Prenatal programming of adult thyroid function by alcohol and thyroid hormones
Jennifer Slone Wilcoxon and
Eva E. Redei
Department of Psychiatry and Behavioral Sciences, Northwestern University Medical School, The Asher Center, Chicago, Illinois 60611
Submitted 15 January 2004
; accepted in final form 22 April 2004
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ABSTRACT
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Increasing evidence associates environmental challenges early in life with permanent alterations of physiological functions in adulthood. These changes in fetal environment can trigger physiological adaptations by the fetus, called fetal programming, which may be beneficial before birth but permanently influence the physiology of the organism. In this study, we investigated the potential connection between alcohol-induced decreased maternal thyroid function and the hypothalamic-pituitary-thyroid (HPT) function of adult rat offspring. Plasma 3,5,3'-triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels were decreased in alcohol-consuming (E) dams on gestational day 21 compared with ad libitum- (C) and pair-fed (PF) controls. No significant differences were found in HPT function in young offspring (3 wk of age) between diet groups. However, adult fetal alcohol-exposed (FAE) offspring had significantly decreased levels of T3 along with elevated TSH compared with control offspring. T4 administration to the mother did not normalize the hypothyroid state of the adult FAE offspring. Interestingly, administration of T4 to control pregnant dams decreased plasma T3 of the adult female offspring only, whereas T4 together with maternal alcohol consumption or pair-feeding led to decreased TSH and T4 in the adult female offspring. Our results suggest that ethanol consumption and T4 administration alter maternal HPT function, leading to prenatally programmed permanent alterations in the thyroid function of the adult offspring.
thyroid-stimulating hormone; 3,5,3'-triiodothyronine; fetal alcohol exposure; thyroxine
THERE IS INCREASING EVIDENCE suggesting that adaptations to the fetal environment that result in low birth weight also "program" physiological changes in the adult. For example, human epidemiological studies suggest that altered maternal nutrition modifies or programs fetal and adult metabolic and endocrine pathways (1, 7, 40). Maternal malnutrition often coincides with alcohol abuse, in which the direct effect of alcohol exposure on the fetus is superimposed on that of malnutrition. Fetal alcohol exposure in humans leads to an array of developmental consequences, including hyperactivity and learning deficits (33), symptoms similar to those observed in congenitally hypothyroid children (18, 26, 35) and those of hypothyroid mothers (19). Human alcoholic patients generally have decreased levels of thyroxine (T4) (16, 24), and decreased plasma thyroid-stimulating hormone (TSH) is found in newborns exposed to alcohol in utero (25). However, others found no change in infant T4 levels in response to prenatal alcohol exposure (22).
It has been proposed that some of the behavioral and perhaps hormonal consequences of prenatal ethanol exposure are due to the effect of ethanol on maternal and fetal thyroid function (17, 49). However, in animals, the findings are conflicting: no change has been shown in peripheral T4 levels of alcohol-consuming dams (31), whereas others have found decreased maternal TSH in rats (13, 31) and decreased maternal 3,5,3'-triiodothyronine (T3) in sheep (10). Neonatal and young fetal alcohol-exposed (FAE) rat offspring exhibit decreased serum T4 levels (21, 43).
There is little information to date on the hypothalamic-pituitary-thyroid (HPT) function of the adult FAE offspring. Thyroid hormones play an important role in the development of the central nervous system. Altered levels of thyroid hormones in the alcohol-consuming mother likely affect the fetal and adult HPT function of the FAE offspring. Indeed, in rats, perinatal hypothyroidism (12, 44) and hyperthyroidism (41, 44) have been shown to alter thyroid function of the adult offspring, but no study to date has systematically investigated the long-term effect of altered maternal thyroid milieu on the adult offspring. Therefore, the purpose of this study was twofold. First, we determined the thyroid status of the alcohol-consuming rat dam and fetus, and how maternal alcohol consumption affected the HPT function of the adult offspring. Then, we investigated the effect of prenatal thyroid hormone treatment in alcohol-consuming mothers on the HPT function of the mother and the adult offspring. We found that adult thyroid function is developmentally regulated by maternal hormonal milieu. Furthermore, the present results demonstrate that, although programming of adult thyroid function occurs in utero, it seems to be manifested only in the mature adult offspring, suggesting the presence of a secondary programming by sex hormones during puberty.
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MATERIALS AND METHODS
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Animals.
All animal procedures were approved by the Northwestern University Animal Care and Use Committee. Adult male and female Sprague-Dawley rats (viral free, 5658 days of age; Harlan, Indianapolis, IN) were housed individually in a temperature- and humidity-controlled vivarium with regular light-dark cycles (light on at 700 and light off at 1900). After 7 days of acclimatization, rats were mated by placing a female in the male cage overnight. Mating was confirmed by microscopic analysis of vaginal smears for the presence of sperm the next morning. The day sperm was found was designated as gestational day 1 (G1).
Experiment 1a.
Pregnant rats were assigned to three experimental groups on G8. Group 1 (E, n = 6) was administered an ethanol liquid diet (Lieber-DeCarli '82, BioServ) adjusted for pregnant rats, which contains 5% (wt/vol) ethanol (35% ethanol-derived calories), and was supplemented with essential vitamins and minerals. The ethanol diet was introduced in stages: 15% ethanol-derived calories was increased to 35% in 5 days, as described previously (51). Group 2 was pair-fed (PF, n = 6) a Lieber-DeCarli '82 isocaloric liquid diet for pregnant rats that contains no ethanol, and group 3 (C, n = 6) was fed lab chow and water ad libitum. The amount of the PF diet presented to the pregnant dams was based on the alcohol diet consumption of the E dams of similar weight on the previous day. Liquid diets were presented daily between 1600 and 1700, and daily intake of diet was recorded. The amount of ethanol-diet consumption is not different from the alcohol intake of E dams reported previously, which produced blood ethanol levels of 80 mg/100 ml (51). Pregnant dams were killed on G21 between 1000 and 1200, and control and experimental animals were time-matched to rule out any diurnal variation in hormone levels. Maternal and fetal blood samples were collected. Plasma TSH, T3, and T4 were measured in individual maternal plasma samples, but only TSH and T4 were measured in fetal plasma, which required pooling the fetal samples within litter by sex.
Experiment 1b.
As in experiment 1a, pregnant rats were assigned to the same three treatment groups (C, n = 5; PF, n = 5; E, n = 5). The diet administration protocol was identical, except on G21 the diets were replaced with laboratory chow and water ad libitum, and the rats were allowed to deliver. Maternal daily alcohol consumption was not different from that in experiment 1a. Pups were weaned at 21 days of age and group-housed by sex and treatment.
At 90100 days of age, adult male and female offspring from all prenatal treatment groups were killed by decapitation under basal conditions between 1000 and 1400, and control and experimental animals were time-matched to rule out any diurnal variation in hormone levels. Trunk blood samples were collected into chilled tubes containing EDTA (0.25 mg/ml whole blood) and kept frozen (80°C) in aliquots.
Experiment 2a.
Pregnant rats were divided into six groups: C, PF, E, C+T4, PF+T4, and E+T4 (n = 5/group). The diet administration protocol for the C, PF, and E groups was identical to that described in experiment 1a. Beginning on G8, the T4 dams received thyroxine (30 µg/ml, Sigma Chemical, St. Louis, MO) in the PF or E liquid diet or in the drinking water for the C+T4 mothers until G18. This thyroid hormone treatment has been shown to alter behavior in adult offspring (11). The average daily diet consumption was 101 ± 4.7 ml, so the dams received
3 mg of T4 per day. Pregnant dams were killed on G18, as described in experiment 1a, and maternal blood samples were collected (n = 5/group).
Experiment 2b.
Pregnant rats were assigned to the same six treatment groups described in experiment 2a (C, PF, E, C+T4, PF+T4, E+T4; n = 5/group). The diet administration protocol was identical, except that on G21 the diets were replaced with laboratory chow and water ad libitum, and the rats were allowed to deliver.
To determine whether the hypothyroid state of adult FAE offspring found in experiment 1b is present throughout development, we measured the HPT function of young offspring in addition to adults. On postnatal day 7 and then again on postnatal day 21, one male and one female from each litter were killed by decapitation, and trunk blood samples were collected as described in experiment 1b.
At 90100 days of age, the remaining adult male and female offspring from all prenatal treatment groups were killed by decapitation. Trunk blood samples were collected as in experiment 1b.
Radioimmunoassays.
All samples were assayed in duplicate, in a single assay when possible.
TSH was measured as described before (45); standards and specific antiserum were obtained from the National Hormone and Pituitary Agency (National Institute of Diabetes and Digestive and Kidney Diseases, Baltimore, MD). Rat TSH RP-2 was used for the iodination and standards. The assay sensitivity was 0.09 ng/ml, and the intra- and interassay coefficients of variation (CV) were 8.1 and 6.7%, respectively.
RIAs for total T3, free T3, free T4, and total T4 were performed using ImmunoChem-coated tubes purchased from ICN Pharmaceuticals (Carson, CA) and following the recommended protocol provided with each kit. Sensitivity of the total T3 assay was 24.7 ng/dl, and the intra-assay CV was 3.6%, with an interassay CV of 6.1%. The free T3 assay sensitivity was 0.8 pg/ml, and the intra-assay CV was 4.2% with an interassay CV of 5.5%. As for total T4, the sensitivity limit was 0.5 µg/dl, with interassay and intra-assay CVs of 4.1 and 5.2%, respectively. For free T4, the assay sensitivity was 0.5 ng/dl, and the intra-assay CV was 5.2% with an interassay CV of 7.3%.
Corticosterone (Cort) concentrations were measured as described previously (52) in unextracted plasma using 125I-labeled Cort RIA (ICN Biomedicals, Carson, CA). For Cort, the assay sensitivity was 16.7 pg/tube. The intra- and interassay CVs were 11.6 and 7.5%, respectively.
Statistics.
The data were analyzed by ANOVA, either a two-way (sex and diet, in experiments 1a and 1b) or a three-way design (sex, diet, and T4 treatment in experiments 2a and 2b). Litter was a nested factor. The Tukey least significant difference test, with a P < 0.05, was used as a post hoc test to locate significant differences among groups. ANOVA results are discussed in RESULTS, and post hoc analyses are shown in Figs. 14.

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Fig. 1. Plasma 3,5,3'-triiodothyronine (T3) and thyroxine (T4) levels in control (C), pair-fed (PF), and ethanol-consuming (E) mothers (n = 6/group) on gestational day 21. Values are means ± SE. *P < 0.01 vs. C and PF.
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Fig. 4. A: plasma TSH and free T3 levels in C, PF, FAE, C+T4, PF+T4, and FAE+T4 adult female offspring at 90100 days of age (n = 68/group). Values are means ± SE of 5 litters/treatment group. *P < 0.01 from C female offspring (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect). B: plasma TSH and free T3 levels in C, PF, FAE, C+T4, PF+T4, and FAE+T4 adult male offspring at 90100 days of age (n = 68/group). Values are means ± SE of 5 litters/treatment group. *P < 0.01 from C male offspring (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect). C: plasma free T4 values for animals described in A and B. Values are means ± SE. *P < 0.05 from C offspring (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect).
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RESULTS
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Diet consumption, maternal and fetal weight, and litter size.
There were no significant differences in the amount of diet consumed by the pregnant dams, regardless of the presence of ethanol or T4 in the diet. The BioServ diets are formulated in such a way that both ethanol and pair-fed diets contain 1 kcal/ml.
Therefore, the volume of liquid diet consumed daily also provides a measure of caloric intake. Diet consumption, maternal weight, and litter size of animals in experiments 1 and 2 have been previously reported (61). Because the current study includes measurements of thyroid function on these animals, we included these data in Table 1 for the sake of completeness. Dams in the ethanol groups consumed 95.2 ± 2.3 ml/day, and dams in the pair-fed groups consumed 104.2 ± 2.7 ml/day of their respective isocaloric diets. In experiment 2, pair-fed dams consumed 102 ± 5.1 ml/day, and ethanol dams consumed 97 ± 6.4 ml/day, and the PF+T4 mothers had 101 ± 4.8 ml/day and the E+T4 mothers 97 ± 7.7 ml/day. The addition of T4 led to significant (P < 0.05) differences in the weight of ethanol dams vs. pair-fed and control dams, and significant differences in weight were found between pair-fed and control dams in experiments 1 and 2 (Table 1). Similar to previous results from our laboratory (61), maternal ethanol consumption led to significantly (P > 0.01) decreased fetal body weight of both males and females compared with control and pair-fed groups (Table 2). At the time animals were killed as adults, there were no significant differences in body weight among the treatment groups, only the typical sex difference (data not shown).
There were no significant differences in fetal litter size, but the size of the surviving ethanol litters was significantly smaller in all experiments (Table 1), a finding similar to data previously reported by our laboratory (61). This difference in litter size occurs between G21 and postnatal day 1 and could be due to stillbirth, a greater death rate of newborns in ethanol litters, or both.
Experiment 1a.
Maternal and fetal blood was collected under basal conditions on G21. Ethanol mothers on G21 had significantly decreased plasma T3 and T4 levels compared with control and pair-fed mothers (Fig. 1), but the decrease in their TSH levels did not reach statistical significance (P = 0.08, data not shown); [T3: main effect of diet: F(2,16) = 16.5; P < 0.001; T4: main effect of diet: F(2,18) = 5.0; P < 0.01]. There were no differences in plasma TSH levels between control, pair-fed, and FAE fetuses (data not shown), and plasma T4 was undetectable in the fetal blood at this time point.
Experiment 1b.
In this experiment adult offspring were evaluated. Basal plasma TSH levels (Fig. 2A) were significantly increased in males compared with females except in FAE animals [main effect of sex: F(1,41) = 11.3; P < 0.01; sex x diet: F(1,41) = 3.8; P < 0.05]. Plasma TSH levels were significantly affected by the prenatal diet [main effect of diet: F(1,41) = 9.8; P < 0.001]. Specifically, FAE females, and both FAE and pair-fed males had significantly (P < 0.05) higher levels of TSH compared with their appropriate controls. Total T3 levels were significantly decreased in both FAE males and females (Fig. 2B), whereas no differences were found in plasma T4 values (Fig. 2C) between diet groups [T3: main effect of diet: F(1,40) = 3.3; P < 0.05].

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Fig. 2. A: plasma thyroid-stimulating hormone (TSH) values in C, PF, and fetal alcohol-exposed (FAE) adult offspring (n = 68/sex/diet) at 90100 days of age. One or two male and female offspring per litter were randomly taken from 5 litters per treatment group. Values are means ± SE. *P < 0.01 from C. B: plasma T3 levels in adult offspring described in A. Values are means ± SE. *P < 0.01 from C. C: plasma T4 values in adult offspring described in A. Values are means ± SE.
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Experiment 2a.
In rats, it has been shown that there is significant maternal to fetal transfer of thyroid hormone from G10 to G17, before the onset of fetal thyroid hormone secretion (34). Ethanol may affect the developing fetal HPT axis via suppressing the maternal HPT axis. Therefore, we administered thyroid hormone replacement to the dams during gestation to determine whether this treatment normalizes the maternal and adult offspring HPT abnormalities after prenatal alcohol exposure.
As in experiment 1a, significantly decreased maternal total T4 (Fig. 3A) and total T3 (Fig. 3B) were found in alcohol-consuming dams on G18 [main effect of diet, T4: F(2,31) = 13.6; P < 0.001; T3: F(2,31) = 8.5; P < 0.001]. Free thyroid hormone levels, both T3 and T4, followed the same pattern [Fig. 3, A and B; main effect of diet, free T4: F(2,31) = 11.6; P < 0.001; free T3: F(2,21) = 14.4; P < 0.001]. However, in this experiment, ethanol dams showed significantly decreased plasma TSH levels as well [Fig. 3C; main effect of diet, F(2,31) = 23.9; P < 0.001].

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Fig. 3. A: plasma total and free T4 values for C, PF, E, control+T4 (C+T4), pair-fed+T4 (PF+T4), and ethanol-consuming+T4 (E+T4) dams on gestational day 18 (n = 5/group). Values are means ± SE. *P < 0.01 from C group (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect). B: maternal plasma total and free T3 values for dams described in A. Values are means ± SE. *P < 0.01 from control dams (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect). C: plasma TSH levels for mothers described in A. Values are means ± SE. *P < 0.01 from C dams (diet effect); #P < 0.01 from respective non-T4-treated group (treatment effect). D: plasma corticosterone (CORT) levels for dams described in A. Values are means ± SE. *P < 0.01 from C dams (diet effect).
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Maternal plasma total and free T4 levels (Fig. 3A) were significantly increased in all three T4-treated groups compared with their respective non-T4-treated "controls" [main effect of treatment, total T4: F(2,31) = 16.3; P < 0.001; free T4: F(2,31) = 12.4; P < 0.001]. However, maternal treatment with T4 increased plasma total and free T3 only in T4-treated pair-fed and ethanol dams [Fig. 3B; total T3, diet x treatment: F(2,31) = 6.2; P < 0.01; free T3, diet x treatment: F(2,29) = 15.8; P < 0.01]. As expected, prenatal T4 treatment significantly decreased maternal TSH (Fig. 3C) regardless of prenatal diet, but the addition of T4 to the restricted pair-fed and ethanol diets led to an even greater decrease in plasma TSH than in the C+T4 group, as shown by post hoc analysis [main effect of treatment: F(2,32) = 5.4; P < 0.01; diet x treatment: F(3,32) = 3.8; P < 0.05].
We have previously shown that alcohol consumption leads to elevated maternal plasma Cort levels (51), and elevated Cort has been shown to suppress plasma TSH levels (6). Therefore, to determine whether the decreased TSH levels of alcohol-consuming dams were, at least in part, related to suppression by Cort, we measured maternal plasma Cort levels. Plasma Cort values (Fig. 3C) were significantly increased in ethanol dams compared with control and pair-fed dams, as previously found in our laboratory (51), and the addition of T4 to the prenatal liquid diets significantly increased maternal plasma Cort values in both PF+T4 and E+T4 groups compared with the C+T4 group [diet x treatment: F(1,31) = 3.1; P < 0.05].
Experiment 2b.
In this experiment, the effect of maternal alcohol consumption and T4 administration on the HPT function of the developing and adult offspring was evaluated. No significant differences were found in TSH and total T4 in FAE young offspring compared with controls at two different time points, postnatal day 7 and postnatal day 21 (Table 3). There were also no significant decreases in plasma TSH in the T4-treated pups on postnatal day 7 (P = 0.1) or on postnatal day 21 (P = 0.4). Levels of T3 were not measured because of insufficient plasma in the young animals.
In adult offspring, plasma TSH levels (Fig. 4, A and B) were significantly lower in females compared with males [main effect of sex: F(1,45) = 7.5; P < 0.001]. Both FAE females and males had significantly elevated TSH compared with pair-fed and control offspring [main effect of diet: F(1,45) = 4.1; P < 0.05]. Corresponding to their elevated plasma TSH, plasma total T3 (Table 4) and free T3 (Fig. 4, A and B) levels were significantly decreased in both male and female FAE offspring compared with their respective control and pair-fed groups [main effect of diet, total T3: F(1,45) = 11.4; P < 0.001; free T3: F(1,45) = 9.8; P < 0.01].
Prenatal T4 treatment altered adult HPT function in a sex- and diet-dependent manner. T4 administration in utero decreased TSH levels in pair-fed and FAE females only [sex x treatment: F(1,45) = 5.34; P < 0.001] and significantly decreased total T3 levels of adult offspring, particularly in the pair-fed and control females [main effect of treatment: F(1,45) = 4.5; P < 0.01]. However, T4 administration did not alter the already lower levels of total T3 in the FAE offspring. A similar pattern was found for plasma free T3 levels [Fig. 4, A and B; main effect of treatment: F(1,45) = 3.9; P < 0.05]. Post hoc analysis demonstrated that plasma total T4 levels (Table 4) were significantly reduced in the PF+T4 and FAE+T4 females only [sex x treatment: F(1,45) = 6.01; P < 0.01]. Plasma free T4 values were also significantly reduced in PF+T4 and FAE+T4 females, whereas no differences were found in the males [Fig. 4C; sex x treatment: F(1,45) = 5.74; P < 0.01].
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DISCUSSION
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The main findings of the present study suggest that prenatal environment permanently imprints adult thyroid function. In our model of fetal alcohol exposure, alcohol-consuming pregnant dams demonstrated suppressed HPT function, which subsequently led to hypothyroidism in the adult FAE offspring. The administration of T4 to the pregnant dams did not normalize the attenuated pituitary-thyroid function of alcohol-consuming mothers or the hypothyroid status of their offspring. Unexpectedly, this treatment resulted in altered HPT function of the offspring depending on sex and age, suggesting that adult thyroid function is programmed in utero but that this programming might be activated by sex steroids in the adult animal.
Maternal HPT function in response to alcohol consumption and T4 administration.
Alterations in maternal hormonal milieu by these manipulations are summarized in Table 5. Suppressed maternal HPT function was consistently found in ethanol dams in both experiments. The finding that plasma free T3 and free T4 are decreased along with total T3 and T4 in FAE dams suggests that alterations in binding proteins are not responsible for the decreased T3 and T4 levels. These findings also indirectly suggest that the 2-wk alcohol exposure paradigm did not provoke any major hepatic changes, as reported previously (23).
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Table 5. Directional differences in maternal hormones compared with controls in response to alcohol or alcohol and T4 administration
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Our reproducible results of decreased maternal T3, T4, and TSH mirror findings in alcohol-consuming pregnant women. The origin of this suppressed HPT function in the alcohol-consuming mother is not known, but it may be related to the TSH or TRH-suppressing effects of alcohol-induced elevated glucocorticoids (51). Previous studies have indicated that cortisol can suppress basal TSH secretion and reduce sensitivity of the thyrotrophs in humans (20, 39) and animals (50). Although we found no change in TSH levels of the FAE fetus, changes in maternal thyroid hormone levels in the rat can play a role in fetal thyroid economy both before and after the onset of fetal thyroid function (34).
The combination of T4 administration and the alcohol- or food-restricted pair-fed diet led to an exaggeratedly hyperthyroid maternal hormonal milieu compared with the T4 administration and diet of C+T4 mothers. Interestingly, the dramatically increased T3 levels of these E+T4 and PF+T4 dams were concomitant with increased plasma Cort levels. These increased Cort levels may have directly caused the maternal HPT responses to T4 administration or, alternatively, conversion of T4 to T3 is differently regulated in the ethanol-consuming and pair-fed dams.
Previous studies have shown that pair-feeding, in addition to providing an essential nutritional control condition for the decreased food intake of the alcohol-consuming dams, may serve as an experimental treatment in itself (15). It has been proposed that pair-fed dams may experience stress derived from their restricted meal-feeding schedule (27). Fasting affects the HPT axis, leading to lower serum T3 and T4 levels, low-to-normal TSH values (32, 47), and lower transthyretin and albumin (54). Thus, in the current study, the pair-fed dams may have experienced a pair-feeding-induced fasting/starvation that in turn affected their HPT function and the programming of the HPT axis of their offspring. This phenomenon of pair-feeding has been found to affect hypothalamic-pituitary-adrenal (HPA) function in offspring of pair-fed dams in some studies, but not in others (27). Interestingly, in the present study, only one experiment showed elevated plasma TSH in pair-fed male offspring. The possibility that the consequences of prenatal ethanol exposure on the adult offspring are a composite effect of FAE and food restriction cannot be excluded.
Restricted feeding or alcohol exposure, coupled with T4 treatment of pregnant dams, may also alter the conversion of T4 and/or T3 in these mothers. The rat thyroid gland is the source of
50% of circulating T3, with the rest generated via extrathyroidal deiodination of T4 by the 5'-deiodinase enzymes (9, 48) type 1 (D1) and type 2 (D2). In the rat, nearly all circulating T3 derived from T4 appears to be produced by D1 (9). D1 is positively regulated by T3 (57) but negatively regulated by glucocorticoids (28) or fasting (36). Thus it is not likely that the increased T3 in PF+T4 and FAE+T4 dams is associated with the elevated levels of plasma Cort in the pair-fed and ethanol dams, because Cort is a negative regulator of D1 activity. Alternatively, D2, the main enzyme in the brain, pituitary, and placenta, which is stimulated by glucocorticoids in a number of tissues (3, 5, 59), may be responsible for elevated T3, at least in the placenta and pituitary. This increased placental T3 should be metabolized by placental or fetal type 3 iodothyronine deiodinase (D3), which protects the fetal brain and other tissues from excessively high levels of maternal thyroid hormones (29, 53). D3 is a T3-regulated gene (53, 58), so D3 mRNA levels are increased during hyperthyroidism (38), reducing the levels of biologically active T3. Therefore, it is feasible that elevated maternal plasma Cort levels may negatively regulate placental D3 activity and, in turn, program the development of the fetal HPT axis via elevated fetal T3. The role of specific deiodinases in the HPT function of the developing or adult offspring exposed to varying maternal glucocorticoid and thyroid hormonal milieu clearly merits further investigation.
Adult offspring HPT function in response to prenatal alcohol exposure.
The effects of maternal treatments on the HPT activity of the adult offspring are summarized in Table 6. The suppressed maternal HPT activity resulted in consistently elevated TSH in adult FAE males and females. However, the distinct hypothyroid profile of elevated TSH and decreased T4 and/or T3 was not consistently observed. Specifically, T4 seemed to be resistant to prenatal alcohol exposure as well as most maternal treatments, whereas T3 was consistently reduced in FAE offspring. Although pair-feeding alone had no effect on maternal HPT function, it resulted in elevated TSH levels in adult pair-fed males only. These findings suggest that, even in the absence of discernible changes in maternal hormonal milieu, food restriction can affect hormonal function of adult offspring, particularly males.
The effect of maternal alcohol consumption on the adult offspring's HPT axis seems to mimic a hypothyroid state. The cause and mechanism of this dysregulation are not known. Increased TSH levels found previously in FAE pups (13) and in the FAE adult offspring of this study are not likely due to enhanced stimulation by TRH, as no change from controls in hypothalamic TRH content was reported in ethanol-exposed pups (13). More likely, the TSH elevation in the FAE adult offspring may be in response to lower free T3 levels, even in the presence of normal T4 levels, because low free T3 is a potent stimulator of TSH secretion. The cause and mechanism of the hypothyroid state in the adult FAE offspring require further examination.
Effect of prenatal T4 treatment on the HPT axis of adult control, pair-fed, and FAE offspring.
The rationale for the prenatal T4 administration was that some of the deleterious effects of maternal hypothyroidism on the offspring thyroid function can be reversed by maternal thyroid hormone replacement (8, 11, 42). However, T4 administration to the alcohol-consuming mother did not reverse the hypothyroid state of the FAE offspring in this study. Thus, although some consequences of alcohol exposure in utero may be mediated by the thyroid hormonal milieu of the developing fetus or neonate (17), the complex effects of ethanol on the developing fetus likely occur via other systems in addition to the thyroid hormonal milieu of the mother.
Unexpectedly, adult HPT function was suppressed by the combination of maternal alcohol consumption or food restriction and T4 treatment in a sexually dimorphic manner. This suppressed HPT function is similar to findings of early postnatal T4 administration (14), even though this treatment suppressed the HPT function in both males and females. In contrast, in the current study, suppressed HPT function was found only in the pair-fed and FAE females in response to prenatal T4 treatment. This female specific vulnerability has been found previously for HPA function of offspring exposed prenatally to alcohol (56, 60) or to stress (30, 55).
The lack of effect of maternal T4 treatments and different diet conditions on the young offspring is intriguing and proposes a testable hypothesis. Namely, is there an activational effect of sex steroids on the HPT function that is similar to that found for the behavioral effects of steroids (4, 46)? Previous studies from our laboratory indicate that there is an organizational effect of alcohol exposure in utero on the developing HPA axis that is manifested in response to developmental effects beyond weaning (2). Others have found underlying activational effects of sex steroids in the gender specificity of the neural regulation of stress responsiveness (37). On the basis of results of this study, activational effects of sex steroids could mediate the sexually dimorphic effects of prenatal alcohol and T4 treatment on adult HPT function.
In summary, the present study identified a significant role for maternal thyroid hormonal milieu and nutritional status in the basal activity of the adult HPT axis. The mechanism by which this prenatal programming occurs is not yet known, but it is likely via alterations in fetal HPT function that are manifested in a sex-specific manner in the offspring after puberty. Further studies are necessary to determine the mechanism of vulnerability of the fetus to prenatal environmental insults, such as alcohol or malnutrition, as well as the mechanism of programming that is manifested in adulthood. Although the link between our rat data and comparable effects in humans is speculative, animal studies of thyroid function may provide mechanistic value to human conditions.
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GRANTS
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This research was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA-07389 (E. Redei) and AA-05587 (J. Slone Wilcoxon).
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ACKNOWLEDGMENTS
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We thank Dr. Peter Kopp and Kelsey Budd for reviewing the manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. Slone Wilcoxon, 303 E. Chicago Ave., Ward 9-190, Chicago, IL 60611 (E-mail: jwilcoxo{at}bsd.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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