Thinning of eggshells in birds by DDE: Mode of action on the eggshell gland

Camp. Biochem. Physiol. Vol. 88C, No. 1, pp. l-22, 1987

0306-4492/87 $3.00 + 0.00 0 1987 Pergamon Journals Ltd

Printed in Great Britain

REVIEW THINNING OF EGGSHELLS IN BIRDS BY DDE: MODE OF ACTION ON THE EGGSHELL GLAND ERIK LUNDHOLM Department

of Pharmacology,

University of Linkiiping, Regionsjukhuset, (Telephone: (013) 19-3456)

S-581 83 Linkoping,

Sweden.

(Received 19 November 1986) Abstract-l. The mode of action of DDE and similar compounds on the thinning of eggshells-often combined with shell breakage-is discussed and related to what is known about their formation and especially the Car+ metabolism of the shell gland. The effect is one of several actions these compounds have on reproduction. 2. The sensitivity of different species of birds to DDE differs. Some are quite sensitive (2 20%); others moderately sensitive (< 20%); others are insensitive. There are also variations among sensitive species as to which of the shell.layers is most affected. 3. In two varieties of ducks with different sensitivities, the et&t of DDE administered in uivo was found to be localized to the translocation of Ca*+ from the mucosa cells of the gland to the shell gland cavity. This DDE effect was only present (moderately sensitive) or most marked (sensitive) in the two varieties 1620 hr before the time of egg laying (oviposition). It was not present in the (insensitive) domestic fowl. 4. The rate of the ATP-dependent or its partly Ca *+ binding of the shell gland mucosa homogenate purified microsomal or mitochondrial subfractions showed functional changes during the shell formation in relation to the value at the time of oviposition of untreated ducks. When DDE has been administered in viuo, the rate of Ca2+ binding, when changed, was reduced and the maximal effect was present 1612 hr before oviposition. At the time of oviposition no DDE effect was present. In the domestic fowl no reduction was present at any time. If the Ca *+ binding of the subcellular fractions was inhibited by the calmodulin antagonist trifluoperazine added in vitro, the DDE effect on this binding was no longer present in ducks. 5. If DDE was added in vitro, it always reduced the specific Ca’+-Mg *+-ATPase activity in the mucosa homogenates from both ducks and domestic fowls. When DDE was administered in vivo, this activity was only occasionally reduced. Instead the mol Ca 2+ bound per mol ATP hydrolyzed (Caz+/P-ratio) was reduced, i.e. DDE had probably had an “uncoupler” effect on the Ca’+-pump. The Ca’+/P ratio also showed functional changes. It was about 0.2 at oviposition and 0.5 during the shell formation. DDE in viuo inhibited this increase during shell formation keeping the Ca*+/P ratio at 0.2. 6. Those observations indicated that DDE administered in uivo mainly influenced the stimulus-secretion mechanism of the shell gland. Among the hormonal factors being most closely involved in the regulation of shell formation, and that may have been influenced by DDE, progesterone and prostaglandins were considered likely candidates. It was observed that DDE in concentrations present in vivo in the shell gland mucosa of the duck inhibited the binding of progesterone to its cytoplasmic receptor. The inhibition was weaker in the domestic fowl since progesterone had a higher affinity to its receptor in the hen than in the duck. DDT and DDE are potent inhibitors of calmodulin. This polypeptide had been suggested to be a part of the progesterone receptor. Other potent calmodulin antagonists such as trifluoperazine and calmoduzaline were as potent as DDE in inhibiting the binding of progesterone. The calmodulin-inhibiting effect of DDE is probably of significance for its shell-thinning action.

1. INTRODUCTION

During the last decades vironmental pollutants

toxicological

actions

of en-

have become of increasing practical importance and have received considerable scientific attention, for a number of reasons. From toxicological and pharmacological points of view, the exposure of many different species of animals (and plants) to various kinds of biologically active substances, especially during long periods of their life cycle, has resulted in sub-chronic or chronic toxic effects which often have not been discovered in acute ‘classical’ toxicological and pharmacological studies. These newer toxic actions of environmental

‘pollutants’ have created serious hazards to several species of animals and even to whole ecological systems. The study and understanding of the mode of action of these pollutants is of increasing importance One such effect which is produced both by chlorinated hydrocarbons-specially compounds such as DDE and PCBs (Ratcliffe, 1970; Hayes, 1975) and dieldrin (Davison and Sell, 1974Fand by some metal compounds for example, Hg2+, methyl-Hg (Stoewsand et al., 1971, 1972) and aluminium (Nyholm, 1981), is thinning of the eggshells in different species of birds. This shell thinning, especially in predatory birds, will lead to cracked or broken eggs and other deleterious consequences for

2

ERIK LUNDHOLM

reproduction, effects which have resulted in near extinction of the populations of several species of birds. In wild birds, in particular, such effects have been correlated to an accumulation of p-p/-DDE in the eggs (Ratcliffe, 1967,197O; Hickey and Anderson, 1968). Other chlorinated substances, for example, p-p’-DDT, and PCBs, which via the food chain become accumulated in the birds and their eggs, will contribute to this action in the wild (Cooke, 1973; Faber and Hickey, 1973; Hayes, 1975; Lundholm, 1985a). Eggshell thinning caused by DDE or other chlorinated hydrocarbons has been observed in several species of birds. Faber and Hickey (1973) estimated that 30 species were affected in this way. King et al. (1978) reported that out of 22 species of Texas aquatic birds, 15 displayed shell thinning related to high egg levels of organochlorine compounds, mostly DDE. Shell thinning does not occur in all kinds of birds after doses which do not affect the health of the egg-laying bird (Cooke, 1973). Bird species can be divided into 3 categories with regard to the maximal shell thinning observed at an optimal concentration of DDE or DDT: (1) those in which the shell thinning reaches a value of 30% or even more, e.g. the peregrine falcon (F&o peregrinus), the brown pelican (Pelecanus occidentalis) (up to 50%) and some species of ducks (2) intermediately sensitive species with shell thinning of 5-15%, such as the American kestrel (Falco sparuerius) and Japanese quail (Coturnix coturnix) and (3) insensitive species such as the domestic fowl (Gallus domesticus) and Bengalese finch (Lonchura striuta), in which DDE may even increase the thickness of the shells. Other chlorinated hydrocarbons, especially PCBs, may cause reproductive disturbances, but their shell thinning action is more moderate (approx. IO%), and in the species whose shells are affected other mechanisms are contributory (Scott et al., 1975). In the wild, high concentrations of DDE are often accompanied by high levels of other pollutants (e.g. dieldrin, aldrin, PCBs, methyl-Hg), which makes it difficult to evaluate the separate effects of these substances. Faber and Hickey (1973) noted that the highest partial correlation coefficient between eggshell thinning and the pesticide content of the eggs in 13 species of fish-eating birds was that referable to p-p/-DDE. The dieldrin content was a contributory variable but not as strong as that of DDE. Only in the common merganeser (Mergus merganeser) and red-breasted merganeser (Mergus serrator) was eggshell thinning correlated to the PCB content but not to the content of DDE. It should be pointed out that many other toxic pollutants are able to cause acute thinning of eggshells lasting a few days, but their effect is mostly associated with a reduction of the intake of food and the appearance of other toxic symptoms (Haegele and Tucker, 1974). In contrast, feeding ofp-p’-DDE to ducks for some weeks resulted in eggshell thinning which persisted for months or even years (Peakall et al., 1975; Longcore and Stendell, 1977) after the DDE administration was stopped and without the occurrence of any other toxic symptoms in the egglaying bird. Part of this long-lasting effect of p-p’DDE could be related to its pronounced ability to

accumulate in the tissues, especially in the adipose tissue and egg yolk, and its slow rate of elimination from the body (Longcore and Stendell, 1977; Lundholm, 1985a). Kolaja and Hinton (1976) reported that the mucosa of the shell gland from DDE-treated ducks showed histological evidence of atrophy, but Friedenbach and Davidson (1977) observed similar changes (smooth cilia) in both the sensitive duck and the insensitive chicken. The DDE concentration in the eggshell gland has been found not to be increased in relation to that of other tissues (Lundholm, 1985a). It is also evident that DDE has a fairly selective effect on the eggshell-forming process, as it has been observed that the o-p’-DDE isomer, which was not accumulated in the body, was nevertheless potent in reducing the eggshell thickness (Bitman et al., 1969; Lundholm, 1980). DDE administration will not reduce the number of eggs laid during a given period when administered in amounts (lG40 ppm) that are sufficient to produce shell thinning (Davison and Sell, 1974; Lundholm, 1985a), and neither will it reduce the weight of the whole egg. In ducks, in high doses such as 200mg/duck, DDE may temporarily inhibit the egg production (Lundholm, 1985a), or in a dose of 100 ppm it may temporarily decrease the weight of the egg (Balasubramanian, 1972). In combination, DDE and PCB (140 ppm of each) reduced the number of eggs laid by ducks after a latency period of 8 weeks (Risebrough and Andersson, 1975). In the chicken l&20 ppm of PCB (Arochlor 1248) given for 68 weeks significantly reduced the egg production, and the hatchability of the eggs (Scott et al., 1975). The main topic of this review will be the mode of action of DDE and related chlorinated hydrocarbons on eggshell formation, with especial regard to Ca2+ metabolism. As is evident from the following discussion, this action is probably mainly located in the mucosal cells of the eggshell gland. However, DDE and similar compounds have several other actions which may reduce the thickness of the eggshell, as pointed out and discussed by Cooke (1973). In order to understand and discuss both the action of DDE on the activity of the eggshell gland and other potential effects of this compound that may influence the shell formation, the main physiological and biochemical processes in the shell formation have to be considered. During the last decade our knowledge about the mechanisms that translocate Ca2+ across membranes and the ways in which they are regulated has increased rapidly. Ca2+ is one of the two main ions involved in the formation of the eggshell. In a shell whose thickness is reduced by DDE, the amount of Ca2+ like that of carbonate, is decreased (Cooke, 19731 Lundholm, 1984b). An effect of DDE on Ca2+ metabolism of the shell gland is therefore a priori a probable mode of action of the pesticide. Both theoretical and methodological problems are involved in the study of the Ca2+ metabolism of the shell gland, however. The earlier observations have not been related to or discussed in the light of more recent observations or theories concerning Ca*+ transport in different cells. To provide the necessary background for discussing the effect and mode of action of DDE, some of the main events during the shell formation are briefly reviewed.

Thinning of eggshells by DDE

3

tion in the albumin simultaneously decreased. The directions of these changes in K+ and Nat were similar to those reported by El Jack and Lake (1967) The egg yolk protein synthesis takes place in the and Mongin and Sauveur (1970) to occur in the fluid liver but the protein is added to the egg in the ovary in the shell gland cavity at ~rresponding points in (McIndol, 1971). The egg albumin is formed and time. added during a period of 2-3 hr to the egg when it is being transported in the magnum part of the oviduct Ninety-eight per cent of the calcified eggshell con(Gilbert, 1971). In the isthmus part of the oviduct, sists of CaCO, in the form of hexagonal crystals of where the egg remains for 1.5-2 hr (Gilbert, 1971), calcite. The remaining 2% is a spongy organic matrix two soft proteineous membranes which will become which according to suggestions by some authors may the inner soft parts of the eggshell are formed. In the function as nucleating sites for the crystallization of isthmus, isolated patches of CaCO, are also formed the mineral components of the shell (Wilbur and on the soft membrane. The former membranes are Simkiss, 1968). The organic matrix contains 3 main probably the origin of the mammillary knobs and the fractions: a sulphur-containing protein (ovo-keratin), crystallization centres of the future CaCO, crystals a glycoprotein, and a sulphated polysaccharide. (calcite) which will be formed in the eggshell gland These fractions are not evenly distributed throughout (Simkiss, 1961; Simkiss and Taylor, 1971). It is of the eggshell (Simkiss and Taylor, 1971). Salevsky and interest that whereas the rate of the ATP-dependent Leach (1980) observed in the domestic fowl that the Ca’+ binding is low in a homogenate of the mucosa organic components of the fluid in the shell gland of the magnum part of the oviduct, it is about 10 cavity during the plumbing stage were quite different times higher than that of the magnum in a homogenfrom those of the organic matrix of the eggshell, ate of the mucosa from the isthmus. In fact, the rate Enclosing the calcified eggshell is a thin (approx. of Ca*+ binding has been found to reach a value 10 pm) proteineous cuticle which is formed by glands similar to that observed in a homogenate from the in the distal part of the shell gland when the egg shell gland mucosa (Lundholm, 1985b). enters the vagina (Taylor and Simkiss, 1971). Electron microscopic investigation of decalcified When the egg reaches the cavity of the eggshell gland, the rate of formation of the calcified eggshell eggshells has shown that the organic matrix contains is quite slow for about 3-5 hr (Burmester 1940; mammillary knobs or cores which are attached to the Bradfield, 1951). During this time and starting al- soft membranes around the albumin and contain ready in the isthmus, there is secretion of water, chondroitin sulphate and protein complex. The electrolytes and glucose from the shell gland mucosa. knobs merge to form a spongy matrix. In the calcified This secretion is absorbed by the egg albumin, the shell 2 main layers are also present-the mammillary volume of which thus increases. The process is called cone layer and the paIisade layer. In the cone layer, ‘plumbing’ and the secreted fluid ‘plumbing fluid’ containing the organic mammillary knobs, the separ(Burmester, 1940). El Jack and Lake (1967) report ate crystals of calcite, which initially originated in the that the composition of this plumbing fluid in hens, isthmus portion where they were attached to the soft in mmol/l, is: Na+ 139, K+ 16, Ca*+ 14, Mg2+ 1.5, shell membranes, do not form a coherent layer. When Cll 80, and CO* 83 (as HCO,), and the pH is 7.65. this occurs, in the organic spongy matrix, the palisade During these initial hours it is mostly the outer layer layer, which is penetrated by the pores, is formed of egg albumin that will swell by absorption of fluid (Simkiss and Taylor, 1971). The stages and the rates (15-16 g, Gilbert, 1971). The total volume of the egg of formation of the different layers in relation to the surrounded by the soft membranes will increase by length of stay of the egg in the eggshell gland of the about 25% (Bradfield, 1951) and will distend the shell domestic hen, have been demonstrated clearly by gland cavity. When the egg has reached its final and Talbot and Tyler (1974). maximal volume after about 5 hr, the secretion of The palisade layer, although it is formed at an Ca*+ by the shell gland mucosa will increase almost constant rate, may display several growth markedly (Bradfield, 1951). In the hen, shell thickness layers or lines (Simkiss and Taylor, 1971), which may and the shell content of CaCO, will then increase at be caused by helical growth of the crystals (Wilbur an almost linear rate for about 13-15 hr, after which and Simkiss, 1968). In the palisade layer just above time the shell formation will cease-l-2 hr before the mammillary layer there is a part poor in organic egg-laying (oviposition) (Talbot and Tyler, 1974). matrix (Talbot and Tyler, 1974). Different layers are According to El Jack and Lake (1967), the compoalso evident in eggshells with a characteristic coloursition of the secreted fluid in the shell gland cavity at ed pattern, as in the quail. Woodard and Mather this time will have changed to the following (mmol/l): (1964) reported that in this species the pigmentation Na+ 43, K+ 75, Ca2+ 26, Mg2+ 10, Cl- 63 and CO, began to appear 3.5 hr before oviposition. In some 91 (as HCO;), with a pH of 7.71. Even when a other species of birds belonging to the genera Pelcompletely calcified eggshell has been formed, ex- ecanus, Sula and Phalacrocorax, the palisade layer of change of electrolytes, water and a protein-containing calcite of the eggshell is covered by a softer layer of gel takes place during the shell formation through the CaCO, (Tyler, 1969) in the form of vaterite (Tullett pores in the eggshell, which in the hen’s egg are et al., 1976). present in a number of up to 17,000 (Gilbert, 1971). 2.1. DDE effect Draper (1966) and Mongin and Sauveur (1979) found The eggshell-thinning effect of DDE shows distinct that there was a continuous increase in the concenspecies differences with regard to the morphology of tration of Kf in the albumin during the period the eggshell. Greenburg et al. (1979) found that in between the entry of the egg into the isthmus and eggshells of ducks treated with 40ppm of DDE, the oviposition taking place, while the Nat concentra2. EGGSHELL

FORMATION

number of mammillary cores per mm2 was reduced by 26% and in those of ducks treated with PCB (4Oppm) it was reduced by 9%; on the other hand, the mammiilae were larger in DDE-treated than in control ducks. In ducks DDE almost exclusively reduced the thickness of the palisade layer of the eggshell (Cooke, 1975). In other species, such as the peregrine falcon (Falco peregrinus), the grey heron (Ardea cinerea), the shag (Phalacrocorax aristotelis) and the great black-backed gull (Lams marims), the thickness of both the mammilfary and the palisade layer was propo~ion3lly reduced (Cooke, 1979). In the eggshells of sparrow hawks (Accipiter nisus), an additional covering of CaCO, is present over the palisade layer. This layer has been found to be deficient in thin eggshells from DDE-exposed birds. In the gannet (&la hassona) and also in the shag, this covering layer was seen to be of a chalky consistency, as the CaCO, existed as vaterite. This chalky covering layer was greatly decreased (by approx. 40%) by DDE in these species of birds. On the other hand, this reduction in shell thickness had scarcely reduced the breaking strength of the eggshell (Cooke, 1979). Peakall er al. (1973) administered 3 ppm of DDE in the diet to the American kestrel (Fhlco ~pa~oe~~~)~ 1Oppm to ring doves ~~t~eptope~jo riroria) and 40 ppm to the white Pekin duck (Anus platyrhynchos) until significant shell thinning had occurred-usually after 4 days. Despite the shell thinning, the rate of water loss from the eggs of DDE-treated birds was slower than that from the corresponding control eggs. On scanning electron micrographs the authors observed that in both the duck and the kestrel the number of pores in the eggshell was reduced after administration of DDE. 2.2, The main biochemical functions ofthe she0 gland The eggshell is formed by the mucosal cells of the shell gland. An effect of a pollutant on the shell is therefore exerted either directly, on the function of the mucosal cells, or more indirectly, on regulatory factors of the activity of these cells. An exception to this is the case in which the pollutant affects the muscle layer of the shell gland and changes the Iength of stay of the egg in the shell gland. With regard to DDE there is no indication that this time is reduced (Cooke, 1973; Lundholm, f985b). The question therefore arises whether DDE directly influences the function of the shell gland mucosa or not. If, instead, this compound affects the shell gland mucosa indirectly, the question is where its primary action is located. In either case it is important, in the attempt to localize the action of DDE, to consider some basal observations regarding the function and structure of the shell gland forming the eggshell. The main component of the eggshell is CaCO, in the form of crystals of calcite. From a physical point of view the formation of these crystals is a fairly simple process which will take place if Ca” and CO:- are present in sufficientiy high concentrations, if the pH is suitable (not too low), and if ‘crystal poisons’ such as Mg2” or PO:- are not present in too high concentrations. It is an interesting observation that hen plasma seems to be supersaturated to such an extent that precipitation of CaCO, would occur if

this were not inhibited by some factor or factors, probably phosphate (Bachra, 1963; Wilbur and Simkiss, 1968). The biological process of the formation of the calcified eggshell is, however, a complicated process which is still only partly understood. Cooke (1973) discussed no fewer than 15 different reactions participating in the process of shell formation which might be influenced by DDE. Several of these possibilities can now be ruled out, as discussed below. There are marked species differences with regard to the shelI-thin~n~ effect of DDE which makes it necessary to report and discuss in some detail the reactions involved in the shell formation and how, when known, they are influenced by DDE. In the formation of the calcified eggshell the following activities of the shell gland mucosa have been demonstrated, or there are reasons to suppose that they are of signi~can~ for the development of the shell. The events are cyclic and of distinct duration. In the domestic fow1 the period between the laying of 2 eggs (oviposition) is about 27 hr (Gilbert, 1971) and in ducks it is about 24 hr (Sheraw, 1975). The following events are repeated at regular intervals in the domestic fowf: 1. During the initial ‘pfumbing period’ (3-5 fir), after the egg has reached the shell gland, water and electrolytes (mostly NaCl) are secreted by the shell gland mucosa and taken up by the more peripheral layers of the egg albumin. 2. The volume of the egg increases (Bradfield, l95l) and there are observations to suggest that it is the consequent distention of the shell gland wall that initiates the formation of the eggshell (Bradfield, 1951; Ogasawara et al., 1974; Eastin and Spaziani, l978a; Lundholm, l985a). 3. Even after a calcified eggshell has been formed, there is still an uptake of water by the egg albumin and exchange of electrolytes between the albumin and the she11gland {Mongin and Sauveur, 1970). Part of this exchange may occur through the pores of the eggshell, which in this way are formed and kept open during the shell formation (Tyler, 1956; Simkiss and Taylor, 1971). During this time reabso~tion of Nat takes place, the concentration of which has been found to be reduced from 158 to 96 mM in the water of the egg albumin during the 18 hr of the shell calcification process; in the fluid of the shell gland cavity there was a reduction from 144 to 48 mM. The concentrations of K+ increased, on the other hand, during the corresponding period from 15 to 39 mM in egg albumin and from 14 to 6OmM in the shell gland fluid (Mongin and Sauveur, 1970). 4. A rapid stage in the shell formation occurs in the domestic fowl between about 5 and 18 hr after the egg has entered the gland (Burmester, 1940; Talbot and Tyler, 1974). During this time several different ions are translocated through the membranes of the shelf gland mucosa from the blood plasma to the shell gland cavity and vice versa. These processes can most logically be discussed in terms of the kinds of ions that are transported and the way in which DDE influences these processes (when known), an ap preach which wiil be used in the following. It should be pointed out, however, that the transport of one ion

Thinning of eggshells by DDE often influences or even regulates that of another. In the formation of the shell so much of one ion, e.g. Ca2+, may be used, or so much of another, e.g. H+, may be produced, that the arterio-venous ionic difference of the blood plasma is changed (Hodges, 1969). The capacity of the shell gland mucosa itself to store ions in free or bound form is low (Simkiss and Taylor, 1971). The ‘plumbing fluid’ that is initially secreted is mostly composed of water, Na+, Cl- and glucose. This secretion may indirectly influence the formation of the calcified eggshell in that it will probably initiate the rapid phase of the shell formation as discussed below. During the formation of the calcified eggshell and of the organic matrix in the shell, there are several separate and partly connected transport processes in the active shell gland which are or may be influenced by DDE (Lundholm, 1985a, b). The following discussion will deal with the established translocation reactions whereby either ions are delivered for the formation of the eggshell or ions are transported away, so that the conditions are made suitable for the formation of CaCO, in the shell. References to recent papers of special interest are given. More recent reviews covering this field of research however, are sparse. The following reactions are of significance in the eggshell formation: 1. Translocation of Ca2+ from the blood plasma to the shell gland cavity, which is able to take place against a concentration gradient or an electrochemical gradient (Hodges, 1969; Simkiss and Taylor, 1971; Eastin and Spaziani, 1978b). 2. Secretion of HCO, by the shell gland mucosal cells in the cavity, mostly against a concentration gradient (El Jack and Lake, 1967). Of the HCO; only a minor part (approx. 20%) originates from the plasma bicarbonate. The rest is probably produced by the shell gland tissue when metabolic CO2 formed by the gland is converted into HCO; by carbonic anhydrase (Simkiss and Taylor, 1971). 3. Resorption of H+ by the shell gland cavity and egg albumin for the formation of the carbonate ion according to the formula H+ + CO:--+HCO; (Hodges, 1969; Simkiss and Taylor, 1971). 4. Secretion of K+ (Draper, 1966; Mongin and Sauveur, 1970; Eastin and Spaziani, 1978b), eventually in exchange for the absorption of H+. 5. Absorption of Na+ (Mongin and Sauveur, 1970) probably coupled to a secretion of Ca2+ and which may take place against an electrochemical gradient (Eastin and Spaziani, 1978b). 6. Absorption of PO:- from the fluid present in the shell gland cavity during the formation of the eggshell (Ogasawara et al., 1974) to make the solution suitable for the crystallization of calcite (Bachra, 1963). This is also to compensate for the increase in plasma phosphate that occurs during shell formation (Ogasawara et al., 1974) by resorption of calcium phosphate from medullary bone (Taylor et al., 1971). 3. RELATION BETWEEN THE STRUCTURE AND BIOCHEMICAL FUNCTION OF THE EGGSHELL GLAND MUCOSA

An important but rather controversial problem is the relation between the morphological structure of

5

the shell gland mucosal cells and their biochemical function. Both DDE and PCB administration have been found to produce degenerative changes of the shell gland mucosal cells in the duck (Kolaja and Hinton, 1976; Friedenbach and Davison, 1977). The latter authors observed a similar effect in the domestic fowl. DDE administration had a selective effect on the weight of the shell gland in the duck. The total weight of the gland in the birds that laid thin-shelled eggs was reduced by 12-18% depending on the duck variety studied. In the domestic fowl, in which DDE caused no shell thinning, the weight of the gland was not changed. DDE did not influence the percentage (83%) of water in the gland tissue (Lundholm, 1985a). Morphologically there are at least 3 different types of epithelial mucosal cells in the shell gland (Aitken, 1971). First, there are the linear epithelial or columnar cells located directly on the basement membrane separating the mucosal and muscular layers. These cells are of 2 types, namely the basal cells, whose nucleus is located close to the basal membrane and which are not ciliated, and the apical cells, which alternate regularly with the basal cells. The apical cells are ciliated and their nucleus lies in the apical half of the cells. The basal cells have secretion granules which are very electron dense and which have a diameter of 0.2pm; they are in close contact with the Golgi apparatus. Their electron microscopic pattern is similar to that of the microsomal fraction FIII (Lundholm, 1985a) and they may be present in the FIII prepared by Lundholm (1984a). The apical cells contain secretory granules with a diameter of 0.6-l pm, which are moderately electron dense. These cells are most numerous 4-6 hr after oviposition, before the calcified eggshell has begun to be formed. They contain a PAS-positive substance, probably a sulphated mucopolysaccharide. Johnstone et al. (1963) have suggested that they participate in the formation of the organic matrix of the calcified eggshell. The third type of secretory epithelial cells consists of the tubular gland cells. These cells are arranged in duct-like structures located mostly under the basement membrane and partly in the muscular layer. The orifice of the duct opens into the shell gland cavity. The tubular gland cells are richly braided with microvilli. In the active gland some of the tips of the microvilli swell up to form vesicles of zero density with a diameter of up to 0.5 pm, which are detached from the ceil and may fill the gland lumen (Johnstone et al., 1963). See section 7. The tubular gland cells themselves contain vesicles of varying size surrounded by smooth membranes, probably originating from the Golgi system. The gland cells lack typical secretory granules, however. The number of mitochondria and their electron density are increased in the active gland cells as compared with resting cells. In the tubular gland cells there are electron dense bodies with a structure showing a resemblence to altered mitochondria, which sometimes accumulate in discrete masses. Similar electron dense secretory material is also present in the lumen of the tubular gland. Johnston et al. (1963) suggest the possibility that these secretory

6

ERIK LUNDHOLM

products originate from the mitochondria. At least part of the secretion from the tubular glands is probably of an apocrine nature. Attempts were made at an early stage to establish which of the mucosal cells of the shell gland secreted the organic matrix or produced the plumbing fluid, and, especially, which of the cells translocated Ca’+. Richardson (1935) provided observations that still have to be taken into consideration today. By microincineration this investigator tried to localize the source of the mineral component in the mucosa that was responsible for the calcification of the eggshell. Although Richardson (1935) found that the columnar cells, in particular, were rich in minerals, he hesitated to draw firm conclusions from his observations. Johnstone et al. (1963), on the other hand, reported that the formation of secretory granules within vacuoles and the swelling of the tips of the microvilli of the tubular gland cells were pronounced during the calcification of the shell and the development of its organic matrix. More conclusive findings were presented by Gay and Schraer (1971) from experiments performed in vivo. 45Ca2+ was injected intravenously into 2 eggshell-producing domestic fowls. The birds were killed after 5 min, whereafter the 4sCa2+ was localized and counted by an autoradiographic technique in different parts of the oviduct mucosa. Most of the 45CaZ+ was present in the shell gland, where its concentration was 2-5 times higher than in the magnum or isthmus. In the shell gland the concentration of “Ca’+ was more than twice as high in the outer third of the columnar epithelium layer than in the tubular gland cells. In 2 birds with no calcifying eggshell in the shell gland, the 4sCa2+ concentration was reduced to the same level as that in the (unchanged) tubular gland cells. Some tests were also performed on shell gland mucosa from shell-forming birds, where the tissue was incubated in vitro for 10 min with 45Ca2C.The uptake was 4-5 times higher in the columnar epithelium than in the tubular glands. The uptake by the former was inhibited to about 50% by dinitrophenol, demonstrating its ATP dependence. A special approach to the elucidation of this problem was used by Nevalainen (1969), who studied the electron microscopic changes in the mucosa of the eggshell gland of calcium-deprived domestic hens. These hens stopped egglaying after l&14 days as a result of failure of the pituitary to secrete gonadotrophin. After 3 weeks the height of the columnar epithelium had decreased and numerous cytoplasmic vacuoles were present in the apical cells. The number of cilia on the apical cells was unaffected, but the microvilli were numerically reduced in both columnar and apical cells. Secretory granules were retained in both cell types. The tubular glands had decreased in size and number and the microvilli surrounding the tubular gland lumen had disappeared. Since exogenous oestrogen was able to partly inhibit these effects of Ca*+ deprivation, the changes in the shell gland mucosa could not be ascribed solely to reduced Ca*+ secretion even if this was contributory. With this reservation the observations of Nevalainen (1969) seem to indicate that both the columnar epithelium and the tubular gland cells are involved in

the secretion of Ca*+. As will be discussed further below, other findings support the idea that more than one mechanism is involved in the secretion of Ca*+ by the mucosal cells of the shell gland. Mongin and Carter (1977) concentrated their studies mainly on the question of which cells of the shell gland mucosa produce the ‘plumbing fluid’. Relying mostly on histological observations made and reviewed by Richardson (1935), they presumed that the tubular gland cells were the origin of this fluid. Mongin and Carter (1977) also hypothesized that the tubular gland cells secrete NaHCO, into the shell gland cavity and reabsorb H+ and Cll from this cavity to the blood plasma. By this process CO:would be produced by the gland and form the anionic part of the eggshell. This suggestion is attractive, but there is no proof that the tubular gland cells have exactly this function. Secretion of Ca(HCO,), would be another possibility (section 4). The authors made the important observation, however, that beginning 6 hr after oviposition in the domestic fowl there was an increase both in the intra- and in the extracellular water content of the eggshell-forming mucosa of the shell gland, which reached a maximum between 10 and 18 hr but was decreased 22 hr after oviposition. The mean increase in the total water content of the shell gland mucosa was 138% of the control value observed 2 hr after oviposition. Of this increase, about 60% was intracellular and 40% extracellular. Of the main monovalent cations, the intracellular concentration of Na+ was markedly elevated, especially between 10 and 18 hr after oviposition and almost reached the intracellular concentration of K+, the main cation, the concentration of which was almost constant in the mucosa during the shell formation. The intracellular concentration of Cl- rose between 18 and 22 hr after oviposition. Two hours after the next oviposition it had regained its basal value. These observations led to the question whether the plumbing fluid might pass from the blood plasma to the albumin of the egg via extracellular channels in the shell gland-a possibility studied by Eastin and Spaziani (1978b). The latter authors also observed that the extracellular space was probably increased in the shell-forming gland of the domestic fowl as compared with that in another part of the oviduct (the magnum). Eastin and Spaziani (1978b) also noted a real increase in the vascular space of the shell-forming gland in comparison with that of the magnum part. The “C-inulin space in the shellforming eggshell gland was 30 ml/100 g, whereas the value in the magnum part of the corresponding oviduct was 6ml/lOOg. The vascular space of the shell-forming gland was twice as large as the shell gland containing no egg. The rate of passage of 14C-inulin from the blood plasma into the shell gland cavity was 4 times higher in the gland forming a shell than in the inactive shell gland. Eastin and Spaziani (1978a) speculate over the possibility of paracellular routes for water and electrolytes between the blood plasma and the cavity of the active shell gland. Johnstone et al. (1963) also observed channels between active tubular gland cells but found that they did not penetrate the basement membrane or (distally) the terminal bar. The oedema of the active shell gland mucosa was probably related to an effect of

Thinning of eggshells by DDE oestrogen, as also was the enhanced blood flow (Eastin and Spaziani, 1978a). This is the case in the mammalian uterus (Spaziani, 1975). Boelkins et al. (1973) reported that the blood flow in the shell gland of the shell-forming domestic hen was increased. Beuving (1971) observed an increase in the oxygen consumption of the active shell gland. Moreover, the electrolyte composition of the fluid in the shell gland cavity is quite different at most times from that of the blood plasma (Hodges, 1969; Mongin and Sauveur, 1970). 4. STUDIES OF THE BIOCHEMICAL MECHANISMS INVOLVED IN EGGSHELL FORMATION

Any investigations of the effects of DDE and other drugs on the formation of the eggshell have to be made with reference to knowledge of the natural process of shell formation. Special problems arise in the study both of the normal process of shell formation and of the questions of when and how it is influenced by drugs. Such problems are encountered in both in vitro and in vivo investigations, although they may differ under the two conditions. In the following the different methods and their application will first be discussed with respect to their significance for shell formation. The effects of DDE administration on the rate of shell formation and on the different biochemical reactions participating in this total process will then be dealt with as far as they are known. The more essential questions which arise in the interpretation of these results will be discussed at the same time. Of the in vitro techniques, that of Ussing and Zerahn (1951) was first used by Ehrenspeck et al. (1967, 1971) on isolated segments of the shell gland of the domestic fowl. It was employed especially to investigate the translocation of Ca2+. More elaborate studies with the same technique were later performed by Pearson and co-workers (1973, 1974, 1977) on shell gland segments of Japanese quail (Coturnix coturnix juponica). The main results in the 2 species of birds were essentially consistent. In the following, the results of Pearson et al. are mostly cited, for two reasons. The shell gland wall of the domestic fowl varied in thickness between 1 and 4 mm (Ehrenspech et al., 1971) and may therefore have hypoxic areas, and the translocation of Ca2+ was greater per cm2 (see below) in the preparations from quail, which have a thinner shell gland wall than the domestic fowl. In the method of Ussing the effect on the ionic flux in the muscular layer of the shell gland was included. About 90% of the factors regulating the rate of Ca*+ flux were located in the mucosal layer, however. Moreover, the flux value in the muscular layer was not dependent on the direction in which it was measured (Ehrenspech et al., 1971), and its influence can therefore be largely neglected. In contrast, the values of the unidirectional flux of most ions in the shell gland wall were usually much greater when measured in the direction from the serosal to the mucosal side (s -+ m) than from the mucosal to the serosal direction (m-+ s). From the difference between these values the investigators calculated the ‘net flux’. With regard to Ca*+ and HCO;, ions which are mostly trapped in viuo on the mucosal side

1

when a shell is formed in the gland cavity, this ‘net flux’ has little physiological significance. This concept is therefore avoided in the present review and the original flux values are discussed. Using the Ussing technique in tests on the shell gland of the eggshell-producing Japanese quail, Pearson and Goldner (1974) observed a Ca2+ flux of 0.17 pEq/cm’/hr in the s+m direction and of 0.01 PEq in the m+s direction when measured with 45Ca2f. The tests were performed in oxygenated solution (95% 0, + 5% C02) in the presence of glucose (22 mM) and with the same Ca2+ concentration (2mM) on both sides. The values were the same whether the experiments were performed on an open or on a short-circuited system. When no glucose was present the s + m influx of Ca2+ fell to 0.07 and the m+s influx to 0.005. Under anaerobic conditions, when 0, was replaced by N, the s + m value was also reduced to 0.09, but the m + s value rose to 0.07. Dinitrophenol (DNP), which inhibited the ATP production in the mitochondria, also decreased the s -+ m flux of Ca2+ to 0.08 and increased the m + s flux moderately to 0.02. The sulphhydryl-blocking agents N-ethylmaleimide and p-chloromercuriphenylsulphonic acid reduced the m + s flux to 0.09 and 0.1 and increased the m + s flux to 0.06 and 0.04 respectively. The electrical potential difference between the s and m sides (the latter negative) was dependent on the ionic composition of the buffer solution. In a sodium bicarbonate buffer it was about 8 mV. It did not co-vary with the rate of Ca2+ flux or vice versa. The potential was not present in the absence of Na+ (Pearson and Goldner, 1973). In sexually mature birds which had not yet started egg laying, the s -+ m flux of Ca2+ was only 0.05; the m-s flux was moderately increased to 0.02. In moulting birds whose egg laying was temporarily inhibited, probably by inhibition of the secretion of gonadotropins (LH, FSH) by an increased production of prolactin (Gilbert and Wood-Gush, 1971), the s +m flux was 0.04 but the m+ s flux was reduced to 0.005 (Pearson et al., 1977). Regarding the effects of anions on the rate of Ca2+ flux, that of HCO; has been found to be the most important. Pearson et al. (1977) reported that when HCO; was replaced by Cl- the s -+ m flux of Ca2+ was reduced to 0.09 mEq/cm2/hr, i.e. to about half of the control value; the m -+ s flux, on the other hand, was doubled to 0.02. The presence of the carbanhydrase inhibitor acetaxolamide (1 mM) had an inhibitory effect on the Ca2+ flux in the presence of HCO; similar to that observed when HCO; was replaced by Cl-. In the absence of HCO; acetazolamide had no effect on the Ca*+ flux. In non-laying quails, whether moulting or not, the presence or absence of HCO; had no influence on the Ca*+ flux. Neither did acetazolamide have any effect on Ca2+ flux, whether HCO; was present or not. The carbonic anhydrase activity of the shell gland of moulting quails was about 50% of that of egg-laying quails. The corresponding value for young quails that had not yet started laying eggs was 25% (Pearson et al., 1977). When Cl- in the buffer solution was replaced by SO:-, or when PO:- was replaced by Tris, the s + m

8

ERIK LUNDHOLM

Ca*+ flux decreased to 0.13, whereas the m + s flux increased to 0.02 and 0.03 respectively (Pearson and Goldner, 1973). With regard to the role of cations other than Ca*+, Pearson and Goldner (1973) observed a flux of Na+ of about 9.1 p Eq/cm*/hr in the m --) s direction and of about 6.2 in the s + m direction. The net flux of Nat was therefore opposite to that of Ca*+. When Nat was replaced by choline the potential over the membrane disappeared, but there was no change in the Ca*+ flux. In the presence of ouabain (0.1 mM) and Na+ the Ca*+ flux in the s --*m direction was reduced to 0.09; in the m * s direction it increased to 0.02. In the absence of Nat, ouabain did not alter the control value for s 4 m Ca*+ flux; it still doubled the m + s flux, however (Pearson and Goldner, 1974). The studies with the Ussing technique on the Ca*+ flux over the shell gland mucosa have provided basal facts and also to some extent have increased the insight into this process. These facts have to be considered in investigations both on the physiological regulation of the Ca*+ secretion and on the way in which this secretion is influenced by drugs. It was evident from the above findings that the translocation of Ca*+ over the shell gland mucosa was favoured in the s --) m direction even when the metabolism was markedly inhibited (compare the tests in N,). With more moderate inhibition of the metabolism (0 glucose, DNP) there was little change in the m--f s flux of CaZ+, whereas the s + m flux was markedly reduced. The latter variable, which was dependent on aerobic energy production, was the main determining factor of the rate of Ca*+ flux into the shell gland cavity. Several observations indicated that the s + m flux of Ca*+ is the sum of at least 2 components. In the egg-laying birds about half of the Ca*+ flux was dependent on the presence of HCO;. This half was also inhibited by the presence of a carbanhydrase inhibitor. In sexually mature birds which were not laying eggs, no HCO;-dependent Ca*+ flux was observed and a carbanhydrase inhibitor had no effect on the Ca*+ flux. The carbanhydrase activity in the shell gland of moulting quails was 50% and in sexually mature non-laying birds 25% of that in egg-laying quails. In sexually mature birds egg-laying is mainly regulated by hormones released from the anterior pituitary, such as LH, FSH or prolactin (Gilbert and Wood-Gush, 1971). These hormones probably ultimately directly or indirectly influence the part of the Ca2+ flux that is dependent on the presence of HCO; . The above experiments indicate that the s-m Ca*+ flux in the egg-laying quail consists of 2 separate parts. One is dependent on the presence of HCO, and blocked by carbanhydrase inhibitors, and is regulated by sex hormones. This part may also be of special importance since it links the carbanhydrase activity not only to the production of HCO; of significance for the shell formation but also to the secretion of Ca’+. A simple explanation for these relationships is that Ca*+ and HCO, are secreted as Ca(HCO,), and not as NaHCO, by the mucosal cells, as suggested by Mongin and Carter (1977). The latter suggestion is also difficult to connect with the above-mentioned observation by Pearson and Goldner (1973) that when Nat in the buffer was replaced by choline there was no change

in the Ca*+ flux. There was an indication, on the other hand, of a relation between the secretion of Ca*+ and absorption of Na+ in the shell gland; this is further discussed below. Hitherto, in the discussion and evaluation of the usefulness of the Ussing method in studying the translocation of Ca*+, in particular, across the shell gland mucosa, qualitative more than quantitative data have been compared. It is of significance and interest to calculate to what extent the values obtained by this method correspond with the ‘true’ physiological secretion of Ca*+ that occurs during the rapid phase of the shell formation. Such calculations have indicated that from the quantitative point of view the Ussing method has marked limitations. In the domestic hen, during the 18-20 hr in which the calcified eggshell is formed in the shell gland about 2 g of Ca*+, or 10% of the total body Ca*+ is secreted by the shell gland mucosa to form the eggshell, 40% of which consists of Ca2+ (Romanoff and Romanoff, 1949). Talbot and Tyler (1974) followed the rate of shell formation in suitable individual living domestic hens by repeated manual extraction of the egg from the shell gland after it had remained for different periods in the gland. During the rapid shell-forming phase 22-2 hr before oviposition, the rate of shell deposition was 4.45 mg/ cm*/hr. Calculated as Ca*+ and in moles, the value was 36pmol Ca*+/cm’/hr. Using the method of Ussing on isolated parts of the shell gland from egg-laying domestic hens, Ehrenspech et al. (1971) found s+rn Ca*+ flux values of O.O42pmol/cm*/hr, or about 0.1% of the expected value. In parts of the thinner eggshell gland of egg-laying Japanese quail, Pearson and Goldner (1973) observed a Ca*+ flux in the s --, m direction of 0.09 pmol/cm*/hr (Eq has been converted to mol). The value was higher but still only 0.25% of the expected maximal value. No investigator has reported whether DDE or other chlorinated compounds influence the ionic flux over the shell gland wall as measured by the Ussing method. The low rate of Ca *+ flux under in vitro conditions in relation to that in vivo has to be taken into consideration if such studies are performed. The reasons why the Ussing method gave such low flux values were investigated by Eastin and Spaziani (1978a, b). These authors also confirmed and extended several of the basal observations made with this method. Eastin and Spaziani (1978a) perfused the shell gland cavity of the nembutal-anaesthetized domestic hen in vivo at a standard rate (2 ml/min) with an electrolyte solution whose composition was varied and to which drugs could be added. By determining the changes in the composition of the outflowing solution, they were able to find out whether an ion was absorbed or secreted under different experimental conditions. They mostly investigated how the rate of Ca*+ secretion was influenced. Experiments were performed in which the egg during shell formation was replaced by an artificial wax egg of the same size. With the Ca2+ secretion arbitrarily set at loo%, removal of the egg without replacing it by such an artificial egg reduced the Ca2+ secretion to 34%. When the gland was not forming a shell and was not distended by an artificial egg, the Ca2+ secretion was 6%; when the gland was

9

Thinning of eggshells by DDE then distended by an artificial egg the Ca2+ secretion rose to 13%. The rate of Ca*+ secretion was also dependent on the time the egg had spent in the shell gland and the length of time before oviposition (Eastin and Spaziani, 1978a). The maximal Ca2+ secretion (approx. 150 pmol/g dry weight/hr) was observed 13-7 hr before oviposition; a marked increase was noted already 19 hr before oviposition. Twenty-four to twenty-two hr before oviposition the secretion was 13% of the maximal value. Under their experimental conditions, Eastin and Spaziani (1978b) observed an ‘active’ net secretion of Ca2+, K+ and HCO; into the cavity of eggshellforming glands even when the concentration of the respective ion in the perfusion solution was higher than that in the plasma. Absorption of Na+ and Cloccurred. When ouabain (0.1 mM) was present in the perfusion solution the secretion of both Ca2+ and HCO; was reduced. The Na+ absorption was changed to a secretion and there was a similar trend with respect to Cl-. The K+ secretion was not altered by ouabain. This would not be expected if the digitalis glycoside had acted solely by inhibiting the Na+ + K+ pump. When the Na+ concentration of the perfusion solution was raised from 25 to 125 mM, and the osmolarity was kept constant by choline, the absorption of Na+ increased, whereas the secretion of Ca2+ rose. When the concentration of HCO; increased from 0 to 40 mmol the rate of secretion of this ion decreased; the secretion of Ca2+ was moderately stimulated, by 20%, however. The presence of acetazolamide (2 mM) totally inhibited the secretion of both Ca2+ and HCO,; the absorption of Na+ and Cl- was reduced by 50%. Eastin and Spaziani (1978a) made the important observation that distention or stretching of the shell gland wall markedly stimulated its Ca2+ secretion, but they also remarked that the maximal Ca2+ secretion that they observed was much lower (approx. 10%) of that needed to produce an eggshell within 20 hr. These investigators noted a maximal steady secretion rate of 150 pmol Ca2+/g dry weight per hr. To convert this value to pmol Ca2+/cm2/hr, some calculations and reasonable assumptions have to be made. The mean wet wt of the shell glands of white Leghorn hens was 17 g and of this, 17% was dry wt (Lundholm, 1985a). The mean area of the eggshell mucosa expanded by an egg was 72 cm2 (Talbot and Tyler, 1974). Using these values, it can be estimated that Eastin and Spaziani (1978a) had observed a secretion rate of 5.0pmol Ca’+/cm’/hr or about 14% of the mean value (approx. 36pmol/cm2/hr) calculated from figures obtained by Talbot and Tyler (1974) when a real egg was present in the shell gland. Eastin and Spaziani (1978a) found a higher rate of Ca2+ secretion during the initial 30 min of the pcrfusion and after the real egg had been exchanged by a wax egg. This rate of secretion of Ca2+ amounted to about 8.7pmol/cm2/hr, or 25% of that observed by Talbot and Tyler (1974). This observation gave reason to consider whether some factor in the egg, especially in its albumin, which exchanges with Ca2+ through the pores throughout the shell-forming period (section 2.2.) has a stimulating effect on the secretion by the shell gland mucosa. A further ques-

tion is whether the anaesthetic agent used had reduced the secretion of the shell gland mucosa. As long as these partly methodological problems are not solved, it is probably most physiologically adequate to follow the secretion of the shell gland by measuring or calculating the rate of eggshell formation in the living unanaesthetized bird as described by Talbot and Tyler (1974). 5. EFFECT OF DDE ADMINISTRATION ON THE RATE OF SHELL FORMATION IN THE DUCK As mentioned in the Introduction, the sensitivity to the maximal shell-thinning effect of DDE and other chlorinated compounds varies considerably among different species of birds. Some species, such as the domestic hen, are insensitive to the shell-thinning action of these compounds (Lundholm, 1985a). In 2 different varieties of ducks, the Indian Runner duck (IRD) was quite sensitive, showing a reduction of the eggshell index of 26% of that of the mature eggshell, whereas a cross-breed of Swedish-Rouen ducks (SRD) displayed intermediate sensitivity and a reduction of 16%. DDE had been administered in a concentration of 40 ppm for 45 days. The thickness of the eggshell was calculated as described by Ratcliffe (1970) from the eggshell index: EI = shell weight (mg)/l x b (mm), where 2 and b are the length and breadth of the shell, respectively. EI is a simplified measure of the shell weight per cm2. The eggshell weight/unit area is closely related to the true eggshell thickness (Tyler and Geake, 1961). EI is easier to measure and is probably a more exact variable to measure than the mean thickness of the shell, since if the true thickness is measured with a micrometer this value will vary in different parts of the eggshell (Tyler, 1961). The rates of shell formation in DDE-treated ducks of the 2 varieties, as reflected by the EI of this process are shown in Fig. 1. The rate of shell formation is presented in per cent/hr of the mean values for the mature eggshells of control IRD and SRD. In Fig. 1

EzlIRD cl

08”” 16””

SRD

16”’ 3,,‘= _

20” 24”

74% OP

Periods Fig. 1. Comparison Swedish-Rouen ducks

of

Indian

Runner

(IRD)

and

(SRD) regarding the effect of DDE administration on the rate of eggshell formation in per cent/hr of the mean values of the mature eggshell of control IRD and SRD. The differences in eggshell index (EI) between different times of day (e.g. 0800 and 1600 hr) are given as a percentage of the corresponding changes in controls.

10

ERIK LUNDHOLM

the changes in the rates of shell formation in the 2 varieties of DDE-treated ducks are compared. In the IRD variety the rates of shell formation were more or less reduced at all periods. The maximal reduction occurred between 1600 and 2000 hr, i.e. 1612 hr before oviposition, when it amounted to 50%. At 2000 hr about half of the mature eggshell had been formed in the control ducks. At the start and end of shell formation the effect of DDE was more moderate. In SRD the effect of DDE was concentrated to the period between 1600 and 2000 hr, when EI was reduced to 30% of that of the control. At other time periods DDE had no significant effect. The reason why the IRD variety was more ‘sensitive’ than SRD seemed to be that DDE reduced the rate of shell formation for a longer period in the former than in the latter birds. In SRD, with ‘intermediate sensitivity’, the effect of DDE on the rate of shell formation was of shorter duration, but its inhibitory action was more marked. 6. EFFECT OF DDE ADMINISTRATION ON THE Ca’+ METABOLISM OF THE SHELL GLAND METABOLISM IN VW0

The action of DDE on the rate of shell formation was strongest (IRD) or occurred almost exclusively (SRD) during the period 1612 hr before oviposition. Of the main components of the eggshell (Ca2+ and CO:-), Ca2+ is transported via the blood plasma to the gland (Simkiss and Taylor, 1971). In previous studies the total Ca2+ stores of the shell gland were found to amount to 3 mg (IRD) or Smg (SRD) (Lundholm, 1985a) and were insufficient to provide the total Ca’+, amounting to 2.7 f 0.1 g (Lundholm, 1984), that is present in a duck (IRD) eggshell. With regard to HCO; , only about 20% is transported via the plasma to the gland, and the rest derives from CO, produced within the gland (Simkiss and Taylor, 1971). The actual concentration of HCO; has to be calculated from the values of pCO,, pH, CO:- and H2C0,, and it is therefore not easy to estimate. The secretions of Ca’+ and HCO, in the gland are related (section 4.). Both from theoretical and practical points of view, it was most rational to investigate first and foremost the effect of DDE on the metabolism of Ca2+ in the shell gland. The supply of Ca2+ to the eggshell involves 3 steps that can be influenced by DDE administration in concentrations that are not sufficiently high to reduce the food and Ca2+ intake. These are: influx of Ca2+ into the blood; translocation of Ca2+ from the blood in the shell gland; transport of Ca2+ into the gland cavity and thence to the shell. The secreting shell gland extracts Ca2+ from the blood plasma at such a rate that a marked Ca2+ difference amounting to up to 4 mM is present between the blood coming to and leaving the gland (Hodges, 1969). The Ca2+ needed for shell formation comes partly from the gut and partly from medullary bone, where the resorption of Ca2+ and PO:- is increased during shell formation. The resorption of Ca*+ both from the gut and medullary bone is stimulated during shell formation. In some varieties of the domestic fowl it has been estimated that 60% of the shell Ca2+ is attributable to increased resorption from the gut (Taylor et al., 1971). For

estimating the increased resorption of Ca2+ both from the gut and from the bone, calcitriol (1 .25-(OH),Dj) is especially involved in mediating these actions (De Luca, 1978). The production of calcitriol from calcifediol (25-OH DJ in the kidney is stimulated by oestrogen, prolactin and parathyroid hormone (PTH) (Haynes and Murad, 1985). Oestrogen, especially in combination with androgens, is able to stimulate the synthesis of medullary bone. When the serum plasma level of Ca2+ is reduced during shell formation, Ca2+ is mobilized from this bone by PTH (Taylor et al., 1971). The blood level of phosphate (Ogasawara et al., 1974) is simultaneously elevated. Oestrogen administration has been found to result in an increased concentration of phosvitin in plasma, a protein that is able to raise the plasma level of Ca2+ by virtue of its high phosphate content (Butler, 1971). DDT administration has been reported to enhance oestrogen degradation in livers of ring doves (Peakall, 1967), but not to influence the uptake of Ca2+ by the gut of male zebra finches (Peakall, 1969). It therefore seemed of interest to investigate whether DDE had an impact on the Ca2+ plasma level during shell formation. In the domestic fowl a slow and moderate decrease in the Ca2+ plasma level was observed during shell formation. Neither in IRD (Lundholm, 1984b) nor in SRD (Lundholm, 1984~) did the mean plasma content of Ca 2f differ significantly between control and DDE-treated birds 13 hr before oviposition-at a time when the reducing effect of DDE on the shell formation was at its maximum. The supply of Ca 2+ to the shell gland was obviously not influenced by DDE. The second step is the uptake of Ca2+ by the shell gland from the blood. The uptake of Ca2+ by the cells is a process that can be influenced by several factors. Some of the more important of these reactions will first be briefly discussed, as they are also of interest in the discussion of step 3. The most important parameters in the regulation of Ca2+ influx are the gradient of Ca*+ over the plasma membrane and the permeability of the membrane to Ca2+. The concentration gradient between ‘free’ ionized Ca2+ in the plasma (l-2 mM) and that in the cytoplasm of the cell (0.1-l PM) lies in the direction of plasma to cytoplasm. Since ‘free’ Ca2+ is a parameter that is not easily determined, the pool of ‘total’ Ca2+ (in the plasma and cytoplasm constituting about lo%), most of which is not in ionized form but is in equilibrium with Ca2+, is usually measured instead (Rasmussen et al., 1984). The low concentration of Ca2+ in the cytoplasm of the cell is due to energy-dependent reactions. By local intracellular reactions Ca2+ is concentrated to special structures in the cell such as the mitochondria and the endoplasmic reticulum. By other translocation processes Ca2+ is transported across the plasma membrane to the extracellular space. This is done by special ‘Ca2+ pumps’. One is driven directly by a specific Ca2+-Mg2+-ATPase. Another acts by the counter-current principle, whereby the energy from the Na+ gradient across the membrane created by the specific Na+-K+-ATPase is used to transport Ca2+ ‘uphill’ against its gradient across the plasma membrane (Carafoli, 1984). The mechanisms of these ‘Ca2+ pumps’ and the influence of DDE upon them are discussed further below in connection with step 3.

Thinning of eggshells by DDE The permeability of the plasma membrane to the downhill gradient of Ca2+ is another controlling factor that is regulated either by specific Ca2+ channels or Ca2+ carriers. The translocation of Ca2+ takes place down its concentration gradient and is therefore not energy-dependent. The semi-permeability of the shell gland wall to Ca2+ is dependent on the energy production (Pearson and Goldner, 1974) as discussed in section 4. The influx of Ca2+ is therefore at least indirectly energy-dependent. Davison (1978) performed experiments with the aim of studying directly the effect of orally administered DDE and DDT on the uptake of intravenously (i.v.) injected 45Ca2+ by the shell gland of ducks and chickens. In acute experiments he gave high doses of the chlorinated compounds (1 g/kg) a few hours before an eggshell could be expected to be produced. Thinning of the eggshells occurred in ducks but not in chickens. After oviposition the amount of “Ca’+ was higher in the shell gland of DDT/DDE-treated birds than in their corresponding controls, in both species. Ducks eliminated less 45Ca2Cin their droppings than chickens. This did not indicate that these high acute doses of DDT or DDE had any inhibitory effect on step 2, i.e. on Ca2+ influx into the shell gland. However, the measurements were not made during an actual shell-formation period. It was therefore considered of interest to study the influence of DDE administration on the Ca2+ content of the shell gland mucosa during the formation of the shell. The amount of Ca2+ contained in the shell gland is only a small fraction of that in the shell (section 6.). Since all the Ca2+ present in the shell has been translocated across the mucosal cells, changes in the Ca2+ content of these cells may be a sensitive indicator of whether the Ca2+ influx or efflux has been affected by DDE. If the influx is selectively decreased by DDE, the Ca2+ content of the gland may be expected to be reduced in relation to that in the corresponding control. If, on the other hand, DDE administration selectively decreases the Ca2+ secretion, the Ca2+ content of the gland should be raised in relation to the controls. Figure 2 shows the

El DDE cl

16”” (23)

Control

20”” (12)

Time Fig. 2. The effect of DDE on the Ca’+ content of the eggshell gland mucosa of ducks in different stages of eggshell formation. The Ca*+ content of the mucosa in control ducks at 0800 hr was set at 100%.

11

mean changes in the Ca2+ content in control and DDE-treated ducks (IRD and SRD combined) between 0800-2000 hr as a percentage of the control value at 0800 hr. When the shell formation was completed at 0800 hr, the DDE value was moderately higher (11%) than in the controls, in accordance with the corresponding observation made by Davison (1978). During the period 1600-2000 hr, when DDE reduced the rate of shell formation to a maximum extent, the Ca2+ content in the controls was decreased by S&40%. The values in the DDE-treated ducks were still higher than in the control birds at these times. The relative difference was similar (l&20%) to that in the resting gland. These observations made it improbable that the DDE effect was caused by a reduced influx of Ca 2+ from the blood into the shell gland at a time when the rate of shell formation was maximally reduced by the compound. The third step that might be influenced by DDE is the release of Ca2+ from the gland cells into the shell gland cavity. The observation illustrated in Fig. 2 of relatively higher Ca2+ levels in the mucosal cells of DDE-treated ducks than in the corresponding controls may already indicate that such an effect may be of significance. Moreover, it was observed by Lundholm (1984b) that in DDE-treated ducks (SRD) the Ca2+ content of the shell gland mucosa was significantly higher at 180&1900 hr than that in the corresponding controls. In contrast, the Ca2+ content of the fluid in the shell gland cavity was lower in DDE-treated than control ducks. In the domestic fowls there was no difference in these variables between control and DDE-treated birds. They were also resistant to the shell-thinning effect of DDE (Lundholm 1984b). These observations indicate that DDE inhibits the translocation of Ca2+ from the mucosal cells into the shell gland cavity. Some findings in tests in uiuo indicated that the effect of DDE has some special features which should be taken into consideration when formulating a hypothesis on the mode of action of this agent. Firstly, some species of birds were found to be insensitive to its shell-thinning effect. Secondly, even in sensitive species the effect of DDE only occurred or was most marked during a limited part of the shell-formation period. When seeking an explanation for these observations concerning DDE it became obvious that its action on the intracellular metabolism of Ca2+ in the shell gland mucosal cells at different stages of the eggshell formation needed to be investigated. Indirect evidence that DDE may influence these parameters had been produced previously. Hohman and Schraer (1966) for instance, investigated the activity of 45Ca2+/mg N in subcellular fractions of the shell gland of hens after single or repeated iv. injections of 45Ca2+.In the mitochondrial fraction of the calcifying shell gland mucosa the activity was reduced, but in the microsomal fraction it was increased, as compared with the corresponding values in non-calcifying glands. As reported in section 4., the unidirectional translocation of Ca2+ across the shell gland mucosa has been found to be an energy-dependent process which was inhibited or reduced when the ATP synthesis was blocked or the ATP hydrolysis decreased. In ducks

ERIK LUNDHOLM

12

fed 40 ppm DDE or in sparrow hawks fed 20 ppm DDE, which produced shell thinning, Miller et al. (1976) and Bird et al. (1983) observed that the Ca’+-activated ATPase (Mg*+ was not added) of a freeze-dried homogenate of the shell gland mucosa was reduced to about 75% of the control value. In the domestic fowl the corresponding variable was not influenced. On addition of DDE in vitro the Ca*+-ATPase activity was again inhibited in a homogenate of the shell gland from the duck (Miller et al., 1976; Kolaja and Hinton, 1977). The carbanhydrase activity was also reduced (Miller et al., 1976). 7.THE EFFECI

OF DDE ADDED IN VITRO OR ADMINISTERED IN VIVO ON THE RATE OF ATP-DEPENDENT Cd+ BINDING BY AND THE Cd+-Mg’+-ATPase ACTIVITY IN DIFFERENT SUBCELLULAR FRACTIONS OF THE SHELL GLAND MUCOSA OF DUCKS AND DOMESTIC FOWLS

Before dealing with the special effects of DDE on the intracellular Ca*+ metabolism in the shell gland mucosa, some of the more important general features of this intracellular Ca*+ metabolism in general and in relation to the present problem have to be discussed (Cheung, 1980; Schuurmans-Stekhoven and Bonting, 1981; Godfraind, 1984; Manalan and Klee, 1984; Rasmussen et al., 1984; Van Belle, 1984). From studies on other tissues it is known that ‘free’ cytoplasmic Ca*+ is a regulatory agent of many important physiological processes. Most of its actions are mediated by the Ca *+-binding protein calmodulin (Cheung, 1980; Manalan and Klee, 1984), the action of which is fairly sensitive to the inhibitory effect of DDT (Hagmann, 1982; Van Belle, 1984). Ca*+ is also a ‘second messenger’ for the effects of many hormones on both biochemical (enzymes) and physiological processes (Rasmussen et al., 1984). It is probable a priori that Ca*+ may control its own translocation across the shell gland mucosa. Too high concentrations of ‘free’ cytoplasmic Ca*+ are harmful to the cell (Bygrave, 1977; Schanne et al., 1979). Both the regulatory role of cytoplasmic Ca*+ and the activity of the mitochondria in producing ATP may be impaired. The latter action of DDE is attributable to the fact that the mitochondria act as a kind of buffer system in the homeostasis of cytoplasmic Ca*+, and if too much Ca*+ is taken up, their ATP production is reduced (Fleckenstein et al., 1967). Through the shell gland mucosa large amounts of Ca*+ are transported across the cell and its plasma membrane during shell formation. The rate and duration of this Ca*+ transport are concomitantly closely regulated. The transport of Ca*+ is almost exclusively unidirectional, occurring into the shell gland cavity. These findings indicate that the substructures in the mucosal cells of the shell gland that control its Ca*+ metabolism probably have specific heterogeneous properties. The above consideration concerning the mode of action of DDE suggest that it is step 3-the translocation of Ca2+ from intracellular pools to the shell gland cavity-that is probably inhibited by DDE. In this process ‘uphill’ energy-dependent Ca*+ transport is obviously involved, since the cytoplasmic Ca*+

concentration is in the range lo-’ M (Rasmussen et al., 1984), whereas the Ca2+ concentration in the shell gland cavity ranges between 4 and 25 x lo-‘M, depending on the stage of shell formation (section 2.). This ‘uphill’ transport of Ca*+ may be driven energetically by a special calmodulin-regulated Ca*+-Mg*+-activated ATPase. An exchange between intracellular Ca*+ and extracellular Na+ operating according to the countercurrent principle also takes place. The energy present in the Nat gradient across the plasma membrane created by the specific Nat--K+-ATPase is used to drive the Ca*+ o3Na+ exchange (Carafoli, 1984). A rate-limiting step in the transport of Ca*+ out of the cell to the extracellular space is the binding of Ca*+ to and the secondary dissociation from a specific carrier in the form of a Ca2+ ionophore probably associated with the specific Ca*+-Mg*+ATPase (Urry, 1978). Observations by Miller et al. (1976) indirectly indicated that DDE may influence the ATP-dependent Ca*+ binding and transport in the shell gland mucosa by inhibiting the ATPase activity. I have investigated this question after adding DDE in vitro both to a mucosal homogenate from ducks or domestic fowls and to some of its purified subfractions (Lundholm, 1985a). Similar studies were also carried out after oral administration of DDE for 45 days. The results of these tests were rather intriguing and were dependent on several factors: firstly, on whether DDE was added in vitro or administered in oiuo, and secondly, in the latter case they were also dependent on which subcellular fraction was investigated and at what stage in the shell-forming process the rate of Ca*+ binding and the specific Ca*+-Mg*+-ATPase activity was studied (Lundholm, 1985a). In an initial series of tests on the ATP-dependent Ca*+ uptake by the mucosal homogenate of egglaying ducks (SRD), the dose-response curve for DDE added in vitro was investigated (Lundholm, 1982). The histidine buffer (pH 7.2) contained 10m4M CaCl, and 10e3 M ATP but also 10m3M MgCl,; 1.2 x 10-l M KCl; lo-*M NaCl; and 5 x 10m3M oxalate, which increased the rate of Ca*+ uptake in the controls. In a concentration of 0.5-l pg/ml, DDE moderately stimulated the Ca*+ uptake, but in the concentration range of 2-16 pgg/ml it progressively diminished the uptake to 20% of the control value. The specific Ca*+-Mg*+-ATPase in the homogenate was also reduced by DDE in a concentrationdependent manner which was fairly similar to that of its inhibitory effect on Ca*+ uptake. The ‘basal’ Mg*+-ATPase activity was not affected by DDE. In a subsequent series of tests in vitro on a homogenate of the shell gland mucosa of egg-laying domestic fowls, studies were made of the dose-response effects of several different chlorinated hydrocarbons (e.g. DDT, DDE and PCBs) on the rate of Ca*+ uptake and Ca*+-Mg*+-ATPase activity. Their potency was compared with the corresponding potency of p-p’DDE and they were found to be fairly similar. Under these experimental conditions the sensitivities of the homogenate from the duck and the domestic fowls showed little difference (Lundholm and Mathson, 1983). It was evident that tests with addition of the compounds to the whole homogenate in vitro gave

Thinning of eggshells by DDE

little reliable information concerning their action in the functioning cell in uivo (Lundholm, 1985a). To gain more reliable facts about the intracellular Ca*+ metabolism of the mucosa of the shell gland and how it was influenced by DDE, some representative subcellular fractions of the mucosal cell had to be prepared. By investigating the effect of DDE at different stages of the shell formation, it was hoped that some of the problems regarding the action of DDE would be elucidated. In tests by Lundholm (1984a) a homogenate of the shell gland mucosa was prepared from those cells that did not penetrate the muscular layer (section 3.). The homogenate was first separated by differential centrifugation into crude ‘nuclear’, ‘mitochondrial’, ‘microsomal’ and ‘supernatant’ fractions. By layering the ‘mitochondrial’ and ‘microsomal’ fractions on discontinuous sucrose gradients, 4 different subfractions of the crude ‘mitochondrial’ (MI-MIV) and 4 of the crude ‘microsomal’ (FI-FIV) fractions were obtained after the centrifugation. The microsomal (FI-FIV) and mitochondrial (MI-MIV) subfractions differed with respect not only to their specific gravity but also to their relative activities of 5’-nucleotidase and cytochrome c oxidase, their rates of ATP-dependent Ca2+ uptake, and the optimal concentration of Ca2+ for its uptake; they also differed with regard to the effects of the presence of oxalate and to their ATPase activities, sensitivity to inhibitors of Ca2+ uptake such as sodium azide, oligomycin and ruthenium red, and electron micrographic appearance. The mitochondrial subfraction MI11 was judged to be most representative of the mitochondria. The microsomal subfraction FI was rich in 5’-nucleotidase but poor in cytochrome c oxidase. In the electron micrograph this subfraction contained vesicles of zero density, with a diameter of up to 0.6 pm, similar to those formed and detached from the microvilli of the tubular cells observed by Johnstone et nl. (1963). See section 3. There were some indications that FI may be mostly derived from the apical part of the plasma membrane of the mucosal cells, whereas FII, with similar relative 5’-nucleotidase and cytochrome c activity and a similar Ca2+-binding capacity (Lundholm, 1984a), may contain more of the basal parts of the plasma membrane (Lundholm, 1985a). The latter suggestion was based on the observation that the Ca2+-Mg2+ATPase activity was stimulated by lO-6 and 10e5 M Ca*+ both in FI and FII. It was completely inhibited by 10e3 M Ca2+ in FI but still stimulated by Ca2+ at this concentration in FII (Lundholm, 1985b). As the concentration of ionized Ca2+ in blood plasma is about l-2 x 10m3M (Eastin and Spaziani, 1978a), such a difference in enzymatic sensitivity may be expected and could explain the unidirectional Ca2+ translocation from the blood plasma to the shell gland cavity (section 4.). The microsomal subfraction FIII contained relatively high activities of both 5’-nucleotidase and cytochrome c oxidase. This subfraction had a marked ability to accumulate Ca2+. In the shell-producing domestic fowl under optimal conditions with respect to the incubation medium, it accumulated Ca2+ linearly during a period of at least 20 min at a rate of 150 p/g protein/5 min. Electron microscopically the structures in this fraction were seen to contain

13

vesicles with a diameter of about 0.2pm, with an electron-dense rough membrane about 0.01 pm thick. About half of the vesicle was filled with an electrondense material which did not have the regular structure observed in the mitochondria which, moreover, had a diameter of 0.6-0.8 pm (Lundholm, 1985a). It was suggested that FIII may be a kind of Ca2+secretory granule formed from the Golgi apparatus (Lundholm, 1984a) similar to those observed by Aitken (1971) in the basal, non-ciliated cells of the columnar epithelium. If this were the case the rate of Ca2+ uptake under optimal conditions of FIII would be expected to be similar to the rate of secretion of Ca2+ by the whole active shell gland. In the secreting domestic fowl this value was higher than in the non-secreting birds, namely 130mg Ca*+/hr per gland or about 36 pmol/cm2 hr (section 4.) when calculated from the rate of shell formation in vivo. The rate of Ca2+ uptake by the purified FIII fraction from the domestic fowl was 150 pmol/g protein per 5 min = 1.8 mmol/g protein per hr (Lundholm 1984b). The total weight of the mucosal layer of the shell gland of the domestic fowl was about 9g wet wt. Of this, about 10% was found to be present as protein (Lundholm, 1985a) when the protein content in the homogenate was determined by the method of Lowry et al. (1951). If all protein were in the form of FIII, the Ca2+ uptake by the shell gland would amount to 1.6 mmol/gland/hr, i.e. about 50% of that observed in viuo. This assumption obviously implies an overestimation of the amount of FIII present in the homogenate (Lundholm, 1985a). When the calculation was based on the rate of the initial Ca’+ uptake by the total homogenate (30pmol/g protein per 5 min, Lundholm, 1984b) a more probable value (4.6 pmol Ca2+/cme2 hr or 13%) of the uptake in uivo was obtained. Preliminary experiments showed that in the control ducks several metabolic variables were altered in the whole homogenate of the mucosal cells during the shell formation, and that similar changes occurred in its purified subfractions. The effect of DDE on these variables also differed in different periods of the shell formation (Lundholm, 1984a). In IRD and SRD the most pronounced effect of DDE on the rate of shell formation was noted between 1600 and 2000 hr (Fig. 1), whereas the secretory activity of the shell gland was at its minimum at 0800 hr in the egg-laying bird (Eastin and Spaziani, 1978a). The value of 0800 hr was therefore chosen as a time point when the mucosal cells of the shell gland were relatively inactive, and 1600 and 2000 hr were chosen as times of active shell formation, when the DDE effect was at its maximum. In IRD the studies were also made at 2400 hr, as DDE was still then exerting its effect in this variety of ducks (Lundholm, 1985b). The rate of Ca2+ uptake, the specific Ca2+-Mg2+ATPase activity and the basal Mg2+-ATPase activity were determined in both control and DDE-treated ducks. In Fig. 3 the rate of ATP-dependent Ca2+ binding by the homogenate and the respective subfractions at 0800 hr in the controls was used as a basal value. The relative changes in these values for the different fractions in the control and DDE-treated ducks at other predetermined stages of the shell

ERIK LUNDHOLM HOM

q

DDE

160

i"

MIT

60

16”” (17)

(14)

20D” (12)

24”

(6)

FI

I

q q

DDE Control

80 8 g x

60

60

16” (17)

24O” (6)

Fig. 3. Comparison between the rate of Ca*+ uptake by a homogenate of the eggshell gland and the mitochondrial fraction and the microsomal subfractions FI and FIII in control and DDE-treated ducks during different periods of eggshell formation. The rate of the Ca*+ uptake in the controls at 0800 hr was set at 100%. a = value in the controls significantly different from the ‘resting’ value at 0800 hr; * = value in the DDE-treated ducks significantly different from the control value at 0800 hr. formation are shown in Fig. 3. Significant changes in control values in relation to the basal control are indicated by ‘a’, and changes in values in DDEtreated birds in relation to those in contemporary controls are indicated by an asterisk. It is seen that in the control birds the rate of Ca2+ binding was increased in the homogenate at 1600 and 2400 hr and in FIII at 1600 hr. The binding was reduced in MI11 at 2000 and 2400 hr, in FI at 1600 hr and in FIII at 2000 hr. The physiological changes in Ca*+ binding during the shell formation process are therefore in themselves quite complicated and should be taken

into consideration when formulating a hypothesis concerning the translocation of Ca2+ across the mucosal cells of the gland. The effect of DDE on the rate of Ca2+ binding was more consistent. In all cases when an effect was observed, DDE had reduced the Ca2+ uptake in relation to that in the contemporary controls, but DDE had almost no effect on the basal values at 0800 hr. Otherwise the inhibitory action was either real in the sense that the value in the DDE-treated animals was lower than that in the contemporary controls, or the reduction was functional in the sense that the increase in the control value in relation to the basal value was inhibited by DDE. With regard to the in uitro tests with DDE (Lundholm, 1982; Lundholm and Mathson, 1983), it could be expected that the specific Ca’+-Mg2+ATPase would be inhibited in parallel with inhibition of Ca*+ binding. This was not generally the case, however. In IRD, DDE significantly inhibited both the functional stimulation of the Ca2+ binding and the Ca2+-Mg2+-ATPase activities in the homogenate at 1600 and 2400 hr, but it had no significant action on either of these basal values at 0800 hr. Such an effect would be expected if the primary mode of action was an inhibition of the enzymatic activity of Ca2+-Mg2+-ATPase. Only in the homogenate from SRD did DDE inhibit the ‘resting’ Ca2+-Mg2+ATPase activity, but here it had no effect on the rate of Ca2+ binding (Lundholm, 1985b). Especially in FI at 1600 and 2000 hr, but also in FIII at 2000 hr, the Ca’+--Mg*+-ATPase activity was increased by DDE despite the fact that the Ca*+ binding in these fractions was reduced at the same time. DDE was therefore able, when the shell formation was mostly reduced, to produce a dissociation between the rate of Ca*+ binding and the specific Ca*+-Mg2+-ATPase activity. This led to the question whether DDE was able to influence the ratio (Ca*+/P) between the Ca2+ bound or taken up by the subcellular fractions of the shell gland mucosa and the simultaneous hydrolysis of ATP by the specific transport ATPase (Ca2+-Mg2+-ATPase). 8. THE 0*+/P RATIO IN THE SUBCELLULAR FRACTIONS OF THE SHELL GLAND MUCOSA AND THE EFFECT OF DDE ADMINISTRATION The ‘true’ Ca2+/P ratio in different tissues is under discussion. In the sarcoplasmic reticulum (SR) of skeletal muscle a value of 2 has been found (Hasselbath, 1974; De Meis and Inesi, 1982). The same value was obtained when the reaction was made to go backwards, i.e. when ATP was synthesized from ADP in the presence of a Ca2+ gradient over SR (Hasselbach, 1974). In the erythrocyte membrane Schatzmann (1982) found a value of 1. He discussed several possible explanations of this lower value. One is that not all of the Ca2+-Mg2+-ATPase activity is involved in the Ca 2+ transport (Sarkadi et al., 1977). When measuring the ‘specific’ Ca2+-Mg2+ATPase activity in biological membranes of various fractions of the cell, this activity is calculated from the difference between the activity of the total ATPhydrolysis and the value obtained in the absence of Ca2+, usually chelated with EGTA. Sometimes oua-

Thinning of eggshells by DDE bain may also be used to block the Na+-K+-ATPase when present. In SR of skeletal muscle about half of the total ATPase activity comprises specific Ca’+Mg’+-ATPase; the rest is ‘basal’ Mg2+-ATPase (Hasselbach, 1974; Duggan, 1977). In the homogenate of the mucosa of the shell gland, only about 6% of the total ATPase activity consisted of ‘specific’ CaZt-Mg2+-ATPase activity (Lundholm, 1985c), in accordance with values obtained in other tissues (Black et al., 1980). By treating the fractions with deoxycholate (DOC) or detergents such as Triton X-100, the activity of the specific Ca2+ -Mg2+-ATPase can be markedly increased (Schatzmann, 1982). In SR of skeletal muscle with a Ca2+/P ratio of 2, Duggan (1977) observed that in the presence of 0.05% deoxycholate the Ca2+/P ratio was reduced, as a result of both a decrease in Ca2+ uptake and of an increase in Ca2+-Mg’+-ATPase-that is, the Ca2+ pump was partly ‘uncoupled’. Freeze-drying, as used by Miller et ul. (1976) in determinations of the inhibitory effect of DDE on ‘Ca2+-ATPase’, also increased the enzymatic activity. When freeze-drying was not performed, no such effect of DDE administration on the ‘Ca2+-ATPase’ activity was observed in the mucosa of DDE treated ducks (Lundholm, 198Sa). The primary aim of the latter investigation was to study the effect of DDE on the binding of Ca2+ by the shell gland mucosa. Its action on the enzyme producing this effect was of secondary importance, especially if it could be suspected that a method was used that increased the enzymatic activity but reduced the Ca2+ uptake by ‘uncoupling’ these processes. At this stage in the investigation enhancement of the activity of the specific Ca2+-Mg2+-ATPase by using DOC, detergents or freeze-drying was therefore intentionally avoided. One exception to this rule was made in view of the fact that the condition probably was of a physiological nature and that bath the Ca’+ uptake and the activity of the specific Ca’+-Mg2+-ATPase activity were increased. It is well known that the presence of Na + and/or K + increases both the enzymatic activity and the rate of Ca2+ transport in the plasma membrane of erythrocytes (Schatzmann, 1982) and other cells (Schuu~ans-Stekhoven and Bonting, 1981). This effect was observed in the subfractions of the shell gland mucosa, of which FIII showed the most pronounced action to the cations. KC and Na+ also markedly enhanced the inhibitory effect of trifluoperazine on the Ca2+ uptake. Trifluoperazine probably acts by inhibiting calmodulin (Lundholm, 1985~). Some methodological problems were encountered in determining the Ca’+/P ratio in the shell gland mucosa homogenate and its different subfractions in untreated controls. At Ca2+ concentrations of > 1 x lO_‘M, Mg2+ and Ca2+ had an antagonistic effect on the ‘basal’ Mg2+-ATPase. Moreover, the presence of K+ and/or Na+ reduced the Ca2+/P ratio. This ratio was also dependent on the functional state of the gland. In a shell gland mucosa homogenate from a shell-forming domestic fowl in the presence of Ca2+ and Mg2+ alone, the Ca2+/P ratio was 0.8. When KC and Na+ were also present, this value fell to 0.4-a significant reduction in paired tests (Lundholm, 1985~).

HOM

j

or/

15

HOM

1

Ff

MIT 1i?*

FIII __i

Fig. 4. Left: Comparison between the effect of DDE on the Ca2+/P ratio in the homogenate (HOM) of ‘resting’ (OS00hr) and ‘active’ (1600hr) shell gland mucosa of ducks (SRD). Right: the Ca2+/P ratios of the subcellular fractions of the ‘active’ (1600 hr) shell gIand mucosa in control and DDE-treated SRD. a = value of the control homogenate at 1600 hr significantly different from the ‘resting’ value at 0800 hr; * = significant differences between the DDE and control values in the homogenate and the subfractions at 1600 hr.

In the tests on ducks after administration of DDE it was considered most physiological to perform the determinations in the presence of Kf and Na+ (Lundholm, 1985~). The main results for SRD are summarized in Fig. 4. In the homogenate from ‘secreting’ control birds (at 1600 hr) the Ca2+/P ratio was 0.46 and significantly higher (a) than that in ‘non-secreting’ ducks (0.19). DDE inhibited this functional increase in the Ca2+jP ratio in the secreting ducks. At 1600 hr the DDE effect was most marked in FI and was also noted in MIII. FIII also showed a reduction, which most probably was real; DDE had reduced the rate of Ca2+ binding, while the Ca2+Mg2+-ATPase activity was unchanged. In IRD the results were the same whether the Ca*“/P ratios were calculated only at 2000 hr or at both 1600 and 2000 hr in IRD. In SRD, DDE also reduced the Ca2+/P ratio in the absence of K+ and Nat (Lundholm, 1985a). Among the results of these studies, 2 findings were of special interest. First, in the control ducks the increase in Ca2+ secretion during shell fo~ation was combined with increased coupling between Ca2+ uptake and the activity of the specific Ca2+Mg’+-ATPase as compared with the ‘non-secreting’ gland. Second, this increase in coupling did not occur after administration of DDE. On the contrary, in several subfractions the Ca2+-Mg’+-ATPase activity was increased during shell formation. This effect was similar to an ‘uncoupling’ effect of DDE on the Ca2+ binding and Ca’+-Mg’+-ATPase activity. When administered in viva, DDE stimulated the basal Mg2+-ATPase. In the shell gland mucosa of ducks this activity was increased by DDE in MI11 and FIII of the resting gland (at 0800 hr). The MgztATPase activity also showed functional changes in the control ducks. The activity was higher at 1600 and 2000 hr than at 0800 hr in FI and FIII. It was lower in MI11 of the ‘secreting’ than of the ‘nonsecreting’ gland (Lundholm, 1985b). The biochemical significance of these changes in Mg’+-ATPase is not clear. The functional changes may be related to the

16

ERIK LUNDHOLM

synthesis of the organic matrix of the eggshell. The DDE-induced changes in the organic matrix (section 2.1.) may be related to altered ATPase activity. The stimulating effect of DDE on the Mg’+-ATPase activity despite a reduction in weight of the shell gland may indicate that even the action of Mg*+ATPase may be ‘uncoupled’ from its biochemical actions. It is of interest that Jefferies (1969) pointed out that DDT induced a state of apparent hyperthyroidism in birds, although Graessle and Biessman (1982) observed no simple relationship between the effect of chlorinated biphenyls and thyroid function. 8.1. Comparison of the effect of DDE administered in vivo on the Ca2+ metabolism of the shell gland of the domestic fowl and ducks

When added in vitro to a homogenate of the shell gland mucosa, the Ca*+ uptake was inhibited in a dose-dependent manner both in ducks and domestic fowls. As judged from the dose-response curves, the hen was more sensitive to DDE than the duck (SRD). The difference was most marked in FI, where in the duck DDE in concentrations of I 8 pg/ml stimulated the Ca*+ binding whereas in the hen it was inhibitory in concentrations of >2pgg/ml (Lundholm, 1985a). When DDE was administered in vivo to the 2 species its effect was quite different. Following addition to the diet for 45 days at a concentration of 40 ppm, which increased the DDE content of the shell gland mucosa to 1.3 f 0.1 pg/g wet wt in ducks and to 2.2 f 0.2 pg/g in hens, eggshell thinning (13%) was only observed in ducks. The Ca*+ content of the shell gland fluid was reduced by DDE as compared with that in control ducks, but no such effect occurred in the domestic fowl. The rate of Ca* + binding in ducks was decreased in the homogenate, MIII, FI and FIII, but was unchanged in the domestic fowl or even stimulated in FI (Lundholm, 1984~). The weight of the total shell gland was decreased by DDE in the ducks, but not in the domestic fowl (Lundholm, 1985a). The absence of these inhibitory effects of DDE on the Ca*+ metabolism of the shell gland of the hen in combination with its lack of reduction of EI supports the assumption of a causal relationship between these variables. The absence of a correlation between the effects of DDE when administered in vivo instead of in vitro indicates that the in vitro results should be interpreted with caution. 9. THE INFLUENCE OF DDE ON THE REGULATION OF THE SECRETORY ACTIVITY OF THE SHELL GLAND: INDICATION OF AN EFFECT ON CALMODULIN

The time-dependent actions of DDE on: (1) the shell formation (section 6.) (2) the Ca*+/P ratios in Ca*+-secreting birds (section 8.) (3) the organic matrix of the eggshell (section 2.1.) (4) the weight of the shell gland (reduction) (section 3.) and (5) the shell gland mucosa (atrophic changes) (section 3.) indicated that the shell-thinning effect of DDE was only one manifestation of a complex of actions of the compound of the shell gland. One important action of DDE that is most probably involved in its shellthinning effect is an influence on the activity of Ca*+-Mg*+-ATPase. In ducks, DDE added in vitro

in relatively high concentrations (2 2 pg/ml) inhibited the enzymatic activity as well as the Ca*+ uptake. When administered in vivo, lower DDE concentrations (approx. 1.2-1.5 rig/g wet wt) were reached in the shell gland mucosa (Lundholm, 1985a), and an ‘uncoupler’ effect was then observed. Lundholm and Mathson (1983) observed that like other chlorinated compounds DDE, when added in vitro to a homogenate of the shell gland mucosa, inhibited the enzymatic activity even in concentrations lower than those that reduced the Ca*+ uptake. The relevance of these observations for the in vivo effect of DDE may be questioned, however. DDE when administered in viuo did not reduce either the Ca*+-Mg*+-ATPase activity, the rate of Ca*+ binding, or the Ca*+/P ratio in the ‘non-secreting’ gland; such effects were only exerted during the shell formation. From these different findings it would seem plausible to draw the following conclusions. Firstly, observations of the in uitro effects of DDE are of limited significance for interpretation of its effects in vivo. Secondly, after administration of DDE in vivo, it is probable that it mainly inhibits some regulating step in the Ca*+ secretion and the shell formation of the active shell gland in ducks. The second probability raises the question how the activity of the shell gland is regulated. The meaning of the term ‘activity’ here is restricted to refer to factors in the sexually mature, egg-producing bird that stimulate eggshell formation. Hormonal agonists are probably involved, since denervation of the shell gland has been found not to influence the shell formation (Eastin and Spaziani, 1978). In birds treated in this way the shell glands are always exposed to different hypophyseal sex hormones such as FSH and LH and ovarian sex hormones such as oestrogen, progesterone, androgens, and sometimes prolactin with anti-gonadotropic activity (Gilbert, 1971). Although these hormones are necessary for the development and function of the oviduct, there are no strong indications that any one of them is the primary hormonal agonist that initiates the great increase in Ca*+ secretion at the rapid stage of shell formation (Eastin and Spaziani, 1978a). On the other hand, several observations have strongly suggested that the stimulation of shell production is a complex phenomenon in which different reactions follow one another in a predestined manner. This possibility is supported by the following: (1) the histological structure and composition of the hard eggshell change during the shell formation. There are also marked species differences in the eggshell structure (section 2.); (2) the rate of Ca*+ translocation over the shell gland mucosa is dependent on the sexual maturity of the bird, and is altered both by mechanical distention of the shell gland by an artificial egg and by the presence of a real egg in shell gland (section 4.); (3) the rate of Ca2+ binding to the subcellular structures of the shell gland mucosa and the specific Ca*+-Mg*+-ATPase activity in these structures each shows a characteristic timedependent pattern (section 7.), as also does the Ca*+ content of the mucosal cells (section 6.) and (4) in SRD the inhibitory effect of DDE on the shell formation was noted only during a fairly short period of this process, whereas in IRD this period was longer

Thinning of eggshells by DDE

(section 5.). This effect was not observed in the domestic fowl (section 8.1.). These considerations lead to the question whether the stimulus-secretion mechanism in the shell formation does depend on a single agonist and does not comprise the actions and/or co-operation of 2 or more hormones whose significance for the shell formation may alter with time. In the following, observations strengthening this latter idea will be reported. The different parts that probably participate in the total stimulus-secretion mechanism, and the way in which they might be inffuenced by DDE, will be discussed. As will be mentioned below, several of these actions may be related to the inhibitory effects of DDT and DDE on the activity of calmodulin. 9.1. Prostaglandins It is possible that the agonist which after the ‘plumbing’ period initiates the rapid stage of Ca2+ secretion and shell formation may be a prostaglandin. As discussed by Lundholm (1985a), there are indications that the increase in egg volume during the ‘plumbing’ stage in the shell gland causes distention of its wall, which starts the rapid phase of shell fo~ation, and that this phase is inhibited if premature oviposition is induced (Ogasawara et ai., 1974). Distention or other mechanical handling of a tissue is able to stimulate the formation and release of prostaglandins (Bakhle and Vane, 1974). The hypothesis that a prostaglandin may be involved in the stimulation of Ca2+ secretion of the shell gland was therefore tested by L~dholm (1985a) by pretreatment of domestic fowls with cycle-oxygenase inhibitors (indomethacin, diclofenac). Depending on the dose, the hens laid eggs with thin calcified or only soft eggshells. Olson et al. (1978) reported that the plasma level of prostaglandin PGE,, which had a very weak contractile effect on the myometrium of the shell gland, was increased at a time point when the rapid stage of shell formation had begun. On the other hand, the plasma level of PGF,, was elevated at the time of oviposition. This prostaglandin has a strong contractile effect on the smooth muscle layer of the shell gland (Hammond et al., 1980). Indomethacin, in particular, made the intervals between the ovipositions irregular (Lundholm, 1985a). If, as suggested, continuous formation of a prostaglandin is required for maximal Ca2+ secretion by the shell gland, this might explain the difficulties in reproducing the rate of Ca2+ secretion calculated from the rate of shell formation (section 4.). Even under the experimental conditions of Eastin and Spaziani (1978), any prostaglandins produced are washed away by the continuous perfusion of the shell gland cavity. The question whether the prostaglandin metabolism is influenced by DDE has not yet been tested. Activation of phospholipase A, is the ratelimiting step in prostaglandin formation. This activation is Ca’+-dependent and is regulated by the cytoplasmic Ca2 + concentration (Samuelsson et ai., 1978), probably via calmodulin (Wong and Cheung, 1979) and it may therefore be expected that it will be inhibited by a calmodulin antagonist such as DDE. 9.2. Calmodulin The activity of the specific Ca2+-Mg*+-ATPase c IU?88,!C--B

is

17

largely attributable to its binding to calmodulin (Schatzman, 1982). Both the dissociation of calmodulin from the enzyme and its reactivation on addition of new calmodulin are comparatively slow reactions. In the homogenate of the shell gland mucosa and its different subfractions from the domestic fowl, the relatively specific calmodulin antagonist trifluoperazine (40 p M), added in vitro, markedly (< 80%) inhibited the Ca2+ binding when K+ and Na+ were present. In the absence of these ions the inhibition was somewhat less marked (Lundholm, 1985c). When t~~uoperazine was administered orally to fowls in a dose of 100 mg for 3 consecutive days, EI was reduced by 19 + 1%. The Ca2+ uptake by the homogenate of the shell gland mucosa of these fowls was decreased by 12 f 3% by trifluoperazine (Lundholm, 1985a). If DDE and trifluoperazine had both acted by the same mechanism, i.e. by calmodulin inhibition, when reducing the Ca2+ uptake by the subcellular fractions from the mucosa, it might have been expected that DDE administration would have interfered with the action of added trifluoperazine. in a DDE-sensitive species the part of the Ca2+ uptake that was inhibited by trifluo~razine could be expected to be reduced by DDE, whereas in an insensitive species the effect of trifluoperazine would not be changed by DDE. Comparative tests supported this hypothesis. In the DDE-sensitive SRD the effect of trifluoperazine was diminished both in the homogenate and in its subfractions by DDE. In the DDE-insensitive domestic fowl the effect of t~fluoperazine was not altered by administration of DDE, supporting the proposed hypothesis (Lundholm, 1985a). It is also possible that the functional variations in the Ca2+-Mg 2+-ATPase activity and the simultaneous changes in the Ca2+ uptake by the subfractions of the mucosa during the shell formation are related to alterations in calmodulin activity. This question needs further investigation. 9.3. The steroid sex hormones It is well known that oestrogen and progesterone are of importance for the function of the oviduct in birds (Eastin and Spaziani, 1978a). Their actions are mostly interrelated. Thus, oestrogen stimulates the development of progesterone receptors in the oviduct (Toft et al., 1972; Walters et al., 1979). In the presence of oestrogen, progesterone is then able to stimulate the formation of the protein avidin in the magnum part of the oviduct. Avidin, in turn, will promote the production and secretion of egg albumin. The significance of oestrogen and/or progesterone for the formation of the eggshell is less clear. Oviposition is related to and probably dependent on a fall in the progesterone level in plasma. By administration of progesterone during shell formation, egglaying can be postponed and the shell thickness increased (Tanaka, 1976). The progesterone level in plasma during shell formation does not start to rise until after the rapid stage of shell formation has begun and therefore in this respect there is no close correlation (Hammond et al., 1980). But there is a possibility that it is not the actual concentration of progesterone in the plasma but rather the number of progesterone receptors in the shell gland or their Kd

18

ERIK LUNDHOLM

values that is of significance for the sensitivity to and effect of progesterone. This question is of interest since there are indirect observations indicating the DDE influences the progesterone receptors (Lundholm, 1985a). It has also been established that o-p’-DDT, in particular, has an affinity for the oestrogen receptor in the uterine cytosol and has oestrogenic actions (Forster et al., 1978; Turner, 1978). It was of interest to investigate whether effects of these sexual hormones on the steroid receptors might in some way be related to the shell-thinning action of DDE. 9.4. Oestrogen In some tests on the domestic fowl, the oestrogen receptors were inhibited by tamoxifen. After a daily dose of 40 mg for 3 consecutive days, egg production was inhibited. During the first 2 days of treatment EI was reduced by 9%. Although the effect was statistically significant, it was weak in relation to that of DDE. Peakall (1970) had observed that the DDEinduced stimulation of oestrogen degradation probably could not explain the shell-thinning effect of DDE. 9.5. Progesterone As yet no specific progesterone receptor antagonist is available that does not also have an affinity for other steroid receptors. Tests on progesterone receptors such as those described with tamoxifen have therefore not been possible. Instead, comparative receptor-binding studies have been performed on the cytosol of the shell gland mucosa from ducks and domestic fowls (Lundholm, 1985a, 1987). The dissociation constant (&) of progesterone to the receptor was about 5 times lower in the fowl than in the duck, i.e. progesterone had a higher affinity for its receptor in fowls than in ducks. The numbers of receptors per mg protein were almost similar. Both p-p/-DDE and o-p’-DDE reduced the binding of progesterone to its receptor. Ducks were significantly more sensitive to this effect of DDE than hens, probably on account of the higher affinity of progesterone for its receptor in the latter species of birds. There was little difference in the effects of p-p/-DDE and o-p/-DDE with regard to the progesterone receptor, whereas o-p/-DDE had a much higher affinity for the oestrogen receptor than the p-p/-isomer (Forster et al., 1975). PCB 1242 was also able to reduce the binding of progesterone and with respect to this compound fowls were again more resistant than ducks (Lundholm, 1985a). The possible significance of the action of DDE on the progesterone receptors in the shell gland mucosa for the shell formation is unclear and needs further investigation. Like other steroid receptors, the progesterone receptor is a phosphoprotein. In the dephosphorylated state the receptor does not bind its agonist (Dougherty et al., 1985). The progesterone receptor is phosphorylated in the presence of Ca*+ and Garcia et al. (1984) have suggested that the reaction is mediated by calmodulin. Calmodulin inhibitors such as trifluoperazine and calmoduzaline were as potent as DDE in inhibiting progesterone binding (Lundholm, 1987). It is thus probable that the inhibitory influence of DDE on the calmodulin

activity may also be of significance for its effect on the progesterone receptor, and that this action of DDE may explain several of its effects on the shell gland. 10. CONCLUSIONS

The reducing action of some chlorinated compounds (p-p/-DDE, p-p’-DDT, PCBs) and other pesticides such as dieldrin on the eggshell thickness and hatchability of the eggs of several species of birds is a serious ecotoxicological problem. The mode of action of these pollutants is therefore of both theoretical and practical interest. A critical review of available observations indicates that the reduction of the eggshell thickness and a decreased Ca*+ content of the shell are 2 of several effects induced by these compounds. These effects also influence the organic matrix of the eggshell and its morphological structure. There is a marked variation between different species of birds regarding both their sensitivity to the shell-thinning action, and the layer of shell that is affected. This review has been focused on the way in which the formation of the calcifying eggshell takes place and how it is influenced by DDE in two sensitive varieties of ducks (IRD and SRD) and in the DDE-insensitive domestic fowl. It was observed in ducks that neither the transport of Ca*+ to the shell gland mucosa nor the influx of Ca*+ from the blood into the mucosal cells was influenced by administration of DDE. On the other hand, the rate of translocation of Ca*+ from the mucosal cells to the shell gland cavity was reduced by this compound. Calculated from the increase in eggshell index (EI), this reduction was most marked in IRD; it was only noted in SRD 16-12 hr before oviposition. It was not observed in the domestic fowl. Associated with or causing this reduction in the rate of shell formation was a decreased content of Ca*+ in the shell gland cavity after administration of DDE. The content of Ca*+ in the shell gland mucosa, which normally fell during the shell formation, decreased at a slower rate after DDE administration. These observations indicated inhibition of the Ca*+ translocation, i.e. of the ‘Ca*+ pump’ across the plasma membranes located close to the shell gland cavity. In studies of pieces of the isolated shell gland wall with the Ussing technique, an almost unidirectional Ca*+ flux from the serosal to mucosal side was observed when the ATP synthesis or utilization was not disturbed. This ‘Ca*+ pump’ was mainly located in the mucosal layer of the shell gland. When Ca*+ was translocated across the mucosa it was first bound or taken up by the particulate fractions of the mucosal cells. The rates of ATP-dependent Ca*+ binding by the whole homogenate of the shell gland mucosa and its purified microsomal or mitochondrial subfractions were investigated in control and DDEtreated ducks at different points in time in relation to oviposition. The subfractions were characterized by marker enzymes and electron microscopy. Timedependent variations in the rate of Ca*+ uptake and ATPase activity in the subfractions were observed during different stages of eggshell calcification. When DDE or other chlorinated hydrocarbons were added in vitro, both the Ca*+ uptake by and the

Thinning of eggshells by DDE Ca2+-Mg2+-ATPase activity in a homogenate or its subfractions of the mucosa from ducks and domestic fowls were inhibited in a concentration-dependent manner, which may indicate inhibition of a ‘Ca2+ pump’. When DDE was administered in uivo, it reduced the Ca2+ uptake by subcellular fractions from shellproducing shell gland mucosa from ducks but not from the domestic fowl. The effect of DDE administration on the specific Ca2 +-Mg2+-ATPase activity varied according to the stage of shell formation and in the different subfractions. In the secreting gland DDE caused an ‘uncoupling’ between the uptake and the stimulation of Ca2+-Mg’+-ATPase, which indicated decreased activity of the ‘Ca2+ pump’. It is well established from tests on other tissues that both the activity of the Ca’+-Mg2+-ATPase and the Ca2+ uptake are stimulated by calmodulin. The fairly antagonist trifluoperazine specific calmodulin markedly inhibited the Ca2+ uptake by and the activity in the shell gland muCa2+-Mg 2f-ATPase cosa homogenate and its subfractions, both in the duck and in the domestic fowl. DDT and DDE are also reported to be potent calmodulin inhibitors. When DDE was administered to ducks it decreased the part of the Ca2+ uptake that was sensitive to trifluoperazine inhibition. In the domestic fowl DDE had no such effect, indicating that in this species no synergistic action occurred between these calmodulin inhibitors. The effect of DDE on Ca2+ uptake and Ca2+Mg2+-ATPase was not seen in the homogenate of the ‘resting’ gland. Even here, however, DDE administration stimulated the ‘basal’ Mg’+-ATPase activity in some subfractions of the eggshell gland mucosa. The question was considered whether DDE may influence the ‘stimulus-secretion’ mechanism in the shell gland, and this possibility was investigated. This mechanism is probably of a hormonal nature, and it is likely that it is the sum of the effects of more than one hormone acting in co-operation and/or in sequence, on the gland. Available observations indicate that the steroid sex hormones, especially progesterone, probably in combination with a prostaglandin, are involved in the stimulation of the secretory activity. It was found that DDE had an affinity for the cytoplasmic progesterone receptor in the shell gland mucosa of both ducks and domestic fowls. The inhibition of progesterone binding to its receptor by DDE was comparatively greater in ducks than in domestic fowls, since the affinity of progesterone for its own receptor was about five times higher in hens than in ducks. This may be of significance in explaining the insensitivity of the fowl to DDE. The binding of progesterone to its receptor is Ca2+dependent and probably mediated by calmodulin. With regard to prostaglandins, their formation is stimulated by Ca2+, the action of which is also mediated by calmodulin. Inhibitors of prostaglandin synthesis, such as indomethacin and diclofenac, reduced the EI or in high doses resulted in laying of eggs devoid of hard shells. The shell-thinning effect of DDE is one of several actions of this compound on the shell gland mucosa. Its direct effect is obviously exerted by a reduction of the secretion or translocation of Ca2+ from the

19

mucosal cells in the shell gland cavity. In some varieties of ducks or species of birds this effect may be limited to a restricted time period during the shell formation. In the duck an ‘uncoupling’ takes place during this period between the Ca2+ uptake and the activity of Ca2+-Mg’+-ATPase. Since both these activities are stimulated by calmodulin, and DDE is a potent calmodulin inhibitor, it is suggested as a working hypothesis that both these and other actions of DDE are attributable to its calmodulin-inhibiting properties. REFERENCES

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