DDE-induced eggshell thinning in birds: Effects of p,p′-DDE on the calcium and prostaglandin metabolism of the eggshell gland

Comp. Biol. Physiol. Vol. 118C, No. 2, pp. 113-128, 1997 Copyright 0 1997 Elsevier Science Inc. All rights reserved.

ISSN 0742-8413/97/$17.00 PI1 SO742-8413(97)00105-9

ELSEVIER

REVIEW

DDE-Induced Eggshell Thinning in Birds: Effects of p,p’-DDE on the Calcium and Prostaglandin Metabolism of the Eggshell Gland C. E. Lundholm DEPARTMENT OF PHARMACOLOGY, FACULTY OF HEALTH SCIENCES,Sz581 85 LINK~PING, SWEDEN, AND ASTRA HASSLE AB, REGULATORY AFFAIRS, S-431 83 M~LNDAL, SWEDEN

ABSTRACT. 1. The focus of this review is the effects and mechanism of action of p,p’-DDE on eggshell formation in birds. Inhibition of prostaglandin synthesis in the eggshell gland mucosa is a probable mechanism for p,p’-DDE-induced 2. The

eggshell thinning.

duck is sensitive

comparing

to p,p’-DDE-induced

shown that eggshell thinning synthetase, content

eggshell

the two species in regard to the calcium induced by p,p’-DDE

reduced levels of prostaglandin

of calcium,

in ducks exhibiting 3. Inhibition

bicarbonate,

chloride,

eggshell thinning.

of prostaglandin

ducks. Neither

regimens with o,p’-DDE,

4. Administration

in ducks is accompanied

sodium, and potassium

by reduced activity of prostaglandin

are also reduced in the eggshell gland lumen

is a specific effect of p,p’-DDE. p,p’-DDT,

prostaglandin

of other compounds

fowl is not, and studies

of the eggshell gland have

the compound

o,p’-DDT,

synthesis

fowl.

The detrimental

with structurally

and p,p’-DDD

effects of p,p’-DDE

related substances,

that do inhibit

prostaglandin

synthesis,

e.g., indomethacin,

thinning

and prostaglandin

gland.

BIOCHEM PHYSIOL 118C;2:113-128,

calcium,

eggshell, eggshell gland, prostaglandins,

duck, chicken,

environmental

pollution,

INTRODUCTION hydrocarbons,

COMP

and the described

does cause

effects on the calcium 1997. 0

1997

Inc.

KEY WORDS. p,p’-DDE, carbons,

of the eggshell

in

in the eggshell gland mucosa.

i.e., eggshell

metabolism

i.e.,

do not cause eggshell thinning

the same effects as those seen with p,p’-DDE, Elsevier Science

Halogenated

metabolism

None of these effects are seen in the domestic

synthesis

do they inhibit

but the domestic

E2, and reduced uptake of 4iCa by the eggshell gland mucosa. The

on the eggshell gland seem to be unique when comparing similar treatment

thinning

and prostaglandin

such

as polychlorinated

bi-

prostaglandin

synthetase,

chlorinated

hydro-

reproduction

the food chain, e.g., peregrine falcons, eagles, eagle owls, white-tailed eagles, seals, otters, minks, alligators, and whales, are especially vulnerable to these pollutants. The of these animals is due not only to high exposure

phenyls (PCBs), DDE and DDT, dioxins and dibenzofurans, produce many different toxicological effects in laboratory

sensitivity

animals, and in some cases, disturbed physiological functions in wild animals has also been correlated to these chemicals. Predatory and fish-eating species at the top of

by a number of physiological and biochemical response to some environmental pollutants.

Address wprint wquests to: C. E. Lundholm, Astra Hkle AB, Regulatory Affairs, S-431 83 Miilndal, Sweden. Abbreviations-p,p’-DDE, l,l-bis-(4-chlorphenyl)-2,2-dichlorethylene; o,p’-DDE, 1-(2-chlorphenyl)-l-(4-chlorphenyl)-2,2~dichl~rethylene; p,p’-DDT, l,l-bis-(4-chlorphenyl)-Z,2,2-trlchlorethane; o,p’-DDT, 2,4’ dichlor-a-trichlormethyl-diphenyl-methane; 2,2-bis-(4P,P’-DDD, chlorphenyl)-l,l-dichlorethane; o,p’.DDD, I-(2-chlorFhenyl)-l-(4chlorphenyl)-2,2-dichloreth ane; p,p’-DDA, 2,2-his (p-chlorphenyl) acetic acid; Methoxychlor, l,l,l-trichlor-2,2-bls(p-merhoxyphenol) ethane; Lindane, y-1,2,3,4,5,6-hexachlorcyclohexane; PCB, polychlorinated biphenyls. Received 24 September 1996; revised 23 April 1997; accepted 24 April 1997.

levels, but also to the species-specific

sensitivity

displayed

functions

in

For almost four decades, scientists have studied birds in regard to reproductive impairment, caused by environmental pollutants. Several species of predatory and fish-eating birds are endangered due to the accumulation of high levels of DDE, DDT, PCB, dioxins, and methyl mercury, particularly in eggs. Exposure to environmental pollutants can lead to reduced egg production, eggshell thinning, decreased fertility and hatchability, malformations, and decreased survival of young. Eggshell thinning elicted by p,p’-DDE represents one of the most serious effects on the reproductive success of many avian species. Although the physiological and biochemical

C. E. Lundholm

114

mechanisms underlying p,p’-DDE-induced eggshell thinning have been the subject of numerous studies, as of yet, they have not been fully clarified. This review is focused on the effects and mechanisms of action of p,p’-DDE on eggshell formation. Results obtained in recent experiments are discussed that suggest that p,p’DDE-induced eggshell thinning is due to inhibition of prostaglandin synthesis in the eggshell gland mucosa. Further information about the effects of chlorinated hydrocarbons, metals, and different therapeutic drugs on eggshell formation and avian reproduction can be found in the following

reviews: (22,31,32,42,79,82,90,104,132,133,

143). The effects of PCBs on avian reproduction has been reviewed hy Barron et aI.(7) and Wassermann et al. (144).

and these were reviewed by Cooke (31), Hayes (53), and Jefferies (66). PCBs does not seem to be involved in eggshell thinning. A recent review (123) comparing the effects of PCBs to DDE found little evidence for involvement of PCBs in eggshell thinning. It was concluded that DDE is the only compound that has caused significant eggshell thinning at environmentally realistic doses. Considering the present situation, the levels of p,p’-DDE and PCB in birds in Europe and the U.S.A. are declining as a result of bans on these chemicals. DDT is still being used in Asia, Africa,

and South

America,

which are the

winter habitats of many migratory birds. The reproductive impact of chlorinated hydrocarbons accumulated by migratory birds in their winter habitats has been discussed by Mora (102). In a study by Burger et al. (21), the eggs of four species of marine birds were collected in the New York Bight during

Brief Ecological Background to @,p’-DDE-induced Eggshell Thinning The widespread decline of birds of prey became evident to ornithologists after the Wisconsin meeting in 1965 (59) although some indications of earlier declines, i.e., the bald eagle in the 1950s had been seen (19). The decline was most prominent among predatory and fish-eating birds such as peregrine falcons (F&o peregrinus), european sparrow hawk (Accipiter nisus), white-tailed eagles (H&ems aI& cilia), eagle owls (Bubo bubo), and brown pelicans (Pelecanus occident&) at the coast of California (31,58,107,129,130).

the 197Os, 1980s and 1990s. For roseate terns (Sterna doug&i), common terns (Sterna hirundo), and black skimmers (Rynchops niger), it was found that eggshell thickness had increased by about 50% from the 1970s to the 199Os, and the corresponding value for least terns (Sterna anti&urn) was 12%; the main increase in eggshell thickness had occurred during the 1980s. King et al. (72) reported 5 to 7% thinning for eggs of Foster’s terns (Sterna forsteri) and black skimmers collected in 1984 in Lavaca Bay, Texas U.S.A.

Ratcliffe ( 128) first noted eggshell breakage in peregrine falcon nests in the 1950s and showed eggshell thinning, starting immediately after the introduction of DDT, in the pere-

Eggshells collected from peregrine falcons breeding in the Canadian arctic were 15% thinner than eggshells produced prior to the introduction of DDT. No improvement in eggshell thickness were observed between the two sampling pe-

grine and the sparrow hawk. Ratcliffe (129) also discovered that the occurrence of broken eggs in nests of the golden eagle (Aquila chrysaetos) in Britain was due to eggshell thin-

riods; 1982-1986 and 1991-1994, respectively (67). Peregrine falcon eggs collected 1990 in Zimbabwe had 10% thinner shells compared to presumed pre-DDT values

ning, and he pointed out that at the same time, DDT had come into general use. In a later paper (130), he presented

(51). Reproductive disturbances are still observed among bald eagles (Haliaeetus leucocephalus) nesting along the shore-

further evidence

to support his first observation

and also

reported a close relationship between eggshell thickness and residue levels of DDT in the eggs of 14 species of birds. Hickey and Anderson (58) found a similar relationship be-

lines of the North American Great Lakes. A combined effect of p,p’-DDE, PCB and, most importantly, dioxins is

tween declines in raptor populations and decreases in eggshell thickness in the United States and they also observed a significant inverse correlation between shell thickness and the content of p,p’-DDE in eggs of herring gulls (Larus UT-

(16). Herring gull eggs collected

gentatus). These initial reports were soon substantiated

and

extended to other species of birds and to other countries. In wild birds, high concentrations of p,p’-DDE is often accompanied by high levels of other pollutants (e.g., PCB, dioxins, dibensofurans, methyl mercury), which makes it difficult to distinguish the effects of the individual compounds. Faber and Hickey (40) concluded that among 13 different environmental pollutants, p,p’-DDE showed the largest partial correlation coefficient between eggshell thinning and the content of pollutant in eggs. Several laboratory studies aimed at elucidating the mechanism of action of p,p’-DDE-induced eggshell thinning were also performed,

thought to be responsible for the reproductive

impairment

in Lake Erie showed an aver-

age eggshell thinning of 6.7%) (148). High levels of p,p’DDE and hexachlorobenzene were detected in eggs from Prairie ialcons (F&o mexicanus) from some regions in California and were associated with reproductive failure (65). In the field situation, eggshell thinning below 10% is not considered to be associated with egg breakage and population decline

Overwiew

(2,13,14,71,109).

of Eggshell Formation

The entire egg laying cycle takes 25 to 27 hr in the domestic For about 19 hr, the egg (i.e., the yolk, white, and shell membranes) resides in the eggshell gland, where shell formation takes place.

fowl.

DDE-Induced

Eggshell Thinning

Eggshell formation

in Birds

115

does not start immediately

after the

din-F?,X-induced premature egg expulsion reduces calbindin

egg has reached the eggshell gland. During an initial period

mRNA and calbindin

of 3-5

seems as if the level of calbindin in the eggshell gland mucosa is, in addition to estrogen, also regulated by the Ca2+

hr, the “plumbing period,” the outer layers of the

albumin absorb water, NaCl, and glucose (17,22,23). Due to this process, the volume of the egg expands so that the membranes come in close contact with the epithelial cells

concentration

levels in the eggshell gland. Thus, it

in the mucosa. The protein

might serve to

in the lumen of the eggshell gland. After the plumbing period, calcium secretion starts and subsequently increases al-

protect the tissue from the high Ca*+ concentrations that prevail during eggshell formation. In contrast to what occurs in the intestinal mucosa, in the eggshell gland mucosa

most linearly for 13-15 hr until it reaches the maximal rate 7-13 hr prior to oviposition; then secretion ceases 1-2 hr

calbindin synthesis (5,106,113).

prior to oviposition.

In a recent study (6), the effects of different steroid hormones were investigated regarding their effects on calbindin

During this period, the composition

of

the fluid in the eggshell gland changes dramatically due to substantial transfer of ions in both directions. This has been shown experimentally (39)

in two laboratories:

analysed the composition

El Jack and Lake

of shell gland fluid at the

is not

regulated

by 1.25

(OH),-D,

and calbindin mRNA levels in eggshell gland mucosa and on eggshell calcium. Administration of progesterone O-2 hr after ovulation increased egg cycle length, reduced eggshell

beginning and the end of shell formation, and Eastin and Spaziani (37,38) perfused the eggshell gland lumen in situ.

calcium, and the level of calbindin gland.

Calcium and bicarbonate are secreted to the shell gland lumen together with potassium and magnesium, whereas sodium, chloride, and protons are absorbed from the lumen.

Dexamethasone increased egg cycle length and eggshell calcium but reduced calbindin mRNA levels in the eggshell gland. Testosterone did not affect any of the parameters.

Calcium

The antiestrogen tamoxifen reduced plasma calcium but did not affect calbindin mRNA levels in the eggshell gland or

is not stored in the shell gland. Instead, the cal-

cium needed for the production of the shell (about 2 g in the domestic fowl) is continuously obtained from the bloodstream.

The

blood calcium

is replenished

by absorption

eggshell calcium. 38486)

increased

mRNA

The antiprogesterone eggshell

calcium

in the eggshell

mifepristone

(RU

but did not changed

from the duodenum and jejunum (65-75%) and by resorption of medullary bone (137). Without this replenishment,

plasma calcium or calbindin mRNA levels in the eggshell gland. The authors suggested that progesterone may act as

blood calcium would be depleted in about 15 min. Carbonate ions are mainly (80%) produced by metabolic activity

depressor of calcium transport across the eggshell gland, bal-

and through the action of carbonic anhydrase in the eggshell gland; only a small fraction (20%) is derived from

ancing the effects of presently unknown stimulators.

plasma bicarbonate (137). Calcium transport from the blood to the shell gland lumen is stimulated by the presence of sodium and bicarbonate in the gland lumen and is inhib-

Some Characteristics of the Ejjects of p,p’#DDE on Eggshell Formation

ited by ouabain and acetazolamid (38,124,125). Excess protons from the reaction CO: + H,O H HzCO, ++ HCOim + H+ are transferred from the shell gland to the blood, which

o,p’-DDT (53). The neurotoxic, insecticidal effect is due primarily to p,p’-DDT (62); by comparison the neurotoxic

creates an arterio-venous pH-difference (60). The proton pump inhibitor omeprazole, dose-dependently decreased eggshell

thickness

and reduced the plasma calcium

level

Technical

DDT consists of about 80% p,p’-DDT

and 20%

effect of p,p’-DDE is about ten times lower (53,105). The o,p’-isomers of DDT and DDE exert estrogenic effects on both

the

mammalian

uterus

and

the

avian

oviduct

(86) (Fig. 1).

(11,20,147). There has been some confusion

During the most intensive period of eggshell formation (i.e., 7-13 hr prior to oviposition), the rate of shell deposi-

DDE is the most important eggshell thinning agent, and also concerning possible differences between the isomers.

tion is 4.45 mg/cm’/hr (= 36 pmol Caz+/cm*/hr). To accomplish such a massive transfer of calcium, the ion must, in some way, be inactivated to avoid damage to the tissue. Experiments by Ieda et al. (63), Nys et al. (112,113), and

Considering the first question, in experiments in which DDT was found to decrease eggshell thickness, either the doses were so high that there was an acute toxic effect on

as to whether

DDT or

Striem and Bar (139) suggest that a calcium binding protein, i.e., calbindin or Ca-BPDK18, participates in this pro-

the birds (18,31), or exposure times were so long that metabolic conversion of DDT to DDE took place (3 1). Administration of p,p’-DDT and o,p’-DDT to ducks did

tective regulation of the calcium level. This protein is found in the tubular gland cells of the eggshell gland mucosa (145), as well as in the intestine. Amounts of the mRNA for this protein increase during egg laying (112,139), but the concentration of the protein in the eggshell gland mucosa does not vary during the ovulatory cycle (113). Sup. pression of eggshell formation for several days by prostaglan-

not result in eggshell thinning, despite treatment regimes directly comparable to p,p’-DDE regimes that produced eggshell thinning (79). Administration of o,p’-DDE to ducks in a comparable treatment regime produced a slight (8%) eggshell thinning as compared to pretreatment values, but no effect as compared to the contemporary controls (76). The o,p’-isomer of either DDE or DDT is readily excreted

C. E. Lundholm

116

and not accumulated

to the same extent as the p,p’-isomer

(53,76). All types of birds are not equally sensitive to p,p’-DDE-induced eggshell thinning. Cooke (3 1) divided avian species into three main categories with SPECIES DIFFERENCES.

regard to the degree of eggshell thinning observed: 1) those in which eggshell thinning reaches 30% or more, e.g., the peregrine

falcon,

the brown pelican,

and some races of

ducks (79) (Indian Runner Ducks, Anas glatyrhynchos var.); 2) intermediately sensitive species, with eggshell thinning between 5-15%,

e.g., the American

kestrel (Falco sparser-

ius) and the Japanese quail (Coturnix coturnix); and 3) insensitive species, e.g., the domestic fowl (Gallus domesticus) and the Bengalese finch (Lonchura striata). The low or nonexistent sensitivity of quails and domestic hens to p,p’DDE-induced eggshell thinning has lead to much confusion in the planning of laboratory experiments aimed at studying the mechanism of action by which p,p’-DDE affects eggshell formation.

These species are readily available as laboratory

animals, but, due to their low or lack of sensitivity, they are not suitable for studying p,p’-DDE-induced eggshell thinning; in this respect, ducks are a much better alternative. Of course the degree of eggshell thinning also depends on the exposure level. Among wild birds, predatory and fisheating species, which are at the top of the food chain, generally accumulate the highest levels of environmental pollutants. However, if the varying residue levels are taken into consideration, differences in sensitivity between species are still apparent. Several species of birds have been experimentally treated with p,p’-DDE under controlled conditions, but, despite comparable treatment regimes and acquired residue levels, different degrees of eggshell thinning were still observed.

crease was statistically significant after 10 days (- 1 l%,p
regime did not induce eggshell thin-

ning in domestic hens (78,91), not even when the level of p,p’-DDE in the food was raised to 100 mg/kg and that amount was given to the birds for an additional period of 25 days (78). After 45 days of giving 40 mg p,p’-DDE/kg food to ducks, the mean residue level in the eggs (yolk + white) was 51.4 -t 6.4 pguglgwet weight (Table l), a level that is in the range of that observed in wild birds exhibiting eggshell thinning (15). The level in the eggshell gland mucosa of the domestic fowl was 1.4 + 0.09 pg/g wet weight. The residue level in eggshell gland mucosa of the domestic fowl was 2.2 ? 0.2 ,ug/g wet weight after 45 days of p,p’-DDE at a level of 40 mg/kg food and 4.9 + 0.9 pg/g wet weight after the extended treatment period (78,79). EGG

PRODUCTION

Administration

AND

EGGSHELL

CHARACTERISTICS.

of 40 mg p,p’-DDE/kg food for 45 days did

not affect either the number of eggs laid by the ducks or the size of the eggs, nor did it alter the weight of eggyolk or eggwhite (Table 2). This is an important observation since other environmental pollutants, e.g., methyl mercury (93,134), petroleum oil (61), PCBs (7,144), and lindane (25,26), also reduce egg production and/or egg size. Administration of estrogen reverses the effect of lindane on eggshell thickness (25) but not that of p,p’-DDE (Lundholm, unpublished). Unlike p,p’-DDE, methyl mercury influences eggshell formation in domestic hens, i.e., shells exhibit a

ning effect of p,p’-DDE is first manifested after only one day

rough surface and indentations, as well as thinning (93). In earlier experiments (79), it was found that the weight

if a sufficiently large dose is administered (120). Large single doses (200 mg) also interfere with ovulation (79) and can

of the eggshell gland was significantly (p < 0.001) reduced, i.e., by 12-18%, in p,p’-DDE-treated ducks, and the weight

be considered as irrelevant in regard to the environmental exposure situation. It should be pointed out that several toxic agents can produce short-term (i.e., lasting only a few

reduction was not due to variation in water content (79). No reduction in shell gland weight was observed in the domestic fowl following a comparable p,p’-DDE treatment re-

days) eggshell thinning (48), probably as the result of a reduced intake of food or general toxic effects. The eggshell thinning effect of p,p’-DDE is, on the other hand, extremely

gime (Table 2). The reduction of eggshell thickness in ducks occurs via a reduction of the palisade layer (32), together with a reduced number of shell pores, which leads to decreased water loss (121) and probably also an altered 02 and CO? exchange. Treatment of ducks with p,p’-DDE also affected eggshell morphology, i.e., the number of mammillary cores was re-

TIME-RESPONSEANDRESIDUELEVELS.

Theeggshell-thin-

long-lasting. Significant eggshell thinning has been observed up to 2 years after discontinuation of p,p’-DDE treatment (74,122). This is due to pronounced accumulation of p,p’-DDE in adipose tissue and to the slow rate of excretion of the compound from the body (74). Administration of p,p’-DDE in food (i.e., 40 mg/kg feed) given ad libitum to ducks has been found to produce maximal eggshell thinning after about 45 days (Table 1) (76,79). This dose-level does not produce any general toxicity to the birds nor does it interfere with eggproduction. Eggshell thickness started to decrease after 4-5 days, and the de-

duced and the mammillae were larger (47). Bebout and Hempleman (8) compared the effects of a calcium-deficient diet with administration of acetazolamide in regard to effects on water vapour conductance through the eggshell of the domestic fowl. A calcium-deficient (0.34% Ca) diet decreased eggshell thickness by 21% and increased water vapour conductance

by 30%. Acetazolam-

DDE-Induced Eggshell Thinning in Birds

117

TABLE 1. The mean Eggshell Index (EI) (n = 80-l 10 eggs/group) in 12 ducks (Swedish X Rouen breed) that were given p,p’-DDE (40 mglkg dry weight) in the diet for 45 days, and 12 control ducks. Standard error of mean (SEM) was +O.Ol0.02 in alI groups. Eggs were collected in periods of 10 days before and during the DDE feeding. The increase (mean + SEM, R = 6 per group) in the p,p’#DDE level in duck eggs (yolk + white) with time during the experiment is also shown. EI Time

p,p’-DDE

Control

DDE

Diff. %

Pretreatment

2.25

2.22

O-10 Days lo-20 Days 20-30 Days 35-45 Days

2.37 2.40 2.41 2.38

2.19 2.07 2.00 1.95

-1.3 -7.6* - 13.8* -17.0” -l&o*

N.D. = not detectable, W.W. = wet weight; f.w. = fat weight (hexaneiether extraction). control and p,p’-DDE treated groups are denoted *p < 0.001. Eggshell Index was calculated from (79).

llglg W.W. N.D. 7.8 k 17.3 2 25.3 2 51.4 -c

0.95 1.14 3.4 6.1

level in eggs uglg f.w.

55.6 121 221 342

N.D. + ? ? ?

8.3 22.3 37.2 38.1

The statistical significance (t-test) of the difference between as: eggshell weight (mg)/length (mm) X hreadth (mm). Data

ide decreased eggshell thickness by 36%, and increased wa-

ATPases

ter vapour conductance by 200% and the total pore area by 89%. This shows that although both treatments lead to

Several studies (73,77,81,100) have shown that p,p’-DDE (and p,p’-DDT ) inhibits Caz+-ATPase or Ca’+-Mg2+

eggshell thinning,

-ATPase activity in the eggshell gland, and it has been suggested that inhibition of ATPase activity represents a mech-

the structure of the eggshell was impaired

in different ways. It has been found that different components of the eggshell are affected by p,p’-DDE in different species (32,33).

to p,p’-DDE

anistic explanation ning. The plasma

treatment

shows greater variation.

for p,p’-DDE-induced eggshell thinmembrane calcium pump is mainly

localized to the microvilli of the tubular gland cells of the eggshell gland and of the isthmus (145,150). The correlaEffects

of p,p'-DDE

Metabolism

on the Calcium

of the Eggshell

Gland

Eggshell thinning induced by p,p-DDE is accompanied by several biochemical changes in the calcium metabolism of the eggshell gland (Tables 2 and 3). These changes are described and discussed in this section. The supply of calcium to the eggshell gland of ducks is not impaired by administration of p,p’-DDE. In a number of studies (77,78,81,91,92,122), such treatment did not affect the blood calcium level and the content of calcium in the eggshell gland mucosa was significantly higher in p,p’DDE-treated ducks than in control animals. In other experiments (Lundholm unpublished), the specific gravity of tibia and femur did not differ between p,p’-DDE-treated ducks and controls, nor was there a difference between treated and untreated animals in regard to the uptake of 4Ca by a homogenate of duodenal or intestinal mucosa. These observations suggest that p,p’-DDE inhibits calcium transport processes in the eggshell gland but does not decrease the availability of calcium to the eggshell gland. The uptake of 45Ca by a homogenate of the eggshell gland mucosa and different subcellular fractions thereof, as well as the Ca*+-Mg *+-ATPase and Mg’+-ATPase activity of these preparations, varies during different periods of eggshell formation (80). The uptake of 45Ca by the homogenate and the different subcellular fractions was significantly inhibited in p,p’-DDE treated ducks, as compared to controls (77,81). This difference between the treated and untreated ducks was observed only during the earlier periods of eggshell formation, i.e., between 16.00 and 20.00. The response of the

tion between Ca’+-ATPase or Ca’+-Mg*+-ATPase activity and calcium transport or the uptake of 45Ca by a homogenate of eggshell gland mucosa and its subcellular fractions has not yet been clarified. As mentioned above, calcium transport across the eggshell gland mucosa is largely dependent on transport of sodium and bicarbonate. It was shown (81) that the main ATPase activity in the eggshell gland mucosa was induced by Mg:+, and addition of Ca2+ at optimal Mgz+-concentrations resulted in a slight (lo-20%) increase in ATP hydrolysis. During the period of active eggshell formation, there was increase in both the activity of Cal+-Mg2+-ATPase and the uptake of 45Ca in a homogenate of the eggshell gland mucosa from ducks, as compared to the corresponding values for eggshell gland mucosa without a calcifying egg. This functional increase was inhibited by p,p’-DDE treatment. In another study (92), the activity of a HCO+timulated ATPase did not change in p,p’-DDE treated ducks. Addition of p,p’-DDE in vitro to a homogenate of the eggshell gland mucosa from ducks (77) and domestic hens (94) has been found to inhibit 4YZa-uptake and Ca’+-Mg’+ATPase activity. The ratio of the reported ICsOvalues for inhibition of Ca*+-Mg *‘-ATPase activity and 4iCa-uptake by p,p’-DDE was 0.29, i.e., the ATPase activity was more sensitive to inhibition by p,p’-DDE than 45Ca uptake. Furthermore, it was noted that these effects are not specifically confined to p,p’-DDE, i.e., they were also observed after addition of p,p’-DDT, o,p’-DDE, p,p’-DDD, PCBs, and several other chlorinated hydrocarbons (76,94). P,p’-DDE had no effect on the activity of calmodulin prepared from eggshell

118

C. E. Lundholm

TABLE 2. Summary of p,p’-DDE-induced changes in egg characteristics, calcium metabolism, and the content of different ions in the eggshell gland lumen. Two species are considered: the p,p’-DDE sensitive duck and the insensitive domestic fowl. P,p’-DDE was administered in the diet at 40 mgl kg food for 45 days. Parameter Eggshell

Domestic fowl

Duck

index

74% (IRD)““” 81% (SRD)““” (76-79, 81)

Egg size

TABLE 3. Summary of p,p’-DDE-induced changes in calcium metabolism and biochemical parameters of importance for eggshell formation. Two species are considered: the p,p’-DDE-sensitive duck and the insensitive domestic fowl. P,p’-DDE was administered in the diet at 40 mg/kg food for 45 days. Parameter

Domestic fowl

Duck

tiCa-uptake hy a homogenate eggshell gland mucosa

of

750/o*“: (77, 78, 81)

‘iCa-uptake hy a homogenate of duodenal or intestinal mucosa

(% Numher of eggs layed

Enzymatic activity in homogenate of eggshell gland mucosa Ca’+-Mg’ ’ -ATPase

(% Time

of laying (;?I

Weight of eggyolk or eggwhite

Mg’+eATPase

Weight

HCO;

68’~o”j: (77, 78, 81) (77,::

of the eggshell gland

Specific graviry of tibia or femur

(7;) 760/O”(*) (91, 92, 96)

PGH-synthetase (:;I

Carbonic

anhydrase

Plasma calcium

g (77,7g&

Calcium in eggshell gland mucoSa Calcium in eggshell gland lumen Bicarbonate Iumcn

in eggshell gland

81)

ATPase

91)

130%* (77, 78, 81, 91)

PGE: content mucosa Calmodulin

in eggshell gland

content

57X*(*) (78, 91, 92) 67Ok,“:“” (91, 92)

Chloride in eggshell gland Iumen

7 1 o,. t * * (92)

Sodium in eggshell gland lumen

8 5 o/* * (92)

Potassium lumen

rectly, but does so indirectly hy blocking a signal for calcium transport. The activity of the ATPases is probably controlled by this signal.

in eggshell gland 85%~~ (92)

gland mucosa either when added in vitro or after administration to ducks at 40 mg/kg food for 45 days (85). Taken together, the available information suggests that instead of directly inhibiting the ion-activated ATPases, p,p’-DDE interferes with translocation of calcium across the eggshell gland during eggshell formation by inhibiting a signal that triggers the translocation itself (81). In other words, p,p’-DDE does not impede the ion-activated ATPases di-

P&‘-DDE-Induced Changes in the Ionic Composition of Eggshell C&d Fluid The calcium content in the eggshell gland lumen is significantly reduced (36-79X) in p,p’-DDE-treated ducks exhibiting eggshell thinning (78,91,92). There are also profound changes in the levels of other ions in the eggshell gland lumen following p,p’-DDE administration (Table 2). In one study (92) it was found that sodium and potassium were both reduced hy 15X, bicarbonate was reduced hy 33%, and the content of chloride was reduced by 29%; the phosphorus content was unchanged. These results raise the following question: did p,p’-DDE primarily impair the transmucosal movement of one particular ion and the observed changes in other ions are secondary to that impairment or did p,p’DDE have an effect on the movement of several different ions. Analysis of the ionic composition of eggwhite and

DDE-Induced Eggshell Thinning in Birds

119

eggyolk did not reveal any significant p,p’-DDE-treated

ducks and controls

differences

between

(unpublished

results).

As already mentioned, calcium transport across the eggshell gland mucosa is coupled to the transport of both sodium and bicarbonate (38,124,125). If the movement of one of these ions is impaired, it will probably have consequences for the movement of other ions as well. That possibility cannot

yet be verified because

knowledge

there

is still insufficient

about the basic mechanisms

of eggshell forma-

tion to give a correct answer to this question. Considering the study cited above (92), the similar magnitudes of the reductions

in bicarbonate

and chloride

suggest that

the

BLOOD

t

:O,+H,O W H,CO, = H++ HCO, -

Ca*’ __ CO,”

movement of these two ions may be coupled (a HCOI--Clexchange?); likewise, reductions in the levels of sodium and

that the Na+-K+-ATPase

El

inhibitor ouabain de-

creased calcium and bicarbonate transport across the eggshell gland mucosa and changed sodium and chloride resorption

to secretion;

the secretion

altered by this treatment

I J CaCO, +H

Fosfolipids 0 AA 0 PGE, *

potassium were similar, which also implies a transport relationship. These latter assumptions are strengthened by the observation

LUMEN

MUCOSA

of potassium was not

I

I

/ P,pDDE

(

EGGSHELL

(38). -

Prostagkmdins

and Eggshell Formation

The interest in the involvement

of prostaglandins

in avian

Ca*’ _

,

reproductive physiology has mainly been focused on the role of these substances in inducing contraction of the eggshell gland muscles during oviposition [review (136)J. Hertelendy (54,55) showed that premature oviposition could be induced by intrauterine injection donic acid, and dibutyryl-CAMP;

of prostaglandins, arachiacetylsalicylic acid and in-

domethacin abolished the effects of injected arachidonic acid. Both in vine (114) and in viuo (135) administration of prostaglandins

induce uterine

contractions.

been shown that plasma concentrations

It has also

Ca’* f

<

K’ -

___ (

’

Na’

‘K

) > -

’ Na’ ,

of prostaglandins

are increased during oviposition (50,115,146) and that the eggshell gland myometrium contains receptors for prostaglandins (3,142). Much less is known about the involvement

of prostaglan-

dins in eggshell formation. Hertelendy and Bieller (57) measured prostaglandin levels in blood from calcifying and non-calcifying domestic hens and found higher levels of prostaglandin E in the former. Blood levels of prostaglandin E and F were highest during oviposition and were also elevated 6-16 hr after oviposition during eggshell formation (50).

Mg”

of the previous egg, i.e., Increased levels of PGFla

and I’GE, in eggshell gland mucosa during eggshell formation has also been reported (131). Conflicting results have been obtained in studies of the effects of inhibitors of prostaglandin synthesis on eggshell formation. Day and Nalbandov (35) administered indomethacin (2.5-50 mg/kg) to domestic hens 4-5 hr prior to the expected time of ovulation and found that oviposition was delayed 15-18 hr. The egg that was ovulated after indo-

FIG. 1. Schematic outline of ion transport across the eggshell gland mucosa during eggshell formation and possible transport mechanisms. Ca ‘+, HCO,-, Mg*+ and K+ are transported from the blood through the mucosa to the calcifying egg in the shell gland lumen. Na+, H+ and Cl- are transported in the opposite direction. HCOjis mainly generated in the mucosa by carbonic anhydrase (CA) from CO2 and H,O. Excess protons are transported to the blood. Ca’+ is continuously removed from blood and there are probably different mechanisms for the transmucosal movement of this ion. (A) = PGEz-stimulated HC03--transport with a coupled Ca’+H transport. P,p’-DDE inhibits this transport by inhibition of Prostaglandin synthetase (PGS; cyklooxygenase + peroxidase) which converts arachidonic acid (AA) to PGE2. Arae chidonic acid is released from membrane fosfolipids by the action of Phospholipase Az (PLA,). (B) = HCOi-/Cl--exchange. (C) = Ca’+-Mg*+.ATPase. (D) = Ca*+/Na+-exThe presence of these transchange. (E) = Na+-K+-ATPase. port mechanisms in the eggshell gland has not been unequivocally demonstrated but is suggested based on the results from experiments presented in the following references: (38,39,41,79,81,85,91,92,96,124,125).

C. E. Lundholm

120

methacin treatment

was laid with a soft shell. Indomethacin

nates of the eggshell gland mucosa from the duck and the

did not interfere with ovulation and had no effect on plasma levels of estrogen, progesterone or testosterone. Hammond

domestic fowl. This inhibition was more marked for the homogenates from the duck (ICC = 4 ,uM) than for the corre-

(49) administered indomethacin (50 mg/kg) to domestic hens 4-6 hr prior to the expected time of oviposition and

sponding preparations from the domestic fowl (I& = 10 PM). In addition, prostaglandin synthesis activity was

found that oviposition was delayed 15 hr; eggshell thickness

higher in the former preparations and was also much higher

was increased, and the plasma calcium level was reduced by 30%.

mogenates

Intramuscular administration of indomethacin (2.2 mg/ kg) to domestic hens 4 and 16 hr after an egg had entered the eggshell gland, delayed oviposition and increased the shell thickness of the egg present in the eggshell gland at the time of administration, but reduced the shell thickness of subsequent eggs laid (4). Nys (110) 10 mg indomethacin

intramuscularly

administered

5 and

to domestic hens 6 hr

after ovulation and found that eggshell thickness duced in eggs that were expelled at the expected

was retime of

oviposition. Oral administration of 100 mg of indomethacin to domestic hens for three consecutive days delayed oviposition three days; two eggs were then laid, one without a shell and the other with a very thin shell (79). Two other inhibitors of prostaglandin

synthesis,

naproxen,

and diclofenac

(100 mg of each, given orally for three consecutive days), delayed oviposition and produced 40% eggshell thinning (79). In the domestic fowl, a lOO-mg oral dose of indomethacin given a few hours prior to the start of eggshell formation (at 17:OO) caused a 21% reduction in the shell thickness

in homogenates

of the eggshell gland mucosa than in ho-

of the magnum and isthmus regions of the ovi-

duct. Only p,p’-DDE interferes with prostaglandin synthesis, i.e., the structurally related chlorinated hydrocarbons 0,~‘. DDE, p,p’-DDT, o,p’-DDT, p,p’-DDA, p,p’-DDD, and methoxychlor do not inhibit the synthesis of prostaglandins, even when added at concentrations homogenates membranes

(95,96).

The

adrenotoxic

published).

Furthermore,

p,p’-DDE-induced

from treated ducks (96). In another study (91) (Table 3), the effects of p,p’-DDE on calcium and prostaglandin metabolism in the eggshell gland of the duck and the domestic fowl were compared. In the duck, which is sensitive to p,p’-DDE, eggshell thinning occurred and was accompanied

cantly reduced both the uptake of Wa of the eggshell gland mucosa (-29%) prostaglandins by the homogenate

also signifi-

by a homogenate

and the synthesis of (-65%). Moreover,

plasma calcium level was decreased by 15%, whereas the calcium level in the eggshell gland mucosa was increased to 153% compared to the controls. In an effort to reduce the number

of soft-shelled

and

shell-less eggs, five different levels of acetylsalicylic acid (ranging from 0.025 to 0.4%) were included in the diet given to domestic hens for a period of 13 months (98). At all levels, acetylsalicylic

acid decreased the shell thickness

and the specific gravity of the eggs. Eggs from hens receiving the largest dose were smaller and had smaller yolks, whereas albumin weight was not affected by any of the dose levels.

eggshell thin-

ning was accompanied by significant inhibition of prostaglandin synthesis in eggshell gland mucosa homogenates

“Ca-uptake

treatment

metabolite

sis when added in concentrations of up to 100 ,uM to a homogenate of duck eggshell gland mucosa (Lundholm un-

treatment

The indomethacin

(75)

MeSOl-DDE (3-methylsulfonyl-2,2-bis(4chlorophenyl) -1 ,l-dichloroethene) did not inhibit prostaglandin synthe-

of the egg present in the eggshell gland the morning after (97).

of up to 100 PM to

of eggshell gland mucosa or human platelet

and prostaglandin

by significant reductions in synthesis in homogenates

of

the eggshell gland mucosa. Significant reductions in the content of prostaglandin E: in the eggshell gland mucosa were also observed, together with decreased levels of calcium and bicarbonate in the eggshell gland lumen. The content of calcium in eggshell gland mucosa was significantly increased. In the domestic fowl, neither eggshell thinning nor any of the other alterations were observed following an identical p,p’-DDE treatment regime (Tables 2 and 3). P,p’-DDE probably inhibits prostaglandin synthesis at the level of PGH-synthetase (cyclooxygenase + peroxidase) since equal inhibition of the formation of prostaglandin Flar prostaglandin El, and thromboxane A1 has been noted after addition of p,p’-DDE in vitro (96). Administration of the phospholipase

A1 inhibitor quina-

In this context, it should also be noted that prostaglandins do not seem to be involved in ovulation in birds since neither prostaglandins nor prostaglandin synthesis inhibitors block ovulation (35,56). This is in contrast to the effects of prostaglandins in mammals.

crine to domestic hens (200 mg/bird) produced slight (7%) eggshell thinning (96). The activity of phospholipase AI is not inhibited by p,p’-DDE added in vitro (unpublished

Effect of p&‘-DDE on the Prostuglandin Metabolism of the Eggshell Cjland Mucosa

has been found that p,p’-DDE added in vitro to a homogenate of eggshell gland mucosa from the domestic fowl had no effect on the binding of prostaglandin E: to its receptors (unpublished results).

In one study (96) it was found that p,p’-DDE inhibited the synthesis of prostaglandins when added in vitro to homoge-

results). Administration of the glucocorticoid prednisolone (10 mg/bird daily for three consecutive days) had no effect on eggshell thickness (96). Also of interest in this context, it

DDE-Induced

Eggshell Thinning

in Birds

121

Possible Effects of Prostaglandins on Ion Transport in the Eggshell @.und Mucosa

thereof (84). Chang and Stockstad (27) studied Japanese quail and reported that p,p’-DDE at a concentration of 50

Little information is available in the literature about the effect of prostaglandins on ion transport in the eggshell gland. Therefore, a hypothesis regarding such transport

mg/kg in a diet administered for 15 weeks increased the carbonic anhydrase activity 2-fold; at a concentration of 200 mg/kg, there was a 20% reduction as compared to the

must be deduced from existing knowledge gained in experiments on other types of tissues.

control value. Bird et al. (10) reported that 20 mg/kg p,p’DDE in the diet administered to the American kestrel (F.

The secretion of bicarbonate

by the gastric and duodenal

mucosa of bullfrogs and mammals is regulated by at leastfour different transport mechanisms (41): 1) a furosemidesensitive Cl-/HCOjexchange; 2) PGE*-stimulated

sparuerius) decreased eggshell thickness by 26% and reduced the activity of Ca’+-Mg’+-ATPase and carbonic anhydrase in the eggshell gland mucosa by 32 and 20%, respectively. It have been suggested (2,12) that inhibition of carbonic

HCOi- transport dependent on the luminal Cl- concentrations; 3) transport via an anion-carrier with some affinity for Cl-; and 4) diffusion of HCOI- through paracellular

anhydrase in the eggshell gland mucosa is a mechanism for p,p’-DDE-induced eggshell thinning, bur other investigators have expressed skepticism regarding that possibility

shunts. As previously described, p,p’-DDE-induced

(3 1,126). Moreover, p,p’-DDE-induced eggshell thinning in ducks is not associated with reduced carbonic anhydrase activity in the eggshell gland mucosa (85) (Table 3).

eggshell

thinning in ducks is accompanied by reduced levels of calcium, bicarbonate, chloride, sodium, and potassium in the eggshell gland lumen during eggshell formation. If PGEIstimulated HCO,transport does occur in the eggshell gland mucosa, inhibition of prostaglandin synthesis would reduce the transport of HCO?- to the shell gland lumen. In turn, because calcium transport is coupled to bicarbonate transport, there would also be a reduction in the transfer of

Steroid Hormones and p,p’-DDE-induced Eggshell Thinning: Possible Effects on the Control of Calcium Secretion in the Eggshell @and

in the egg-

ESTROGEN. A common hypothesis among environmental toxicologists is that p,p’-DDE-induced eggshell thinning in birds is caused by increased liver metabolism of steroid hormones. Estrogen is a prerequisite for completion of

shell gland mucosa has been described by (100,111). This enzyme might be involved in the transfer of bicarbonate across rhe eggshell gland. The activity of this ATPase was

many stages in the reproductive physiology of the female bird, for instance, growth and development of the oviduct, synthesis of yolk material, and egg albumin (44,99). Deposi-

calcium

to the eggshell gland lumen, which has, in fact,

been reported (91,92) (Fig. 1). The presence of a HCOim-stimulated

ATPase

not inhibited in eggshell gland mucosa of p,p’-DDE-treated

tion of medullary bone is also stimulated

ducks (92). At present, it is not clear whether inhibition of prostaglandin synthesis in the eggshell gland mucosa directly af-

combination with androgens (137). There is, however, no support for the suggestion that estrogen is the primary regulator of calcium secretion in the eggshell gland during shell

by estrogen

in

fects the transport of one ion only and other changes in the eggshell gland lumen are secondary to that, or if other ion transport mechanisms are affected as well. In other organs,

formation. Administration of the estrogen receptor antagonist tamoxifen to the domestic fowl (100 mg/bird daily for three consecutive days) has been found to cause a slight reduction in eggshell thickness (-9%) and to inhibit egg

for example, frog cornea (9), toad bladder (118), rabbit kidney thick ascending limb and collecting tubule (64,138), and canine tracheal mucosa (l), prostaglandins are known

production (79). The findings of Chakravarty and Lahiri (25,26) suggest that lindane-induced eggshell thinning in

to influence ion and water movement, i.e., they increase water and Cl- transport and attenuate Na+ transport. Furthermore, prostaglandin Ez inhibits Na+-K+-ATPase in the

ducks is due to decreased estrogen levels since eggshell thinning was reversed by administration of diethylstilbestrol. Petroleum compounds may interfere with the synthesis of

distal segment of the mammalian

estradiol, estrone, progesterone, and luteinizing hormone in female mallards (24). In addition, several authors have reported that adrenocortical activity is suppressed after expo-

nephron

(34).

Carbonic Anhydrase The supply of bicarbonate for eggshell formation could also be reduced by inhibition of carbonic anhydrase. The carbonic anhydrase inhibitor acetazolamide has been noted to decrease calcium transfer across the mucosa of the eggshell gland that were mounted in an Ussing chamber (125) or were perfused in situ (38). Administration of acetazolamide to the domestic hen also induces eggshell thinning (84,103) and reduces 45Ca-uptake and Ca”-Mg’+-ATPase activity in eggshell gland mucosa homogenate and subcellular fractions

sure to petroleum compounds (45,46,52). Qin and Klandorf (127) studied the domestic fowl and found that eggshell thickness and oviduct and egg weight were not affected by treatment with estradiol. Chen et al. (28) noticed that hens given phenobarbital (100 mg) for 7 days exhibited reductions in eggshell thickness, egg production, plasma calcium, and plasma estradiol content. In the same investigation, hepatic cytochrome P450 was increased and inversely correlated to estradiol levels in plasma, and there was a positive correlation between

C. E. Lundholm

122

calcium and estradiol in plasma. In a subsequent study on

stimulates the secretion of eggwhite in the magnum region

the domestic fowl (29), it was observed that oral administra-

of the oviduct (116,117,119) and has also been found to increase eggshell thickness and the duration of eggshell for-

tion of DDT (technical) at a daily dose of 40 mg for five consecutive days increased hepatic cytochrome P-450 and

mation when injected into domestic fowls 4 and 10 hr after

decreased the plasma level of estradiol; no effect on plasma calcium or eggshell thickness was observed. Oral administration of the PCB Arochlor 1254 (10 or 25 mg daily for 5

ovulation (110). Injection of progesterone 16 hr after ovulation caused premature expulsion of the egg being formed and decreased

days) decreased plasma calcium and estradiol and egg production and induced hepatic cytochrome P-450; no effect on eggshell thickness was seen. Although the induction of

eggshell thickness (110). Tanaka (141) reported that administration of progesterone delayed oviposition and in-

liver metabolizing enzymes can certainly impair avian reproduction in several ways, it could not be concluded that this is the mechanism of p,p’-DDE-induced eggshell thinning. The most obvious reason for this is that neither plasma calcium nor egg production are reduced by doses of p,p’-DDE

creased eggshell thickness and is has been noted that the plasma concentration of progesterone shows a distinct peak at the time of ovulation (43,50). The plasma concentration of progesterone was also increased 6-16 hr after oviposition, i.e., during formation of the eggshell. This increase was almost similar to a simultaneous

increase in the plasma con-

that cause eggshell thinning. P,p’-DDE does not bind to estrogen receptors in significant amounts (20,147). Further-

centration of PGEI and PGF!, (50). This biphasic increase in plasma concentration of progesterone was not observed

more, administration of estrogen, progesterone or corticosterone does not reverse p,p’-DDE-induced eggshell thin-

in Khaki Campbell ducks (149). There are large numbers of progesterone receptors in both the eggwhite-secreting magnum region and in the eggshell gland mucosa (83,116,117). These receptors are located on the nuclei of epithelial apical cells and on the nuclei of basal cells of the

ning (79). TESTOSTERONE. Receptors for testosterone are present in the eggshell gland mucosa of ducks and domestic fowls (88), but the involvement of testosterone in the function of the

eggshell gland is poorly understood. Testosterone injected into domestic fowls 4 hr after oviposition increased the duration of shell formation and eggshell weight (110). The treatment also increased plasma progesterone concentration for about 3 hr. Recent studies has shown that p,p’-DDE is a potent antiandrogen. Kelce et al. (68) reported that p,p’-DDE inhibited androgen-receptor binding with an ICj, = 5 ,uM. The structurally related compounds p,p’-DDT, o,p’-DDT, and p,p’DDL> were 12-20-fold less effective than p,p’-DDE in this respect. P,p’-DDE

also

inhibited

androgen-induced

transcrip-

tional activity, and androgen action in developing, pubertal, and adult male rats. p,p’-DDE did not inhibit the binding of estrogen to the estrogen receptor or the activity of the enzyme 5 R-reductase, which converts testosterone to 5a-dihydrotestosterone. In a following study (69), the authors investigated the effects of p,p’-DDE on androgenreceptor-dependent gene expression in e’iuo. P,p’-DDE induced a decline in the weight of seminal vesicles and prostate of rats and altered the expression of androgenreceptor-dependent genes in a similar way as the fungicide vinclozolin. This specific inhibitory effect of p,p’-DDE on androgenreceptor binding was not observed in tests of the binding of testosterone to receptors prepared from eggshell gland mucosa (88). No differences were seen between control and p,p’DDE-treated ducks in the binding of testosterone to receptors prepared from eggshell gland mucosa. PROGESTERONE.

terone

is involved

It is tempting

in eggshell

to speculate that progesformation. This hormone

surface epithelium of the eggshell gland mucosa ( 15 1); these two cell types are supposed to be responsible for calcium and bicarbonate transport. Fewer receptors are found on tubular gland cells of the eggshell gland mucosa. MECHANICAL

STIMULI.

The

presence

of an egg in the

eggshell gland could also stimulate calcium secretion. During the plumbing period, the egg absorbs water, glucose, and ions, and the shell gland is distended. Calcium secretion starts after this swelling is completed. By stretching tissue, prostaglandins are released (101). The nature of the stimuli that triggers calcium secretion in the eggshell gland was investigated in Eastin and Spaziani (37). Maximal calcium secretion was obtained when a surrogate wax egg was inserted into the eggshell gland during shell formation in replacement for the spontaneously ovulated egg. With the calcium secretion arbitrarily set at 100%) when an eggshell was being calcified, removal of the egg without replacing it by such an artificial egg reduced the calcium secretion to 34%. When the gland was not forming a shell and was not distended by an artificial egg, the calcium secretion was 6%; when the gland was then distended by an artificial egg the calcium secretion rose to 13%. This shows that at least two stimuli, ovulation and distension of the eggshell gland, are necessary for maximal calcium secretion. It must be emphasized that maximal calcium secretion under these experimental conditions was only about 10% of the calcium secretion necessary for eggshell formation (82,140). PROGESTERONEANDPROSTAGLANDINS. Administration of progesterone to estrogen-primed chickens has been found to stimulate PGH-synthetase activity in a homogenate of eggshell gland mucosa and to decrease the K,, value for the

DDE-Induced Eggshell Thinning in Birds

123

binding of PGE2 to its receptors in this tissue (89). This is in

terone receptor,

accordance

the inhibitory

with a proposed role for progesterone

in eggshell

this effect might operate in concert

effect of p,p’-DDE

on prostaglandin

with

synthe-

formation. Further evidence for a connection between progesterone and prostaglandins in the avian oviduct has been

tase and contribute to the reduced calcium transport across the eggshell gland. Further studies in this area are certainly

obtained

needed in order to clarify the regulation of calcium transport in the eggshell gland mucosa and a possible role for

in experiments

by Niemela

(108).

Using cultured

cells from the avian oviduct, it was found that progesteroneinduced synthesis of the protein avidin could be inhibited by indomethacin, and that the effects of progesterone prostaglandin on avidin synthesis were not additive.

Steroid Hormone Receptors The o,p’-isomers have estrogenic

and

re-

Species Difference to p,p’-DDE-induced Eggshell Thinning

and DDEIDDT

of DDE and DDT have been shown to effects on the avian oviduct, the mamma-

lian uterus and on the liver of juvenile

p,p’-DDE as a antagonist or agonist on the progesterone ceptor.

rainbow

trout

(11,20,36,147), and these effects are mediated through binding to estrogen receptors. P,p’-DDE, on the other hand,

A possible explanation

for the species difference

DDE-induced

thinning

eggshell

is greater

to p,p’-

sensitivity

of

PGH-synthetase in the eggshell gland mucosa to p,p’-DDE, as seen in the duck. The increased sensitivity may also en-

has no estrogenic effects and does not bind to estrogen receptors in significant amounts (20,147). When added in

tail a greater dependence of prostaglandin-stimulated calcium and bicarbonate transport in this species since it is possible that multiple transport mechanisms for calcium

vitro to preparations of eggshell gland mucosa, p,p’-DDE and

and bicarbonate

several other chlorinated hydrocarbons has been found to inhibit the binding of progesterone, testosterone, and corti-

importance and contribution of each of these transport mechanisms could vary between different species of birds,

costerone to their respective receptors (83,88). Receptors prepared from duck eggshell gland mucosa were about three times more sensitive to inhibition by p,p’-DDE than such

and also differ at different times during eggshell formation. This could explain the observed variation in species sensitivity. For example, some species could have a more “prosta-

receptors prepared from the domestic fowl. However, there was no inhibition of the binding of progesterone, corticoste-

glandin-dependent” calcium and bicarbonate transport (as seen in the duck), and would therefore be sensitive to p,p’-

rone, or testosterone

DDE-induced

to their respective

receptors in a ho-

mogenate of eggshell gland mucosa from ducks that had been treated with p,p’-DDE and were laying thin-shelled eggs. Inhibition of binding of these hormones to their receptors is not a specific effect of p,p’-DDE since such inhibition is also induced by structurally similar compounds such as p,p’-DDT, o,p’-DDT, and o,p’-DDE (83,88). Hypothetically, the transport of calcium and bicarbonate across the

exist in the eggshell gland mucosa. The

eggshell

shown that prostaglandin

thinning.

Indeed,

studies

have

synthesis in eggshell gland mu-

cosa is greater in the duck than in the domestic fowl (91,96). The prostaglandin-dependent transport would also explain the finding that p,p’-DDE had a more pronounced effect on eggshell formation and calcium metabolism during the early stages of eggshell formation stages, additional transport mechanisms

(81); during later may come into op-

eggshell gland mucosa could be stimulated by progesterone through increased PGH-synthetase activity that results in

eration. It is unclear

an increase in prostaglandin production. The peak increase in the plasma level of progesterone at the time of ovulation

interaction

eggshell gland mucosa. No difference in progesterone

would be a signal, not only for coming eggwhite secretion by the magnum region, but also for activation of ion transport

ing between p,p’-DDE-treated ducks and control ducks has been observed (88). This does not necessarily exclude the

mechanisms in the eggshell gland. However, some authors have suggested that progesterone may in fact act as a depressor of eggshell formation (6) and administration of the progesterone receptor antagonist mi-

possibility that p,p’-DDE has an effect that is exerted through interference with the binding of progesterone to its receptor. At least in the case of the estrogen receptor, the degree of tissue response is dependent not only on the

fepristone was shown to increase eggshell calcium in the domestic hen (6,87). This raises the possibility that p,p’DDE acts as a weak agonist or partial agonist on the proges-

number of hormone-receptor complexes, but also on the extent to which these complexes occupy nuclear binding sites for a sufficient length of time (30). The observed reduction in the weight the eggshell glands from p,p’-DDE-treated ducks (79) indicates that hormonal effects might occur as well.

terone receptor producing the effects of progesterone described in Bar et al. (6), i.e., reduced calcium transport across the eggshell gland and reduced calbindin mRNA levels. P,p’-DDE-induced eggshell thinning was not accompanied by an increase in the length of the egg cycle (79) as was observed after administration of progesterone (6). If p,p’-DDE acts as an agonist or partial agonist on the proges-

whether

of p,p’-DDE

these effects are amplified by an on progesterone

receptors in duck bind-

To reproduce the true physiological effects of exogenously administered steroid hormones, two things must be taken into account: that the correct dose is given and that it is administered at the right time in the ovulatory cycle.

C. E. Lundholm

124

Altered hormonal mic-hypophyseal

balance might interfere with hypothalafunction

the effects of physiological

and give false results regarding concentrations

of the hormones.

is an effect specifically produced by p,p’-

DDE at least at low residue levels that do not cause general toxicity to the birds. There is substantial experimental evidence that p,p’-DDE-induced eggshell thinning involves inhibition of prostaglandin synthesis in the eggshell gland mucosa. This possibility is supported by the following findings: 1. Only p,p’-DDE produces eggshell thinning; p,p’-DDT, o,p’-DDT and o,p’-DDE have no such effect when administered regimens.

to ducks in directly

comparable

treatment

2. p,p’-DDE, but not p,p’-DDT, o,p’-DDT, or o,p’-DDE inhibits prostaglandin synthetase when added in vitro to homogenates of eggshell gland mucosa. The activity of prostaglandin synthetase in eggshell gland mucosa from p,p’-DDE-treated ducks is significantly reduced. 3. The level of prostaglandin from p,p’-DDE-treated

E2 in eggshell gland mucosa

ducks is significantly

reduced.

4. Administration of p,p’-DDE, in a treatment regime comparable to that used for the duck, does not produce eggshell thinning in the domestic fowl. No effect on prostaglandin synthetase activity, prostaglandin Ez content, or the calcium metabolism of the eggshell gland mucosa is observed in this species after treatment with p,p’-DDE. 5. Prostaglandin

synthetase

prepared from eggshell gland

mucosa of the duck is more sensitive to inhibition by p,p’-DDE than the corresponding preparation from the domestic fowl. 6. Indomethacin produces eggshell thinning and about the same effects on the calcium and prostaglandin metabolism of the eggshell gland as p,p’-DDE. 7. The levels of calcium, bicarbonate, chloride, sodium, and potassium in the lumen of the eggshell gland during shell formation

reason might be that the level of p,p’-DDE is insufficient to effectively inhibit the production of the great amount of prostaglandins that are released during oviposition but that the continues production of PGEl that stimulates ion-trans-

CONCLUSIONS Eggshell thinning

the pre- and postovulatory follicles in the ovary, together with contractions of abdominal muscles (70,136). Another

are significantly

reduced in p,p’-DDE-

treated ducks. 8. Prostaglandin E2 probably stimulates calcium transport across the eggshell gland mucosa via a mechanism coupled to bicarbonate transport. Prostaglandin-El-dependent bicarbonate transport has been demonstrated in other organs, for example, gastric and duodenal mucosa. Bicarbonate has been shown to stimulate calcium transport across the eggshell gland mucosa. Since prostaglandins are intimately involved in oviposition, it would be expected that p,p’-DDE would interfere also with egglaying. High doses of p,p’-DDE inhibits egglaying but repeated low-dose administration does not (79). One reason might be that oviposition requires the additional influence of prostaglandins, arginine-vasotocin, and an unidentified oviposition-inducing factor released from

port in the eggshell gland is more effectively p,p’-DDE.

inhibited

by

Inhibition of prostaglandin synthesis in eggshell gland mucosa by p,p.-DDE is a novel mechanism of action of this compound.

Inhibition

of prostaglandin

for this isomer and gives a probable

synthesis is specific explanation

to the

mechanism of action of p,p-DDE-induced eggshell thinning. Based on the presented experimental results, the “prostaglandin hypothesis” gives a better explanation to the mechanism of p,p’-DDE-induced eggshell thinning than the “estrogen-receptor-interaction hypothesis” or the “enzyme-induction hypothesis.” However, a contribution from an interaction possible.

of p,p’-DDE

with progesterone

receptors is

The presence and control of different calcium transport mechanisms, as well as the relative importance of these mechanisms during various stages of eggshell formation and in different species of birds, are of crucial importance in the understanding

of the shell formation

disturbed. Further investigation subjects.

process and how it is

is needed to clarify these

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