0013-7227/78/1036-2283$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 6 Printed in U.S.A.
Phylogenetic Distribution of Aromatase and Other Androgen-Converting Enzymes in the Central Nervous System* GLORIA V. CALLARD, ZOLTAN PETRO, AND KENNETH J. RYAN Department of Obstetrics and Gynecology, Harvard Medical School, Laboratory of Human Reproduction and Reproductive Biology, Boston, Massachusetts 02115 ABSTRACT. Metabolism of [3H]androstenedione was studied in brain tissue homogenates of opossum, bird, snake, sea turtle, urodele amphibian, teleost, shark, skate, hagfish, and lobster. Estrone, 17/3-estradiol, or 17a-estradiol was formed by central neural tissues of all species, with the exception of the opossum, hagfish, and lobster. Aromatase activity was concentrated in the forebrain, although some estrogen was synthesized by mid- or hindbrain homogenates of two lower vertebrates (teleost and skate) and the newly hatched chick. 5a-
^ H E PRIMARY sites of aromatization in ^ . mammals are the gonads or placenta, but
small quantities of estrogen are also synthesized in the central nervous system (CNS) and other peripheral nonendocrine tissues. Estrogen formed in the brain itself is thought to mediate androgen actions on sex behavior, gonadotropin secretion, and brain sex differentiation (1). Aromatase activity in the brain of mammals is found predominantly in the phylogenetically ancient components (the hypo thalamus, preoptic area, and limbic lobe), suggesting that estrogen synthesis may be a primitive characteristic of the CNS that can be traced through vertebrate development. Comparative studies in nonmammalian species support this hypothesis. The data reported here and in previous investigations (2, 3) show that estrogen synthesis from labeled precursor in vitro is detectable in the brain of representatives of each major vertebrate group, with the exception of the Agnatha. In Received April 3, 1978. Address requests for reprints to: Dr. Gloria V. Callard, Department of Obstetrics and Gynecology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115. * This work was supported by NSF-PCM 76-19970 (GVC) and NIH Grant HD 07923-05 (KJR).
Androstanedione (5a-androstane-3,17-dione) or 5adihydrotestosterone were products of metabolism in several nonmammalian vertebrates and in the invertebrate central nervous system also. 5a-Reductase was found in all major brain divisions. These and previously reported comparative studies indicate that the ability to aromatize and otherwise transform androgen substrates is a primitive characteristic of the brain that has been widely conserved phylogenetically. (Endocrinology 103: 2283, 1978)
addition, central androgen-transforming enzymes other than aromatase are present in nonmammalian species and can be identified in invertebrate CNS also. Materials and Methods Animals Opossums (Didelphis virginiana), birds {Gallus domesticus), snakes (Natrix taxispilota), mudpuppies {Necturus maculosus), and lobsters (Homarus americanus) were purchased from commercial suppliers. Newly hatched (plastron length, 7.5-10 cm) green sea turtles (Chelonia mydas) were obtained through the courtesy of Dr. James Wood (Cayman Turtle Farm, Grand Cayman Island, British West Indies). Longhorn sculpin (Myoxocephalus octadecimspinosus), winter skates (Raja ocellata), and spiny dogfish (Squalus acanthias) were collected locally in New England by the supply department of the Marine Biological Laboratories (Woods Hole, MA). Of the sculpin collected in the relatively shallow waters near Woods Hole, mature females predominate and only two specimens were males. Hagfish (Myxine glutinosa) were purchased from Ma rine Research Associates (Lord's Cove, Deer Island New Brunswick, Canada). Optic glands were sur gically removed from a 40- to 50-lb. spawning fe male Octopus in the New England aquarium. Ii most experiments, tissues were obtained 24-48 I
2283
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2284
CALLARD, PETRO, AND RYAN
Endo • 1978 Vol 103 • No 6
hatched birds, and in addition, 17a-oxidoreductase activity was detected in the frontal cortex of laying hens. No aromatization was found in muscle homogenates, but mature and immature ovary and immature testis all synthesized Ei and E2/?. Substantial amounts of E20: were also found in chick gonadal homogenates. Forebrain areas of 3-month-old green sea turtles (Chelonia) and mature female Natrix aromatized androgens to estrogens, but no activity was detected in mid- or hindbrain tissues, muscle, or in the ovaries of mature snakes (Table 2). Streak gonads of newly hatched sea turtles could not be sexed macroscopically or dissected. 17a-Oxidoreductase Incubation and steroid analysis activity was identified in the snake CNS, but For assay of aromatase and other androgen-me- no 17/?-hydroxylated estrogen was found in tabolizing enzymes, tissue homogenates were incu- either species. 5a-A was present in all brain bated with [7-H3]androstenedione (A4A). Substrate areas assayed from both reptilian species and concentration, specific activity, and the incubation was also synthesized in muscle, but Natrix temperature judged to be within the physiological ovary had no 5a-reductase activity. range for each species are indicated in Tables 1-5. In the urodele amphibian Necturus, estroDetails of the assay and procedure for identification gen was synthesized from labeled androsteneof steroid products, based on repeated recrystallidione by the brain segment containing the zation to constant specific activity, have been reported (2). Estrone (Ei) and 17^-estradiol (E2y6) hypothalamus, preoptic area, and amygdala, were routinely measured in all experiments. In but no detectable aromatization occurred in some experiments, conversion of substrate to 17a- remaining forebrain, midbrain, or hindbrain estradiol (E2
2286
Endo • 1978 Vol 103 • No 6
CALLARD, PETRO, AND RYAN
TABLE 2. Aromatase and 5a-reductase activity in the reptiles, Chelonia and Natrix Product yield (pmol/g) of immature Chelonia mydas (n = 10)"
E, Hypothalamus/preoptic area Amygdala Remaining forebrain
1.38 (212/218)6 0.94 (149/152) 0
Mid/hindbrain
0
Muscle
0
Product yield (pmol/g) of mature Natrix taxispilota females (n = 7)"
5a-A 22.2 (1139/1129) 5.9 (457/474) 16.1 (838/871) 22.6 (1159/1195) 7.8 (417/455)
E, NMC
E2a
5a-A
NM
NM
NM
NM
NM
1.77 (272/280)" 0
3.88 (593/624)d 0
0
0
6.27 (968/101l) rf 21.0 (3228/3583) 4.16 (641/617)
Homogenates were incubated with [3H]androstenedione (233 nM; 11.4 Ci/mmol) at 27 C for 1 h (Chelonia) or 2 h (Natrix). No estrogen or 5a-A was synthesized by Natrix ovary, and streak gonads of Chelonia were not incubated. " Yield (picomoles per g wet tissue incubated) was calculated after correction for procedural losses. * Specific activities of final crystals and mother liquor in disintegrations per min/mg. c Not measured. d Sample includes entire forebrain. TABLE 3. Aromatase activity in the urodele amphibian, Necturus Product yield (pmol/g) of mature males (n = 13) a
Hypothalamus/preoptic area/amygdala Gonad
Product yield (pmol/g) of mature females ( n = 14)°
E,
E2/3
E,
E20
1.001 (229/237)* 641.0 (149,989/146,678)
0
1.649 (378/389) 0.788 (181/170)° 0.648 (149/151) d
0
260.387 (59,706/59,273)
48.494 (11,120/10,803)° 23.644 (5422/5294)''
Homogenates were incubated with [ 3 H]androstenedione (349 nM; 8.2 Ci/mmol) at 25 C for 1 h. No estrogens were synthesized by remaining forebrain tissues, midbrain, or hindbrain. 0 Yield (picomoles per g wet tissue incubated) was calculated after correction for procedural losses. * Specific activities of final crystals and mother liquor in disintegrations per min/mg. c Preovulatory. d
Postovulatory.
skate. In contrast to the brain, ovarian aromatase activity was 10-fold greater in the two elasmobranch species than in the sculpin. Testicular aromatization was detected only in Squalus, and muscle of all species was negative. Estrogen synthesis was absent or below the limits of detection in pooled brain tissues from mature female and undifferentiated hagfish (Myxine). The cerebral ganglion of mature lobsters of both sexes (Homarus) and the optic gland of a spawning female Octopus were aromatase negative also; however, 5a-reductase was identified in the same tissues of all three species and in the lobster, 17/?-oxidoreductase activity was present also (Table 3).
Neither aromatase nor 5a-reductase were detected in muscle, but the hagfish ovary converted A4A to 5a-A. A single experiment in a metatherian mammal, the opossum (Didelphis virginiana), failed to detect Ei or E2/8 in homogenates of dorsolateral cortex, dorsomedial cortex, posterior ventrolateral cortex, hypothalamus/ preoptic area, mid/hindbrain, ovary, or muscle. All opossums were pregnant females. Discussion Among mammals, androgen is converted to estrogen in vitro in the CNS of the rat, mouse, rabbit, monkey, human (1), cow (10), cat (11), and hamster (Callard, unpublished). In this
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 May 2015. at 06:55 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN-CONVERTING ENZYMES IN BRAIN
and prior studies (2, 3), we identified aromatase activity in the brain of representatives of each major vertebrate group, with the exception of the Agnatha (jawless vertebrates). These include cartilaginous and bony fishes, urodele and anuran amphibians, chelonian and squamate reptiles, and an avian species. From the animals surveyed to date, the presence of aromatizing activity seems to be a primitive characteristic of the CNS that has been widely conserved through vertebrate phylogeny. Unless this enzyme arose independently many times during the course of evolution, we can assume that estrogen-biosynthesizing potential of the brain is an inheritance from a common ancestor. The oldest vertebrates which have detectable central aromatase activity are the cartilaginous fishes {Raja and Squalus), suggesting an origin for this character possibly before the adaptive radiation of jawed vertebrates (gnathostomes) 400-450 x 106 yr ago. This conclusion is based on examination of the brain of one agnathan (Myxine) and one invertebrate (Homarus), both of which are aromatase negative. It is still possible that aromatase will be identified in another jawless species, since in some respects, the brain of Myxine is considered degenerate (12). In addition, there is no evidence for a functioning hypothalamus-pituitary-gon-
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Vol. 103, No. 6 Printed in U.S.A.
Phylogenetic Distribution of Aromatase and Other Androgen-Converting Enzymes in the Central Nervous System* GLORIA V. CALLARD, ZOLTAN PETRO, AND KENNETH J. RYAN Department of Obstetrics and Gynecology, Harvard Medical School, Laboratory of Human Reproduction and Reproductive Biology, Boston, Massachusetts 02115 ABSTRACT. Metabolism of [3H]androstenedione was studied in brain tissue homogenates of opossum, bird, snake, sea turtle, urodele amphibian, teleost, shark, skate, hagfish, and lobster. Estrone, 17/3-estradiol, or 17a-estradiol was formed by central neural tissues of all species, with the exception of the opossum, hagfish, and lobster. Aromatase activity was concentrated in the forebrain, although some estrogen was synthesized by mid- or hindbrain homogenates of two lower vertebrates (teleost and skate) and the newly hatched chick. 5a-
^ H E PRIMARY sites of aromatization in ^ . mammals are the gonads or placenta, but
small quantities of estrogen are also synthesized in the central nervous system (CNS) and other peripheral nonendocrine tissues. Estrogen formed in the brain itself is thought to mediate androgen actions on sex behavior, gonadotropin secretion, and brain sex differentiation (1). Aromatase activity in the brain of mammals is found predominantly in the phylogenetically ancient components (the hypo thalamus, preoptic area, and limbic lobe), suggesting that estrogen synthesis may be a primitive characteristic of the CNS that can be traced through vertebrate development. Comparative studies in nonmammalian species support this hypothesis. The data reported here and in previous investigations (2, 3) show that estrogen synthesis from labeled precursor in vitro is detectable in the brain of representatives of each major vertebrate group, with the exception of the Agnatha. In Received April 3, 1978. Address requests for reprints to: Dr. Gloria V. Callard, Department of Obstetrics and Gynecology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115. * This work was supported by NSF-PCM 76-19970 (GVC) and NIH Grant HD 07923-05 (KJR).
Androstanedione (5a-androstane-3,17-dione) or 5adihydrotestosterone were products of metabolism in several nonmammalian vertebrates and in the invertebrate central nervous system also. 5a-Reductase was found in all major brain divisions. These and previously reported comparative studies indicate that the ability to aromatize and otherwise transform androgen substrates is a primitive characteristic of the brain that has been widely conserved phylogenetically. (Endocrinology 103: 2283, 1978)
addition, central androgen-transforming enzymes other than aromatase are present in nonmammalian species and can be identified in invertebrate CNS also. Materials and Methods Animals Opossums (Didelphis virginiana), birds {Gallus domesticus), snakes (Natrix taxispilota), mudpuppies {Necturus maculosus), and lobsters (Homarus americanus) were purchased from commercial suppliers. Newly hatched (plastron length, 7.5-10 cm) green sea turtles (Chelonia mydas) were obtained through the courtesy of Dr. James Wood (Cayman Turtle Farm, Grand Cayman Island, British West Indies). Longhorn sculpin (Myoxocephalus octadecimspinosus), winter skates (Raja ocellata), and spiny dogfish (Squalus acanthias) were collected locally in New England by the supply department of the Marine Biological Laboratories (Woods Hole, MA). Of the sculpin collected in the relatively shallow waters near Woods Hole, mature females predominate and only two specimens were males. Hagfish (Myxine glutinosa) were purchased from Ma rine Research Associates (Lord's Cove, Deer Island New Brunswick, Canada). Optic glands were sur gically removed from a 40- to 50-lb. spawning fe male Octopus in the New England aquarium. Ii most experiments, tissues were obtained 24-48 I
2283
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 May 2015. at 06:55 For personal use only. No other uses without permission. . All rights reserved.
2284
CALLARD, PETRO, AND RYAN
Endo • 1978 Vol 103 • No 6
hatched birds, and in addition, 17a-oxidoreductase activity was detected in the frontal cortex of laying hens. No aromatization was found in muscle homogenates, but mature and immature ovary and immature testis all synthesized Ei and E2/?. Substantial amounts of E20: were also found in chick gonadal homogenates. Forebrain areas of 3-month-old green sea turtles (Chelonia) and mature female Natrix aromatized androgens to estrogens, but no activity was detected in mid- or hindbrain tissues, muscle, or in the ovaries of mature snakes (Table 2). Streak gonads of newly hatched sea turtles could not be sexed macroscopically or dissected. 17a-Oxidoreductase Incubation and steroid analysis activity was identified in the snake CNS, but For assay of aromatase and other androgen-me- no 17/?-hydroxylated estrogen was found in tabolizing enzymes, tissue homogenates were incu- either species. 5a-A was present in all brain bated with [7-H3]androstenedione (A4A). Substrate areas assayed from both reptilian species and concentration, specific activity, and the incubation was also synthesized in muscle, but Natrix temperature judged to be within the physiological ovary had no 5a-reductase activity. range for each species are indicated in Tables 1-5. In the urodele amphibian Necturus, estroDetails of the assay and procedure for identification gen was synthesized from labeled androsteneof steroid products, based on repeated recrystallidione by the brain segment containing the zation to constant specific activity, have been reported (2). Estrone (Ei) and 17^-estradiol (E2y6) hypothalamus, preoptic area, and amygdala, were routinely measured in all experiments. In but no detectable aromatization occurred in some experiments, conversion of substrate to 17a- remaining forebrain, midbrain, or hindbrain estradiol (E2
2286
Endo • 1978 Vol 103 • No 6
CALLARD, PETRO, AND RYAN
TABLE 2. Aromatase and 5a-reductase activity in the reptiles, Chelonia and Natrix Product yield (pmol/g) of immature Chelonia mydas (n = 10)"
E, Hypothalamus/preoptic area Amygdala Remaining forebrain
1.38 (212/218)6 0.94 (149/152) 0
Mid/hindbrain
0
Muscle
0
Product yield (pmol/g) of mature Natrix taxispilota females (n = 7)"
5a-A 22.2 (1139/1129) 5.9 (457/474) 16.1 (838/871) 22.6 (1159/1195) 7.8 (417/455)
E, NMC
E2a
5a-A
NM
NM
NM
NM
NM
1.77 (272/280)" 0
3.88 (593/624)d 0
0
0
6.27 (968/101l) rf 21.0 (3228/3583) 4.16 (641/617)
Homogenates were incubated with [3H]androstenedione (233 nM; 11.4 Ci/mmol) at 27 C for 1 h (Chelonia) or 2 h (Natrix). No estrogen or 5a-A was synthesized by Natrix ovary, and streak gonads of Chelonia were not incubated. " Yield (picomoles per g wet tissue incubated) was calculated after correction for procedural losses. * Specific activities of final crystals and mother liquor in disintegrations per min/mg. c Not measured. d Sample includes entire forebrain. TABLE 3. Aromatase activity in the urodele amphibian, Necturus Product yield (pmol/g) of mature males (n = 13) a
Hypothalamus/preoptic area/amygdala Gonad
Product yield (pmol/g) of mature females ( n = 14)°
E,
E2/3
E,
E20
1.001 (229/237)* 641.0 (149,989/146,678)
0
1.649 (378/389) 0.788 (181/170)° 0.648 (149/151) d
0
260.387 (59,706/59,273)
48.494 (11,120/10,803)° 23.644 (5422/5294)''
Homogenates were incubated with [ 3 H]androstenedione (349 nM; 8.2 Ci/mmol) at 25 C for 1 h. No estrogens were synthesized by remaining forebrain tissues, midbrain, or hindbrain. 0 Yield (picomoles per g wet tissue incubated) was calculated after correction for procedural losses. * Specific activities of final crystals and mother liquor in disintegrations per min/mg. c Preovulatory. d
Postovulatory.
skate. In contrast to the brain, ovarian aromatase activity was 10-fold greater in the two elasmobranch species than in the sculpin. Testicular aromatization was detected only in Squalus, and muscle of all species was negative. Estrogen synthesis was absent or below the limits of detection in pooled brain tissues from mature female and undifferentiated hagfish (Myxine). The cerebral ganglion of mature lobsters of both sexes (Homarus) and the optic gland of a spawning female Octopus were aromatase negative also; however, 5a-reductase was identified in the same tissues of all three species and in the lobster, 17/?-oxidoreductase activity was present also (Table 3).
Neither aromatase nor 5a-reductase were detected in muscle, but the hagfish ovary converted A4A to 5a-A. A single experiment in a metatherian mammal, the opossum (Didelphis virginiana), failed to detect Ei or E2/8 in homogenates of dorsolateral cortex, dorsomedial cortex, posterior ventrolateral cortex, hypothalamus/ preoptic area, mid/hindbrain, ovary, or muscle. All opossums were pregnant females. Discussion Among mammals, androgen is converted to estrogen in vitro in the CNS of the rat, mouse, rabbit, monkey, human (1), cow (10), cat (11), and hamster (Callard, unpublished). In this
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 May 2015. at 06:55 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN-CONVERTING ENZYMES IN BRAIN
and prior studies (2, 3), we identified aromatase activity in the brain of representatives of each major vertebrate group, with the exception of the Agnatha (jawless vertebrates). These include cartilaginous and bony fishes, urodele and anuran amphibians, chelonian and squamate reptiles, and an avian species. From the animals surveyed to date, the presence of aromatizing activity seems to be a primitive characteristic of the CNS that has been widely conserved through vertebrate phylogeny. Unless this enzyme arose independently many times during the course of evolution, we can assume that estrogen-biosynthesizing potential of the brain is an inheritance from a common ancestor. The oldest vertebrates which have detectable central aromatase activity are the cartilaginous fishes {Raja and Squalus), suggesting an origin for this character possibly before the adaptive radiation of jawed vertebrates (gnathostomes) 400-450 x 106 yr ago. This conclusion is based on examination of the brain of one agnathan (Myxine) and one invertebrate (Homarus), both of which are aromatase negative. It is still possible that aromatase will be identified in another jawless species, since in some respects, the brain of Myxine is considered degenerate (12). In addition, there is no evidence for a functioning hypothalamus-pituitary-gon-
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