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Masculinization of females of the isopod crustacean, Armadillidium vulgare, following injections of an active extract of the androgenic gland
GENERAL
AND
COMPARATIVE
48, 51-62, (1983)
ENDOCRINOLOGY
asculinization of Females of the lsopo are, following Injections of an Androgenic Gla YASUTOSHI Department
of Biology,
KATAKURA Keio
AND YURIKO HASEGAWA
University,
Yokohama,
Kanagnwa
223. Japan
Accepted January 6, 1982 Injections of 64 U of an active extract of the androgenic gland into young females of vulgare induced masculinization of the external sexual characteristics and transformation of the internal female reproductive organs into testes, seminal vesicles, and vasa deferentia. Following injections of 10-U doses, the internal organs were hardly affected in 8 of 10 females although the external characteristics underwent mascuhnization.
Armadillidiurn
In the amphipod, Orchesfia gammarella, Charniaux-Cotton (1952, 1954) first reported that the androgenic glands, so named by this author, were responsible for male differentiation and development. Since then, glands of similar function have been identified in a variety of species from nearly all orders of Malacostraca (Charniaux-Cotton et al., 1966). However, the active principle of the androgenic glands has not yet been thoroughly characterized. ecently, Katakura et al. (1975) exted an active thermolabile protein from male reproductive organs including the androgenic glands of the isopod, Armadillid&m vuigare. A single injection of the extract induced masculinization of the secondary sexual characteristics in females of A~mad~~~idi~rn.The molecular weight of the protein was estimated to be about 16,000 altons. Juchault et ai. (1978) extracted an active substance from the androgenic glands of intersexual individuals of A. vulgare and orted that it was thermostable and ysable, but destroyed by Pronase. In the present paper, we report further details of masculinization of external and internal sexual characteristics in females given injections of an extract similar to that
reported previously and support our previous conclusion that the androgenlc gland hormone is a protein.
Injection materials. The reproductive organs including androgenic glands from 6000 mature males of Armadillidium vulgare, measuring more than 9 mm in body length were used as materials for extraction. Procedures of extraction and partial purification of the proteinacous active principle were described previously (Katakurm et al., 1975). The activity of the fraction obtained by the second gel fiitration fir5ccdon IV) was bioassayed on sexually inactive ~~rn~dii~~d~~4rn females. Then, 160 units (U)l or 1280 U ef the fraction was taken in 124 separate vessels and iyophilized and stored. Just before injection. the two doses were dissolved in 8 or 10 /*I of redistilled water, respectively. Injections. The standard volume of a singPe injection was 0.5 ~1 per 18.5 mg body wt. At ihe first injection, young females (5.74 mg in average body wt, 4~0-4.5 mm in body length) at the intermolt stage were used as recipients. Thereafter, the extract with a fixed activity was injected once per cycle for four consecutive molting cycles. The extract was injected through the intersegmental membrane between the steraite of the sixth and seventh thoracic segments by means of a microsyringe.
’ One ArmadiUidium Unit of the active principk wzs defined as the minimum amount necessary to cause masculinization, i.e., the elongation of endopodites of the first pair of abdominal legs, in one female assay (18.5 mg in average body wt. 6-7 mm in body length:.
57 0~16-64g0~~~/010057-06$01.~0/~ Copyright All rights
Q 1983 by Academic Fvess. Inc. of reproduction in any form resewed.
KATAKURA
58
AND HASEGAWA TABLE
RESULTS
Injection
OF INJECTIONS
Injected females
1
OF 10 AND
Surviving females
64 U EXTRACT Masculinized females
Fixed females
10u 1st 2nd 3rd 4th
471 194 101 44
335 143 68 25
204 113 53 23
10 10 6 10
64 U 1st 2nd 3rd 4th
167 104 48 15
110 53 25 6
104 48 25 6
0 0 10 0
Observations of masculinized females. The external characteristics of the females were examined under a dissecting microscope after each postinjection molt. The length of the endopodites of the first pair of abdominal legs and the width of the fourth thoracic segment were measured. The endopodite length/width of the thoracic segment was used for comparison of the degree of masculinization between individuals of different size groups (see Fig. 1). Two or three days after molt, some of the masculinized specimens were fixed in Bouin’s solution for histological study. Sections were stained with Delafield’s hematoxylin and eosin to study the state of the internal sexual organs. The maximum diameter of the vasa deferentia relative to the width of the fourth thoracic segment was measured (vide szqxa). In addition to these subjects, young females (4.0-4.5 mm in body length) implanted with three fresh androgenic glands from mature males and young males of similar size were used for the study of external and internal sexual characteristics.
RESULTS AND DISCUSSION
Table 1 shows the numbers of injected, surviving, and successfully masculinized females which had received 10 or 64 U of the extract at each injection. The numbers of females fixed and studied histologically are also given. Figure 1 presents the changes of abdominal appendages in females masculinized following injections the extract. The penial buds appeared between the endopodites of the first pair of abdominal legs at the molt after the second injection and fully devel-
oped at the third molt. The tip of elongated endopodites of the first pair of abdominal legs curved outward at the third or the fourth molt. In the females given implants of three androgenic glands, the formation of the penial buds occurred at the first postoperative molt and the tip of the endopodites curved outward at the second or the third molt. In the normal males measuring 4.0-4.5 mm in body length, the penial buds are already present and the curving of the abdominal appendages is occurring. Masculinization of female external sexual characteristics following periodic injections of the active extract advanced with molt, the 64-U injections being more effective than 10-U injections. Masculinization progressed more slowly after injections than after implantation of three androgenic glands. In the females given a single injection of the extract, only a slight masculinization was visible after the second postinjection molt. The internal reproductive organs remained unaffected in 8 of 10 females given injections of 10 U of the extract; only the remaining two specimens showed the development of testes, seminal vesicles, and vasa deferentia, one after the third injection and the other after the fourth (Fig. 2). In 7 of 10 females which were externally
MASCULINIZATION
OF
WITH
A. w&are
ANDROGENIC
GLAND
EXTRACT‘S
Normal 3AG
6 6 6 5 d
59
8 imp.
64U
inj.
1ou
inj.
0.1 Norma! 1st injection
1st
2nd Molt
during
4th
3rd injec:ion
9
period
FIG. 1. Masculinization of the endopodites of the first pair of abdominal legs following injections cf extract. Body length at first injection was 4.0-4.5 mm.
took place as after injections of 10 U. In the developed testes, spermatozoa were not found although spermatogenesis eccurre (Figs. 314, >. The apical region the developed testes bore a grou
masculinized following injections of 64 U of the extract, the testes, seminal vesicles, and vasa deferentia were developed (Fig. 3D), whereas in the remaining 3 only slight masculinization of the reproductive organs
1st injection
2nd
1 st Molt
during
3rd injection
4th perbd
FIG. 2. Development of vasa deferentia following injections of extract. Body length at first injection was 4.0-4.5 mm.
60
KATAKURA
FIG. 3. Transverse sections of extract. (A) Well-developed testis atrophied ovary (0) are seen. (C) deferens (vd) is seen. Scale = 100
AND
HASEGAWA
masculinized female Armadillidium following injections of 64 U is visible (t). (B) Developing testis (t), seminal vesicle (sv), and Developing androgenic gland (ag) is visible. (D) Developed vas pm.
suggestive of a developing an androgenic gland (Fig. 3C). All of the masculinized females retained the oviducts like those of the normal females but ovarian growth was totally inhibited in them. In the females implanted with three androgenic glands, the testes, seminal vesicles, and vasa deferentia were well developed.
In conclusion, injections of 64 U of the extract into young females of ArmadiElidium vulgare brought about masculinization of the external sexual characteristics and transformation of the internal reproductive organs into the testes, seminal vesicles, and vasa deferentia. Injections of 10 U of the extract were hardly effective in in-
MASCULINIZATION
OF A. vulgare
WITH
ducing masculinization of internal organs of externally maswlinized females. These findings suggest that an active proteinaceous principle extracted from the reproductive organs male Armadillidium including androgenic glands is the androgenic gland hormone as previously concluded by Katakura et al. (1975). The results also demonstrate that the external sexual characteristics are more sensitive to the androgenic gland hormone than the internal sexual srgans.
ANDROGENIC
GLAND
ae
EXTRACTS
I-E
$1
T
We wish to express our sincere thanks to Dr. Takewaki for help in preparing the manuscript.
K.
Charniaux-Cotton, H. (1952). Castration chirurgicale chez un crustact amphipode (Oxhestia gummarella) et dberminisme des caracteres sexuels secoudaires. Premiers resultats. C,R. Acad. Sci. 234, 2570-2572.
62
KATAKURA
AND HASEGAWA
Charniaux-Cotton, H. (1954). Decouverte chez un crustace amphipode (Ouchestin gammarella) d’une glane endocrine responsable de la differenciation de caracteres sexuels primaires et secondaires males. C.R. Acad. Sci. 239, 780-782. Charniaux-Cotton, H., Zerbib, C., and Meusy, J. J. (1966). Monographie de la glande androgene des crustaces superieurs. Crustaceana 10, 113-136. Juchault, P., Maissiat, J., and Legrand, J. J. (1978).
Caracterisation chimique d’une substance ayant les effets biologiques de l’hormone androgene chez le crustace isopode terrestre Aramadillidium vulgare Latreille. C.R. Acad. Sci. 286, 73-76. Katakura, Y., Fujimaki, Y., and Unno, K. (1975). Partial purification and characterization of androgenic gland hormone from the isopod crustacean,Armadillidium 48, 203-209.
vulgare.
Annot.
Zool.
Japan.
AND
COMPARATIVE
48, 51-62, (1983)
ENDOCRINOLOGY
asculinization of Females of the lsopo are, following Injections of an Androgenic Gla YASUTOSHI Department
of Biology,
KATAKURA Keio
AND YURIKO HASEGAWA
University,
Yokohama,
Kanagnwa
223. Japan
Accepted January 6, 1982 Injections of 64 U of an active extract of the androgenic gland into young females of vulgare induced masculinization of the external sexual characteristics and transformation of the internal female reproductive organs into testes, seminal vesicles, and vasa deferentia. Following injections of 10-U doses, the internal organs were hardly affected in 8 of 10 females although the external characteristics underwent mascuhnization.
Armadillidiurn
In the amphipod, Orchesfia gammarella, Charniaux-Cotton (1952, 1954) first reported that the androgenic glands, so named by this author, were responsible for male differentiation and development. Since then, glands of similar function have been identified in a variety of species from nearly all orders of Malacostraca (Charniaux-Cotton et al., 1966). However, the active principle of the androgenic glands has not yet been thoroughly characterized. ecently, Katakura et al. (1975) exted an active thermolabile protein from male reproductive organs including the androgenic glands of the isopod, Armadillid&m vuigare. A single injection of the extract induced masculinization of the secondary sexual characteristics in females of A~mad~~~idi~rn.The molecular weight of the protein was estimated to be about 16,000 altons. Juchault et ai. (1978) extracted an active substance from the androgenic glands of intersexual individuals of A. vulgare and orted that it was thermostable and ysable, but destroyed by Pronase. In the present paper, we report further details of masculinization of external and internal sexual characteristics in females given injections of an extract similar to that
reported previously and support our previous conclusion that the androgenlc gland hormone is a protein.
Injection materials. The reproductive organs including androgenic glands from 6000 mature males of Armadillidium vulgare, measuring more than 9 mm in body length were used as materials for extraction. Procedures of extraction and partial purification of the proteinacous active principle were described previously (Katakurm et al., 1975). The activity of the fraction obtained by the second gel fiitration fir5ccdon IV) was bioassayed on sexually inactive ~~rn~dii~~d~~4rn females. Then, 160 units (U)l or 1280 U ef the fraction was taken in 124 separate vessels and iyophilized and stored. Just before injection. the two doses were dissolved in 8 or 10 /*I of redistilled water, respectively. Injections. The standard volume of a singPe injection was 0.5 ~1 per 18.5 mg body wt. At ihe first injection, young females (5.74 mg in average body wt, 4~0-4.5 mm in body length) at the intermolt stage were used as recipients. Thereafter, the extract with a fixed activity was injected once per cycle for four consecutive molting cycles. The extract was injected through the intersegmental membrane between the steraite of the sixth and seventh thoracic segments by means of a microsyringe.
’ One ArmadiUidium Unit of the active principk wzs defined as the minimum amount necessary to cause masculinization, i.e., the elongation of endopodites of the first pair of abdominal legs, in one female assay (18.5 mg in average body wt. 6-7 mm in body length:.
57 0~16-64g0~~~/010057-06$01.~0/~ Copyright All rights
Q 1983 by Academic Fvess. Inc. of reproduction in any form resewed.
KATAKURA
58
AND HASEGAWA TABLE
RESULTS
Injection
OF INJECTIONS
Injected females
1
OF 10 AND
Surviving females
64 U EXTRACT Masculinized females
Fixed females
10u 1st 2nd 3rd 4th
471 194 101 44
335 143 68 25
204 113 53 23
10 10 6 10
64 U 1st 2nd 3rd 4th
167 104 48 15
110 53 25 6
104 48 25 6
0 0 10 0
Observations of masculinized females. The external characteristics of the females were examined under a dissecting microscope after each postinjection molt. The length of the endopodites of the first pair of abdominal legs and the width of the fourth thoracic segment were measured. The endopodite length/width of the thoracic segment was used for comparison of the degree of masculinization between individuals of different size groups (see Fig. 1). Two or three days after molt, some of the masculinized specimens were fixed in Bouin’s solution for histological study. Sections were stained with Delafield’s hematoxylin and eosin to study the state of the internal sexual organs. The maximum diameter of the vasa deferentia relative to the width of the fourth thoracic segment was measured (vide szqxa). In addition to these subjects, young females (4.0-4.5 mm in body length) implanted with three fresh androgenic glands from mature males and young males of similar size were used for the study of external and internal sexual characteristics.
RESULTS AND DISCUSSION
Table 1 shows the numbers of injected, surviving, and successfully masculinized females which had received 10 or 64 U of the extract at each injection. The numbers of females fixed and studied histologically are also given. Figure 1 presents the changes of abdominal appendages in females masculinized following injections the extract. The penial buds appeared between the endopodites of the first pair of abdominal legs at the molt after the second injection and fully devel-
oped at the third molt. The tip of elongated endopodites of the first pair of abdominal legs curved outward at the third or the fourth molt. In the females given implants of three androgenic glands, the formation of the penial buds occurred at the first postoperative molt and the tip of the endopodites curved outward at the second or the third molt. In the normal males measuring 4.0-4.5 mm in body length, the penial buds are already present and the curving of the abdominal appendages is occurring. Masculinization of female external sexual characteristics following periodic injections of the active extract advanced with molt, the 64-U injections being more effective than 10-U injections. Masculinization progressed more slowly after injections than after implantation of three androgenic glands. In the females given a single injection of the extract, only a slight masculinization was visible after the second postinjection molt. The internal reproductive organs remained unaffected in 8 of 10 females given injections of 10 U of the extract; only the remaining two specimens showed the development of testes, seminal vesicles, and vasa deferentia, one after the third injection and the other after the fourth (Fig. 2). In 7 of 10 females which were externally
MASCULINIZATION
OF
WITH
A. w&are
ANDROGENIC
GLAND
EXTRACT‘S
Normal 3AG
6 6 6 5 d
59
8 imp.
64U
inj.
1ou
inj.
0.1 Norma! 1st injection
1st
2nd Molt
during
4th
3rd injec:ion
9
period
FIG. 1. Masculinization of the endopodites of the first pair of abdominal legs following injections cf extract. Body length at first injection was 4.0-4.5 mm.
took place as after injections of 10 U. In the developed testes, spermatozoa were not found although spermatogenesis eccurre (Figs. 314, >. The apical region the developed testes bore a grou
masculinized following injections of 64 U of the extract, the testes, seminal vesicles, and vasa deferentia were developed (Fig. 3D), whereas in the remaining 3 only slight masculinization of the reproductive organs
1st injection
2nd
1 st Molt
during
3rd injection
4th perbd
FIG. 2. Development of vasa deferentia following injections of extract. Body length at first injection was 4.0-4.5 mm.
60
KATAKURA
FIG. 3. Transverse sections of extract. (A) Well-developed testis atrophied ovary (0) are seen. (C) deferens (vd) is seen. Scale = 100
AND
HASEGAWA
masculinized female Armadillidium following injections of 64 U is visible (t). (B) Developing testis (t), seminal vesicle (sv), and Developing androgenic gland (ag) is visible. (D) Developed vas pm.
suggestive of a developing an androgenic gland (Fig. 3C). All of the masculinized females retained the oviducts like those of the normal females but ovarian growth was totally inhibited in them. In the females implanted with three androgenic glands, the testes, seminal vesicles, and vasa deferentia were well developed.
In conclusion, injections of 64 U of the extract into young females of ArmadiElidium vulgare brought about masculinization of the external sexual characteristics and transformation of the internal reproductive organs into the testes, seminal vesicles, and vasa deferentia. Injections of 10 U of the extract were hardly effective in in-
MASCULINIZATION
OF A. vulgare
WITH
ducing masculinization of internal organs of externally maswlinized females. These findings suggest that an active proteinaceous principle extracted from the reproductive organs male Armadillidium including androgenic glands is the androgenic gland hormone as previously concluded by Katakura et al. (1975). The results also demonstrate that the external sexual characteristics are more sensitive to the androgenic gland hormone than the internal sexual srgans.
ANDROGENIC
GLAND
ae
EXTRACTS
I-E
$1
T
We wish to express our sincere thanks to Dr. Takewaki for help in preparing the manuscript.
K.
Charniaux-Cotton, H. (1952). Castration chirurgicale chez un crustact amphipode (Oxhestia gummarella) et dberminisme des caracteres sexuels secoudaires. Premiers resultats. C,R. Acad. Sci. 234, 2570-2572.
62
KATAKURA
AND HASEGAWA
Charniaux-Cotton, H. (1954). Decouverte chez un crustace amphipode (Ouchestin gammarella) d’une glane endocrine responsable de la differenciation de caracteres sexuels primaires et secondaires males. C.R. Acad. Sci. 239, 780-782. Charniaux-Cotton, H., Zerbib, C., and Meusy, J. J. (1966). Monographie de la glande androgene des crustaces superieurs. Crustaceana 10, 113-136. Juchault, P., Maissiat, J., and Legrand, J. J. (1978).
Caracterisation chimique d’une substance ayant les effets biologiques de l’hormone androgene chez le crustace isopode terrestre Aramadillidium vulgare Latreille. C.R. Acad. Sci. 286, 73-76. Katakura, Y., Fujimaki, Y., and Unno, K. (1975). Partial purification and characterization of androgenic gland hormone from the isopod crustacean,Armadillidium 48, 203-209.
vulgare.
Annot.
Zool.
Japan.