GENERAL

AND

COMPARAITVE

ENDOCRINOLOGY

28, 143-1.50 (1976)

Hormonal Control of Vitellogenesis Hypophysectomized Winter FI (Pseudopleuronectes americanus C. M. CAMPBELL~

AND

D.

R.

i

IDLER

Memorial University of Newfoundland, Marine Sciences Research Laboratov, John’s, Newfoundland

St.

Accepted September 19, 1975 A surgical method of hypophysectomy employing a lateral approach to the brain is described; this technique results in total hypophysectomy and 90% survival. Bypophysectomy decreased the incorporation of 33P0, or [W]leucine into yolk by the liver and abolished the accumulation of labeled yolk by the gonad. Estradiol treatment of hypophysectomized fish stimulates growth of the liver and hepatic synthesis of yolk proteins. No incorporation of yolk into the gonads was seen after estradioi treatment, and yolk protein accumulated in serum to higher levels than those seen in shamoperated fish. A glycoprotein preparation from teleost pituitaries, which should contain any classical gonadotropin, failed to stimulate gonadal incorporation of the yolk which accumulated in the serum. In contrast, the nonglycoprotein fraction of these pituitaries stimulated yolk incorporation into the ovary.

A hypothesis developed for amphibia (Follett et al., 1968; Wallace and Dumont, 1968) proposes that vitellogenesis consists of at least two phases. A primary phase is t&e production of a serum lipophosphoprotein, vitellogenin, by the liver under estrogen stimulation. The secondary phase is stimulated by gonadotropin and involves incorporation of vitellogenin into the k of oocytes. Gonadotropins are assumed to be responsible for both phases by esis of estradiol and by the

Much of the evidence for estrogen involvement in teleost vitellogenesis was circumstantial (Bailey, 19571,but t roles of gonadotropins and the control of yolk incorporation in had not been investigated at all injections inhibited viteilogenic intact fish (Egami, 19

manner similar t estrogen effect was ex feedback at the tion in the am The role of the pituitary gland in teleost leased. Intact 4;a vitellogenesis has been reviewed by de esized yolk prote Vlaming (1974). Hypophysectomy inhibited eins accumulate vitellogenesis, and various mammalian or serum, but there was no i~~~r~~rat~o~ mto piscine gonadotropic preparations were the ovary (Hack et al., capable of restoring the process (Anand and gonadal i~~or~~ration of Sundararaj, 1974). sulted from the absence

1 Present address: Laboratoire de Physiologie des p&sons, Institut National de la recherche agronomique, 78350 Jouy-en-Josas, France.

ericanus were used to ~~arni~~ ne separate roles of estrogen and ~~t~it~~ mone (or hormones) on vite liver and in the gonad. 143

Copyright AI1 ngbts

@

1976 by Academic

of reproduction

Press,

in any form

Inc. reserved.

144

CAMPBELL

MATERIALS

AND

IDLER

AND METHODS

Fish collected by divers using hand nets between September and January were maintained, without feeding, at the Marine Sciences Research Laboratory in 230-I tanks of running seawater. Water temperatures were 8-12” for Experiments I, III, and IV; -0.5” in Experiment II. Tricaine methane sulfonate (125 ppm at 8”, 250 ppm at -0.5”; Kent Laboratories) was used to anesthetize the fish and during operations the gills were irrigated with a 31-ppm solution in seawater. Fish were sexed prior to operation by visualization of the gonadal silhouette using a sealed beam lamp. A 2- to 3-cm skin incision between the hinge of the operculum and the angle of the jaws on the lower (left) side of the fish allows the adductor mandibulae to be split and retracted so that the ventro-lateral surface of the cranium can be exposed. At the posterior end of the wound a facial nerve emerges from the cranium. A 2- to 3-mm hole was drilled anterior to this point (Fig. 1), using a number 8 round burr, and bone debris and fluids were aspirated from the opening. A probe was inserted into the cranial cavity and used to lift the optic nerves dorsally, pulling the brain into a position where the pituitary gland could be seen clearly. In shamoperated fish the cranial fluids were aspirated, while in others, hypophysectomy was performed by aspiration of the gland and confirmed by its appearance in the aspiration pipet. The cranial opening was plugged using Achromycin grease (Cyanamid of Canada Ltd.), and the wound was sutured with Ethicon plain gut (000). The fish were allowed to recover for approximately 3 weeks before injection of 2 ,&i of H,33P0,/100 g of fish (in addition, 2&i/100 g of [3H]leucine was given in Experiment I). Intramuscular injections of estradiol benzoate (1.5 mg/kg) in peanut oil, or vehicle alone, were made at this time (a second injection was given 3 FIG. 1. (I) Hypophysectomy technique. Completed days later in Experiment I), and 3 days later the intraperitoneal treatment with pituitary material was operation showing orientation of incision relative to made and repeated for four to six daily injections. opercular hinge and angle of the jaws (arrowed). (2) Each injection was equivalent to an extract of 13 Open wound showing hole drilled in cranium through pituitary glands/100 g of fish in Experiment I or 5 which the pituitary gland is aspirated. (3) Pituitary glands/100 g in other experiments. Three days after the gland in aspirator pipet confirms success of operation. last injection, blood was collected from a caudal blood vessel into a syringe and allowed to clot for preparation of a serum sample. The total body, gonad, and liver weights were recorded, and samples of the organs washed from the dialysis bags into scintillation vials using distilled water. After low-speed centrifugation were frozen. A yolk-containing preparation was made by the supematant was aspirated off, and the precipitate homogenizing aliquots of liver or ovary with 4 volumes was redissolved in the extraction saline and mixed of 0.5 M NaCl, 5 mM EDTA (Plack et al., 1971). Pre- with 10 ml of Aquasol (New England Nuclear) for scinliminary centrifugation for 45 min at 50,OOOgyielded a tillation counting in a Packard 2003, using external supernatant from which, after 45-90 min at lOO,OOOg, standarization to correct for efficiency. Disintegrations an aliquot could be removed for dialysis against two per minute per gram of tissue were calculated and corchanges of more than 10 volumes of distilled water for rected for decay of radioactivity. Statistical compariat least 24 hr. Aliquots of serum were dialyzed against sons were made by two-level nested analysis of varaqueous EDTA (0.2g/l). iance or by least significant difference of arcsine transThe yolk which precipitated upon dialysis was formed percentage data (Sokal and Rohlf, 1969).

VITELLOGENESIS

IN WINTER

FLOUNDER

845

Pituitary glands were collected from iced Hippoglossoides platessoides (up to 9 days postmortem) at

yolk was seen, though in bot a lower incorporation Bonavista Cold Storage, Fermeuse, Newfoundland, hypophysectomized animals (Fig. 3); no efCanada, and stored at -70” until used. Purification of k content was detected hormonal preparations was performed at 4”. 3P in the yolk extracts of The method of preparation (Idler ef al., 1975) was based on the separation on Con-A-Sepharose (Pharmacia Fine Chemicals) of glycoprotein and nonglycoprotein material which had been extracted by 0.05 M Tris-Cl, 0.5 M NaCl, 2 x lo-* M dithiothreitol. The nonglycoprotein preparation was further fractionated by chromatography on two 90 x 2.5cm columns of Sephadex G-75 (Pharmacia Fine Chemicals) using 0.05 iw Tris-Cl, 0.9% NaCl, 1 mM EDTA, and 1VM dithiothreitol. The profile of optical density (280 nm) of the eluate, and description of the subfractions A,B,C,D is seen in Fig. 2.

was 4.2 t 0.5.

RESULTS

and sixty fish have been subjected to this hypophysectomy with an overall mortality of only 12%. Each operation takes less than 10 min. Hypophysectomized fish incorporated less 33P04(P < 0.01) into the yolk material of the gonad than did sham fish. No statistically significant effect of hypophysectomy on the incorporation of isotope into liver

360

440

520

sham-operatedfish tban in those whi been ~ypophysectomi~ed for more weeks (Fig. 3). 1) an Clear differences in HSI into the yolk fraction ived two ~~jecti~~sof erime~t I, esestradiol (Table 1). In tradiol treatment increas tion of33P04 into the yolk fr above that found in sham-o (Fig. 41, but the change was not statist1 significant. In Experiment

600

Elution volume (ml)

Fat. 2. Elution profile of partial separation of H. platessoides pituitary nonglycoprotein on Sephadex G-75. Optical density to ultraviolet light (A = 280 nm) of eluates from two 90 x 2.5cm columns of Sephadex G-75 superfine when eluted with 0.05 M Tris-Cl, 0.9% N&l, I m!i4 EDTA, 10m4DTT buffer.

SHHY S” HY GONAD

SH HYSHHY LIVER

0

SH HY SF HY G S.I.

SHHI sn HY H.S.I.

FIG. 3. The effects of hypophysectomy on 33PGqincorporation into liver and gonad yolk fractions, hepatosomatic (WI) and gonadosomatic (GSI) indices. (Confidence intervals, 2 SEM; number of fish in parentheses; SH, sham; HY, hypophysectomized.)

146

CAMPBELL

AND

TABLE EFFECT OF

Two

INJECTIONS

OF ESTRADIOL

Treatment

BENZOATE

IDLER

1

ON THE LIVER

OF HYPOPHYSECTOMIZED

P. americanus

Number of fish

HSI (% body wt f SEM)

dpm in yolk (extract of 1 g of liver f SEM)

6 4

2.11 + 0.95 2.57 + 0.14

21.50 2 300 5220 k 340

Peanut oil Estradiol benzoate

EXPERIMENT

I

EXPERlMENT III

EXPERIMENT Ip

(4) (3)

(6) h

14)

(5)

(6)

it SH HYW

Ml

:

%A n SH Tqzc

G NG

.

FI B

NG HY, ES

C

D,

_

FIG. 4. 33P0, disintegrations per minute in the yolk fraction of 1 ml of serum collected from fish after hypophysectomy and after replacement therapy with estradiol (ES) and pituitary fractions (G, glycoprotein; NG, nonglycoprotein; A,B,C,D, subfractions of NG; confidence intervals, 2 SEM; number of fish in parentheses.)

yolk 33P04 concentration was greater (P < 0.01) than that of sham animals. Estradiol treatment did not stimulate growth of the gonads in hypophysectomized fish (Table 2) but a slight stimulation (P < 0.05) of incorporation of labeled yolk into the gonad was seen in one experiment (Fig. 5). The nonglycoprotein pituitary preparation abolished (P < 0.05, Fig. 4) the estradiol stimulated increase of labeled yolk in serum and induced uptake (Z’< 0.01, Fig. 5) of this material into the gonad with a concurrent stimulation of gonadal growth (P < 0.001, Table 2). In contrast, the

glycoprotein preparation had neither of these effects, and both pituitary hormone fractions did not effect the labeling of liver yolk. Fraction C of the nonglycoprotein pituitary preparation retained the gonadotropic activity (Figs. 4 and 5; Table 2) after gel chromatography of the cruder preparation. DISCUSSION

Hypophysectomy resulted in little bleeding if the hole was drilled in such a position that the pituitary had to be moved to bring it into sight. If the hole was drilled in a more ventral position, extensive bleeding re-

.-_^

-

(‘2

(3) -

H+E k 0.7

(4) 6.7 f 1 (5) .._-

(4) 2.3 2 0.3

7

--

-

(6)

(6) -

2.8 -+ 0.3

H+E+NG -

(4) 2 f 0.1

H+E+G 6 t 0.3

Treatments

-

H+E+B -

9 2 1.5 7.4 -c 1 (4) 14) ~__lll_”

-

H+E+A -

11.7 I 1 (3 .-

-

H+E+C -

7.8 4 1.5 (3

--

H+E+D -

n $ = sham; M = hypox; E = estradiol benzoate; G = pituitary glycoprotein; NG = nonglycoprotein; A, B, C, D = ~o~glycoprotein subfractions.

IV

(6) -

2.1 k 0.1

III

H 5.3 f 0.8

S kl.4

9

I

Experiment

TABLE 2 GONADOSOMATIC INDICES OF FISH TREATED WITH ESTRADIOL AND PITUITARY PREPARATIONS. (PERCENTAGE OF BODY WEIGHT -C SEM: NUMBER OF FISH IN PARENTHESES~

c

3 vl I m 2 < z ;;i 0 ;! 2 2 ti E

8

s

7 F

148

CAMPBELL

6000

SH

HY; ES

FIG. 5. 33P0, Disintegrations per minute in the yolk fraction of 1 g of gonad from estradiol-treated hypophysectomized fish after treatment with piscine pituitary fractions. (Confidence intervals, 2 SEM; number of fish in parentheses.)

sulted. The rapidity with which the operation can be performed, good survival, and on the spot evidence of success makes hypophysectomy a powerful tool for investigations into the physiology of this fish. Hypophysectomy significantly reduced the incorporation of labeled yolk into the gonads of P. americanus. The effect of hypophysectomy on vitellogenesis of several teleosts has been assessedby histological means. Vivien (1941) in Gobius paganellus, Barr (1963) in Pleuronectes platessa, Yamazaki (1965) in Carassius auratus, and Sundararaj and Goswami (1968) in Heteropneustes fossilis found that upon hypophysectomy of these fish, the yolky oocytes underwent atresia. Belsare and Murthy (1968) briefly reported that hypophysectomy of Clarias batrachus resulted in a decrease in free cholesterol levels in the ovary but an increase in esterified cholesterol. These changes were probably part of the atretic changes induced in yolky oocytes by hypophysectomy. The data from P. americanus suggested that the pituitary gland is necessary to maintain

AND

IDLER

synthesis and/or accumulation of the yolk materials in addition to the maintenance of yolky oocytes proposed by other workers. In Experiment I, a higher 3HT3P isotope ratio was found in the yolk extracted from the gonads of sham-operated fish than in those of fish which were hypophysectomized and treated with hormones. Wallace et al. (1972) found no evidence for direct incorporation of Na,H33POa or rH]leucine into yolk material of X. laevis oocytes. They demonstrated no in vitro uptake of isotope labeled yolk into oocytes, and proposed that partial dephosphorylation occurs during the rearrangement of vitellogenin to form lipovitellin and phosvitin. The approximately constant isotope ratio seen in all yolk extracts in Experiment I suggested that the material found in the three body compartments of hypophysectomized P americanus was similar, but in the sham fish some dephosphorylation may have occurred during normal absorption of yolk material from the blood stream. No double isotope labeling experiments were performed using the nonglycoprotein pituitary preparations. The slight depression in levels of radioactive yolk in liver of hypophysectomized P. americanus suggested that the removal of the pituitary may have resulted in a reduced synthesis of yolk material by the liver. The failure to demonstrate a statistically significant effect of hypophysectomy on the concentration of yolk in serum may have resulted, because in the absence of the pituitary gland, yolk incorporation by the gonad ceased more rapidly than did liver synthesis, which was only indirectly controlled by the pituitary. The estradiol-stimulated increase in the HSI of hypophysectomized P. americanus was similar to that noted after estradiol treatment of intact Misgurnus anguillicaudatus (Kobayashi, 1953), Oryzias latipes (Egami, 1955), and Gasterosteus aculeatus (Oguro, 1956). The liver of normal vitellogenic Oncorhynchus nerka is

VITELLOGENESIS

IN

larger than immature of m a le fish (Ishii, 1971),which suggestedthat enlargementof the liver induced by estradiol may be the result of stimulation of vitellogenic processes. The increasedincorporation of 33P0, into the yolk fraction of P. americanus liver by estradiol treatment is evidence that the yolk synthesis was stimulated by this steroid. Livers from vitellogenic G . morhua synthesized yolk proteins in vitro (Plack and Frazer, 1971)and estradiol injection of m a le or female G . morhua @ ‘lacket al, 1971)inuced accumulation of yolk in serum. Serum levels of 33P0,-labeled yolk were higher in estradiol-treated hypophysectomized P. americanus than in sham or hypophysectomizedfish. The accumulation of yolk proteins in serum of intact or hypophysectomized fish after treatment ‘tb estrogenscan be explained by a lack of nadotropic stimulation of yolk uptake by the ovaries. Several workers have proposed a negative feedback by estrogens on pituitary gonadotropins to explain an inhibition of gonadalgrowth by estrogens(Sundararaj Goswami, 1968; Egami, 1954; Egami I&ii, 1962), and histological study of the pituitary supports this hypothesis (Sun’ and Goswami, 1968). kers using a m p h ibia found that an ol-induced accumulation of yolk in be abolished by treatment an gonadotropins (Emmersen 4). In P. americanus only the uo~glycoprotei~ fraction C (Fig. 4), prepared from N. platessoides pituitaries, stimulated removal of estradiol-induced yolk from the serum and accumulation in the ovary. The inability of the glycoprotein fraction from these pituitaries to stimulate yolk incorporation was surprising since this fraction had gonadotropic activity in inducing oocyte maturation and ovulation in ypophysectomizedP. americanus (Campell, 1975). Further evidence that the glyrotein fraction of teleost pituitaries

WINTER

149

FLOUNDER

contains a classical go~adotro~~~ came from Idler et a2. (1975)who prepare hormone from 0. nerka pituitarie affinity chromatography method us present study. Further studies to characterize the nonglycoprotein vitelloge tor in fraction C of H. plafe pituitaries are now in progress. It appears that vitellogenesis in americanus consists of a yolk synthe mechanism in the liver which is $~irn~~~~~ by estradiol and a yolk uptake rn~cba~i~rn in the gonad which responds to t~~atrn~~~ e with a ~o~glycoprotei~ resent i pituitary gland. ACK

LEBGME

This work was part of a thesis submitted in partial fulfillment ofthe requirements for the degree of Doctor of Philosophy to Memorial University of newfoundland. The first author was supported during this work by a Canadian Commonwealth Scholarship, M.S.R.L. Contribution #216.

REFERENCES Anand, T. C., and Sundararaj, B. I. (1974). Ovarian maintenance in the hypophysectomised catfish, Heteropneustesfossifis (Bloch), with mammalian hypophyseal and placental hormones and gonadaf and adrenocortical steroids. Gcn. Camp. Endocrinol. 22, 154-168. Bailey, R. E. (1957). The effect of estradiol on s,erum calcium, phosphorus and protein of goldfish. J. Exp. Zoo/. 136, 455-470. Barr, W. A. (1963). The endocrine control of the sexua? cycle in plaice. II. The endocrine control of oogenesis. 6en. Camp. Endocrinol. 3, 205- 215. Belsare, D. K., and Mm-thy, P. S. R. (19683.The effects of hypophysectomy and goitrogen treatment on ovarian compensatory hypertrophy in CIavias batrachus Linnaeus. A&an. Abstr. Contrib. Fish. Aquat. Sci. India. Campbell, C. M. (1975). Physiological control of rein female W inter Flounder production (Pseudopeuronectes americanus Walbaumj. Ph.D. Thesis, Memorial University of Newfoundland, Canada. de Vlaming, V. L. i974. Environmental and endocrine control of teleost reproduction in: “Control of Sex in Fishes” (C. B. S&reck, ed.). Extension Division, Virginia Polytechnic Institute and State University. Egami, N. (1954). Inhibitory effect of estrone benzoate on ovarian growth in the loach, Misgurnus anguil-

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CAMPBELL

J. Fat. Sci. Univ. Tokyo. 7(l) 113-119. Egami, N. (1955). Effect of estrogen and androgen on the weight and structure of the liver of the fish licaudatus.

Orzias latipes. Annot. Zool. Jap. 28,(2), 79-85.

Egami, N., and Ishii, S. (1962). Hypophyseal control of reproductive functions in teleost fishes. Gen. Comp. Endocrinol. Suppl. 1, 248-253. Emmersen, B., and Kjaer, K. (1974). Seasonally and hormonally induced changes in the serum level of the precursor protein vitellogenin in relation to ovarian vitellogenic growth in the toad Bufo bufo b&o (L.). Gen. Comp. Endocrinol. 22, 261-267. Follett, B. K., Nicholls, T. J., and Redshaw, M. R. (1968). The vitellogenic response in the South African clawed toad (Xenopus laevis Daudin.) J. Cell Physiol. 72 Suppl. 1, 91- 102. Idler, D. R., Bazar, L. S., and Hwang, S. J. (1975). Fish gonadotropin(s). II. Isolation and purification of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography. Endocrine Res. Comm. 2 (3), 215-235. Ishii, K. (1971). Morphological changes in the liver of the Kokanee, Oncorhynchus nerka, accompanied with sexual maturation. Bull. Fat. Fish. Hokkaido Univ. 22(3), 215-230. Kobayashi, H. (1953). Effect of estrone upon the structure, weight and fat content of the liver in the fish, Misgurnus anguillicaudatus. Annot. Zool. Jap. 26, 213-216. Oguro, C. (1956). Some observations on the effect of estrogen upon the liver of the three-spined stickleback, Gasterosteus aculeatus L. Annot.

AND

IDLER

Zool. Jap. 29, 19-22. Plack, P. A., and Frazer, N. W. (1971). Incorporation of L-(‘4C) leucine into egg proteins by liver slices from cod. Biochem. J. 121, 857-862. Plack, P. A., Pritchard, D. J. and Frazer, N. W. (1971). Egg proteins in cod serum. Natural occurrence and induction by injection of oestradiol-3-benzoate. Biochem. J. 121,847-856. Sokal, R. R., and Rohlf, F. J. (1969). “Biometry.” W. H. Freeman and Company, San Francisco. Sundararaj, B. I., and Goswami, S. V. (1968). Effects of estrogen, progesterone and testosterone on the pituitary and ovary of the catfish, Heteropneustes fossilis. J. Exp. Biol. 169(2) 21 l-228. Vivien, J. H. (1941). Contribution a l’etude de la physiologie hypophysaire dans ses relations avec l’appareil genital, la thyroide et les interrenaux chez les poissons selaciens et teleosteens. Bull. Biol. de la France et de la Belgique. 7.5,258-309.

Wallace, R. A., and Dumont, J. N. (1968). The induced synthesis and transport of yolk proteins and their accumulation by the oocyte in Xenopus laevis. J. Cell. Physiol. 72 Suppl. 1, 73-90. Wallace, R. A., Nikol, J. M., Ti Ho, and Jared, D. W. (1972). Studies on amphibian yolk. X. The relative roles of antosynthetic and heterosynthetic process during yolk protein assembly by isolated oocytes. Dev. Biol. 29, 255-272. Yamazaki, F. (1965). Endocrinological studies on the reproduction of the female goldfish, Carassius auratus L., with special reference to the pituitary gland. Mem. Fat. Fish. Hokkaido University. 13(l), l-64.