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Effects of teleost gonadotropins and their antibodies on gonadal histology in winter flounder
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Effects of Teleost
Gonadotropins and Their Antibodies Histology in Winter Flounder’
T. BUNNG, Marine
Sciences
42, 355-364 (1980)
D.R. IDLER, AND M.P.
Research Laboratory, Memorial St. John’s, Newfoundland
University AIC 5S7
on Gonadal
BURTON of Newfoundland,
Accepted May 13, 1980 The gonadotropic actions of teleost vitellogenic and maturational hormones were studied in flounder hypophysectomized shortly after reinitiation of vitellogenesis and spermatogenesis in August. Maturational hormone alone was able to stimulate formation of yolky oocytes, but vitellogenic hormone required the action of estrogen before it could stimulate production of yolky oocytes. An antiserum to vitellogenic hormone induced atresia of yolky oocytes and lowered the gonadosomatic index in vitellogenic flounder whereas an antiserum to maturational hormone did not affect ovarian histology or gonadosomatic index to any great extent. Preincubation of flounder ovarian sections with vitellogenic hormone, followed by incubation with antiserum to vitellogenic hormone and fluorescein isothiocyanate-labeled goat-anti-rabbit y-globulin in succession, resulted in immunofluorescence in ooplasm of both large immature and vitellogenic oocytes, and in follicular envelopes of vitellogenic oocytes. Preincubation of ovarian sections with maturational hormone resulted in fluorescence in the interstitial tissue, and the follicular envelopes and the perinuclear regions of yolky oocytes. Maturational hormone stimulated spermatogenesis in hypophysectomized male flounder. The significance of these results in relation to previous studies was discussed.
Both types of teleost gonadotropins, viteilogenic hormone and maturational hormone, could reinitiate incorporation of vitellogenin into ovarian yolk (phosvitin and lipovitellin) in hypophysectomized flounder which were actively undergoing vitellogenesis (from November to February) prior to hypophysectomy (Idler and Ng, 1979; Ng and Idler, 1979). In nature spawning occurs in May and June and vitellogenesis, is reinitiated in August. It was decided to investigate the actions of the two teleost gonadotropins on the histological appearance of ovaries of flounder hypophysectomized at the beginning of the vitellogenic season. Hopefully this would provide a clue to the hormonal induction of vitellogenesis. Vitellogenic flounder was treated with antisera to vitellogenic and maturational hormones, and subsequently ovarian his' MSRL Contribution
No. 394.
tology was examined, in order to gain an insight into the relative roles played by the two gonadotropins in maintaining and continuing vitellogenesis after the process has been induced. The binding of vitellogenic and maturational hormones to the ovary was studied with the immunofluorescence technique. Salmon maturational hormone, which is absorbed on concanavalin A- Sepharose (Idler et al., 1975; Breton et al., 1978), induced spermatogenesis in immature trout (Upadhyay, 1977) but its effect in hypophysectomized adult male teleosts has not been studied. The actions of vitellogenic and maturational hormones were therefore studied in adult male flounder hypophysectomized at the beginning of the reproductive cycle. MATERIALS Experiment I
AND METHODS
Adult female winter flounder Pseudopleuronectes were collected by divers early in August
americanus
355 0016-6480/80/110355-10$01.00/O Copyri&t @ 1980 by Academic Press, Inc. All rightsof reproductionin any form reserved.
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IDLER
and hypophysectomized on August 20. Three weeks after hypophysectomy the flounder were divided into three groups of five to seven each and injected intraperitoneally, respectively, with (a) buffer, (b) flounder vitellogenic hormone, 0.5 mg/kg/injection, and (c) salmon maturational hormone, 0.5 mg/kg/injection. The vitellogenic hormone was prepared by chromatography of the MW 28,000 subfraction of flounder pituitary extract unadsorbed on Con A-Sepharose (Con AI), on DEAE Bio-Gel A (Bio-Rad), and then on CM Bio-Gel A (Bio-Rad). The fraction adsorbed on DEAE Bio-Gel A (DEAE III, and unadsorbed on CM Bio-Gel A (CMI) was the purified vitellogenic hormone used for injection and it did not possess thyroid-stimulating activity. The salmon maturational hormone was isolated by chromatography of the MW 40,000 subfraction of chum salmon pituitary extract adsorbed on Con A-Sepharose (Con AH), on DEAE Bio-Gel A, and then on CM Bio-Gel A. The fraction adsorbed on DEAE Bio-Gel A (DEAE II) and unadsorbed on CM Bio-Gel A (CMI), devoid of bioassayable thyroidstimulating activity, was utilized for injection. The salmon hormone was given because the flounder maturational hormone was not available in sufficient quantity. Treatments were continued three times per week for 10 injections. The flounder were killed 48 hr after the last injection and the gonads were fixed in Bouin’s fluid for a week before they were processed for light microscopic examination.
Experiment
2
Early in September, adult female flounder were hypophysectomized and divided into two groups of five to seven each, which were injected, respectively, with (a) estradiol benzoate, 4 mg/kg/injection. (b) estradiol benzoate, 4 mg/kg/injection, and flounder vitellogenic hormone, 0.5 mg/kg/injection. Treatments were continued three times per week for 10 injections. Flounder were killed 48 hr after the last injection. Ovaries were fixed in Bouin’s fluid for 1 week before being processed for microscopic examination.
Experiment
3
Adult male winter flounder were hypophysectomized on August 20. Three weeks after hypophysectomy they were divided into three groups of five to seven each which received the same treatments as the females in Experiment 1. The flounder were sacrificed and the gonads processed as described in Experiment 1.
Experiment
4
A New Zealand white rabbit was immunized with flounder Con AI MW 28,000 DEAE III CM1 fraction (vitellogenic hormone), and another rabbit was immunized with flounder Con AI1 MW 62,000 DEAE I CM1 fraction (maturational hormone). The hormones
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were homogenized in Freund’s complete adjuvant before being injected into the rabbits. The rabbits received a primary intradermal injection at multiple sites on the back which was followed 2 weeks later with an intramuscular booster injection into the hind limbs. Booster injections were continued until a reasonably high-antibody titer was attained. The antiserum to flounder vitellogenic hormone (designated anti-f Con AI GTH) utilized in this study showed 35% binding at 1:8000 dilution to iodinated vitellogenic hormone and only 3% binding at 1: 160 dilution to iodinated maturational hormone. The antiserum to flounder maturational hormone (designated anti-f Con AI1 GTH) showed 50%~ binding at 1: 10,000 dilution to iodinated maturational hormone and only 1% binding at 1:64 dilution to iodinated vitellogenic hormone. In September, vitellogenic female flounder were divided into three groups and injected, respectively, with 0.2 ml/injection of (a) normal rabbit serum NRS, (b) anti-f Con AI GTH, or (c) anti-f Con AI1 GTH. Injections were given twice a week for 7 weeks. Flounder were killed 48 hr after the last injection and the ovaries processed as described above.
Immunojluorescence Flounder Ovary
Study on the
Ovaries from vitellogenic flounder collected in October were fixed in Bouin’s fluid, dehydrated, cleared, and embedded. Sagittal sections were cut at 6 pm and mounted serially on microscopic slides. Following hydration, sections were washed in phosphate-buffered saline (PBS) and preincubated with either (a) flounder vitellogenic hormone or (b) flounder maturational hormone at room temperature for 1 hr, then washed with PBS before being incubated for 1 hr with, respectively, (a) anti-f Con AI GTH and (b) anti-f Con AI1 GTH, at room temperature. The slide was then washed with PBS and incubated at room temperature for 1 hr with fluorescein isothiocyanate-labeled goat antirabbit y-globulin (Antibodies Inc., Calif.). The slide was washed with PBS and mounted in glycerine for examination using a Zeiss epifluorescent microscope. Control sections were checked for autofluorescence and fluorescence after incubation with normal rabbit serum or second antibody alone.
RESULTS
The histology of winter flounder gonads conforms in general to the description for the plaice Pleuronectes platessa (Barr, 1963a,c). Certain features are of particular interest to the present investigation. The ovary of flounder during the vitellogenic phase of the seasonal reproductive cycle also contains “large” and “small” immature oocytes (Dunn and Tyler, 1969) con-
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sidered to represent batches of oocytes due to mature 1 and 2 years, respectively, after the vitellogenic oocytes. The maturing oocytes of the current reproductive season pass from a previtellogenic stage to the vitellogenic stage in which there is a prominent zona radiata around the oocyte. It was difficult to differentiate granulosa cells from the theta cells of the follicular envelope because of its extreme thinness. In the vitellogenic stage yolk accumulation occurs in successive phases, in the early phase (Barr’s stage 3) a ring of “vacuoles,” in standard wax preparations, is present in the periphery of the ooplasm. There is then a development of eosinophilic yolk inclusions initially at the periphery (Barr’s stage 4) which eventually extends through the ooplasm to the nuclear membrane region with a subsequent growth in the oocyte and continuing yolk deposition. We propose to refer to the vitellogenic oocytes as follows: previtellogenic, basophilic ooplasm, nucleoli round periphery of nucleus; vitellogenic A, zona radiata and vacuoles at the periphery of ooplasm; vitellogenic B, eosinophilic inclusions at the periphery of ooplasm, vitellogenic C, eosinophilic inclusions extend from the periphery of the ooplasm to the nucleus. In Experiment 1, the ovaries of every flounder treated with maturational hormone contained many oocytes with stage B yolk (Fig. 1) whereas ovaries of the control group injected with buffer contained oocytes either at the previtellogenic stage or with stage A yolk (Fig. 2). Ovaries of the group injected with vitellogenic hormone showed variation in the response but 60% had no oocytes beyond stage A (Fig. 3). In Experiment 2, the ovaries of fish treated with estradiol benzoate contained either previtellogenic oocytes or oocytes at stage B or oocytes which had undergone atresia (Fig. 4). The ovaries of fish treated simultaneously with both estrogen and vitellogenic hormone all contained oocytes beyond previtellogenesis (Fig. 5), and 60%
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had oocytes with abnormally large yolk inclusions (Fig. 6). Many of these yolky oocytes were undergoing changes indicative of early atresia. In Experiment 3, testes of fish injected with buffer (Fig. 7) and of fish treated with vitellogenic hormone (Fig. 8) contained regressed lobules with only spermatogonia. Testes of fish injected with maturational hormone were better developed and germ cells in the lobules developed to as far as spermatocytes (Fig. 9). In Experiment 4, ovaries of flounder injected with normal rabbit serum contained large stage C yolky oocytes (Fig. 10) and the gonadosomatic index for the group was 14.0 +- 1.9 (N = 5). Many vitellogenic oocytes in ovaries of flounder treated with anti-f Con AI GTH had undergone atresia (Fig. 11) and the gonadosomatic index of the group (6.8 ? 0.8, N = 6) was significantly lower than that of the control group (P < 0.01). In contrast, ovaries of flounder treated with anti-f Con AI1 GTH were not much different from the ovary of control fish either in histology (Fig. 12) or in gonadosomatic index (11.4 ? 2.2, N = 5). Immunofluorescrnce
Sections of ovaries from flounder with stage C vitellogenesis (Fig. 13) were examined. Incubations with normal rabbit serum showed some fluorescence in the ooplasm, particularly in the autofluorescent yolk, of the maturing oocytes but little fluorescence elsewhere. In ovarian sections which were preincubated with vitellogenic hormone before incubation with anti-f Con AI GTH, the ooplasm of vitellogenic oocytes showed increased fluorescence. There was also fluorescence from the follicular envelope of the oocyte outside the zona radiata and also from ooplasm of large immature oocytes (Fig. 14). In contrast, when ovarian sections were preincubated with maturational hormone before incubation with anti-f Con AI1 GTH the ooplasm of vitellogenic oocytes had low fluorescence. There was a distinct perinuclear response as well as flu-
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AND
GONADAL
HISTOLOGY
orescence from the follicular envelope and the interstitial tissue and the ooplasm of large immature oocytes (Figs. 15, 16). A similar distribution of target tissue was obtained from flounder ovary at stage B in vitellogenesis except that there was no perinuclear fluorescence.
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the consequence that the stimulatory effect of vitellogenic hormone on the second step of the process of vitellogenesis, incorporation of vitellogenin into ovarian yolk, was not readily observable. When estrogen was injected simultaneously with vitellogenic hormone, yolk accumulation occurred in the ovary. This observation can be exDISCUSSION plained by the stimulating effect of estroThe present study revealed that in Au- gen on hepatic vitellogenin production (Campbell and Idler, 1976) and the stimgust and September, when vitellogenesis had just been reinitiated, estrogen was re- ulating effect of vitellogenic hormone on quired before vitellogenic hormone could vitellogenin incorporation into the ovary exert its gonadotropic action on oocytes. (Campbell, 1978; Campbell and Idler, 1976; However, the oocytes could accumulate Idler and Ng, 1979; Ng and Idler, 1978a; yolk under the stimulation of maturational 1978b; 1979). Estrogen alone was not able hormone, indicating that maturational hor- to maintain vitellogenesis in flounder mone stimulated production of estrogen hypophysectomized shortly after the beand therefore vitellogenin, and facilitated ginning of the vitellogenic season. The efincorporation of vitellogenin into oocytes. fect of estrogen in inhibiting atresia of oocytes after hypophysectomy is controversial This finding is consistent with the observation of Campbell and Idler (1979) in the im- (see Lam et al., 1978). The dose of estrogen mature rainbow trout that salmon matura- given was possibly excessive: the abnormal tional hormone stimulated the first step in appearance of many oocytes treated with the process of vitellogenesis, i.e., producestrogen and vitellogenic hormone suggests tion of estrogen and vitellogenin, but that that the combination used overpromoted salmon Con AI fraction containing the vi- uptake of vitellogenin thus leading to disortellogenic hormone was not capable of doing ganization of the enlarged oocytes. so. Hypophysectomy at the beginning of Salmon maturational hormone (Breton et the vitellogenic season probably led to a al., 1978) induced accumulation of lipid low circulating level of vitellogenin, with bodies in oocytes of intact juvenile trout,
FIG. 1. Section of ovary from hypophysectomized flounder treated with maturational hormone (Experiment 1). L, large immature oocyte. B, maturing oocyte, Stage B vitellogenesis. Scale bar, 45 pm. FIG. 2. Section of ovary from hypophysectomized flounder treated with buffer (Experiment 1). 0, regressed maturing oocyte. Magnification as for Fig. 1. FIG. 3. Section of ovary from hypophysectomized flounder treated with vitellogenic hormone (Experiment 1). A, maturing oocyte, stage A vitellogenesis. Magnification as for Fig. 1. FIG. 4. Section of ovary from hypophysectomized flounder treated with estrogen (Experiment 2). AT, atretic follicles. Magnification as for Fig. 1. FIG. 5. Section of ovary from hypophysectomized flounder treated with estrogen and vitellogenic hormone (Experiment 2). B, maturing oocyte, stage B vitellogenesis. Magnification as for Fig. 1. FIG. 6. Section of ovary from hypophysectomized flounder treated with estrogen and vitellogenic hormone (Experiment 2). AB, abnormal enlarged oocyte with extensive disorganization. Magnification as for Fig. 1. FIG. 7. Section of testis from hypophysectomized flounder treated with buffer (Experiment 3) showing spermatogonia (arrows). Scale bar = 8 Wm. FIG. 8. Section of testis from hypophysectomized flounder treated with vitellogenic hormone (Experiment 3) showing spermatogonia (arrows). Magnification as for Fig. 7.
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suggestive of endogenous vitellogenesis. However, a pituitary extract of mature salmon was able to stimulate formation of zona radiata and cytoplasmic vesicles, and an extensive pinocytotic activity on the oocyte plasma membrane, indicative of exogenous incorporation, i.e., incorporation of vitellogenin (Upadhyay et al., 1978). This observation suggests the existence of a hormone in the pituitary Con AI fraction capable of stimulating incorporation of vitellogenin. Campbell (1978) found that salmon pituitary Con AI1 fraction did not stimulate incorporation of vitellogenin into oocytes from intact juvenile trout in vitro whereas salmon pituitary Con AI fraction was active. In the present study salmon maturational hormone stimulated in ovary of hypophysectomized flounder formation of yolky oocytes with a prominent zona radiata, and in previous studies (Idler and Ng, 1979; Ng and Idler, 1979) maturational hormones from various teleost species promoted incorporation of 33P into ovarian lipovitellin and phosvitin, suggesting that maturational hormone stimulated incorporation of vitellogenin into the ovary. When taken together these observations suggest that oocytes of hypophysectomized adult flounder were able to incorporate vitellogenin under the stimulation of maturational hormone, because they had been primed before hypophysectomy by a hormone
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present in the pituitary Con AI fraction which was absent in intact juvenile trout, and this hormone made the oocytes responsive to stimulation by maturational hormone to incorporate vitellogenin. Binding of vitellogenic hormone to large immature oocytes in the flounder ovary indicates that vitellogenic hormone may prepare the nonyolky oocytes for the vitellogenic phase. It is likely that priming of oocytes by vitellogenic hormone accounts for the discrepancy in response to maturational hormone between juvenile trout and adult flounder. Salmon gonadotropic preparation SGGlOO (Donaldson et al., 1972) was potent in reinitiating production of yolky oocytes in hypophysectomized adult goldfish (Yamazaki and Donaldson, 1968) and catfish (Sundararaj et al., 1972). It is known that SG-GlOO contained about 33% Con AI material (Pierce ef al., 1976) but the extent of contamination of the preparation with the vitellogenic hormone is not known. Vitellogenic activity of the preparation could be due to a combination of the effect of maturational hormone on hepatic vitellogenin production and the effect of vitellogenic hormone on vitellogenin incorporation into the ovary. Even if SG-GlOO was minimally contaminated with vitellogenic hormone maturational hormone in the preparation might still be able to stimulate
FIG. 9. Section of testis from hypophysectomized flounder treated with maturational hormone (Experiment 3). Arrow, spermatogonium. S, spermatocytes. Magnification as for Fig. 7. FIG. 10. Section of ovary from intact flounder injected with normal rabbit serum (Experiment 4), showing large stage C oocytes. Magnification as for Fig. I. FIG. 11. Section of ovary from intact flounder injected with antiserum to vitellogenic hormone (Experiment 4). AT, atretic oocyte. Magnification as for Fig. 1. FIG. 12. Section of ovary from intact flounder injected with antiserum to maturational hormone (Experiment 4) showing large stage C oocytes. Magnification as for Fig. 1. FIG. 13. Section of ovary from intact flounder consecutive to the sections in Figs. 14- 16, showing stage C oocyte. Magnification as for Fig. 1. FIG. 14. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with vitellogenic hormone, first antibody anti-f Con AI GTH. L, large immature oocyte. Scale bar = 55 Frn. FIG. 15. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with maturational hormone, first antibody anti-f Con AI1 GTH. Scale bar = 140 Frn. FIG. 16. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with maturational hormone, first antibody anti-f Con AI1 GTH. Magnification as for Fig. 14.
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incorporation of vitellogenin into the oocytes which had been primed by vitellogenic hormone before hypophysectomy. An antiserum to vitellogenic hormone induces atresia in yolky oocytes and a decrease in gonadosomatic index. No marked changes were observed in either histological details or weight of ovaries of flounder treated with an antiserum to maturational hormone. Atresia in yolky oocytes occurred after hypophysectomy in the plaice Pleuronectes platessa (Barr, 1963b) and in the goldfish (Lam et al., 1978). It seems that the vitellogenic hormone alone is able to maintain and continue vitellogenesis after the process has been initiated. It has to be borne in mind that treatment with a specific antiserum ties up only the appropriate hormone (if present in circulation) leaving the other pituitary hormones to exert their biological actions, whereas hypophysectomy removes the source of all pituitary hormones. Results of hormone replacement therapy are not necessarily the same as the normal physiological action of the hormone in vivo. The maturational hormone might initiate vitellogenesis after spawning by stimulating estradiol and vitellogenin production but it does not appear to play a significant role in regulating incorporation of yolk into the ovary of flounder undergoing vitellogenesis. In salmonids the maturational hormone is not readily detectable in circulation until about the spawning time (Crim et al., 1975), indicating that its level is very low even if present. A very low level of the maturational hormone might be sufficient to maintain ovarian estradiol and hence hepatic vitellogenin production but not sufficient to maintain vitellogenin incorporation into the ovary. Hence it is quite reasonable to think that in the flounder ovarian yolk deposition is maintained and continued by the vitellogenic hormone after the process of vitellogenesis has been initiated by the maturational hormone through its action on ovarian estradiol and hepatic vitellogenin production.
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The binding of a hormone to its target tissue revealed by the immunofluorescence technique, after preincubation of the tissue with the hormone, shows the location of hormone receptors in the tissue. Binding of vitellogenic hormone to ooplasm of large immature oocytes of flounder ovary collected in October is indicative of the capacity for facilitation and preparation (priming) for the vitellogenic phase. Binding of vitellogenic hormone to ooplasm of yolky oocytes and to the follicular envelope surrounding the oocytes suggests that vitellogenic hormone acts on the yolky oocytes to stimulate incorporation of vitellogenin. Binding of maturational hormone to ovarian interstitial tissue and follicular envelope of oocytes implicates the hormone in stimulating steroidogenesis and vitellogenesis. Binding of the maturational hormone to a perinuclear region of the vitellogenic oocyte after preincubation with the hormone may reflect a future role of the hormone in oocyte maturation (resumption of meiosis). The results of this immunofluorescence study suggest that the hormones have distinct binding sites and distinct actions. The stimulating action of maturational hormone on spermatogenesis may be related to its steroidogenic activity (Idler and Ng, 1979) because androgens are known to promote spermatogenesis (e.g., Sundararaj et al., 1971; Idler et al., 1961; 1971). Mammalian-luteinizing hormone stimulates steroidogenesis as well as spermatogenesis in mammals while follicle-stimulating hormone stimulates spermatogonial proliferation. A role of vitellogenic hormone in spermatogenesis is not obvious in the present study. Previous assays for gonadotropic activities in pituitary hormone preparations were short-term assays, for instance, in the assay for vitellogenic activity (Ng and Idler, 1978a) the assay parameter was the incorporation of newly synthesized 33P-labeled vitellogenin into ovarian yolk of flounder,
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which were actively undergoing vitellogenesis before hypophysectomy, over a 7-day period. The present study involved prolonged hormonal treatment of fish hypophysectomized at the beginning of the reproductive cycle, and assessment of overall structural changes in the gonad with the histological technique; the study provided both additional and corroborative information. REFERENCES Barr, W. A. (1963a). The endocrine control of the sexual cycle in the plaice, Pleuronectesplatessa (L.). I. Cyclical changes in the normal ovary. Gen. Camp.
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Barr, W. A. (1963b). The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (L.). II. The endocrine control of oogenesis. Grn. Camp.
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Barr, W. A. (1963~). The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (L.). III. The endocrine control of spermatogenesis. Gen. Comp. Endocrinol. 3, 216-225. Breton, B., Prunet, P., and Reinaud, P. (1978). Sexual differences in salmon gonadotropin. Ann. Biol. Anim. B&hem. Biophys. l(l) 25-36. Campbell, C. M. (1978). In vitro stimulation of vitellogenin incorporation into trout oocytes by salmon pituitary extracts. Ann. Biol. Anim. Biochem. Biophys. 18(4), 1013- 1018. Campbell, C. M., and Idler, D. R. (1976). Hormonal control of vitellogenesis in hypophysectomized winter flounder (Pseudopleuronectes americanus Walbaum). Gen. Comp. Endocrinol. 28, 143- 150. Campbell, C. M., and Idler, D. R. (1979). Gonadotropin stimulation of estrogen and yolk precursor synthesis in juvenile rainbow trout. Biol. Reprod. 20, 1, 16. Grim, L. W., Watts, E. Cl., and Evans, D. M. (1975). The plasma gonadotropin profile during sexual maturation in a variety of salmonid fishes. Gen. Comp.
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Donaldson, E. M., Yamasaki, F., Dye, H. M., and Philleo, W. W. (1972). Preparation of gonadotropin from salmon (Oncorhynchus tshawytscha) pituitary glands. Gen. Camp. Endocrinol. 18, 469-481. Dunn, R. S., and Tyler, A. V. (1969). Aspects of the anatomy of the winter flounder ovary with hypotheses on oocyte maturation time. J. Fish. Res. Bd. Canad. 26, 1943-1947. Idler, D. R., Bazar, L. S., and Hwang, S. J. (1975). Fish gonadotropin(s). III. Evidence for more than
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one gonadotropin in chum salmon pituitary glands. Endocrine Res. Commun. 2(3), 237-249. Idler, D. R., Bitners, I. I., and Schmidt, P. J. (1961). II-Ketotestosterone. An androgen for sockeye salmon. Canad. J. Biochem. Physiol. 39. l737- 1742. Idler, D. R., Horne, D. A., and Sangalang, G. B. (1971). Identification and quantification of the major androgens in testicular and peripheral plasma of Atlantic salmon (Salmo salar) during sexual maturation. Gen. Comp. Endocrinol. 16, 257-267. Idler, D. R., and Ng, T. B. (1979). Studies on two types of gonadotropins from both salmon and carp pituitaries. Gen. Comp. Endocrinol. 38,421-440. Lam, T. J., Pandey, S., Nagahama, Y., and Hoar, W. S. (1978). Endocrine control of oogenesis, ovulation and oviposition in goldfish. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 55-64. ElsevieriNorth-Holland, Amsterdam. Ng, T. B., and Idler, D. R. (1978a). “Big” and “little” forms of plaice vitellogenic and maturational hormones. Gen. Comp. Endocrinol. 34, 408-420. Ng, T. B., and Idler, D. R. (1978b). A vitellogenic hormone with a large and a small form from salmon pituitaries. Gen. Camp. Endocrinol. 35, 189- 195. Ng, T. B., and Idler, D. R. (1979). Studies on two types of gonadotropins from both American plaice and winter flounder pituitaries. Gen. Comp. Endocrinol.
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Pierce, J. G., Faith, M. R., and Donaldson, E. M. (1976). Antibodies to reduced S-carboxymethylated alpha subunit of bovine luteinizing hormone and their application to study of the purification of gonadotropin from salmon (Oncorhynchus tshawyrscha) pituitary glands. Gen. Comp.
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Sundararaj, B. I., Nayyar, S. K., Anand, T. C., and Donaldson, E. M. (1971). Effects of salmon pituitary gonadotropin, ovine luteinizing hormone and testosterone on the testes and seminal vesicles of hypophysectomized catfish, Heteropneustes fossilk (Bloch). Gen. Camp. Endocrinol. 17, 73-82. Sundararaj, B. I., Anand, T. C., and Donaldson, E. M. (1972). Effects of partially purified salmon pituitary gonadotropin on ovarian maintenance, ovulation and vitellogenesis in hypophysectomized catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endoc,rinol. 18, 102- 114. Upadhyay, S. N. (1977). Morphologie des gonades immatures et etude experimente de l’induction de la gametogenese chez la truite arc-en-ciel juvenile (Salmo gairdnerii R.). Th. Doct. Etat. Univ. Paris VI. Upadhyay, S. N., Breton, B., Billard, R. (1978). Ultrastructural studies on experimentally induced
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COMPARATIVE
ENDOCRINOLOGY
Effects of Teleost
Gonadotropins and Their Antibodies Histology in Winter Flounder’
T. BUNNG, Marine
Sciences
42, 355-364 (1980)
D.R. IDLER, AND M.P.
Research Laboratory, Memorial St. John’s, Newfoundland
University AIC 5S7
on Gonadal
BURTON of Newfoundland,
Accepted May 13, 1980 The gonadotropic actions of teleost vitellogenic and maturational hormones were studied in flounder hypophysectomized shortly after reinitiation of vitellogenesis and spermatogenesis in August. Maturational hormone alone was able to stimulate formation of yolky oocytes, but vitellogenic hormone required the action of estrogen before it could stimulate production of yolky oocytes. An antiserum to vitellogenic hormone induced atresia of yolky oocytes and lowered the gonadosomatic index in vitellogenic flounder whereas an antiserum to maturational hormone did not affect ovarian histology or gonadosomatic index to any great extent. Preincubation of flounder ovarian sections with vitellogenic hormone, followed by incubation with antiserum to vitellogenic hormone and fluorescein isothiocyanate-labeled goat-anti-rabbit y-globulin in succession, resulted in immunofluorescence in ooplasm of both large immature and vitellogenic oocytes, and in follicular envelopes of vitellogenic oocytes. Preincubation of ovarian sections with maturational hormone resulted in fluorescence in the interstitial tissue, and the follicular envelopes and the perinuclear regions of yolky oocytes. Maturational hormone stimulated spermatogenesis in hypophysectomized male flounder. The significance of these results in relation to previous studies was discussed.
Both types of teleost gonadotropins, viteilogenic hormone and maturational hormone, could reinitiate incorporation of vitellogenin into ovarian yolk (phosvitin and lipovitellin) in hypophysectomized flounder which were actively undergoing vitellogenesis (from November to February) prior to hypophysectomy (Idler and Ng, 1979; Ng and Idler, 1979). In nature spawning occurs in May and June and vitellogenesis, is reinitiated in August. It was decided to investigate the actions of the two teleost gonadotropins on the histological appearance of ovaries of flounder hypophysectomized at the beginning of the vitellogenic season. Hopefully this would provide a clue to the hormonal induction of vitellogenesis. Vitellogenic flounder was treated with antisera to vitellogenic and maturational hormones, and subsequently ovarian his' MSRL Contribution
No. 394.
tology was examined, in order to gain an insight into the relative roles played by the two gonadotropins in maintaining and continuing vitellogenesis after the process has been induced. The binding of vitellogenic and maturational hormones to the ovary was studied with the immunofluorescence technique. Salmon maturational hormone, which is absorbed on concanavalin A- Sepharose (Idler et al., 1975; Breton et al., 1978), induced spermatogenesis in immature trout (Upadhyay, 1977) but its effect in hypophysectomized adult male teleosts has not been studied. The actions of vitellogenic and maturational hormones were therefore studied in adult male flounder hypophysectomized at the beginning of the reproductive cycle. MATERIALS Experiment I
AND METHODS
Adult female winter flounder Pseudopleuronectes were collected by divers early in August
americanus
355 0016-6480/80/110355-10$01.00/O Copyri&t @ 1980 by Academic Press, Inc. All rightsof reproductionin any form reserved.
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and hypophysectomized on August 20. Three weeks after hypophysectomy the flounder were divided into three groups of five to seven each and injected intraperitoneally, respectively, with (a) buffer, (b) flounder vitellogenic hormone, 0.5 mg/kg/injection, and (c) salmon maturational hormone, 0.5 mg/kg/injection. The vitellogenic hormone was prepared by chromatography of the MW 28,000 subfraction of flounder pituitary extract unadsorbed on Con A-Sepharose (Con AI), on DEAE Bio-Gel A (Bio-Rad), and then on CM Bio-Gel A (Bio-Rad). The fraction adsorbed on DEAE Bio-Gel A (DEAE III, and unadsorbed on CM Bio-Gel A (CMI) was the purified vitellogenic hormone used for injection and it did not possess thyroid-stimulating activity. The salmon maturational hormone was isolated by chromatography of the MW 40,000 subfraction of chum salmon pituitary extract adsorbed on Con A-Sepharose (Con AH), on DEAE Bio-Gel A, and then on CM Bio-Gel A. The fraction adsorbed on DEAE Bio-Gel A (DEAE II) and unadsorbed on CM Bio-Gel A (CMI), devoid of bioassayable thyroidstimulating activity, was utilized for injection. The salmon hormone was given because the flounder maturational hormone was not available in sufficient quantity. Treatments were continued three times per week for 10 injections. The flounder were killed 48 hr after the last injection and the gonads were fixed in Bouin’s fluid for a week before they were processed for light microscopic examination.
Experiment
2
Early in September, adult female flounder were hypophysectomized and divided into two groups of five to seven each, which were injected, respectively, with (a) estradiol benzoate, 4 mg/kg/injection. (b) estradiol benzoate, 4 mg/kg/injection, and flounder vitellogenic hormone, 0.5 mg/kg/injection. Treatments were continued three times per week for 10 injections. Flounder were killed 48 hr after the last injection. Ovaries were fixed in Bouin’s fluid for 1 week before being processed for microscopic examination.
Experiment
3
Adult male winter flounder were hypophysectomized on August 20. Three weeks after hypophysectomy they were divided into three groups of five to seven each which received the same treatments as the females in Experiment 1. The flounder were sacrificed and the gonads processed as described in Experiment 1.
Experiment
4
A New Zealand white rabbit was immunized with flounder Con AI MW 28,000 DEAE III CM1 fraction (vitellogenic hormone), and another rabbit was immunized with flounder Con AI1 MW 62,000 DEAE I CM1 fraction (maturational hormone). The hormones
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were homogenized in Freund’s complete adjuvant before being injected into the rabbits. The rabbits received a primary intradermal injection at multiple sites on the back which was followed 2 weeks later with an intramuscular booster injection into the hind limbs. Booster injections were continued until a reasonably high-antibody titer was attained. The antiserum to flounder vitellogenic hormone (designated anti-f Con AI GTH) utilized in this study showed 35% binding at 1:8000 dilution to iodinated vitellogenic hormone and only 3% binding at 1: 160 dilution to iodinated maturational hormone. The antiserum to flounder maturational hormone (designated anti-f Con AI1 GTH) showed 50%~ binding at 1: 10,000 dilution to iodinated maturational hormone and only 1% binding at 1:64 dilution to iodinated vitellogenic hormone. In September, vitellogenic female flounder were divided into three groups and injected, respectively, with 0.2 ml/injection of (a) normal rabbit serum NRS, (b) anti-f Con AI GTH, or (c) anti-f Con AI1 GTH. Injections were given twice a week for 7 weeks. Flounder were killed 48 hr after the last injection and the ovaries processed as described above.
Immunojluorescence Flounder Ovary
Study on the
Ovaries from vitellogenic flounder collected in October were fixed in Bouin’s fluid, dehydrated, cleared, and embedded. Sagittal sections were cut at 6 pm and mounted serially on microscopic slides. Following hydration, sections were washed in phosphate-buffered saline (PBS) and preincubated with either (a) flounder vitellogenic hormone or (b) flounder maturational hormone at room temperature for 1 hr, then washed with PBS before being incubated for 1 hr with, respectively, (a) anti-f Con AI GTH and (b) anti-f Con AI1 GTH, at room temperature. The slide was then washed with PBS and incubated at room temperature for 1 hr with fluorescein isothiocyanate-labeled goat antirabbit y-globulin (Antibodies Inc., Calif.). The slide was washed with PBS and mounted in glycerine for examination using a Zeiss epifluorescent microscope. Control sections were checked for autofluorescence and fluorescence after incubation with normal rabbit serum or second antibody alone.
RESULTS
The histology of winter flounder gonads conforms in general to the description for the plaice Pleuronectes platessa (Barr, 1963a,c). Certain features are of particular interest to the present investigation. The ovary of flounder during the vitellogenic phase of the seasonal reproductive cycle also contains “large” and “small” immature oocytes (Dunn and Tyler, 1969) con-
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sidered to represent batches of oocytes due to mature 1 and 2 years, respectively, after the vitellogenic oocytes. The maturing oocytes of the current reproductive season pass from a previtellogenic stage to the vitellogenic stage in which there is a prominent zona radiata around the oocyte. It was difficult to differentiate granulosa cells from the theta cells of the follicular envelope because of its extreme thinness. In the vitellogenic stage yolk accumulation occurs in successive phases, in the early phase (Barr’s stage 3) a ring of “vacuoles,” in standard wax preparations, is present in the periphery of the ooplasm. There is then a development of eosinophilic yolk inclusions initially at the periphery (Barr’s stage 4) which eventually extends through the ooplasm to the nuclear membrane region with a subsequent growth in the oocyte and continuing yolk deposition. We propose to refer to the vitellogenic oocytes as follows: previtellogenic, basophilic ooplasm, nucleoli round periphery of nucleus; vitellogenic A, zona radiata and vacuoles at the periphery of ooplasm; vitellogenic B, eosinophilic inclusions at the periphery of ooplasm, vitellogenic C, eosinophilic inclusions extend from the periphery of the ooplasm to the nucleus. In Experiment 1, the ovaries of every flounder treated with maturational hormone contained many oocytes with stage B yolk (Fig. 1) whereas ovaries of the control group injected with buffer contained oocytes either at the previtellogenic stage or with stage A yolk (Fig. 2). Ovaries of the group injected with vitellogenic hormone showed variation in the response but 60% had no oocytes beyond stage A (Fig. 3). In Experiment 2, the ovaries of fish treated with estradiol benzoate contained either previtellogenic oocytes or oocytes at stage B or oocytes which had undergone atresia (Fig. 4). The ovaries of fish treated simultaneously with both estrogen and vitellogenic hormone all contained oocytes beyond previtellogenesis (Fig. 5), and 60%
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had oocytes with abnormally large yolk inclusions (Fig. 6). Many of these yolky oocytes were undergoing changes indicative of early atresia. In Experiment 3, testes of fish injected with buffer (Fig. 7) and of fish treated with vitellogenic hormone (Fig. 8) contained regressed lobules with only spermatogonia. Testes of fish injected with maturational hormone were better developed and germ cells in the lobules developed to as far as spermatocytes (Fig. 9). In Experiment 4, ovaries of flounder injected with normal rabbit serum contained large stage C yolky oocytes (Fig. 10) and the gonadosomatic index for the group was 14.0 +- 1.9 (N = 5). Many vitellogenic oocytes in ovaries of flounder treated with anti-f Con AI GTH had undergone atresia (Fig. 11) and the gonadosomatic index of the group (6.8 ? 0.8, N = 6) was significantly lower than that of the control group (P < 0.01). In contrast, ovaries of flounder treated with anti-f Con AI1 GTH were not much different from the ovary of control fish either in histology (Fig. 12) or in gonadosomatic index (11.4 ? 2.2, N = 5). Immunofluorescrnce
Sections of ovaries from flounder with stage C vitellogenesis (Fig. 13) were examined. Incubations with normal rabbit serum showed some fluorescence in the ooplasm, particularly in the autofluorescent yolk, of the maturing oocytes but little fluorescence elsewhere. In ovarian sections which were preincubated with vitellogenic hormone before incubation with anti-f Con AI GTH, the ooplasm of vitellogenic oocytes showed increased fluorescence. There was also fluorescence from the follicular envelope of the oocyte outside the zona radiata and also from ooplasm of large immature oocytes (Fig. 14). In contrast, when ovarian sections were preincubated with maturational hormone before incubation with anti-f Con AI1 GTH the ooplasm of vitellogenic oocytes had low fluorescence. There was a distinct perinuclear response as well as flu-
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orescence from the follicular envelope and the interstitial tissue and the ooplasm of large immature oocytes (Figs. 15, 16). A similar distribution of target tissue was obtained from flounder ovary at stage B in vitellogenesis except that there was no perinuclear fluorescence.
359
the consequence that the stimulatory effect of vitellogenic hormone on the second step of the process of vitellogenesis, incorporation of vitellogenin into ovarian yolk, was not readily observable. When estrogen was injected simultaneously with vitellogenic hormone, yolk accumulation occurred in the ovary. This observation can be exDISCUSSION plained by the stimulating effect of estroThe present study revealed that in Au- gen on hepatic vitellogenin production (Campbell and Idler, 1976) and the stimgust and September, when vitellogenesis had just been reinitiated, estrogen was re- ulating effect of vitellogenic hormone on quired before vitellogenic hormone could vitellogenin incorporation into the ovary exert its gonadotropic action on oocytes. (Campbell, 1978; Campbell and Idler, 1976; However, the oocytes could accumulate Idler and Ng, 1979; Ng and Idler, 1978a; yolk under the stimulation of maturational 1978b; 1979). Estrogen alone was not able hormone, indicating that maturational hor- to maintain vitellogenesis in flounder mone stimulated production of estrogen hypophysectomized shortly after the beand therefore vitellogenin, and facilitated ginning of the vitellogenic season. The efincorporation of vitellogenin into oocytes. fect of estrogen in inhibiting atresia of oocytes after hypophysectomy is controversial This finding is consistent with the observation of Campbell and Idler (1979) in the im- (see Lam et al., 1978). The dose of estrogen mature rainbow trout that salmon matura- given was possibly excessive: the abnormal tional hormone stimulated the first step in appearance of many oocytes treated with the process of vitellogenesis, i.e., producestrogen and vitellogenic hormone suggests tion of estrogen and vitellogenin, but that that the combination used overpromoted salmon Con AI fraction containing the vi- uptake of vitellogenin thus leading to disortellogenic hormone was not capable of doing ganization of the enlarged oocytes. so. Hypophysectomy at the beginning of Salmon maturational hormone (Breton et the vitellogenic season probably led to a al., 1978) induced accumulation of lipid low circulating level of vitellogenin, with bodies in oocytes of intact juvenile trout,
FIG. 1. Section of ovary from hypophysectomized flounder treated with maturational hormone (Experiment 1). L, large immature oocyte. B, maturing oocyte, Stage B vitellogenesis. Scale bar, 45 pm. FIG. 2. Section of ovary from hypophysectomized flounder treated with buffer (Experiment 1). 0, regressed maturing oocyte. Magnification as for Fig. 1. FIG. 3. Section of ovary from hypophysectomized flounder treated with vitellogenic hormone (Experiment 1). A, maturing oocyte, stage A vitellogenesis. Magnification as for Fig. 1. FIG. 4. Section of ovary from hypophysectomized flounder treated with estrogen (Experiment 2). AT, atretic follicles. Magnification as for Fig. 1. FIG. 5. Section of ovary from hypophysectomized flounder treated with estrogen and vitellogenic hormone (Experiment 2). B, maturing oocyte, stage B vitellogenesis. Magnification as for Fig. 1. FIG. 6. Section of ovary from hypophysectomized flounder treated with estrogen and vitellogenic hormone (Experiment 2). AB, abnormal enlarged oocyte with extensive disorganization. Magnification as for Fig. 1. FIG. 7. Section of testis from hypophysectomized flounder treated with buffer (Experiment 3) showing spermatogonia (arrows). Scale bar = 8 Wm. FIG. 8. Section of testis from hypophysectomized flounder treated with vitellogenic hormone (Experiment 3) showing spermatogonia (arrows). Magnification as for Fig. 7.
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suggestive of endogenous vitellogenesis. However, a pituitary extract of mature salmon was able to stimulate formation of zona radiata and cytoplasmic vesicles, and an extensive pinocytotic activity on the oocyte plasma membrane, indicative of exogenous incorporation, i.e., incorporation of vitellogenin (Upadhyay et al., 1978). This observation suggests the existence of a hormone in the pituitary Con AI fraction capable of stimulating incorporation of vitellogenin. Campbell (1978) found that salmon pituitary Con AI1 fraction did not stimulate incorporation of vitellogenin into oocytes from intact juvenile trout in vitro whereas salmon pituitary Con AI fraction was active. In the present study salmon maturational hormone stimulated in ovary of hypophysectomized flounder formation of yolky oocytes with a prominent zona radiata, and in previous studies (Idler and Ng, 1979; Ng and Idler, 1979) maturational hormones from various teleost species promoted incorporation of 33P into ovarian lipovitellin and phosvitin, suggesting that maturational hormone stimulated incorporation of vitellogenin into the ovary. When taken together these observations suggest that oocytes of hypophysectomized adult flounder were able to incorporate vitellogenin under the stimulation of maturational hormone, because they had been primed before hypophysectomy by a hormone
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present in the pituitary Con AI fraction which was absent in intact juvenile trout, and this hormone made the oocytes responsive to stimulation by maturational hormone to incorporate vitellogenin. Binding of vitellogenic hormone to large immature oocytes in the flounder ovary indicates that vitellogenic hormone may prepare the nonyolky oocytes for the vitellogenic phase. It is likely that priming of oocytes by vitellogenic hormone accounts for the discrepancy in response to maturational hormone between juvenile trout and adult flounder. Salmon gonadotropic preparation SGGlOO (Donaldson et al., 1972) was potent in reinitiating production of yolky oocytes in hypophysectomized adult goldfish (Yamazaki and Donaldson, 1968) and catfish (Sundararaj et al., 1972). It is known that SG-GlOO contained about 33% Con AI material (Pierce ef al., 1976) but the extent of contamination of the preparation with the vitellogenic hormone is not known. Vitellogenic activity of the preparation could be due to a combination of the effect of maturational hormone on hepatic vitellogenin production and the effect of vitellogenic hormone on vitellogenin incorporation into the ovary. Even if SG-GlOO was minimally contaminated with vitellogenic hormone maturational hormone in the preparation might still be able to stimulate
FIG. 9. Section of testis from hypophysectomized flounder treated with maturational hormone (Experiment 3). Arrow, spermatogonium. S, spermatocytes. Magnification as for Fig. 7. FIG. 10. Section of ovary from intact flounder injected with normal rabbit serum (Experiment 4), showing large stage C oocytes. Magnification as for Fig. I. FIG. 11. Section of ovary from intact flounder injected with antiserum to vitellogenic hormone (Experiment 4). AT, atretic oocyte. Magnification as for Fig. 1. FIG. 12. Section of ovary from intact flounder injected with antiserum to maturational hormone (Experiment 4) showing large stage C oocytes. Magnification as for Fig. 1. FIG. 13. Section of ovary from intact flounder consecutive to the sections in Figs. 14- 16, showing stage C oocyte. Magnification as for Fig. 1. FIG. 14. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with vitellogenic hormone, first antibody anti-f Con AI GTH. L, large immature oocyte. Scale bar = 55 Frn. FIG. 15. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with maturational hormone, first antibody anti-f Con AI1 GTH. Scale bar = 140 Frn. FIG. 16. Section of ovary from intact flounder. Immunofluorescent procedure: preincubation with maturational hormone, first antibody anti-f Con AI1 GTH. Magnification as for Fig. 14.
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incorporation of vitellogenin into the oocytes which had been primed by vitellogenic hormone before hypophysectomy. An antiserum to vitellogenic hormone induces atresia in yolky oocytes and a decrease in gonadosomatic index. No marked changes were observed in either histological details or weight of ovaries of flounder treated with an antiserum to maturational hormone. Atresia in yolky oocytes occurred after hypophysectomy in the plaice Pleuronectes platessa (Barr, 1963b) and in the goldfish (Lam et al., 1978). It seems that the vitellogenic hormone alone is able to maintain and continue vitellogenesis after the process has been initiated. It has to be borne in mind that treatment with a specific antiserum ties up only the appropriate hormone (if present in circulation) leaving the other pituitary hormones to exert their biological actions, whereas hypophysectomy removes the source of all pituitary hormones. Results of hormone replacement therapy are not necessarily the same as the normal physiological action of the hormone in vivo. The maturational hormone might initiate vitellogenesis after spawning by stimulating estradiol and vitellogenin production but it does not appear to play a significant role in regulating incorporation of yolk into the ovary of flounder undergoing vitellogenesis. In salmonids the maturational hormone is not readily detectable in circulation until about the spawning time (Crim et al., 1975), indicating that its level is very low even if present. A very low level of the maturational hormone might be sufficient to maintain ovarian estradiol and hence hepatic vitellogenin production but not sufficient to maintain vitellogenin incorporation into the ovary. Hence it is quite reasonable to think that in the flounder ovarian yolk deposition is maintained and continued by the vitellogenic hormone after the process of vitellogenesis has been initiated by the maturational hormone through its action on ovarian estradiol and hepatic vitellogenin production.
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The binding of a hormone to its target tissue revealed by the immunofluorescence technique, after preincubation of the tissue with the hormone, shows the location of hormone receptors in the tissue. Binding of vitellogenic hormone to ooplasm of large immature oocytes of flounder ovary collected in October is indicative of the capacity for facilitation and preparation (priming) for the vitellogenic phase. Binding of vitellogenic hormone to ooplasm of yolky oocytes and to the follicular envelope surrounding the oocytes suggests that vitellogenic hormone acts on the yolky oocytes to stimulate incorporation of vitellogenin. Binding of maturational hormone to ovarian interstitial tissue and follicular envelope of oocytes implicates the hormone in stimulating steroidogenesis and vitellogenesis. Binding of the maturational hormone to a perinuclear region of the vitellogenic oocyte after preincubation with the hormone may reflect a future role of the hormone in oocyte maturation (resumption of meiosis). The results of this immunofluorescence study suggest that the hormones have distinct binding sites and distinct actions. The stimulating action of maturational hormone on spermatogenesis may be related to its steroidogenic activity (Idler and Ng, 1979) because androgens are known to promote spermatogenesis (e.g., Sundararaj et al., 1971; Idler et al., 1961; 1971). Mammalian-luteinizing hormone stimulates steroidogenesis as well as spermatogenesis in mammals while follicle-stimulating hormone stimulates spermatogonial proliferation. A role of vitellogenic hormone in spermatogenesis is not obvious in the present study. Previous assays for gonadotropic activities in pituitary hormone preparations were short-term assays, for instance, in the assay for vitellogenic activity (Ng and Idler, 1978a) the assay parameter was the incorporation of newly synthesized 33P-labeled vitellogenin into ovarian yolk of flounder,
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which were actively undergoing vitellogenesis before hypophysectomy, over a 7-day period. The present study involved prolonged hormonal treatment of fish hypophysectomized at the beginning of the reproductive cycle, and assessment of overall structural changes in the gonad with the histological technique; the study provided both additional and corroborative information. REFERENCES Barr, W. A. (1963a). The endocrine control of the sexual cycle in the plaice, Pleuronectesplatessa (L.). I. Cyclical changes in the normal ovary. Gen. Camp.
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Barr, W. A. (1963~). The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (L.). III. The endocrine control of spermatogenesis. Gen. Comp. Endocrinol. 3, 216-225. Breton, B., Prunet, P., and Reinaud, P. (1978). Sexual differences in salmon gonadotropin. Ann. Biol. Anim. B&hem. Biophys. l(l) 25-36. Campbell, C. M. (1978). In vitro stimulation of vitellogenin incorporation into trout oocytes by salmon pituitary extracts. Ann. Biol. Anim. Biochem. Biophys. 18(4), 1013- 1018. Campbell, C. M., and Idler, D. R. (1976). Hormonal control of vitellogenesis in hypophysectomized winter flounder (Pseudopleuronectes americanus Walbaum). Gen. Comp. Endocrinol. 28, 143- 150. Campbell, C. M., and Idler, D. R. (1979). Gonadotropin stimulation of estrogen and yolk precursor synthesis in juvenile rainbow trout. Biol. Reprod. 20, 1, 16. Grim, L. W., Watts, E. Cl., and Evans, D. M. (1975). The plasma gonadotropin profile during sexual maturation in a variety of salmonid fishes. Gen. Comp.
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