Stress responses in avian embryos

American Zoologist, Dec 1997 by Epple, August, Gower, Barbara, Busch, Marc Ten, Gill, Tejendra, Et al

Stress Responses in Avian Embryos1

AUGUST EPPLE,2 BARBARA GOWER2, MARC TEN BUSCH, TEJENDRA GILL, LOUIS MILAKOFSKY^, RALF PIECHOTTA, BARBARA NIBBIO, T. HARE^^, AND MILTON H. STETSON(secs)

SYNOPSIS. The day 13-14 chicken embryo is a useful model for studies on prenatal stress responses. Free dopamine, norepinephrine and epinephrine in its plasma, amniotic and allantoic fluid respond to a variety of stresses. The allantoic fluid also contains conjugated catecholamines and conjugated steroids. However, a blood/allantois barrier excludes free thyroid hormones and free steroids, and insulin. On the other hand, the allantoic fluid contains at least 40 amino acids (including six excitatory amino acids) and related compounds. Most, possibly all, components of the allantoic fluid are regulated at specific blood/allantois and amnion/allantois barriers, and they respond to ethanol stress and metabolite loading differentially. The avian allantois is a depot for important metabolites and messenger substances which seems to be controlled by as yet unidentified hormones.

INTRODUCTION

If one were to design a creature for the study of prenatal stress responses of hormones and metabolites, one would probably come up with something similar to the 13day chicken embryo. The many advantages of this "model" include: (1) The stress responses are exclusively due to the embryo, without maternal interference. (2) Plasma, amniotic and allantoic fluids have volumes large enough for measurements of hormones and metabolites (cf., Romanoff, 1960). (3) The allantois can be cannulated, and its fluid withdrawn without handling stress (Epple et al., 1992). (4) The compositions of plasma, amniotic and allantoic fluids differ, due to the presence of specific barriers (Smoczkiewiczowa, 1959; Romanoff, 1960, 1967; Graves et al., 1986; Murphy, 1997; Murphy et al., 1982, 1986, 1991; Davis et al., 1988; Ten Busch et al., 1997a, b; Piechotta et al., 1998). (5) Amnion and allantois are not innervated (Romanoff, 1960), indicating a purely endocrine regulation of these barriers.

Despite the investigative challenges provided by these features, there is only a limited number of endocrine studies on the avian allantois (Boucek and Bourne, 1962; Woods et al., 1971; D.V. Gill et al., 1983; Murphy, 1997; Murphy et al., 1982, 1986; Koide and Tuan, 1989; Clark et al., 1990). Probably, a major reason for the dearth of data has been the wide-spread belief that the allantois is merely a respiratory organ and a dumping ground for metabolic wastes.

During the past five years, we have analyzed the presence of messenger substances and metabolites in blood and amniotic fluid, and in particular in the allantoic fluid of the chicken embryo (Epple et al., 1992; Gill et al., 1994; Ten Busch et al., 1997 a, b; Piechotta et al., 1998). In the following, we will concentrate on two rather unexpected results of this work: (1) the exquisite sensitivity of the chicken embryo to certain types of stress; (2) the function of the allantois as a dynamic depot of messenger substances and hormones. MATERIALS AND METHODS

The eggs were purchased from Truslow Farms (Chestertown, Maryland) and incubated at 37.5 deg C in a "Humidaire" incubator. Samples of allantoic fluid, amniotic fluid and blood (chorioallantoic vein) were centrifuged and cleared by passage through a Star II filter (Costar Corporation, Cambridge, Massachusetts). Total thyroxine, total triiodothyronine, cortisol, corticosterone and total testosterone were measured with Coat-A-Count RIA reagents purchased from Diagnostic Products Corporation (DPC, Los Angeles, California). Estradiol was measured with a double-antibody RIA kit, also from DPC. The catecholamines were measured by radioenzymatic assay (CAT-A-KIT, Amersham). Glucose was determined by the hexokinase technique (Sigma), and the amino acids and related compounds by an HPLC-fluorometric technique (Milakofsky et aL, 1984). The latter identifies the following 41 substances in 0.050.1 ml samples of the three fluids studied: alpha-aminoadipic acid (AAA), alpha-aminobutyric acid (ABA), alanine (ALA), anserine (ANS), arginine (ARG), asparagine (ASN), aspartic acid (ASP), beta-aminoisobutyric acid (BAIBA), beta-alanine (BALA), carnosine (CAR), citrulline (CIT), cystine (CYS), cystathionine (CYST), ethanolamine (EA), gamma-aminobutyric acid (GABA), glutamine (GLN), glutamic acid (GLU), glycine (GLY), reduced glutathionine (GSH), oxidized glutathionine (GSSG), Histidine (HIS), homocarnosine (hoCAR), hydroxylysine 1 (HYLYS1), hydroxylysine 2 (HYLYS 2), isoleucine (ILE), leucine (LEU), lysine (LYS), 1-methylhistidine (MEHIS1), 3-methylhistidine (MEHIS3), methionine (MET), ornithine (ORN), phosphoethanolamine (PEA), phenylalanine (PHE), phosphoserine (PSER), serine (SER), taurine (TAU), threonine (THR), tryptophan (TRP), tyrosine (TYR), valine (VAL). The total amounts of substances in the three fluids studied were estimated assuming that the volumes of allantoic fluid, amniotic fluid and plasma were 6 ml, 3 ml and 1.5 ml, respectively (cf Romanoff, 1960). RESULTS