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Estrogen-induced vitellogenin production by triploid coho salmon (Oncorhynchus kisutch), and its effect on plasma and pituitary gonadotropin
GENERAL
AND
COMPARATIVE
75,83-87 (1989)
ENDOCRINOLOGY
Estrogen-Induced Vitellogenin Production by Triploid Coho Salmon (Oncorhynchus kisutch), and Its Effect on Plasma and Pituitary Gonadotropin’ TILLMANN
J.
BENFEY,’
HELEN
M.
DYE,
AND EDWARD
M.
DONALDSON
Department of Fisheries and Oceans, Biological Sciences Branch, West Vancouver Laboratory, Drive, West Vancouver, British Columbia, Canada V7V IN6
4160 Marine
Accepted October 24, 1988 Vitellogenin production was induced in immature diploid and triploid coho salmon by the weekly injection of 17S-estradiol at 1 mgikg body wt. There was no significant diierence between diploids and triploids for any of the results obtained, i.e., change in plasma vitellogenin and gonadotropin levels, hepatosomatic index, or pituitary gonadotropin content. Plasma vitellogenin levels were significantly higher in 17Q-estradiol-treated fish than in sham-injected fish within a week of the first injection, and continued to rise with each subsequent injection. Plasma gonadotropin levels, on the other hand, were slightly (but signilicantly) depressed. The 17g-estradiol-treated fish had higher hepatosomatic indices and pituitary gonadotropin contents than sham-injected fish by 3 weeks after the first treatment. These data suggest that the occasional postmeiotic oocytes observed in triploids do not grow to full maturity due, in part, to an absent or diminished estrogen stimulus from the ovary on hepatic vitellogenin production. 0 1989 Academic PESS, Inc.
pink salmon, Oncorhynchus gorbuscha (Benfey et al., 1988b), as well as in the newt, Pleurodeles waltl (Cayrol et al., 1985). Hepatic production of the protein vitellogenin in teleosts is mediated by the ovarian estrogens, including 17@estradiol (Ng and Idler, 1983; Scott and Sumpter, 1983; Wallace, 1985). Hence, one would expect triploid females to have much lower plasma vitellogenin levels than maturing diploid females, and this appears to be the case (Sumpter et al., 1984; Benfey et al., 1988b). Furthermore, triploid females tend to have smaller livers than maturing diploids, which is likely an indication of diminished or completely absent vitellogenin production (Lincoln and Scott, 1984; Johnson et al., 1986). In coho salmon, 0. kisutch, a difference in vitellogenin levels between diploid and triploid females is apparent even before there are noticeable differences in plasma levels of 17@estradiol (Johnson et al., 1986). The aim of the present study was to at-
INTRODUCTION
Ovarian development is greatly retarded in triploid fish because triploid cells cannot undergo meiotic division (Thorgaard, 1983; Purdom, 1984), and hence most oogonia do not become transformed into oocytes. Small numbers of postmeiotic cells are produced by triploid salmonids, but these only develop to the mature stage in males (i.e., functional spermatozoa). Because of diminished ovarian development, 17p-estradiol levels are much lower in triploid females than in maturing diploid females of the same age, as has been demonstrated in rainbow trout, Salmo gairdneri (Lincoln and Scott, 1984; Sumpter et al., 1984; Nakamura et al., 1987; Benfey et al., 1988b), and ’ Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John’s, Newfoundland, August 2-7, 1987. 2 Present address: IvIinistry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk, NR33 OHT, UK. 83
001~6480/89 $1.50 Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in my form mservcd.
84
BENFEY,
DYE,
tempt to induce vitellogenin production in triploid coho salmon by 17P-estradiol therapy, a technique known to stimulate vitellogenesis in normal immature salmonids (Haux and Norberg, 1985). We also examined the effect of this treatment on gonadotropin levels, since estrogens and aromatizable androgens can exert feedback controls on gonadotropin synthesis and release in immature salmonids (e.g., Crim et al., 1981), and gonadotropin itself mediates vitellogenin uptake by oocytes (Wallace, 1985). MATERIALS
AND METHODS
Fish. The fish used in this study were immature 29month-old diploid and triploid coho salmon raised at the West Vancouver Laboratory. The eggs from which this stock came originated from the Capilano River Salmonid Enhancement Program Hatchery (Department of Fisheries and Oceans). Tripioids were produced by applying a hydrostatic pressure shock of 4 min at 10,000 psi to the eggs 15 min after fertilization and incubation at lO.Y, using the laboratory-scale apparatus described by Benfey ei nl. (1988a). All the fish were marked with external numerically coded tags, and were positively identified as diploid or triploid by propidium-iodide flow cytometry (Allen, 1983). Induced vitellogenin production. To induce vitellogenin production, fish were injected intraperitoneally with 17B-estradiol at 1 mg/kg body wt. The 17B-estradiol was first dissolved in 10% ethanol at 12.5 mg/ml, and then a fivefold dilution was made in peanut oil to give a concentration of 2.5 mg/ml. Thus, for an average fish (0.56 kg), the injection volume was 0.22 ml. Controls were sham injected with the same solution of ethanol and peanut oil, but lacking the 178-estradiol. The starting weights were 0.56 kg (20.15 kg SD, n = 10) for diploid-sham, 0.61 kg (20.20 kg, n = 7) for triploid-sham, 0.56 kg (20.23 kg, n = 10) for diploid-treated, and 0.53 kg (-CO.21 kg, n = 10) for triploid-treated fish. Fish were anaesthetized, injected, and bled once a week for 3 weeks. One week after the third injection, all the fish were killed, bled, and had their gonads and livers removed and weighed for the calculation of gonadosomatic index (GSI) and hepatosomatic index (HSI), and pituitaries were removed for the measurement of pituitary gonadotropin content. The gonads were kept for histology. Radioimmunoassays. Blood samples were centrifuged immediately after their collection and were stored at - 30” until they were assayed. Fresh pituitaries were homogenized in 1 ml of radioimmunoassay
AND
DONALDSON
buffer and were similarly stored. Vitellogenin was measured using the homologous radioimmunoassay described by Benfey et al. (1989). The radioimmunoassay for gonadotropin was essentially as that described by Pickering et al. (1987), but using an immunologically less potent form of purified hormone.
RESULTS There was no apparent histological difference in the development of diploid and triploid testes. Most cells were still at the spermatogonial stage, but some spermatocytes were present in all the fish. There was no difference in GSI among any of the groups of males (Table 1). Diploid ovaries were much further advanced than triploid ovaries: diploid ovaries were full of oocytes at “endogenous” vitellogenesis (i.e., “yolk vesicle” or cortical alveoli stage) whereas those of triploids were completely devoid of oocytes. Diploid females had a significantly higher GSI than triploid females (Table 1). Two of the seven diploid females treated with 17P-estradiol had some “yolk globules” in their oocytes, indicative of the start of “exogenous” or true vitellogenesis. The data for this experiment are summarized in Fig. 1. Of the sham-injected fish, only the diploid females had measurable levels of vitellogenin. These levels remained constant at 3 to 4 &ml (mean value) throughout the experiment. Vitellogenin levels were below assay detectability (0.05 ~&ml) for all the sham-injected males and triploid females. There was no difference between sexes for any of the remainTABLE
1
GONADOSOMATIC INDICES OF DIPLOID TRIPLOID COHO SALMON
Diploid Triploid Diploid Triploid
males males females females
Sham 0.15 5 0.03 (4) 0.13 f 0.04 (4) 0.64 k 0.45 (6) 0.04 f 0.01 (3)
AND
l7~-estradiol 0.14 f 0.14 f 0.69 f 0.05 f
0.03 (2) 0.02 (5) 0.44 (7) 0.01 (5)
Note. Values are. given as percentages, mean + standard deviation; sample size is given in parentheses.
VITELLOGENESIS
IN TRIPLOID
SALMON
85
diol-treated fish had lower levels than their respective shams at Week 2 (P < 0.01 in each case), as well as at Week 3 in the case of the triploids (P < 0.05). There was no difference in pituitary gonadotropin content between diploid and triploid sham-injected fish or between diploid and triploid 17pestradiol-treated fish at the end of the experiment. However, 17p-estradiol-treated fish had significantly higher pituitary gonadotropin levels than their respective sham-injected controls (P < 0.001). DISCUSSION
WEEK
FIG. 1. Change in plasma vitellogenin (Vtg) and gonadotropin (GtH) over 3 weeks, and final values for hepatosomatic index and pituitary GtH content (0, diploid sham; A, triploid sham; 0, diploid 17@-estradiol; A, triploid 17P-estradiol; plotted as mean + standard deviation).
ing results, so males and females have been combined. Treatment with 17@estradiol caused a rapid and significant increase in plasma vitellogenin levels. Within 1 week of the first injection, vitellogenin levels were significantly higher than in sham-injected fish (P < 0.001) and continued to rise with each subsequent injection. There was no significant difference between the vitellogenin levels of 17P-estradiol-treated diploids and triploids at any given date. Diploids treated with 17@estradiol had a significantly higher HSI than sham-injected diploids by the end of the experiment (P < 0.05); treated triploids also had a higher HSI than shaminjected triploids, but this difference was not significant. At any given date, there was no difference between diploids and triploids of either sham-injected fish or 17@-estradiol treated fish for plasma gonadotropin. However, both diploid and triploid 17l3-estra-
Our data demonstrate that normal vitellogenin production can occur in triploid fish if the appropriate stimulus is provided. This suggests that the occasional postmeiotic oocytes observed in triploid females may not reach full maturity because of insufftcient vitellogenin production by the liver. This, in turn, would likely be due to the greatly diminished number of estrogenproducing follicle cells in the triploid ovary. The obtainment of fully mature oocytes from triploids would be of great interest for the production of polyploid lines (Purdom, 1984), and may thus be possible with longterm therapy to induce vitellogenin synthesis. However, long-term estrogen therapy may not be suitable because it causes the asynchronous development of oocytes (Lessman and Habibi, 1987). In fact, none of these particular triploid females had any postmeiotic oocytes, so true vitellogenesis (i.e., hepatic vitellogenin synthesis and its uptake by oocytes) could not be observed. However, the treatment did appear to be sufficient to initiate vitellogenin uptake by the oocytes of at least some of the diploid females. The absence of oocytes in the triploids makes it impossible to state conclusively that a lack of vitellogenin production is the cause for the failure of postmeiotic oocytes to grow to maturity in triploid females. Certainly there is a closely related factor which may be in-
86
BENFEY,
DYE,
volved, i.e., the synthesis and secretion of gonadotropin. Clearly, triploid females were able to synthesize the “ovulatory” gonadotropin that was measured in this study, so one can assume that they should also be able to produce other gonadotrogonadotropin(s), such as “vitellogenic” pin. Gonadotropin plays an important role in mediating vitellogenesis in fish (Wallace, 1985). Another possibility, not examined in this study, is that oocyte growth is blocked earlier than vitellogenesis. Nothing is known about the meiotic mechanism which allows occasional oocytes to develop beyond the normal initial disfunction in triploids, but if chromosomal tertiary structure is abnormal then the production of RNA may be affected. Thus, it is quite possible that such oocytes have chromosomes that are too condensed to allow DNA transcription for the production of critical proteins involved in the initial postmeiotic growth of oocytes prior to true vitellogenesis. The significant rise in pituitary gonadotropin content in the 17P-estradiol-treated fish clearly demonstrates the positive effect of estrogens on gonadotropin synthesis in immature fish (Crim et al., 1981; Peter, 1982; Kah, 1986). However, there was no increase in plasma gonadotropin levels; in fact, the levels of this hormone in circulation were slightly (but significantly) depressed by estrogen treatment. This is probably a sign that gonadotropin release from the pituitary was inhibited; estrogens appear to have such an effect on the pituitary (Peter, 1982; Kah, 1986). ACKNOWLEDGMENTS We sincerely thank John Sumpter for his comments on this manuscript. We also thank Cathy ClancyBenfey and Mark Graham for their help in collecting the gonads, livers, and pituitaries; Andy Lamb for rearing the fish; Gary de Jong for performing the flow cytometry; and John Sumpter and Terry Owen for providing the materials for the gonadotropin and vitellogenin radioimmunoassays, respectively. T.J.B. was
AND
DONALDSON
supported by a NSERC postgraduate scholarship and a Quebec FCAR graduate fellowship.
REFERENCES Allen, S. K., Jr. (1983). Flow cytometry: Assaying experimental polyploid fish and shellfish. Aquuculture 33, 317-328. Benfey, T. J., Bosa, P. G., Richardson, N. L., and Donaldson, E. M. (1988a). Effectiveness of a commercial-scale pressure shocking device for producing triploid salmonids. Aquact. Eng., 7, 147-154. Benfey, T. J., Donaldson, E. M., and Owen, T. G. (1989). An homologous radioimmunoassay for coho salmon (Oncorhynchus kisurch) vitellogenin, with general applicability to other Pacific salmonids. Gen. Camp. Endocrinol. 75, 78-82. Benfey, T. J., Dye, H. M., Solar, I. I., and Donaldson, E. M. (1988b). The growth and reproductive endocrinology of adult triploid Pacific salmonids. Fish Physiol. Biochem., in press. Cayrol, C., Gamier, D. H., and Deparis, P. (1985). Comparative plasma levels of androgens and I7B-estradiol in the diploid and triploid newt, Pleurodeles waltl. Gen. Comp. Endocrinol. 58, 342-346. Grim, L. W., Peter, R. E., and Billard, R. (1981). Onset of gonadotropic hormone accumulation in the immature trout pituitary gland in response to estrogen or aromatizable androgen steroid hormones. Gen. Comp. Endocrinol. 44, 374-381. Haux, C., and Norberg, B. (1985). The influence of estradiol-17S on the liver content of protein, lipids, glycogen and nucleic acids in juvenile rainbow trout, Salmo gairdnerii. Comp. Biochem. Physiol. B 81, 275-279. Johnson, 0. W., Dickhoff, W. W., and Utter, F. M. (1986). Comparative growth and development of diploid and triploid coho salmon, Oncorhynchus kisutch. Aquaculture 57, 329-336. Kah, 0. (1986). Central regulation of reproduction in teleosts. Fish Physiol. Biochem. 2. 25-34. Lessman, C. A., and Habibi, H. R. (1987). Estradiol-17B silastic implants suppress oocyte development in the brook trout, Satvelinus fontinab Gen. Comp. Endocrinol. 67, 311-323. Lincoln, R. F., and Scott, A. P. (1984). Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson. .I. Fish Biol. 25, 385-392. Nakamura, M., Nagahama, Y., Iwahashi, M., and Kojima, M. (1987). Ovarian structure and plasma steroid hormones of triploid female rainbow trout. Nippon Suisan Gakkaishi 53, 1105. Ng, T. B., and Idler, D. R. (1983). Yolk formation and differentiation in teleost fishes. In “Fish
VITELLOGENESIS
IN TRIPLOID
Physiology” (W. S. Hoar, D. J. Randall, and E. M. Donaldson, Eds.), Vol. 9A, Chap. 8. Academic Press, New York. Peter, R. E. (1982). Neuroendocrine control of reproduction in teleosts. Canad. J. Fish. Aquat. Sci. 39, 48-55. Pickering, A. D., Pottinger, T. G., Carragher, J., and Sumpter, J. P. (1987). The effects of acute and chronic stress on the level of reproductive hormones in the plasma of mature male brown trout, Salmo trutta L. Gen. Camp. Endocrinol. 68,249259. Purdom, C. E. (1984). Atypical modes of reproduction in fish. In “Oxford Reviews of Reproductive Biology” (J. R. Clarke, Ed.), Vol. 6, Chap. 7. Oxford Univ. Press, Oxford. Scott, A. P., and Sumpter, J. P. (1983). The control of trout reproduction: Basic and applied research on
SALMON
87
hormones. In “Control Processes in Fish Physiology” (J. C. Rankin, T. J. Pitcher, and R. T. Duggan, Eds.), Chap. 11. Croom Helm, London. Sumpter, J. P., Scott, A. P., Baynes, S. M., and Witthames, P. R. (1984). Early stages of the reproductive cycle in virgin female rainbow trout (Salmo gairdneri Richardson). Aquaculture 43, 235-242. Thorgaard, G. H. (1983). Chromosome set manipulation and sex control in fish. In “Fish Physiology” (W. S. Hoar, D. J. Randall, and E. hi. Donaldson, Eds.), Vol. 9B, Chap. 8. Academic Press, New York. Wallace, R. A. (1985). Vitellogenesis and oocyte growth in non-mammalian vertebrates. In “Developmental Biology” (L. W. Browder, Ed.), Vol. 1, Chap. 3. Plenum, New York.
AND
COMPARATIVE
75,83-87 (1989)
ENDOCRINOLOGY
Estrogen-Induced Vitellogenin Production by Triploid Coho Salmon (Oncorhynchus kisutch), and Its Effect on Plasma and Pituitary Gonadotropin’ TILLMANN
J.
BENFEY,’
HELEN
M.
DYE,
AND EDWARD
M.
DONALDSON
Department of Fisheries and Oceans, Biological Sciences Branch, West Vancouver Laboratory, Drive, West Vancouver, British Columbia, Canada V7V IN6
4160 Marine
Accepted October 24, 1988 Vitellogenin production was induced in immature diploid and triploid coho salmon by the weekly injection of 17S-estradiol at 1 mgikg body wt. There was no significant diierence between diploids and triploids for any of the results obtained, i.e., change in plasma vitellogenin and gonadotropin levels, hepatosomatic index, or pituitary gonadotropin content. Plasma vitellogenin levels were significantly higher in 17Q-estradiol-treated fish than in sham-injected fish within a week of the first injection, and continued to rise with each subsequent injection. Plasma gonadotropin levels, on the other hand, were slightly (but signilicantly) depressed. The 17g-estradiol-treated fish had higher hepatosomatic indices and pituitary gonadotropin contents than sham-injected fish by 3 weeks after the first treatment. These data suggest that the occasional postmeiotic oocytes observed in triploids do not grow to full maturity due, in part, to an absent or diminished estrogen stimulus from the ovary on hepatic vitellogenin production. 0 1989 Academic PESS, Inc.
pink salmon, Oncorhynchus gorbuscha (Benfey et al., 1988b), as well as in the newt, Pleurodeles waltl (Cayrol et al., 1985). Hepatic production of the protein vitellogenin in teleosts is mediated by the ovarian estrogens, including 17@estradiol (Ng and Idler, 1983; Scott and Sumpter, 1983; Wallace, 1985). Hence, one would expect triploid females to have much lower plasma vitellogenin levels than maturing diploid females, and this appears to be the case (Sumpter et al., 1984; Benfey et al., 1988b). Furthermore, triploid females tend to have smaller livers than maturing diploids, which is likely an indication of diminished or completely absent vitellogenin production (Lincoln and Scott, 1984; Johnson et al., 1986). In coho salmon, 0. kisutch, a difference in vitellogenin levels between diploid and triploid females is apparent even before there are noticeable differences in plasma levels of 17@estradiol (Johnson et al., 1986). The aim of the present study was to at-
INTRODUCTION
Ovarian development is greatly retarded in triploid fish because triploid cells cannot undergo meiotic division (Thorgaard, 1983; Purdom, 1984), and hence most oogonia do not become transformed into oocytes. Small numbers of postmeiotic cells are produced by triploid salmonids, but these only develop to the mature stage in males (i.e., functional spermatozoa). Because of diminished ovarian development, 17p-estradiol levels are much lower in triploid females than in maturing diploid females of the same age, as has been demonstrated in rainbow trout, Salmo gairdneri (Lincoln and Scott, 1984; Sumpter et al., 1984; Nakamura et al., 1987; Benfey et al., 1988b), and ’ Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John’s, Newfoundland, August 2-7, 1987. 2 Present address: IvIinistry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk, NR33 OHT, UK. 83
001~6480/89 $1.50 Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in my form mservcd.
84
BENFEY,
DYE,
tempt to induce vitellogenin production in triploid coho salmon by 17P-estradiol therapy, a technique known to stimulate vitellogenesis in normal immature salmonids (Haux and Norberg, 1985). We also examined the effect of this treatment on gonadotropin levels, since estrogens and aromatizable androgens can exert feedback controls on gonadotropin synthesis and release in immature salmonids (e.g., Crim et al., 1981), and gonadotropin itself mediates vitellogenin uptake by oocytes (Wallace, 1985). MATERIALS
AND METHODS
Fish. The fish used in this study were immature 29month-old diploid and triploid coho salmon raised at the West Vancouver Laboratory. The eggs from which this stock came originated from the Capilano River Salmonid Enhancement Program Hatchery (Department of Fisheries and Oceans). Tripioids were produced by applying a hydrostatic pressure shock of 4 min at 10,000 psi to the eggs 15 min after fertilization and incubation at lO.Y, using the laboratory-scale apparatus described by Benfey ei nl. (1988a). All the fish were marked with external numerically coded tags, and were positively identified as diploid or triploid by propidium-iodide flow cytometry (Allen, 1983). Induced vitellogenin production. To induce vitellogenin production, fish were injected intraperitoneally with 17B-estradiol at 1 mg/kg body wt. The 17B-estradiol was first dissolved in 10% ethanol at 12.5 mg/ml, and then a fivefold dilution was made in peanut oil to give a concentration of 2.5 mg/ml. Thus, for an average fish (0.56 kg), the injection volume was 0.22 ml. Controls were sham injected with the same solution of ethanol and peanut oil, but lacking the 178-estradiol. The starting weights were 0.56 kg (20.15 kg SD, n = 10) for diploid-sham, 0.61 kg (20.20 kg, n = 7) for triploid-sham, 0.56 kg (20.23 kg, n = 10) for diploid-treated, and 0.53 kg (-CO.21 kg, n = 10) for triploid-treated fish. Fish were anaesthetized, injected, and bled once a week for 3 weeks. One week after the third injection, all the fish were killed, bled, and had their gonads and livers removed and weighed for the calculation of gonadosomatic index (GSI) and hepatosomatic index (HSI), and pituitaries were removed for the measurement of pituitary gonadotropin content. The gonads were kept for histology. Radioimmunoassays. Blood samples were centrifuged immediately after their collection and were stored at - 30” until they were assayed. Fresh pituitaries were homogenized in 1 ml of radioimmunoassay
AND
DONALDSON
buffer and were similarly stored. Vitellogenin was measured using the homologous radioimmunoassay described by Benfey et al. (1989). The radioimmunoassay for gonadotropin was essentially as that described by Pickering et al. (1987), but using an immunologically less potent form of purified hormone.
RESULTS There was no apparent histological difference in the development of diploid and triploid testes. Most cells were still at the spermatogonial stage, but some spermatocytes were present in all the fish. There was no difference in GSI among any of the groups of males (Table 1). Diploid ovaries were much further advanced than triploid ovaries: diploid ovaries were full of oocytes at “endogenous” vitellogenesis (i.e., “yolk vesicle” or cortical alveoli stage) whereas those of triploids were completely devoid of oocytes. Diploid females had a significantly higher GSI than triploid females (Table 1). Two of the seven diploid females treated with 17P-estradiol had some “yolk globules” in their oocytes, indicative of the start of “exogenous” or true vitellogenesis. The data for this experiment are summarized in Fig. 1. Of the sham-injected fish, only the diploid females had measurable levels of vitellogenin. These levels remained constant at 3 to 4 &ml (mean value) throughout the experiment. Vitellogenin levels were below assay detectability (0.05 ~&ml) for all the sham-injected males and triploid females. There was no difference between sexes for any of the remainTABLE
1
GONADOSOMATIC INDICES OF DIPLOID TRIPLOID COHO SALMON
Diploid Triploid Diploid Triploid
males males females females
Sham 0.15 5 0.03 (4) 0.13 f 0.04 (4) 0.64 k 0.45 (6) 0.04 f 0.01 (3)
AND
l7~-estradiol 0.14 f 0.14 f 0.69 f 0.05 f
0.03 (2) 0.02 (5) 0.44 (7) 0.01 (5)
Note. Values are. given as percentages, mean + standard deviation; sample size is given in parentheses.
VITELLOGENESIS
IN TRIPLOID
SALMON
85
diol-treated fish had lower levels than their respective shams at Week 2 (P < 0.01 in each case), as well as at Week 3 in the case of the triploids (P < 0.05). There was no difference in pituitary gonadotropin content between diploid and triploid sham-injected fish or between diploid and triploid 17pestradiol-treated fish at the end of the experiment. However, 17p-estradiol-treated fish had significantly higher pituitary gonadotropin levels than their respective sham-injected controls (P < 0.001). DISCUSSION
WEEK
FIG. 1. Change in plasma vitellogenin (Vtg) and gonadotropin (GtH) over 3 weeks, and final values for hepatosomatic index and pituitary GtH content (0, diploid sham; A, triploid sham; 0, diploid 17@-estradiol; A, triploid 17P-estradiol; plotted as mean + standard deviation).
ing results, so males and females have been combined. Treatment with 17@estradiol caused a rapid and significant increase in plasma vitellogenin levels. Within 1 week of the first injection, vitellogenin levels were significantly higher than in sham-injected fish (P < 0.001) and continued to rise with each subsequent injection. There was no significant difference between the vitellogenin levels of 17P-estradiol-treated diploids and triploids at any given date. Diploids treated with 17@estradiol had a significantly higher HSI than sham-injected diploids by the end of the experiment (P < 0.05); treated triploids also had a higher HSI than shaminjected triploids, but this difference was not significant. At any given date, there was no difference between diploids and triploids of either sham-injected fish or 17@-estradiol treated fish for plasma gonadotropin. However, both diploid and triploid 17l3-estra-
Our data demonstrate that normal vitellogenin production can occur in triploid fish if the appropriate stimulus is provided. This suggests that the occasional postmeiotic oocytes observed in triploid females may not reach full maturity because of insufftcient vitellogenin production by the liver. This, in turn, would likely be due to the greatly diminished number of estrogenproducing follicle cells in the triploid ovary. The obtainment of fully mature oocytes from triploids would be of great interest for the production of polyploid lines (Purdom, 1984), and may thus be possible with longterm therapy to induce vitellogenin synthesis. However, long-term estrogen therapy may not be suitable because it causes the asynchronous development of oocytes (Lessman and Habibi, 1987). In fact, none of these particular triploid females had any postmeiotic oocytes, so true vitellogenesis (i.e., hepatic vitellogenin synthesis and its uptake by oocytes) could not be observed. However, the treatment did appear to be sufficient to initiate vitellogenin uptake by the oocytes of at least some of the diploid females. The absence of oocytes in the triploids makes it impossible to state conclusively that a lack of vitellogenin production is the cause for the failure of postmeiotic oocytes to grow to maturity in triploid females. Certainly there is a closely related factor which may be in-
86
BENFEY,
DYE,
volved, i.e., the synthesis and secretion of gonadotropin. Clearly, triploid females were able to synthesize the “ovulatory” gonadotropin that was measured in this study, so one can assume that they should also be able to produce other gonadotrogonadotropin(s), such as “vitellogenic” pin. Gonadotropin plays an important role in mediating vitellogenesis in fish (Wallace, 1985). Another possibility, not examined in this study, is that oocyte growth is blocked earlier than vitellogenesis. Nothing is known about the meiotic mechanism which allows occasional oocytes to develop beyond the normal initial disfunction in triploids, but if chromosomal tertiary structure is abnormal then the production of RNA may be affected. Thus, it is quite possible that such oocytes have chromosomes that are too condensed to allow DNA transcription for the production of critical proteins involved in the initial postmeiotic growth of oocytes prior to true vitellogenesis. The significant rise in pituitary gonadotropin content in the 17P-estradiol-treated fish clearly demonstrates the positive effect of estrogens on gonadotropin synthesis in immature fish (Crim et al., 1981; Peter, 1982; Kah, 1986). However, there was no increase in plasma gonadotropin levels; in fact, the levels of this hormone in circulation were slightly (but significantly) depressed by estrogen treatment. This is probably a sign that gonadotropin release from the pituitary was inhibited; estrogens appear to have such an effect on the pituitary (Peter, 1982; Kah, 1986). ACKNOWLEDGMENTS We sincerely thank John Sumpter for his comments on this manuscript. We also thank Cathy ClancyBenfey and Mark Graham for their help in collecting the gonads, livers, and pituitaries; Andy Lamb for rearing the fish; Gary de Jong for performing the flow cytometry; and John Sumpter and Terry Owen for providing the materials for the gonadotropin and vitellogenin radioimmunoassays, respectively. T.J.B. was
AND
DONALDSON
supported by a NSERC postgraduate scholarship and a Quebec FCAR graduate fellowship.
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