Camp. Biochem. Physiol. Vol. 106A, No. I, pp. 57-60, 1993

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OF ESTRADIOL- 178, ESTRONE, CATECHOL ESTROGENS, [D-ALA6]-LUTEINIZING

HORMONE RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN ON SERUM LEVELS OF

LIPIDS AND VITELLOGENIN IN THE IMMATURE DUCKLING (ANAS PLATY~HYNCHOS)

T. B. Nc and V. E. C. Got

apartments of Bi~hernis~~ and Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong (Fax 852-695-0958)

(Received 3 Ntwember 1992; accepted 2 December 1992)

Abstract-l. The effects of administration of estradiol-17fl (E,), es&one (E,), catechol estrogens, [D-Ala6]-luteinizing hormone releasing hormone (LHRH) and human chorionic gonadotropin (HCG) on serum levels of vitellogenin and lipids were examined in immature ducklings.

2. E, brought about an elevation in serum levels of total lipid, triglyceride, cholesterol and vitellogenin. 3. Treatment with both LHRH and E2 resulted in an increase in serum levels of total lipid, triglyceride,

cholesterol and vitellogenin. 4. Treatment with LHRH alone, HCG, E, or catecho estrogen failed to exert any effect on serum lipid

or vitellogenin Ievel. 5. The results indicate that, of the various hormones examined including estrogens, LHRH and HCG,

only E, was abIe to affect serum lipid and viteuogenin con~ntrations in the duckling.

INTRODUCTION

In oviparous vertebrates ovarian follicular growth is achieved by the process of vitellogenesis. Under the influence of estrogens the liver synthesizes the yolk precursor vitellogenin which is released into the blood circulation. The ovarian follicles sequester vitel- Iogenin which is subsequently cleaved into the yolk proteins Iipovitellin and phosvitin. Lipovitellin is a Iipophosphoprotein and phosvitin is a phospho- protein (Follett and Redshaw, 1974; Tata and Smith, 1979; Browder, 1985; Ng and Idler, 1983; Wallace, 1985). The process of vitellogenin incorpor- ation into the ovary is facilitated by pituitary gon- adotropins. Estradiol-induced vitellogenesis is accompanied by profound metabolic changes in the amphibian (Follett and Redshaw, 1968) and teleost (Ng et al., 1984). Tata and Smith (1979) and Tata (1988) have studied the regulation of Xenopus vitel- logenin genes.

The hormonal regulation of vitellogenesis in birds has been studied using the chicken as an example (Beuving and Gruber, 1971; Jailkhani and Talwar, 1972; Wang and Williams, 1980). The molecular mechanism of estrogenic action in the avian liver has been elucidated (Mester and Baulieu, 1972; Gshwendt and Kittstein, 1974; Dower and Ryan, 1976; Schneider and Schwendt, 1977; Lazier and Haggarty, 1979; Jost et al., 1984, 1986). The chicken vitellogenin gene has been sequenced (Burch, 1984). The purpose of the present study was to extend the

investigation to another avian species, the duck. Although estrone has been demonstrated to be effec- tive in stimulating vitellogenin production in teleosts (van Bohemen et al., 1982), its action and those of catechol estrogens which are metabolites of estrogens and biologically active in mammals (Booth, 1979; Fishman and Tulchinsky, 1980; Jones and Hsueh, 1981), have not been investigated in birds. Hence we examined the actions of the aforementioned estro- genie ho~ones, together with those of [D-Ala&]- luteinizing hormone releasing hormone (LHRH) which is a potent LHRH analog (Monahan et al., 1973), and human chorionic gonadotropin (HCG) which has a prolonged circulatory half-life, on metab- olism and vitellogenin production in immature duck- lings. It is well documented that avian pituitaries except that of the duck secrete two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Licht et al., 1977; Follett and Robinson, 1980). In the duck only one chromato- graphic fraction designated FSH/LH could be iso- lated (Licht et al., 1977). Since LH is steroidogenic in birds (Follett and Robinson, 1980; Wingfield and Farner, 1980) HCG was used in the investjgation to mimic the action of LH.

MATERIALS AND METHODS

Estradiol-I 78, estrone, 2-hydroxyestradiol, 2- hydroxyestrone, human chorionic gonadotropin, [D- Ala61_luteinizing hormone releasing hormone and

57

58 T. B. No and V. E. C. 001

Table I. Effects of estradial-17fi and rstrone on serum lipids in duckling

Total lipid ‘Triglyceride Cholesterol (mgiml) (mg;ml) (rng,‘rnl)

Oil 16.42 & 0.32 0.03 2 0.004 4.8 3: 0.13 Estrone 15.66 i 0.41 0.026 + 0.002 4,02 i 0.18 Estradiol-$78 18.66 * 0.02* 0.048 i 0.003* 6.50 & 0. I!‘* The doses of estrone and estradiol were both 0.25 n&injection for

a lotal of six injections. Results are preserited as means 2 SE. The asterisk* indicates statistically significant difference from oil-treated group by students’ r-test.

polyethylene giycol were purchased from Sigma Chemical Company. St Louis, MO, U.S.A.

Ducklings (Ana.s ~~~~~~~2.~~~~~~~~~1 weighing about 5Og were used for investigation. They were kept under a photoperiod of 13L: 1 ID and received food and water ad lib. Seven ducklings were assigned to each experimental group.

In experiment I, a control group received injections of corn oil. One group received estrone (0.25 mg per injection in corn oil), and another group received cstradiol-I 7fi (0.25 mg per injection in corn oil). The ducklings received a total of six injections spread over 2 weeks.

In experiment 2, a control group received saline injections. One group received human chorionic gonadotropin (3 pg per injection in 200~1 saline). Another group received [D”Ala6]-~uteinizing hormone releasing hormone (1 pg per injection in 200 ~1 saline). The fourth group received estradiol (0.25 mg per injection in corn oil) while the fifth group received both estradiol(0.25 mg per injection dissolved in corn oil) and [D-Aia6]-Iuteinizing hormone releasing hor- mone (1 gg per injection in 200 ~1 saline). The duck- lings received a total of six injections spread over 2 weeks. Experiment 2 was conducted at the same time and place as experiment 1.

In experiment 3, a control group received polyethylene glycol. The second group received hy-

droxyestradiol at a dose of 0.25 mg per injection in polyethyIene glycol. The third group received hydrox- yestrone (0.25 mg per injection in polyethylene gly- col). The ducklings received a total of six injections spread over 2 weeks. At the end of the experiments the ducklings were bled. The serum samples were stored at - 12*C until analysis. Serum total lipid tevel was determjn~d according to Woodman and Price (1972), trigly~e~de according to Galletti (1971). cholesterol using a coupled cholesterol oxidasu- peroxidase reaction (Sigma), and ~~ite~l~~enin by the measurement of alkaJ~-labile phosphorus (Ghan rp/ rrl., 19%).

RESULTS

In experiment I, the serum concentrations of total lipid, triglyceride and cholesterol were heightened by estradioLi7fi injections. Estrone was ineffective (Table I). In experiment 2. neither [D-Ala”]-luteiniz- ing hormone releasing hormone nor chorionic gonadotropin was able to bring about an increase in serum lipid or glucose concentration. Treatment with estradiol-17~ increased serum levels of total lipid. triglyceride, cholesterol and vitellogenin. The same changes occurred when the ducklings were treated with both estradiol- 17/I and [~-Ala*]-luteini%ing hor- mone releasing hormone (Table 2). In experiment 3, neither 2-hydroxyestradiol nor 2-hydroxyestrone had any effect on serum concentrations of total lipid, triglyceride, cholesterol and v~tellogen~n (Table 3).

DfSCUSSlON

Of the various estrogens tested in the present investigation, only estradiol-17~ was able to eIicit an increase in serum concentrations of total lipid,

Table 2. Effects of [tr-Ala6]-luteinizil~~ hormone releasing hormone (LHRH). estradioi-I 78 (E,), and human chorionic gonadotropm (HCG) on serum lipids, glucose and vitellogenm

(Vn) m ducklings

Total lipid ‘Triglyceride Cholesterol Glucose (mg~ml) (rng@ll) M%!m0 (mgiml) VY

Saline 17.6 rf 0.6 0.046 5 0.001 4.36 & 0.86 1.47 * 0.05 22.3 & i.0 E2 21.2 i- 0.9% ..,. 0.061 k 0.002* 7.98 + 0.92* i.2.~io,18 33.9?: 1.1* LHRH 17.8 2 1.8 0.045 2 0.003 4.35 * 0.58 I .34 + 0.08 20.2 & 1.6 LHRH + E, 20.8 * I .o* O.OSQ I_ O-003* 8.431 1.11* 0.95*0.13 31,8*0.9* HCG 16.4 rf: 1 .o 0.04s ? 0.005 3.18L0.34 I.40 t_ 0.03 29.8 1 3.0

The doses of Ez, LHRH and HCG were respectively 0.25 mg. I pg and 3 gg per injection for a total of six injections. Results are presented as means & SE. The asterisk *indicates statistically significant difference from satine-treated group by students’ r-test. Vg was measured in terms of alkali-labile protein-bound phosphorus, *g/ml serum.

Table 3. Effects of ?-hydroxycstradiol and 2-hydroxyestrooe on serum lepid and viteliogenin We) levels in ducklinns

Total lipid Triglyceride Cholesterol (m&ml) (w/ml) (wlml) VY

Polyethylene glycol 19.79” 1.80 0.04 + 0.004 4.66 i: 0.60 18.0 e 3.2 2-Hydroxycstradiol 21.11 & 3.06 0.045 + wXJ6 4.10* 1.12 22.1 i: 2.6 2-Hydroxyestrone 20.99 $; I .85 0.049 * 0.01 5.21 $1 1.03 20.2 2 3.4

The doses of bydraxyestrad~ol and hydroxyestrone were both 0.25 mg per injection for a told

of six injections. Vg was measured in terms of alkali-iabilc protein-bound phosphorus. p&l serum. Results are presented as means 2 SE.

Hormone regulatior 1 of vitellogenesis 59

triglyceride and cholesterol. Estrone and the catechol estrogens 2-hydroxyestradiol and 2-hydroxyestrone were inactive in this regard. It has been shown in the rainbow trout that estrone was much less potent than estradiol- 17p in stimulating vitellogenin production (van Bohemen er al., 1982). The inactivity of human chorionic gonadotropin in influencing lipid metab- olism in the duckling was probably because the level of estradiol-I 78 secreted by the immature duck gonad in response to the gonadotropin was too low. The lack of an effect of [D-Ala’~-lute~nizing hormone releasing hormone in stimulating lipid metabolism was perhaps attributed to the fact that the hypothala- mic hormone is at the top of the hypotha- lame-adenohypophyseal-gonadal axis and it had to stimulate pituitary gonadotropin secretion before ovarian estradiol production could be enhanced. The activity of estradiol-178 in stimulating lipid metab- olism and vitellogenin production in the duckling is similar to observations on the immature pullet (Heald and McLachlan, I964) and other oviparous ver- tebrates (Follett and Redshaw, 5974; Ho et ul., I982; Gavaud, 1986; Ho, 1987; Burzawa-Gerard and Dumas-Vidal, 1991).

Acknorvledgemenu-The expert secretarial assistance of MS Doris Yeung is gratefully acknowledged.

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