatography
(HPLC).
HPLC was performed using a Beckman Model 1lOA pump, a Waters Model U6K septumless injector, a Vydac 201TP (S-brn particle size, 4.6 mm x 25 cm) column, and a Schoeffel Model SF770 variable-wavelength detector. The ultraviolet absorbance of the testosterone in the effluent was measured at 241 nm. The solvent systems used were (I) 45% acetonitrile (isocratic); (II) 55% acetonitrile in water (isocratic); (III) a linear gradient of 30 to 70% acetonitrile in water (30 min) and then held at 70% acetonitrile in water. The flow rates of all systems was 1.0 ml/min. The linear gradient was obtained by pumping acetonitrile, at a constant rate under controlled conditions, into a mixing cylinder containing acetonitrile and water, which was the solvent reservoir for the Beckman pump. Radioactive chemicals. [14C]- and [3H]testosterone and r3H]acetic anhydride were purchased from New England Nuclear Corporation, Boston, Massachusetts. The radioactive steroids were purified by chromatography before use. Radioactive measurements. Radioactivity was measured using a Packard Tri-Garb, Model 3225, liquid scintillation spectrometer equipped with automatic external standardization. Isolation of steroid fractions. One-tenth volume of each of the extracts (serum and testes) was applied to a TLC plate and was developed twice in system I and once in system II. Steroids were detected by ultraviolet (uv) absorption (240 nm) and sulfuric acid:ethanol (1: 1) spray followed by heating at 105” for a few minutes for color development. The remaining nine-tenths of each extract was run similarly but steroids were located only by (uv) absorption. Three major areas were eluted: one isopolar with testosterone, one less polar (RT = 1.19), and one more-polar (RT = 0.77). Each of the above eluted areas were divided into two parts (one-tenth and nine-tenths). Each part was rechromatographed by TLC III, the steroids were located, and the area isopolar with testosterone was eluted as in the first chromatography. The testosterone area was further purified and quantified using HPLC (system I). One-third of the testosterone purified by HPLC was rechromatographed by HPLC with a trace of standard [3H]testosterone (4000 dpm). The remaining two-thirds was acetylated with [3H]acetic anhydride as described by Idler et al. (1972). Radioimmunoassay. The radioimmunoassay procedure for testosterone was as described by Sangalang and Freeman (1977). Double-isotope derivative assay (DZDA). The DIDA
procedure was similar to that reported by Idler et al. (1972). A trace quantity of [14C]testosterone (5000 dpm) was added to quantify 3H-acetylation as well as correct for losses during the procedure. The chromatographic systems used and sequences were TLC system IV, TLC system V, HPLC system II, TLC system VI, and HPLC system III. Approximately 20 pg of authentic nonradioactive testosterone acetate was added as carrier to the [14C]testosterone[3H]acetate before chromatography. Purification of the acetate was discontinued when ‘H/14C isotope ratios were constant for three consecutive chromatographic procedures. The identity of the isolated testosterone as testosterone acetate was then confirmed by recrystallization with nonradioactive standard testosterone acetate to constant 3H/‘4C ratios as described by AXelrod et al. (1965).
RESULTS AND DISCUSSi6N
Testosterone was isolated from lobster serum and its concentration was 0.3 r&ml by HPLC. Serum testosterone was homogeneous with standard testosterone in HPLC system I and its [3H]acetate derivative was isopolar with standard testosterone acetate in TLC system IV and HPLC system I, indicating that the isolited steroid was testosterone. The isolated testosterone reacted positively and specifically with testosterone antiserum when quantified by RIA. Its concentration by RIA was also 0.3 @ml. The testosterone in lobster serum was isolated and also quantified by DIDA and its concentration was again confirmed at 0.3 rig/ml. The final 3H/14C ratios of 3H-acetylated serum sample plus [‘4C]testosterone tracer and of [14C]testosterone tracer only were 2.83 and 0.79, respectively (Table 1). The specific activity of the [3H]acetic anhydride was 25.8 &i/pmol. Testosterone was also similarly isolated and quantified from the lobster testes. The concentration of testosterone in the lobster testes was 14.3 rig/g. Previous investigations have shown that lobster testes possess many of the enzyme systems necessary for steroidogenesis (Burns et al., 1982; Gilgan and Idler, 1967; Kanazawa and Teshma, 1971); however, to
TESTOSTERONE TABLE ‘H/i4C RATIOS OF 3H-A~~~~~~~~~
431
IN LOBSTER 1
ISOLATED TESTOSTERONE PLUS AUTHENTIC [‘4C]T~~~~~~~~~~~ ONLY
[L4C]T~~~~~~~~~~
AND
3H/‘4C ratio System TLC System IV TLC System V LC System II TLC System VI LC System III Recrystallization Ia Recrystallization IIc
Isolated + authentic testosterone acetate
Authentic testosterone acetate
261 24.5 2.81 2.81 2.83 2.83(2.83)b (2.83)(2.82)b
173 20.9 0.80 0.78 0.79 (0.79)(0.79)b (0.79)(0.79)b
a Acetonelhexane. b Ratios of mother liquor. c Acetonelpentane.
the authors’ knowledge, no steroids, other than the molting hormones, have been positively isolated and identified from the hemolymph of the American lobster. The level of testosterone in the serum of the lobster is low compared to the concentrations reported in the plasma of most fishes, amphibians, and birds where the values are approximately 100 times higher (Ozon, 1972). The plasma levels of testosterone in 90 vivaparous lizards during one annual cycle, however, ranged approximately two to six times higher than the level in lobster serum (Courty and Dufaure, 1982). The testosterone levels in the testes of the lizards ranged from 14.0 to 1472.9 rig/g during one annual cycle (Courty and Dufaure, 1982). The lowest concentration of testosterone in the lizard testes is close to the value of 14.3 rig/g found in the testes of the lobster in this study. The levels of testosterone in both lobster testes and serum may also have seasonal fluctuations as in the lizard, and the sampling of the lobsters in the present investigation could have been done at a time when testosterone levels were low. The occurrence of testosterone in the testes of the American lobster indicates that this steroid may function as an androgen as it does in
vertebrates. The role of testosterone in the reproductive physiology of the lobster deserves investigation. REFERENCES Axelrod, L. R., Matthijssen, C., Goldzieher, J. W., and Pulliam, J. E. (1965). Definitive identification of microquantities of radioactive steroids by recrystallization to constant specific alterity. Acta Endocrinol. Copenhngen 49 (Suppl. 99). Burns, B. G., Sangalang, G. B., Freeman, H. C., and McMenemy, M. (1982). In vitro bioconversion of steroids by the testes of the American lobster Homarus americanus. In preparation. Courty, II., and Dufaure, J. P. (1982). Circannual testosterone, dihydrotestosterone and androstanediols in plasma and testes of Lacerta vivipara, a seasonally breeding vivaparous lizard. Steroids 39, 517-529. Donahue, J. K. (1948). Fluorometric and biological determination of estrogens in the eggs of the American lobster (Homarus americanus). Proc. Sot. Exp. Biol. Med. 69, 179-181. Donahue, J. K. (1952). “Studies on Ecdysis in the American Lobster (Homarus americanus). I. The Lobster Egg as a Source of Estrogenic Hormone.” State of Maine, Department of Sea and Shore Fisheries Res. Bull. No. 8. Donahue, J. K. (1957). -“Chromatographic Identification of Lobster Egg Estrogen. State of Maine, Department of Sea and Shore Fisheries Res. Bull. No. 28. Idler, D. R., Horne, D. A., and Sangalang, G. B. (1971). Identification and quantification of the major androgens in testicular and peripheral
432
BURNS ET AL.
plasma of Atlantic salmon (Salmo salar) during sexual maturation. Gen. Comp. Endocrinol. 16, 251-267. Idler, D. R., Sangalang, G. B., and Truscott, B. (1972). Corticosteroids in the South American lungfish. Gen. Comp. Endocrinol. Suppl. 3, 238244. Kanazawa, A., and Teshima, S. (1971). In vivo conversion of cholesterol to steroid hormones in the spiny lobster, Panulirus japonica. Bull. Japan. Sot. Sci. Fish. 37, 891-898. Lisk, R. D. (1961). Estradiol-17 in the eggs of the American lobster, Homarus americanus. Canad. J. Biochem.
Physiol.
39, 659-662.
Nikitina, S. M., Savchenko, 0. N., Kogan, M. E., and Ezhkova, N. S., (1977). Preparative isolation of progesterone, testosterone, and estrogens from tissues of marine invertebrates. Zh. Evol. Biokhim. Fiziol. 13, 443-447.
Ozon, R. (1972). In “Steroids in Nonmammalian Vertebrates” (D. R. Idler, ed.), pp. 328-389. Academic Press, New York. Sangalang, G. B., and Freeman, H. C. (1977). A new sensitive and precise method for determining testosterone and 1I-ketotestosterone in fish plasma by radioimmunoassay. Gen. Comp. Endocrinol. 32, 432-439. Schwerdtfeger, H. (1932). Beitrlge zum Vorkomen und zur Wirkung der weiblichen Sexual-hormone. Arch. Exp. Pathol. Pharmakol. 163, 487-492. Steidle, H. (1930). Uber die Verbreitung des weiblichen sexual Hormones. Arch. Exp. Pathol. Pharmakol. 157, 89. Teshima, S., and Kanazawa, A. (1970). Production of 1 I-ketotestosterone and other steroids by the sliced ovaries of the crab Portunus trituberculatus.
Bull.
Japan.
Sot.
Sci. Fish.
36, 246-249.