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Estradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one production by isolated ovarian follicles of amago salmon (Oncorhynchus rhodurus) in response to mammalian pituitary and placental hormones and salmon gonadotropin
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
52, 329-335
(1983)
Estradiol-l7P and 17~~,20P-Dihydroxy-4-pregnen-3-one Production by Isolated Ovarian Follicles of Amago Salmo (Oncorhynchus rhodurus) in Response to Mammalian Pituitary an Placental Hormones and Salmon Gonadotropin GRAHAMYOUNG,~ Laboratory
HIROSHI
UEDA,AND~OSHITAKA
NAGAHAMA~
of Reproductive Biology, National Institute for Basic Biology, Okazaki 444. Japan Accepted December 14, 1982
The effects of mammalian pituitary and placental hormones and dibutyryl cyclic AM? (CAMP) on the production of estradiol-17g and 17cu,2Ql3-dihydroxy-4-pregnen-3-one (17a,20@-diOHprog) by amago salmon (Oncorhynch~~ rhodurus) ovarian follicles in vitro were compared with partially purified chum salmon .(Oncorhynchus keta) gonadotropin @GA). EstradioI-17P production was examined using midvitellogenic ovarian follicles; SGA was the most effective hormone tested; at a dose of 1 pg/ml it stimulated the production of approximately 4 rig/ml estradiol-17S. Ovine LH (o-LH) was about 0.5% as effective as SGA. Taking into account the relative purity of the preparations used, ovine FSH (o-PSH) was almost as effective as o-LH. Roth HCG and bovine TSH (b-TSH) stimulated estradiol-178 production at high doses. Forcine ACTH (p-ACTH), ovine GH (o-GE& and ovine PRL (oPRL) induced very modest increases in estradiol-17R; CAMP was highly effective. l7~r,2OpDiOHprog production was exam&d using full-grown immature oocytes which were capable of maturing in vitro in response to 1 pg/ml SGA. o-LH was about 0.5% as effectiv’e as SGA. o-FSH, b-TSH, and HCG appeared to be less effective in stimulating 17cr208diOHprog production, compared to estradioKl7p. o-GH and o-PRL induced marginal increases in production and p-ACTH had a very modest inverse dose-response effect. Dibutyryl CAMP stinmiated the production of approximately 0.9 and 3.8 r&ml 17a.20@-diOHprog at concentrations of I and 10 W, respectively. These data are discussed in relation to studies on gonadotropin stimulation of gnrmdal steroidogenesis in other teleosts.
S~everaL laboratories have now examined s,teroId production by gonadal &sue of teleos~tts ik I&W in response to, gon&otropins and the information which has, resulted mdicates. th& these techniques wi3J make an’ imp,ortant contribution to thle nn’derstanchng, of the &uitary-gona&f axis in fishes. l&%rzd~~-I7p production has been exami.ned iin the ovary of plaice,. PZeUroWC&S: J&Z.%SSQ~@aron and Barton, 1980), titapia, Sjar&&oldon aweus (Yams et al., 19X?),,. and amago salmo;n, Oncorhynchws t Present. address;, Department of Zoology, Univers&p of’CXifornia, Berkeley, Calif. 94720. 2 To w&m correspondence should be addressed: taboratary of Reproductive Biology, National Institute for, Basic Biology, 38 Nishigonaka, Myodaijicho, Qkazaki 444, Japan.
rhodurus (Kagawa et al., 1982a, b; Young et al., 1982b). Progesterone and 17~~,20/3 hydroxy-4-pregnen-3-one (1701,20@OHprog) production has been investigate during oocyte maturation and in the postovulatory follicles of the latter species (Nagahama and Kagawa, 1982; Young et al., 1982a, 1983). 17cx,20@-DiOHprog production has also been reported in rainbow trout, Salmo gairdneri, follicles (Fostier eb al., 1981). In4 V&XI gonadal estradiol-17P productive has been suggested as a suitable bioassay system for determining the gonad~tr~~ic activity of agonists (Yaron et al., 19821, and Bona&allo and Licht (1981) made use nrf the production of testosterone by the testes of several teleost species to determine the 3-29: 001&6480/83 Copyright AZ1 rights
$1.50
@ 1983 by Academic Press, Inc. of reproductioil in any form reserved.
330
YOUNG,
UEDA,
relative potency of various tetrapod gonadotropins. Considering the recent controversy about the number and role(s) of gonadotropins in teleosts, it seems appropriate, with the advent of specific radioimmunoassays, to examine the relative effectiveness of mammalian pituitary and placental hormones in stimulating the production of different classes of gonadal steroids in the same species. We report here the effects of mammalian pituitary and placental hormones on estradiol-17P and 17q20l3-diOHprog production by ovarian tissue of amago salmon, employing partially purified chum salmon (0. beta) gonadotropin as a standard. The effects of dibutyryl CAMP have also been assessed. MATERIALS
AND METHODS
Female amago salmon were obtained from the Gifu Prefectural Fisheries Experimental Station. Ovarian follicles from midvitellogenic females obtained in August 1981 (nonglycoprotein hormones and CAMP) and 1982 (glycoprotein hormones) were used to examine estradiol-17s production; 17a,20B-diOHprog production was examined using full-grown, immature ovarian follicles from a female obtained in October 1981. These latter follicles underwent germinal vesicle breakdown when incubated with salmon gonadotropin (see Young et al. (1982a), for the incubation procedure) . Ovarian follicles were separated by dissection and incubated in plastic tissue culture dishes containing 1 ml Ringer (10 follicles per dish) with various doses of salmon gonadotropin (SGA, Syndel Laboratories, Vancouver, Canada), mammalian hormones (ovine LH, NIAMDD,o-LH-21; ovine FSH, NIAMDD,oFSH-13; human chorionic gonadotropin, Sigma, HCG; bovine TSH, Sigma, b-TSH; porcine ACTH, Sigma, p-ACTH; ovine PRL, NIAMDD, o-PRL-NIH-P-S13; ovine GH, NIAMDD, o-GH-NIH-GH-Sll) or dibutyryl CAMP (Boehringer-Mannheim, GmbH, Mannheim, West Germany). Contamination of most NIAMDD preparations with other pituitary hormones was less than 1%; the purity of the other preparations is discussed later. Incubations were carried out in a humidified incubator at 15” and were terminated after 18 hr, when incubation medium was collected and stored frozen until radioimmunoassays for estradiol178 (Kagawa et al., 1982a) and 17a,20B-diOHprog (Young et al., 1982a, 1983) were conducted. The choice of an 18-hr incubation period was based on the data of Kagawa et al. (1982b) for estradid-17P and of
AND NAGAHAMA Kagawa (1982) for 17a,ZOB-diOHprog. Full details of the incubation procedures have been given in a previous paper (Kagawa et al., 1982a). Resulting data were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls multiple range test. The relative potencies of hormones in each system were calculated graphically from the steepest portions of the dose-response curves; however, due to nonparallelism of the response, in some cases these values are only approximate.
RESULTS (I) EstradioLI7p Satisfactory dose-response curves forestradiol-17P were obtained for all the mammalian glycoprotein pituitary and placental hormones and for SGA (Fig. 1). Basal control levels were about 1.5 @ml. SGA at concentrations of 1 and 10 kg/ml stimulated the production of between 4 and 5 @ml estradiol-17P. A plateau in the response appears to have been reached at estradiol-17P levels of approximately 5 rig/ml, and this value was taken to be the maximal capacity of the follicle in this system. LH at concentrations of between 1 and 100 kg/ml stimulated a similar pattern of response, but LH was only about 0.5% as effective as SGA in terms of dose response. FSH was 15% as effective as LH, the relatively huge dose of 500 pg/ml stimulating the production of about 5 rig/ml estradiol-17P. A similar response was obtained with TSH. A similar dose-response curve was obtained with HCG at concentrations of between 1 and 100 IU/ml, but no further significant increase occurred with 500 III/ml. In a separate experiment dose-response curves for the other mammalian pituitary hormones tested, GH, PRL, and ACTH, were not obtained, although estradiol-17P levels were modestly but significantly elevated (P < 0.05) to between 0.5 and 0.7 ng/ ml compared to the basal control values of 0.4 rig/ml (Fig. 2). SGA caused a significant elevation (P < 0.01) of estradiol-17P levels to about 2 &ml; there was no significant difference in values between the concentra-
GTH AND SALMON
I
I
001
. . .
I
.I
#
OVARIAN
.
I
I
333.
STEROIDS
.I
0.1
. ..I
,....I
1000
100 Dose'(pg/ml
O"d'&lrnl)
FIG. 1. Effects of SGA and mammalian glycoprotein pituitary and placental hormones on estradiol176 production by amago salmon ovarian follicles (August 1982; diameter 3.5 mm). Each point with bar represents the mean t SEM of three replicate incubations. R, Ringer control.
tions of SGA tested (0.1-10 rig/ml). The lower levels of estradiol-17P, compared to the previous experiment, may be related to the smaller size of the follicles (3.2 mm diameter); Ringer control levels were also lower. Kagawa et al. (1983) discuss the relationship between follicle size and estradiol- 17g production further. Dibutyryl CAMP at concentrations of I and 10 mM significantly (P < 0.01) increased estradiol-17P production, the highest value, with 10 mM CAMP, representing a fivefold increase in production (Fig. 3). (2) 17a,20f3-DiOHprog Figure 4 shows the effects of SGA and mammalian glycoprotein pituitary and placental hormones on 17a,20P-diOHprog production. Basal control values were low, at approximately 0.1 rig/ml. Satisfactory dose-response curves were obtained for all the hormones tested. Maximum production, with 10 yg/ml SGA, was 6.1 nglml 17at,20@diOHprog. LH was about 0.5% as effective as SGA, and the maximum pro-
duction, with 100 pgiml, was about 3 ~~lrn~ 17a,20@diOHprog. PSH was only about 5% as effective as LH; 500 p&nl FSH was calculated to elicit the same response as about. 25 pgiml LH. The response to TSH and HCG was almost identical as that to FSH. The other mammalian pituitary hormones (Fig. 5), similar to the case of estradiol-f7P production, elicited modest in2 3 E zl d E -7 2. .o D e zl w
1
O-PRL
I R 0.1
10
1
$03
Dose @g/ml)
FIG. 2. Effects of SGA and nonglycoproteiein mammalian pituitary hormones on estradiol-17P production by amago salmon ovarian follicles (August 1981; diameter 3.2 mm). Each point with bar represents the mean * SEM of three replicate incubation. R, Ringer control.
332
YOUNG,
1
0.1
Dose
UEDA, AND NAGAHAMA
10
(mM)
FIG. 3. Effects of dibutyryl CAMP on estradioL17P production by amago salmon ovarian follicles (August 1981; diameter 3.2 mm). Each point with bar represents the mean 2 SEM of three replicate incubations. R, Ringer control.
creases in production of 17a,20@diOHprog (P < 0.05) although dose-response curves were not obtained apart from ACTH, which showed a very modest inverse dose response (P < 0.05). Dibutyryl CAMP at concentrations of 1 and 10 mM significantly enhanced 17~,2OpdiOHprog production (P < 0.01) to 1.4 and 3.8 rig/ml, respectively (Fig. 6).
DISCUSSION The present study extends the observations of Kagawa et al. (1982b) on the effects of gonadotropins on estradiol-l7fi production by ovarian follicles of amago salmon. In that study, only a limited comparison of the effects of mammalian gonadotropins could be made, since full dose-response curves were not obtained. We confirmed that LH was much less effective than SGA, a partially purified gonadotropin prepared from chum salmon pituitaries, with a reported activity of 1.82 times that of partially purified chinook salmon (Oncorhynchus tshawytscha) gonadotropin (SG-GlOO) (determined by augmentation of CAMP levels in ovarian tissue of immature salmonids; see Idler et al. (1975)). Yaron et al. (1982) found that in S. aureus, LH is almost three times more potent than SG-GlOO in stimulating estradiol-17p production by ovarian fragments, suggesting that SC-G100 (and SGA) is less similar to tilapia gonadotropin than to LH.
,i/_..,Jg;; 01
1 Dose
10 (pglml
and
100
1000
IUlml)
FIG. 4. Effects of SGA and mammalian glycoprotein pituitary and placental hormones on 17u,2OpdiOHprog production by amago salmon ovarian follicles (October 1981; diameter 5.0 mm). Each point with bar represents the mean i SEM of three replicate incubations. R, Ringer control.
GTH AND SALMON
0
OVARIAN
STEROIDS
333
eL -“---...” ......i ............H:,z:G& o-PRL 100
1 Dose
Q$nl)
5%. 5. Effects of SC&k and mammaliannonglycopro‘pituitary hormones on 17cx,2Op-diOHprog product&n by amago salmm ovarian follicles @ctober 1981; &meter 5.0 mm). E&h point with barlrepresents the mrean + SEM ofthwe replicate incuba%ns. R, Ringer fnontrol.
FIG. 6. Effects of dibutyryl CAMP on 17cx,20@-diOHprog production by amago salmon ovarian follicles (October 1981; diameter 5.0 mm). Each point represents the mean I SEM of three replicate incubations. R, Ringer control.
on the effects of mammalian or fetra TSHs also suggest that TSH may have intrinsic gonadotropic activity in certain teWe found @hat in terms &f mass t&s leost species (see Fontaine and BurzawaFSH e asLHan t1 Gerard, 1977). Yaron et al. (1982) found the s of that b-TSH was about twice as effec However~, tie o-FSH is only about SG-GIN3 and 65% as effective as Qpure, whereas the o-LB has a purity (of stimulating estradiol-l7l3 pro about 95% (information from Dr. A. F. ovarian tissue-of S. au-em, and Parlow, NIAMDD); alkowing for this Bis- and Licht (1981) reported that sea turtle crepanoy, we calculated that the ,gonado- TSH was Xl-100% as :effective as ‘sea turtle tropic potency of o-F.SH on estradiol-17p LH in stimulating Letiosterone ~~od~ct~o~ produ’ction in the amago salmon ovarian by the testes of G. mirabilis; hwever, in follicle approaches X-90% that of ,o-LH. C. tiirtinellum testes, sea turtle FSH had Bona-Gallo and Licht (1981), using about 20% of the a&G&y of the LH. Interhighly purified preparakons, found that o- pretation of the present (data on the stimuFSH was of the same poZ.ency or was more lation of estradiol-l7@ production by bpotent than o-LH in st&nulating TSH reqtires extreme care since precise interone production by the &stes of a &n-mation $on the purity of the pre~~at~o~ Sarothers~don mossambi~us; a and the percentage contamination with LH Cichiasoma Icitrinellum; and rainbow is not available. Thus, the que$ti~~ of S. gairdneri.; however, the tistes of the goI whether mammalian TSH molecule has inbiid, ~ill~c~~~s mirubilis, exhibited a high trinsic gonadotropic activity in amago degree of specificity for LH. Paradoxisalmon remains open. In the range of l-100 ccally, o-LH was reported to be much more IU/ml, the dose--response curve for active than o-XBH in stimulat& estradiolwas strikingly similar to that of l-100 pg/ 7p production by the ovary of another, ti- ml TSH and FSH. However, no further si ia, S. aureus (Yaron et al., 1982). It ap- nificant increase in estradiol-17P producpears from these studies that generalization occurred with SO0 IU/ml. According to tions as to the specificity of heterologous Sigma, 1 IU of the HCG preparation used hormones in teleosts should be made with is equivalent to about 0.33 kg HCG. Since extieme caution, even in closely related the contamination levels and the purity of species. Results from several laboratories the WCC preparation are not known, fur-
334
YOUNG,
UEDA,
AND
ther comparison of HCG with the other hormones is not possible. HCG has been reported to be less effective than LH in stimulating estradiol-17P production by the ovary of S. aureus (Yaron et al., 1982). p-ACTH, o-GH, and o-PRL had marginal but significant effects on estradiol-17P production. Contamination of these preparations with about 1% LH may have been enough to elicit a threshold response. This study shows that in amago salmon ovarian follicles SGA and mammalian glycoprotein pituitary and placental hormones can stimulate the production of both estradiol-17@ and 17a,20@diOHprog, the steroid implicated as the maturation-inducing steroid of several species (see Nagahama et al., 1983; Young et al., 1982a,1983). For the production of both steroids, LH had about the same relative potency compared to SGA, based on the dose-response curves; it is notable, however, that while 100 pg/ml LH stimulated the production of estradiol17p to levels approaching the maximum elicited by SGA, the same dose of LH only stimulated the production of 17cw,20P-diOHprog to about 50% of the SGA maximum. Compared to the estradiol-17P study, FSH appeared to be less effective, relative to &H in the stimulation of 17a,20J3-diOHprog production: taking into account the difference in purity, FSH was about 20% ai effective as LH, although this figure is very tipproximate since the response curves ‘for FSH and LH were not parallel. The dose-response curves for TSH and HCG were similar to that for FSH (uncorrected for purity). GH, PRL, and ACTlj only marginally stimulated 17cz,20@-diOHprog production. ACTH had a very modest inverse dose-response effect for which no explanation can be offered at present. The dose-response curve for estradiol17p production in response to SGA essentially reached a plateau between 0.1 and 10 pg/ml, whereas for 17cY,2O@diOHprog the curve was still quite steep in this concentration range. This is almost certainly related to the existence in the vitellogenic fol-
NAGAHAMA
licle of the biosynthetic pathway for estradiol-17/3 prior to the incubation, as indicated by the relatively high basal control levels. We have shown that amago salmon follicles acquire the capacity to produce 17ol,20@-diOHprog in response to gonadotropin immediately prior to final oocyte maturation, when germinal vesicle breakdown follows as a consequence of gonadotropin treatment (Young et al., 1982a, 1983). In the present study follicles were removed from the ovary before the gonadotropin surge and thus the pathway for 17cy,2O@diOHprog was presumably not functional. The relatively higher threshold for the stimulation of maximal 17a,20PdiOHprog production, in the 18-hr incubations observed in the present study, is therefore probably due to a latency in the response during which the pathway for 17a,20@diOHprog was established. Relative to LH, the other glycoprotein hormones (FSH, TSH, and HCG) were less effective in the stimulation of 17a,20PdiOHprog production compared to their effects on estradiol-17P production. These data therefore lead to the important point that the results of studies assessing the relative effectiveness of gonadotropic hormones on the production of a specific gonadal steroid may not necessarily be applicable to some other steroid, even in the same species. Dibutyryl CAMP stimulated both estradiol-17P and 17a,20@diOHprog production by follicles of amago salmon. This observation suggests that as in other vertebrates, CAMP acts as the intracellular mediator of gonadotropin and relates to studies showing increased CAMP levels or adenyl cyclase activity in ovarian homogenates from several teleosts after incubation with gonadotropin (Idler et al., 1975; Fontaine et al., 1970, 1978; Kuo and Watanabe, 1978). Exogenous CAMP, although stimulating the production of 17cy,20P-diOHprog, does not induce final oocyte maturation; inhibitors of phosphodiesterase also prevent germinal vesicle breakdown in response to gonadotropin (Young, unpublished obser-
GTH AND SALMON
vations). Cyclic AMP probably inhibits the formation within the ooplasm of maturation-promoting factor (MPF) which promotes germinal vesicle breakdown (Schorderet-Slatkine et al., 1982). ACKNOWLEDGMENTS We are grateful to Mr. F. Tashiro and the staff of the Gifu Prefectural Fisheries Experimental Station for providing amago salmon and laboratory space. Puritied mammalian pituitary hormones were kindly provided by NIAMDD; we thank Dr. A. F. Parlow for additional information on the purity of the hormones. This study was supported in part by grants-in-aid from the Ministry of Education, Japan, and the award of a Japan Society for the Promotion of Science-Royal Society (Great Britain) Fellowship.
REFERENCES Bona-Gallo, A., and Licht, P. (1981). Gonadotropin specificity of in vitro testosterone secretion by fish testes. Gen. Comp. Endocrinol. 43, 467-478. Fontaine, Y. A., and Burzawa-Gerard, E. (1977). Esquisse de l’evolution des hormones gonadotropes et thyreotropes des vertebres. Gen. Comp. Endocrinol. 32, 341-347. Fontaine, Y. A., Burzawa-Gerard, E., and Delerue-Le Belle, N. (1970). Stimulation hormonale de l’activite ad&y1 cyclasique de l’ovaire d’un Poisson teleosteen, le cyprin (Carassius auratus L.). C.R. Hebd. Seances Acad. Sci. Ser. D 271, 780-783. Fontaine-Bertrand, E., Salmon, C., and Fontaine, Y. A. (1978). Effet d’hormones gonadotropes, in vitro, sur la concentration de l’adenosine monophosphate cyclique dam I’ovaire de l’anguille (Anguilla anguilla Blopkys. 18,
Fostier, and vitro
par
L.).
Ann.
Biol.
Anim.
Biockim.
805-811. A., Jalabert, B., Campbell, C., Terqui, M., Breton, B. (1981). Cinetique de liberation in de 17~ hydroxy-2OS dihydroprogesterone des follicles de Truite arc-en-ciel, Salmo gaird-
nerii. C.R. 777-780.
Hebd.
Seances
Acad.
Sci. Ser. D 292,
Idler, D. R., Hwang, S. J., and Bazar, L. S. (1975). Fish gonadotropin(s). 1. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads. Endocrine Res. Commun. 2, 199-213. Kagawa, H. (1982). “Investigation of Steroidogenesis in the Ovarian Follicle of Amago Salmon, Oncorkynckus rkodurus.” Ph.D. thesis, Hokkaido University. Kagawa, H., Young, G., Adachi, S., and Nagahama, Y. (1982a). Estradiol-17B production in amago salmon (Oncorkynckus rkodurus) ovarian follicles: Role of the thecal and granulosa cells. Gen. Comp.
Endoerinol.
47, 440-448.
OVARIAN
STEROIDS
335
Kagawa, H., Young, G., and Nagahama, Y. (1982b). Estradiol-17P production in isolated amago salmon (Oncorkynckus rkodurus) ovarian follicles and its stimulation by gonadotropins. @en. Camp. Endocrinol. 41, 361-365. Kagawa, H., Young, G., and Nagahama, Y. (1983). Relationship between seasonal plasma estradiol178 and testosterone levels and in vitro production by ovarian follicles of amago salmon (Oncorkynckus rkodurus). Biol. Reprod., in press. Kuo, C. M., and Watanabe, W. 0. (1978). Circadian responses of teleostean oocytes to gonadotropins and prostaglandins determined by cyclic AMP concentration. Arm. Biol. Anim. Biochim. Biopkys. 18, 949-956. Nagahama, Y., and Kagawa, H. (1982). In vitro steroid production in the postovulatory follicles of the amago salmon, Oncorkynckus rkodurus, in response to salmon gonadotropin. .?. Exp. Zool. 219, 105-109. Nagahama, Y., Young, G., and Kagawa, H. (1983). Steroidogenesis in the amago salmon (Oncorkynckus rkodurus) ovarian follicle: A two cell-type model. In “Proc. IXth Int. Symp. Comp. Endocrinol.,” in press. Schorderet-Slatkine, S., Schorderet, M., and Baulieu, E.-E. (1982). Cyclic AMP-mediated control of meiosis: Effects of progesterone, cholera toxin, and membrane-active drugs in Xenopus laevis oocytes. hoc. Natl. Acad. Sci. USA 79, 850-854. Yaron, Z., and Barton, C. (1980). Stimulation of estradiol-178 output from isolated ovarian fragments of the plaice (Z’leuronectes platessa) by homologous pituitary extract. Gen. Camp. Endocrinol. 42, 1.51-154.
Yaron, Z., Bogomoinaya, A., and Donaldson, E. M. (1982). The stimulation of estradiol-17@ secretion by the ovary of Sarotkerodon aureus as a bioassay for fish gonadotropin. In “Proc. IXth Int. Symp. Comp. Endocrinol.“, in press. Young, G., Crim, L. W., Kagawa, Il., Kambegawa, A., and Nagahama, Y. (1983). Plasma l&208dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorkynckas rkodurus): Correlation with plasma gonadotropin and in vitro production by ovarian follicles. Gen. Comp.
Endocrinol.
51, 96-105.
Young, G., Kagawa, II., and Nagahama, Y. (1982a). Oocyte maturation in the amago salmon (Oizcorkynckus rkodurus): In vitro effects of salmon go nadotropin, steraids and cyanoketone (an inhibitor of 3S-hydroxy-As-steroid dehydrogenase). 3. Exp.
Zool.
224, 264-275.
Young, G., Kagawa, II., and Nagahama, ‘pi. (1982b). Inhibitory effect of cyanoketone on salmon gonadotropin-induced estradiol-I7B production by ovarian follicles of the amago salmon (Oncorkynckus rkodurus) 47, 357-360.
in vitro.
Gen.
Comp.
~~docr~~o~.
AND
COMPARATIVE
ENDOCRINOLOGY
52, 329-335
(1983)
Estradiol-l7P and 17~~,20P-Dihydroxy-4-pregnen-3-one Production by Isolated Ovarian Follicles of Amago Salmo (Oncorhynchus rhodurus) in Response to Mammalian Pituitary an Placental Hormones and Salmon Gonadotropin GRAHAMYOUNG,~ Laboratory
HIROSHI
UEDA,AND~OSHITAKA
NAGAHAMA~
of Reproductive Biology, National Institute for Basic Biology, Okazaki 444. Japan Accepted December 14, 1982
The effects of mammalian pituitary and placental hormones and dibutyryl cyclic AM? (CAMP) on the production of estradiol-17g and 17cu,2Ql3-dihydroxy-4-pregnen-3-one (17a,20@-diOHprog) by amago salmon (Oncorhynch~~ rhodurus) ovarian follicles in vitro were compared with partially purified chum salmon .(Oncorhynchus keta) gonadotropin @GA). EstradioI-17P production was examined using midvitellogenic ovarian follicles; SGA was the most effective hormone tested; at a dose of 1 pg/ml it stimulated the production of approximately 4 rig/ml estradiol-17S. Ovine LH (o-LH) was about 0.5% as effective as SGA. Taking into account the relative purity of the preparations used, ovine FSH (o-PSH) was almost as effective as o-LH. Roth HCG and bovine TSH (b-TSH) stimulated estradiol-178 production at high doses. Forcine ACTH (p-ACTH), ovine GH (o-GE& and ovine PRL (oPRL) induced very modest increases in estradiol-17R; CAMP was highly effective. l7~r,2OpDiOHprog production was exam&d using full-grown immature oocytes which were capable of maturing in vitro in response to 1 pg/ml SGA. o-LH was about 0.5% as effectiv’e as SGA. o-FSH, b-TSH, and HCG appeared to be less effective in stimulating 17cr208diOHprog production, compared to estradioKl7p. o-GH and o-PRL induced marginal increases in production and p-ACTH had a very modest inverse dose-response effect. Dibutyryl CAMP stinmiated the production of approximately 0.9 and 3.8 r&ml 17a.20@-diOHprog at concentrations of I and 10 W, respectively. These data are discussed in relation to studies on gonadotropin stimulation of gnrmdal steroidogenesis in other teleosts.
S~everaL laboratories have now examined s,teroId production by gonadal &sue of teleos~tts ik I&W in response to, gon&otropins and the information which has, resulted mdicates. th& these techniques wi3J make an’ imp,ortant contribution to thle nn’derstanchng, of the &uitary-gona&f axis in fishes. l&%rzd~~-I7p production has been exami.ned iin the ovary of plaice,. PZeUroWC&S: J&Z.%SSQ~@aron and Barton, 1980), titapia, Sjar&&oldon aweus (Yams et al., 19X?),,. and amago salmo;n, Oncorhynchws t Present. address;, Department of Zoology, Univers&p of’CXifornia, Berkeley, Calif. 94720. 2 To w&m correspondence should be addressed: taboratary of Reproductive Biology, National Institute for, Basic Biology, 38 Nishigonaka, Myodaijicho, Qkazaki 444, Japan.
rhodurus (Kagawa et al., 1982a, b; Young et al., 1982b). Progesterone and 17~~,20/3 hydroxy-4-pregnen-3-one (1701,20@OHprog) production has been investigate during oocyte maturation and in the postovulatory follicles of the latter species (Nagahama and Kagawa, 1982; Young et al., 1982a, 1983). 17cx,20@-DiOHprog production has also been reported in rainbow trout, Salmo gairdneri, follicles (Fostier eb al., 1981). In4 V&XI gonadal estradiol-17P productive has been suggested as a suitable bioassay system for determining the gonad~tr~~ic activity of agonists (Yaron et al., 19821, and Bona&allo and Licht (1981) made use nrf the production of testosterone by the testes of several teleost species to determine the 3-29: 001&6480/83 Copyright AZ1 rights
$1.50
@ 1983 by Academic Press, Inc. of reproductioil in any form reserved.
330
YOUNG,
UEDA,
relative potency of various tetrapod gonadotropins. Considering the recent controversy about the number and role(s) of gonadotropins in teleosts, it seems appropriate, with the advent of specific radioimmunoassays, to examine the relative effectiveness of mammalian pituitary and placental hormones in stimulating the production of different classes of gonadal steroids in the same species. We report here the effects of mammalian pituitary and placental hormones on estradiol-17P and 17q20l3-diOHprog production by ovarian tissue of amago salmon, employing partially purified chum salmon (0. beta) gonadotropin as a standard. The effects of dibutyryl CAMP have also been assessed. MATERIALS
AND METHODS
Female amago salmon were obtained from the Gifu Prefectural Fisheries Experimental Station. Ovarian follicles from midvitellogenic females obtained in August 1981 (nonglycoprotein hormones and CAMP) and 1982 (glycoprotein hormones) were used to examine estradiol-17s production; 17a,20B-diOHprog production was examined using full-grown, immature ovarian follicles from a female obtained in October 1981. These latter follicles underwent germinal vesicle breakdown when incubated with salmon gonadotropin (see Young et al. (1982a), for the incubation procedure) . Ovarian follicles were separated by dissection and incubated in plastic tissue culture dishes containing 1 ml Ringer (10 follicles per dish) with various doses of salmon gonadotropin (SGA, Syndel Laboratories, Vancouver, Canada), mammalian hormones (ovine LH, NIAMDD,o-LH-21; ovine FSH, NIAMDD,oFSH-13; human chorionic gonadotropin, Sigma, HCG; bovine TSH, Sigma, b-TSH; porcine ACTH, Sigma, p-ACTH; ovine PRL, NIAMDD, o-PRL-NIH-P-S13; ovine GH, NIAMDD, o-GH-NIH-GH-Sll) or dibutyryl CAMP (Boehringer-Mannheim, GmbH, Mannheim, West Germany). Contamination of most NIAMDD preparations with other pituitary hormones was less than 1%; the purity of the other preparations is discussed later. Incubations were carried out in a humidified incubator at 15” and were terminated after 18 hr, when incubation medium was collected and stored frozen until radioimmunoassays for estradiol178 (Kagawa et al., 1982a) and 17a,20B-diOHprog (Young et al., 1982a, 1983) were conducted. The choice of an 18-hr incubation period was based on the data of Kagawa et al. (1982b) for estradid-17P and of
AND NAGAHAMA Kagawa (1982) for 17a,ZOB-diOHprog. Full details of the incubation procedures have been given in a previous paper (Kagawa et al., 1982a). Resulting data were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls multiple range test. The relative potencies of hormones in each system were calculated graphically from the steepest portions of the dose-response curves; however, due to nonparallelism of the response, in some cases these values are only approximate.
RESULTS (I) EstradioLI7p Satisfactory dose-response curves forestradiol-17P were obtained for all the mammalian glycoprotein pituitary and placental hormones and for SGA (Fig. 1). Basal control levels were about 1.5 @ml. SGA at concentrations of 1 and 10 kg/ml stimulated the production of between 4 and 5 @ml estradiol-17P. A plateau in the response appears to have been reached at estradiol-17P levels of approximately 5 rig/ml, and this value was taken to be the maximal capacity of the follicle in this system. LH at concentrations of between 1 and 100 kg/ml stimulated a similar pattern of response, but LH was only about 0.5% as effective as SGA in terms of dose response. FSH was 15% as effective as LH, the relatively huge dose of 500 pg/ml stimulating the production of about 5 rig/ml estradiol-17P. A similar response was obtained with TSH. A similar dose-response curve was obtained with HCG at concentrations of between 1 and 100 IU/ml, but no further significant increase occurred with 500 III/ml. In a separate experiment dose-response curves for the other mammalian pituitary hormones tested, GH, PRL, and ACTH, were not obtained, although estradiol-17P levels were modestly but significantly elevated (P < 0.05) to between 0.5 and 0.7 ng/ ml compared to the basal control values of 0.4 rig/ml (Fig. 2). SGA caused a significant elevation (P < 0.01) of estradiol-17P levels to about 2 &ml; there was no significant difference in values between the concentra-
GTH AND SALMON
I
I
001
. . .
I
.I
#
OVARIAN
.
I
I
333.
STEROIDS
.I
0.1
. ..I
,....I
1000
100 Dose'(pg/ml
O"d'&lrnl)
FIG. 1. Effects of SGA and mammalian glycoprotein pituitary and placental hormones on estradiol176 production by amago salmon ovarian follicles (August 1982; diameter 3.5 mm). Each point with bar represents the mean t SEM of three replicate incubations. R, Ringer control.
tions of SGA tested (0.1-10 rig/ml). The lower levels of estradiol-17P, compared to the previous experiment, may be related to the smaller size of the follicles (3.2 mm diameter); Ringer control levels were also lower. Kagawa et al. (1983) discuss the relationship between follicle size and estradiol- 17g production further. Dibutyryl CAMP at concentrations of I and 10 mM significantly (P < 0.01) increased estradiol-17P production, the highest value, with 10 mM CAMP, representing a fivefold increase in production (Fig. 3). (2) 17a,20f3-DiOHprog Figure 4 shows the effects of SGA and mammalian glycoprotein pituitary and placental hormones on 17a,20P-diOHprog production. Basal control values were low, at approximately 0.1 rig/ml. Satisfactory dose-response curves were obtained for all the hormones tested. Maximum production, with 10 yg/ml SGA, was 6.1 nglml 17at,20@diOHprog. LH was about 0.5% as effective as SGA, and the maximum pro-
duction, with 100 pgiml, was about 3 ~~lrn~ 17a,20@diOHprog. PSH was only about 5% as effective as LH; 500 p&nl FSH was calculated to elicit the same response as about. 25 pgiml LH. The response to TSH and HCG was almost identical as that to FSH. The other mammalian pituitary hormones (Fig. 5), similar to the case of estradiol-f7P production, elicited modest in2 3 E zl d E -7 2. .o D e zl w
1
O-PRL
I R 0.1
10
1
$03
Dose @g/ml)
FIG. 2. Effects of SGA and nonglycoproteiein mammalian pituitary hormones on estradiol-17P production by amago salmon ovarian follicles (August 1981; diameter 3.2 mm). Each point with bar represents the mean * SEM of three replicate incubation. R, Ringer control.
332
YOUNG,
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0.1
Dose
UEDA, AND NAGAHAMA
10
(mM)
FIG. 3. Effects of dibutyryl CAMP on estradioL17P production by amago salmon ovarian follicles (August 1981; diameter 3.2 mm). Each point with bar represents the mean 2 SEM of three replicate incubations. R, Ringer control.
creases in production of 17a,20@diOHprog (P < 0.05) although dose-response curves were not obtained apart from ACTH, which showed a very modest inverse dose response (P < 0.05). Dibutyryl CAMP at concentrations of 1 and 10 mM significantly enhanced 17~,2OpdiOHprog production (P < 0.01) to 1.4 and 3.8 rig/ml, respectively (Fig. 6).
DISCUSSION The present study extends the observations of Kagawa et al. (1982b) on the effects of gonadotropins on estradiol-l7fi production by ovarian follicles of amago salmon. In that study, only a limited comparison of the effects of mammalian gonadotropins could be made, since full dose-response curves were not obtained. We confirmed that LH was much less effective than SGA, a partially purified gonadotropin prepared from chum salmon pituitaries, with a reported activity of 1.82 times that of partially purified chinook salmon (Oncorhynchus tshawytscha) gonadotropin (SG-GlOO) (determined by augmentation of CAMP levels in ovarian tissue of immature salmonids; see Idler et al. (1975)). Yaron et al. (1982) found that in S. aureus, LH is almost three times more potent than SG-GlOO in stimulating estradiol-17p production by ovarian fragments, suggesting that SC-G100 (and SGA) is less similar to tilapia gonadotropin than to LH.
,i/_..,Jg;; 01
1 Dose
10 (pglml
and
100
1000
IUlml)
FIG. 4. Effects of SGA and mammalian glycoprotein pituitary and placental hormones on 17u,2OpdiOHprog production by amago salmon ovarian follicles (October 1981; diameter 5.0 mm). Each point with bar represents the mean i SEM of three replicate incubations. R, Ringer control.
GTH AND SALMON
0
OVARIAN
STEROIDS
333
eL -“---...” ......i ............H:,z:G& o-PRL 100
1 Dose
Q$nl)
5%. 5. Effects of SC&k and mammaliannonglycopro‘pituitary hormones on 17cx,2Op-diOHprog product&n by amago salmm ovarian follicles @ctober 1981; &meter 5.0 mm). E&h point with barlrepresents the mrean + SEM ofthwe replicate incuba%ns. R, Ringer fnontrol.
FIG. 6. Effects of dibutyryl CAMP on 17cx,20@-diOHprog production by amago salmon ovarian follicles (October 1981; diameter 5.0 mm). Each point represents the mean I SEM of three replicate incubations. R, Ringer control.
on the effects of mammalian or fetra TSHs also suggest that TSH may have intrinsic gonadotropic activity in certain teWe found @hat in terms &f mass t&s leost species (see Fontaine and BurzawaFSH e asLHan t1 Gerard, 1977). Yaron et al. (1982) found the s of that b-TSH was about twice as effec However~, tie o-FSH is only about SG-GIN3 and 65% as effective as Qpure, whereas the o-LB has a purity (of stimulating estradiol-l7l3 pro about 95% (information from Dr. A. F. ovarian tissue-of S. au-em, and Parlow, NIAMDD); alkowing for this Bis- and Licht (1981) reported that sea turtle crepanoy, we calculated that the ,gonado- TSH was Xl-100% as :effective as ‘sea turtle tropic potency of o-F.SH on estradiol-17p LH in stimulating Letiosterone ~~od~ct~o~ produ’ction in the amago salmon ovarian by the testes of G. mirabilis; hwever, in follicle approaches X-90% that of ,o-LH. C. tiirtinellum testes, sea turtle FSH had Bona-Gallo and Licht (1981), using about 20% of the a&G&y of the LH. Interhighly purified preparakons, found that o- pretation of the present (data on the stimuFSH was of the same poZ.ency or was more lation of estradiol-l7@ production by bpotent than o-LH in st&nulating TSH reqtires extreme care since precise interone production by the &stes of a &n-mation $on the purity of the pre~~at~o~ Sarothers~don mossambi~us; a and the percentage contamination with LH Cichiasoma Icitrinellum; and rainbow is not available. Thus, the que$ti~~ of S. gairdneri.; however, the tistes of the goI whether mammalian TSH molecule has inbiid, ~ill~c~~~s mirubilis, exhibited a high trinsic gonadotropic activity in amago degree of specificity for LH. Paradoxisalmon remains open. In the range of l-100 ccally, o-LH was reported to be much more IU/ml, the dose--response curve for active than o-XBH in stimulat& estradiolwas strikingly similar to that of l-100 pg/ 7p production by the ovary of another, ti- ml TSH and FSH. However, no further si ia, S. aureus (Yaron et al., 1982). It ap- nificant increase in estradiol-17P producpears from these studies that generalization occurred with SO0 IU/ml. According to tions as to the specificity of heterologous Sigma, 1 IU of the HCG preparation used hormones in teleosts should be made with is equivalent to about 0.33 kg HCG. Since extieme caution, even in closely related the contamination levels and the purity of species. Results from several laboratories the WCC preparation are not known, fur-
334
YOUNG,
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AND
ther comparison of HCG with the other hormones is not possible. HCG has been reported to be less effective than LH in stimulating estradiol-17P production by the ovary of S. aureus (Yaron et al., 1982). p-ACTH, o-GH, and o-PRL had marginal but significant effects on estradiol-17P production. Contamination of these preparations with about 1% LH may have been enough to elicit a threshold response. This study shows that in amago salmon ovarian follicles SGA and mammalian glycoprotein pituitary and placental hormones can stimulate the production of both estradiol-17@ and 17a,20@diOHprog, the steroid implicated as the maturation-inducing steroid of several species (see Nagahama et al., 1983; Young et al., 1982a,1983). For the production of both steroids, LH had about the same relative potency compared to SGA, based on the dose-response curves; it is notable, however, that while 100 pg/ml LH stimulated the production of estradiol17p to levels approaching the maximum elicited by SGA, the same dose of LH only stimulated the production of 17cw,20P-diOHprog to about 50% of the SGA maximum. Compared to the estradiol-17P study, FSH appeared to be less effective, relative to &H in the stimulation of 17a,20J3-diOHprog production: taking into account the difference in purity, FSH was about 20% ai effective as LH, although this figure is very tipproximate since the response curves ‘for FSH and LH were not parallel. The dose-response curves for TSH and HCG were similar to that for FSH (uncorrected for purity). GH, PRL, and ACTlj only marginally stimulated 17cz,20@-diOHprog production. ACTH had a very modest inverse dose-response effect for which no explanation can be offered at present. The dose-response curve for estradiol17p production in response to SGA essentially reached a plateau between 0.1 and 10 pg/ml, whereas for 17cY,2O@diOHprog the curve was still quite steep in this concentration range. This is almost certainly related to the existence in the vitellogenic fol-
NAGAHAMA
licle of the biosynthetic pathway for estradiol-17/3 prior to the incubation, as indicated by the relatively high basal control levels. We have shown that amago salmon follicles acquire the capacity to produce 17ol,20@-diOHprog in response to gonadotropin immediately prior to final oocyte maturation, when germinal vesicle breakdown follows as a consequence of gonadotropin treatment (Young et al., 1982a, 1983). In the present study follicles were removed from the ovary before the gonadotropin surge and thus the pathway for 17cy,2O@diOHprog was presumably not functional. The relatively higher threshold for the stimulation of maximal 17a,20PdiOHprog production, in the 18-hr incubations observed in the present study, is therefore probably due to a latency in the response during which the pathway for 17a,20@diOHprog was established. Relative to LH, the other glycoprotein hormones (FSH, TSH, and HCG) were less effective in the stimulation of 17a,20PdiOHprog production compared to their effects on estradiol-17P production. These data therefore lead to the important point that the results of studies assessing the relative effectiveness of gonadotropic hormones on the production of a specific gonadal steroid may not necessarily be applicable to some other steroid, even in the same species. Dibutyryl CAMP stimulated both estradiol-17P and 17a,20@diOHprog production by follicles of amago salmon. This observation suggests that as in other vertebrates, CAMP acts as the intracellular mediator of gonadotropin and relates to studies showing increased CAMP levels or adenyl cyclase activity in ovarian homogenates from several teleosts after incubation with gonadotropin (Idler et al., 1975; Fontaine et al., 1970, 1978; Kuo and Watanabe, 1978). Exogenous CAMP, although stimulating the production of 17cy,20P-diOHprog, does not induce final oocyte maturation; inhibitors of phosphodiesterase also prevent germinal vesicle breakdown in response to gonadotropin (Young, unpublished obser-
GTH AND SALMON
vations). Cyclic AMP probably inhibits the formation within the ooplasm of maturation-promoting factor (MPF) which promotes germinal vesicle breakdown (Schorderet-Slatkine et al., 1982). ACKNOWLEDGMENTS We are grateful to Mr. F. Tashiro and the staff of the Gifu Prefectural Fisheries Experimental Station for providing amago salmon and laboratory space. Puritied mammalian pituitary hormones were kindly provided by NIAMDD; we thank Dr. A. F. Parlow for additional information on the purity of the hormones. This study was supported in part by grants-in-aid from the Ministry of Education, Japan, and the award of a Japan Society for the Promotion of Science-Royal Society (Great Britain) Fellowship.
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Idler, D. R., Hwang, S. J., and Bazar, L. S. (1975). Fish gonadotropin(s). 1. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads. Endocrine Res. Commun. 2, 199-213. Kagawa, H. (1982). “Investigation of Steroidogenesis in the Ovarian Follicle of Amago Salmon, Oncorkynckus rkodurus.” Ph.D. thesis, Hokkaido University. Kagawa, H., Young, G., Adachi, S., and Nagahama, Y. (1982a). Estradiol-17B production in amago salmon (Oncorkynckus rkodurus) ovarian follicles: Role of the thecal and granulosa cells. Gen. Comp.
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Kagawa, H., Young, G., and Nagahama, Y. (1982b). Estradiol-17P production in isolated amago salmon (Oncorkynckus rkodurus) ovarian follicles and its stimulation by gonadotropins. @en. Camp. Endocrinol. 41, 361-365. Kagawa, H., Young, G., and Nagahama, Y. (1983). Relationship between seasonal plasma estradiol178 and testosterone levels and in vitro production by ovarian follicles of amago salmon (Oncorkynckus rkodurus). Biol. Reprod., in press. Kuo, C. M., and Watanabe, W. 0. (1978). Circadian responses of teleostean oocytes to gonadotropins and prostaglandins determined by cyclic AMP concentration. Arm. Biol. Anim. Biochim. Biopkys. 18, 949-956. Nagahama, Y., and Kagawa, H. (1982). In vitro steroid production in the postovulatory follicles of the amago salmon, Oncorkynckus rkodurus, in response to salmon gonadotropin. .?. Exp. Zool. 219, 105-109. Nagahama, Y., Young, G., and Kagawa, H. (1983). Steroidogenesis in the amago salmon (Oncorkynckus rkodurus) ovarian follicle: A two cell-type model. In “Proc. IXth Int. Symp. Comp. Endocrinol.,” in press. Schorderet-Slatkine, S., Schorderet, M., and Baulieu, E.-E. (1982). Cyclic AMP-mediated control of meiosis: Effects of progesterone, cholera toxin, and membrane-active drugs in Xenopus laevis oocytes. hoc. Natl. Acad. Sci. USA 79, 850-854. Yaron, Z., and Barton, C. (1980). Stimulation of estradiol-178 output from isolated ovarian fragments of the plaice (Z’leuronectes platessa) by homologous pituitary extract. Gen. Camp. Endocrinol. 42, 1.51-154.
Yaron, Z., Bogomoinaya, A., and Donaldson, E. M. (1982). The stimulation of estradiol-17@ secretion by the ovary of Sarotkerodon aureus as a bioassay for fish gonadotropin. In “Proc. IXth Int. Symp. Comp. Endocrinol.“, in press. Young, G., Crim, L. W., Kagawa, Il., Kambegawa, A., and Nagahama, Y. (1983). Plasma l&208dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorkynckas rkodurus): Correlation with plasma gonadotropin and in vitro production by ovarian follicles. Gen. Comp.
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Young, G., Kagawa, II., and Nagahama, Y. (1982a). Oocyte maturation in the amago salmon (Oizcorkynckus rkodurus): In vitro effects of salmon go nadotropin, steraids and cyanoketone (an inhibitor of 3S-hydroxy-As-steroid dehydrogenase). 3. Exp.
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Young, G., Kagawa, II., and Nagahama, ‘pi. (1982b). Inhibitory effect of cyanoketone on salmon gonadotropin-induced estradiol-I7B production by ovarian follicles of the amago salmon (Oncorkynckus rkodurus) 47, 357-360.
in vitro.
Gen.
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