The Effects of Acth, Phytoestrogens and Estrogens on Corticosterone Secretion by Gander Adrenocortical Cells in Breeding and Nonbreeding Seasons

Acta Biologica Hungarica 59 (2), pp. 173–184 (2008) DOI: 10.1556/ABiol.59.2008.2.4

THE EFFECTS OF ACTH, PHYTOESTROGENS AND ESTROGENS ON CORTICOSTERONE SECRETION BY GANDER ADRENOCORTICAL CELLS IN BREEDING AND NONBREEDING SEASONS

BARBARA KAMINSKA,* M. OPALKA and L. DUSZA

Department of Animal Physiology, Faculty of Biology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-718 Olsztyn, Poland

(Received: November 2, 2006; accepted: April 16, 2007)

The aim of this study was to investigate the effects of ACTH, phytoestrogens (genistein, daidzein, biochanin A and coumestrol), and animal estrogens (estradiol and estrone) on corticosterone secretion by isolated adrenocortical cells of the ganders in breeding (April) and nonbreeding seasons (July). ACTH stimulated corticosterone output in the breeding season. In July (photorefractoriness and postbreeding molt) ACTH had no effect on corticosterone production. Coumestrol reduced corticosterone secretion by the cells obtained in nonbreeding season. Other examined phytoestrogens did not affect corticosterone production. Estrogens showed differentiated effects. Estradiol stimulated the corticosterone output in breeding season; estrone inhibited corticosterone release in July. The season can probably affect sensi- tivity of isolated gander adrenal cells, especially to ACTH. It seems that goose adrenocortical cells, in contrast to the mammalian cells, can be weakly sensitive to phytoestrogens.

Keywords: Phytoestrogens – adrenals – breeding season – ganders

INTRODUCTION

Phytoestrogens are plant-derived compounds with estrogen-like activity. Based on their chemical structure three main classes of phytoestrogens were separated: isoflavones, coumestans and lignans [15]. Phytoestrogens were identified in fruits, vegetables and legumes and they are present in human diet and animal fodder [12, 13, 25]. These compounds can act on animals and human organisms by a number of different mechanisms: binding with estrogen receptors (estrogenic and antiestrogenic activity), inhibition of tyrosine autophosphorylation, tumor growth and angiogenesis inhibition, antioxidant activity, interaction with key steroidogenic enzymes and stim- ulation of sex hormone binding protein [for review see 15]. Up to now, only a few

* Corresponding author; e-mail: [email protected]

0236-5383/$ 20.00 © 2008 Akadémiai Kiadó, Budapest 174 BARBARA KAMINSKA et al. studies have been published on the phytoestrogen influence on avian organisms. Leopold et al. [9] observed a negative effect of natural feed with high concentration of genistein and formononetin (isoflavones) on offspring number in free-living California quail (Callipepla californica). On the other hand, Lien et al. [10] did not find the negative influence of biochanin A (an isoflavone) on reproductive perfor- mance in Bobwhite quails (Colinus virginianus). In our earlier studies [19] were noted that dietary phytoestrogens had a slight effect on in vitro testosterone secretion in Bilgoraj ganders (Anser anser). In contrast, in vitro treatment with isoflavones (genistein, daidzein and equol), and coumestrol (a coumestan) inhibited testosterone production by isolated Leydig cells. Studies concerning the influence of phytoestrogens on adrenal cortex are not numerous. They were performed first of all on humans and laboratory animals but, to our knowledge, there are no data on their effect on avian adrenal steroidogenesis. Geese seem to be interesting objects of studies, not only with regard for a kind of taken feed, rich in phytoestrogens (soya, red clover and alfalfa). Corticosterone secretion in this species (estimated on the basis of the steroid concentration in feces) reveals seasonal rhythmicity connected with annual changes in breeding activity [6]. Annual rhythms of plasma glucocorticoid concentrations and adrenal sensitivity to stress and exogenous ACTH seems to be common in most of free living birds. During breeding season, plasma corticosterone level and response to stress are usu- ally higher, while the lowest plasma concentration of the steroid is observed in the nonbreeding period [26]. Therefore in geese, like in many other species of birds, annual changes in gonadal steroids’ concentration may be one of the important fac- tors regulating hypothalamo-pituitary-adrenal axis function. One can assume that in vitro response of goose adrenocortical cells to ACTH, estrogens and phytoestrogens changes in dependence to physiological state of birds. The aim of this study was to examine seasonal changes in gander adrenocortical cell function in vitro and the effect of ACTH, estrogen and phytoestrogen treatment.

MATERIALS AND METHODS Chemicals

The phytoestrogens: genistein, daidzein, biochanin A, coumestrol and other reagents were purchased from Sigma (Germany). Synacthen (ACTH1–24) was obtained from Ciba-Geigy (Switzerland). Radiochemicals were purchased from Amersham (UK) and Du Pont NEN (USA).

Animals

The unpaired males of Bilgoraj geese, a domestic form of Greylag geese (Anser anser), were kept at the experimental farm of the Department of Poultry Breeding, University of Warmia and Mazury in Olsztyn. Animals were housed in a building

Acta Biologica Hungarica 59, 2008 Seasonal changes in adrenal steroidogenesis 175 with light-regulated windows and restricted access to the open field without water area in accordance with accepted standards. Lighting was partly controlled. Goslings (from 3 d old) were kept on natural light for no longer than 8 h. At 36 weeks (January), ganders were given stimulatory, artificial lighting (10 h/d), until the nat- ural daylight was 10 h. Thereafter, natural daylight was provided to the end of exper- iment. Diets were balanced (crude protein, metabolisable energy, exogenous amino acids and minerals) in accordance with the nutrient requirements of domestic fowls [31]. Diets contained grass meal, bruised grain, amino acids, vitamins and minerals. Ganders were given free access to water and food during the reproductive season. Before the reproductive season diets were restricted. All experiments were performed in accordance with the principles and procedures of the Animal Ethics Committee of the University of Warmia and Mazury in Olsztyn.

Adrenocortical cell isolation and incubation

Adrenals were obtained from ganders (n = 5) at two different times of the season: the middle of reproductive season (April) and the beginning of photorefractoriness (July). The mean weights of animals and weights of their adrenals were 4.1 ± 0.2 kg and 0.56 ± 0.04 g in April, and 4.3 ± 0.36 kg and 0.57 ± 0.06 g in July, respectively. The adrenals were taken following decapitation and cleaned of the adherent tissue. Glands were transported to the laboratory in ice-cold Ham’s F-12 medium. Dispersed cells were obtained by mechanical disaggregation using the 80 μm pore-size steel sieve (Cell Dissociation Sieve-Tissue Grinder Kit, Sigma, Germany). Then the steroidogenic cells were counted by haemocytometer, diluted to density of 1.5×105/mL and seeded (1 mL) into 24-well culture plates. The steroidogenic cells contained many lipid droplets, therefore they are easily identified in the light micro- scope [3]. Additionally, the steroidogenic cells content (38–61%) was examined by a histochemical test for 3β-hydroxysteroid dehydrogenase [2, 30]. Ham’s F-12 (pH 7.4) containing 2% BSA and supplemented with 25 mM Hepes buffer, 1.2 g/L NaHCO3 and antibiotics, was used as an incubation medium. All incubations were performed under the water-saturated atmosphere of 95% air and 5% CO2 at 37 °C. Cell viability, determined by trypan blue exclusion method, ranged from 95% to 100%. None of the solvents or treatments, in concentration used in this study, affect- ed the viability of cells.

Experimental design

After 30 min of preincubation the cells were incubated for additional 20 h with or without ACTH1–24 (10 or 20 nM, a positive control), genistein, daidzein, biochanin A and coumestrol (0.5, 5 or 50 mM), estradiol and estrone (5, 50 or 500 pg/mL). The concentration of treatments used in this experiment was established in our dose- response preliminary studies and based on the corresponding in vivo [12] and in vitro

Acta Biologica Hungarica 59, 2008 176 BARBARA KAMINSKA et al.

[14, 18] experiments. After the end of incubation medium was collected and stored at –20 °C. Concentration of corticosterone was determined by radioimmunoassay (RIA).

Corticosterone assay

Corticosterone level in the medium was measured using specific RIA as described previously [7]. The specificity of antibodies against corticosterone, has previously been reported [32]. The sensitivity of assay was 15 pg/tube. The intra- and interas- say coefficients of variation for corticosterone were less than 7%.

Statistical analysis

All data are presented as mean ± S.E.M. Data from the two in vitro experiments (n = 5; n = 5) were submitted to one-way analysis of variance for repeated measure- ments followed by LSD-test. The influence of season on basal and ACTH-stimulat- ed corticosterone secretion was analysed by two-way analysis of variance and LSD test (Statistica, StatSoft Inc., Tulsa, OK., USA). Significance is reported at P < 0.05.

Fig. 1. The effect of ACTH (10 and 20 nM) on corticosterone secretion by adrenocortical cells (1.5×105/mL, after 20 h incubation) obtained from ganders (n = 5) in breeding (A) and nonbreeding (B) season. Bars without common superscripts are statistically different (P < 0.05)

Acta Biologica Hungarica 59, 2008 Seasonal changes in adrenal steroidogenesis 177 M) and (D) coumestrol (0.5, 5, μ < 0.05) P M); (C) daidzein (0.5, 5 or 50 μ /mL, after 20 h incubation) obtained from ganders (n = 5) in breeding 5 μ M); (B) biochanin A (0.5, 5 or 50 season. Bars without common superscripts are statistically different ( season. Bars without common superscripts are statistically different M) on corticosterone secretion by adrenocortical cells (1.5×10 The effect of (A) genistein (0.5, 5 or 50 The effect μ or 50 Fig. 2.

Acta Biologica Hungarica 59, 2008 178 BARBARA KAMINSKA et al. M) and (D) coumestrol (0.5, 5 or μ < 0.05) P μ M); (C) daidzein (0.5, 5 or 50 /mL, after 20 h incubation) obtained from ganders (n = 5) in nonbreeding season. 5 μ M); (B) biochanin A (0.5, 5 or 50 Bars without common superscripts are statistically different ( Bars without common superscripts are statistically different The effect of (A) genistein (0.5, 5 or 50 The effect μ M) on corticosterone secretion by adrenocortical cells (1.5×10 Fig. 3. 50

Acta Biologica Hungarica 59, 2008 Seasonal changes in adrenal steroidogenesis 179

RESULTS

Addition of ACTH (10 or 20 nM, a positive control) was followed by a significant (P < 0.05) increase in corticosterone secretion by adrenocortical cells obtained from ganders (n = 5) in breeding season – April (Fig. 1A). In July (the photorefractoriness and postbreeding molt) ACTH had no significantly effect on corticosterone output (Fig. 1B). The basal corticosterone secretion by adrenocortical cells obtained in April was significantly less (P<0.05) than in July (0.86 ± 0.2 vs. 1.99 ± 0.3 ng/mL). Next, the effect of phytoestrogens, estradiol and estrone on the steroid secretion by the cells during the 20 h incubation was studied. Increasing doses of genistein, daidzein, biochanin A, coumestrol (0.5; 5 and 50 μM), and estradiol or estrone (5; 50; and 500 pg/mL) were added to the cultures. Corticosterone release by adrenocortical cells was not altered by treatment with phytoestrogens in breeding season (Fig. 2). In July the inhibitory effect of coumestrol (50 μM; P < 0.05, n = 5) on corticosterone production was observed (Fig. 3D). In April corticosterone secretion by the cells exposed to estradiol (50 and 500 pg/mL) was significantly (P < 0.05, n = 5) enhanced, estrone at the highest dose tend- ed to diminish (P<0.0516) corticosterone release (Fig. 4A and B). In nonbreeding season (July) the influence of estradiol was not observed (Fig. 4C), but estrone (5; 50; and 500 pg/mL) significantly (P < 0.05, n = 5) suppressed the steroid release (Fig. 4D).

DISCUSSION

In the present study the effect of ACTH, phytoestrogens and estrogens on cortico- sterone secretion by gander adrenocortical cells in breeding (April) and nonbreeding (July) seasons was examined. The basal and ACTH-stimulated in vitro corticosterone secretion during these two periods was also compared. ACTH enhanced corticos- terone output by isolated adrenocortical cells obtained from ganders only in breeding season. Additionally, seasonal changes in basal and ACTH-stimulated medium cor- ticosterone concentration were observed. Basal and stimulated corticosterone output were approx. twice higher in July than in April. However, lack of significant response to ACTH (vs. control) in nonbreeding season limits the interpretation. In studies in vivo Barna et al. [1] shown that in White Hungarian domestic gan- ders (Anser anser) the highest daily mean value of the plasma immunoreactive ACTH was measured in middle June. It may at least partially explain the high basal corticosterone secretion in vitro and reduction of adrenocortical cells sensitivity to exogenous ACTH observed in our study in July. In a flock of semi-tame, free rang- ing males of greylag geese (Anser anser) it was examined annual corticosterone lev- els in feces [6]. All birds, independently from social status had higher corticosterone level in feces in breeding, although its concentration was also affected by possession of a partner and offspring. In domestic ganders (Hungarian White) testosterone achieves its maximal plasma level in the middle of March [23]. Thus, changes in cor-

Acta Biologica Hungarica 59, 2008 180 BARBARA KAMINSKA et al. < 0.05) P common superscripts are statistically different ( common superscripts are statistically different /mL, after 20 h incubation) obtained from ganders (n = 5) in breeding (A and B) nonbreeding season (C D). Bars without 5 The effect of (A and C) estradiol (5, 50 or 500 pg/mL) and (B and D) estrone (5, 50 or 500 pg/mL) on corticosterone secretion by adrenocor- of (A and C) estradiol (5, 50 or 500 pg/mL) (B D) estrone on corticosterone secretion by The effect Fig. 4. tical cells (1.5×10

Acta Biologica Hungarica 59, 2008 Seasonal changes in adrenal steroidogenesis 181 ticosterone level in ganders are proportional to variations of gonadal steroids’ con- centration. These data indicate that in Bilgoraj ganders adrenal functions changes seasonally and probably are connected with reproductive system activity. Similar phenomenon takes place in other species. Romero and Wingfield [28] observed in free-living Gambel’s white-crowned sparrows (Zonotrichia leucophyrs gambelii) seasonal changes in plasma corticosterone concentration in response to ACTH cou- pled with capture and handling. In contrast to breeding season, exogenous ACTH had no effect on plasma corticosterone during winter and fall migration. Interestingly, in the Alaskan Arctic free-living redpolls (Carduelis flammea) corticosterone release is modulated under the influence of exogenous ACTH depending upon the breeding site [27]. On the other hand, in outdoor-kept domestic ganders (Hungarian White), both baseline and ACTH-stimulated fecal corticosterone metabolites concentrations were not different between seasons. ACTH significantly enhanced corticosterone metabolites concentration during breeding (March), photorefractoriness and post- breeding molt (July), and during fall sexual reactivation in November [5]. Similarly, corticosterone responses to stress and ACTH in molting and nonmolting free-living pigeons (Columba livia) did no differ [29]. Thus, it seems that seasonal changes in corticosterone secretion, and response of the steroid production to stress and ACTH are species-dependent and modulated upon the physiological, social and ecological state of animals. Up till now, in vivo or in vitro studies focused on phytoestrogen and estrogen influ- ence on goose or other avian adrenals have not been published. There are no studies concerning seasonal changes in sensitivity of adrenocortical cells to these agents either. In presented experiment most of examined phytoestrogens had negligible effect on corticosterone secretion, independently from season. Only coumestrol at the highest dose used (50 μM) inhibited the steroid secretion in the nonbreeding season. These data seem to be, to some extent, surprising, because isoflavones used (espe- cially genistein and daidzein) belong to the strongest phytoestrogens possessing pro- portionally high affinity to estrogen receptors [8]. In vivo and in vitro studies per- formed on mammalian adrenals indicate an inhibitory effect of phytoestrogens on glucocorticoid secretion and steroidogenic enzymes activities. Treatments of rats with genistein (subcutaneous) caused a decrease in plasma corticosterone concentra- tion [17]. Genistein and daidzein inhibited also ACTH-induced cortisol production by post- natal and fetal human adrenocortical cells. In H295 (adult adrenocortical cell line) cells genistein, daidzein and estradiol suppressed the steroid secretion stimulated by cAMP. Interestingly, it was observed an increase in basal and stimulated adrenal androgen secretion: dehydroepiandrosterone (DHEA) or dehydroepiandrosterone sulphate (DHEAS) by these cells as a consequence of adrenal 21-hydroxylase inhi- bition by genistein and daidzein [14]. In other studies it was revealed that genistein, daidzein, biochanin A and formononetin inhibited cortisol release from H295R cells [18]. Isoflavones depressed also bovine adrenal 3β-hydroxysteroid dehydrogenase activity [33].

Acta Biologica Hungarica 59, 2008 182 BARBARA KAMINSKA et al.

In turn, estrogen influence on corticosterone secretion was more differentiated. Estrone (all doses), like coumestrol, inhibited the corticoid secretion in nonbreeding season; we observed also the inhibitory tendency in April. In contrast, estradiol stim- ulated corticosterone secretion in the breeding season and had no effect during the nonbreeding season. Unfortunately, there are no studies on adrenals of other birds. To our knowledge, there is also lack of papers describing the in vitro influence of estrone on adrenocortical cells. However, numerous in vivo experiments indicate that the plasma corticosterone concentration is directly (or via thyroid activity) regulated by gonadal steroids both in male and female birds [20–22]. For example, testosterone decreased plasma corticosterone levels in male Pekin duck (Anas domestica), domes- tic pigeon (Columba livia domestica), Japanese quail (Coturnix coturnix japonica), and cock (Gallus gallus domesticus), whereas in female Japanese quail progesterone, estrone and estradiol administration increased corticosterone concentration [20, 24]. Mesiano et al. [14] showed inhibitory effect of estradiol on cAMP-induced corti- sol secretion in H295 cells parallel with an increase in DHEA output. In other stud- ies priming of adrenocortical cells of male and female rats with estradiol resulted in an enhancement of basal corticosterone release [16]. Treatment of ovariectomized rats with estradiol benzoate (EB) caused an increase of plasma corticosterone level and potentiation of its basal and ACTH-stimulated in vitro output [11]. Similarly, Kau et al. [4] observed an augmentation of plasma aldosterone concentration and an enhancement of its in vitro secretion in ovariectomized female rats under influence of EB. The obtained results indicate seasonal differences in ACTH and estrogens action on adrenals of ganders. It seems that in contrast to the mammalian adrenocortical cells, sensitivity of goose adrenocortical cells to phytoestrogens is limited.

ACKNOWLEDGEMENT

This study was supported by State Committee for Scientific Research, Poland (projects No 528 0206 0805).

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