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Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia, 6907, Australia
Address all correspondence and requests for reprints to: Dr. Brendan J. Waddell, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Western Australia 6907, Australia. E-mail: bwaddell{at}anhb.uwa.edu.au
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Abstract |
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Introduction |
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Exposure of the fetus to endogenous maternal glucocorticoids is normally limited by placental metabolism via the 11ß-hydroxysteroid dehydrogenase (11ß-HSD) enzymes that form the placental glucocorticoid barrier (for review see Ref. 11). Placental 11ß-HSD catalyses the interconversion of active and inactive forms of glucocorticoids, specifically corticosterone and 11-dehydrocorticosterone (DHC) in the rat. The physiological importance of this placental glucocorticoid barrier is highlighted by the observation that placental 11ß-HSD activity is positively correlated with birthweight in the rat (5). Moreover, treatment of adrenal-intact pregnant rats with the 11ß-HSD inhibitor carbenoxolone reduces birthweight and leads to development of hypertension (6) and hyperglycemia in adult life (7). In the present work, therefore, the timing of puberty onset was measured in male and female rats that had been subjected to excess glucocorticoid exposure in utero by maternal treatment with either dexamethasone (which is poorly metabolized by 11ß-HSD) or carbenoxolone from day 13 of pregnancy (term = day 23). The onset of puberty was also determined following reduced fetal glucocorticoid exposure effected by treatment of pregnant mothers from day 13 with metyrapone, an inhibitor of glucocorticoid synthesis. All offspring were cross-fostered to untreated mothers at birth, and plasma levels of leptin, the product of the ob gene produced by adipocytes (12, 13), and LH were measured before and after puberty onset. It was hypothesized that any alteration in the timing of puberty may be mediated via changes in leptin because this hormone provides a key signal for puberty onset (14, 15) and its secretion and action are regulated by glucocorticoids in adults (16, 17, 18).
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Materials and Methods |
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Experimental treatments
Metyrapone (MET) treatment was administered from day 13 of
pregnancy to term in physiological saline (in place of drinking water)
at a concentration of 500 µg/ml, which has previously been shown to
enhance fetal growth (19). Dexamethasone acetate was administered in
drinking water at two different doses (HDEX: 1 µg/ml; LDEX: 0.25
µg/ml) over the same period. Carbenoxolone was administered twice
daily (10 mg in 4% ethanol-saline, 0.1 ml sc injection at 0700 h
and 1730 h) to intact (CBX) and adrenalectomized (CBX/ADX)
pregnant rats from day 13 to term. Adrenalectomy had been performed via
two dorso-lateral incisions 2 days earlier (day 11 of pregnancy) under
halothane:nitrous oxide anesthesia. An additional group of otherwise
untreated adrenalectomized rats (ADX) was included to assess the
effects of adrenalectomy alone; both adrenalectomized groups were
provided with physiological saline in place of drinking water.
Litter management
Within 12 h of birth, offspring sex was identified by
examination of external genital morphology and males and females
weighed separately. Within 24 h of birth, each litter was
cross-fostered to an untreated mother, including one untreated group
(CONf); in a second untreated control group (CONnf) pups remained with
their own mother to assess any effect of fostering alone. Offspring
were subsequently separated from mothers after pup independence was
clearly evident (at approximately 4 weeks) and litters were weighed
weekly from birth to week 8. Individual rats were also weighed at the
time of puberty onset.
From 30 days of age all offspring were examined daily for changes in genital morphology. Puberty onset in females was defined as the time of vaginal opening (20), and in males as the capacity for preputial separation, determined by manual retraction of the prepuce (21). Once deemed to have reached puberty, each rat was labeled and weighed. To give an indication of fertility in female offspring, vaginal smears were obtained (from approximately 80 days of age) for three consecutive estrous cycles from one member of each litter of control and MET groups and all groups in which puberty was delayed (LDEX, HDEX, CBX).
Blood sampling and tissue collection
Blood samples (2 ml) were collected from offspring at age 31
days (prepubertal) and 52 days (postpubertal) for subsequent
measurement of plasma leptin and LH. One male and one female were
randomly chosen from each litter, anesthetized with halothane and a
blood sample obtained from the dorsal aorta into a heparinized syringe.
Samples were centrifuged at 11,000 rpm for 5 min and plasma was stored
at -20 C until assay.
RIAs
Plasma leptin and LH concentrations were measured in pre- and
postpubertal blood samples using specific RIAs; leptin was assayed
using a kit supplied by Linco Research, Inc. (St. Charles,
MO), and LH using materials from The National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK-rLH-I-9 antigen;
NIDDK-anti-rLH-S-11 antiserum; NIDDK-r-LH-RP3 for reference
preparation) as previously described (22).
Statistical analysis
All data are expressed as mean ± SEM, with
each litter representing an ‘n’ of one, and most comparisons were
made by single factor analyses of variance (one-way ANOVAs). When the
F-test for the ANOVA reached statistical significance
(P < 0.05), differences among specific means were
assessed by least significant difference (LSD) test (23). The effects
of age, sex and treatment on offspring plasma leptin and LH levels were
assessed by three-way ANOVAs. Treatment and age effects on offspring
body weight were assessed separately for males and females by two-way
ANOVA of logarithm-adjusted values (due to unequal variance among
groups). To account for nonnormality of data, variation in the sex
ratio among groups was analyzed using arcsine transformed data.
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Results |
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). Specifically, compared with the CONf
group, birthweight was reduced by maternal dexamethasone treatment
(LDEX: 9%, P < 0.05; HDEX: 27%, P <
0.01, LSD tests), CBX (8%, P < 0.05) and maternal ADX
alone (5%, P < 0.05), but was increased by MET
treatment (5%, P < 0.05). The effect of CBX required
intact maternal adrenals, since no reduction in birthweight was
observed in offspring of CBX/ADX mothers.
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) but not
thereafter, except for elevated weight in the CBX/ADX group at weeks 6
and 7 (see Fig. 2). Male offspring weight also varied with treatment
(P < 0.001) and age (P < 0.001), but
unlike observations in females there was no significant interaction
(P = 0.82).
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).
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), being delayed in
offspring of LDEX (44.8 ± 0.7 days of age), HDEX (48.5 ±
0.4) and CBX (43.6 ± 0.5) compared with both control groups
(CONf: 41.4 ± 0.5; CONnf: 41.0 ± 0.4) (P <
0.05, LSD test). The delay in puberty following CBX treatment was not
observed in ADX mothers (40.8 ± 0.9), and neither ADX alone
(41.1 ± 0.4) nor treatment with MET (41.1 ± 0.8) had any
effect on puberty onset. Weight at puberty in females also varied among
groups (P < 0.01), being clearly higher in those that
showed delayed puberty onset (see Fig. 3b). After puberty, estrous
cycle length was 4 days in effectively all offspring except those from
MET-treated mothers, in which 4 of 6 animals exhibited cycles longer
than 4 days (4.33 ± 0.12 days; group mean ±
SEM).
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). Although there appeared to be a trend for delayed
puberty in the DEX and CBX groups, this did not reach statistical
significance. Weight at puberty in males also varied among groups
(P < 0.01), but specific differences did not appear to
be directly linked to the timing of puberty (see Fig. 3b).
Effects of age, sex, and treatment on postnatal plasma
leptin
Offspring plasma leptin concentrations varied with treatment
(P < 0.001), sex (P < 0.001) and age
(P < 0.001) and there was significant interaction
between age and sex (P < 0.001; see Table 2). Analysis of male leptin
concentrations demonstrated a clear effect of age (e.g.
increasing 2-fold in the CONf group from pre- to postpuberty,
P < 0.001), whereas only a marginal increase was
observed in females (P < 0.05). Accordingly, plasma
leptin levels were similar in males and females before puberty
(P = 0.9), but were considerably higher in males after
puberty (P < 0.001). The age effect on leptin was
apparent regardless of treatment (i.e. no significant
interaction between treatment and age effects). Plasma leptin also
varied among treatment groups before puberty in both sexes, and after
puberty in males. In each case this variation primarily reflected
higher leptin levels in the offspring of CBX/ADX mothers
(P < 0.05, LSD-test).
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). The major treatment effects
on plasma LH were observed in male offspring, primarily reflecting
higher levels in the MET group. Regardless of treatment, plasma LH was
consistently higher in males than females, and in both sexes plasma LH
increased with age. Although plasma LH and leptin both increased with
age, analyses of covariance at each age in each sex demonstrated no
significant overall relationship (data not shown). |
Discussion |
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Fetal exposure to maternally administered dexamethasone provided a potent and dose-related delay in the timing of puberty in female offspring. Dupouy et al. (24) had previously shown that dexamethasone acetate administered in drinking water reduces fetal corticosterone levels, indicative of transplacental dexamethasone passage. Our data provide the first direct evidence that abnormally high concentrations of prenatal glucocorticoids program a delay in the subsequent timing of puberty. Previous studies had established that excess fetal glucocorticoid exposure can predispose to certain disease states including hypertension (5, 6); the present work extends these observations to show that glucocorticoid exposure can influence the timing of normal postnatal development. Because dexamethasone exhibits high biological potency relative to endogenous glucocorticoids, it was important to ascertain whether maternal glucocorticoids could also influence the timing of puberty in offspring. This was examined by treatment of mothers with carbenoxolone, an inhibitor of the glucocorticoid-metabolizing enzyme 11ß-HSD, placental expression of which inactivates maternal glucocorticoids during their passage to the fetus and thereby constitutes a placental glucocorticoid barrier (for review see Ref. 11). Inhibition of placental 11ß-HSD with carbenoxolone, which has been shown to enhance passage of maternal corticosterone to the fetal compartment (7), reduced birthweight as previously reported (7), and most importantly mimicked the effect of dexamethasone on the timing of puberty onset in female offspring. Although maternally administered carbenoxolone is likely to reach the fetus, any direct effect on the subsequent timing of puberty seems unlikely since its effects on both birthweight and puberty onset were observed only in the offspring of adrenal-intact mothers. Moreover, although control animals in the present study received no sham treatment, we have recently reported that twice-daily sham injections over the same treatment period had no effect on placental or fetal growth (25).
Reduced exposure of the fetus to endogenous glucocorticoids (by maternal metyrapone treatment) was also shown to enhance birthweight, and this subsequently led to advanced puberty onset in males. Metyrapone reduces endogenous glucocorticoid synthesis via inhibition of 11ß-hydroxylase, and this effect occurs in the adrenal of both mother and fetus (26). The resultant, minimal fetal glucocorticoid exposure is thus likely to account for the enhanced fetal growth and subsequent advanced puberty in males. In contrast, fetuses of adrenalectomized mothers are likely to be exposed to higher glucocorticoid levels (due to fetal adrenal compensatory hypertrophy; 26) than those of metyrapone-treated mothers, and this may account for the slight reduction in birthweight following maternal adrenalectomy. A similar effect of maternal adrenalectomy on birthweight was recently reported by Lindsay et al. (7).
Two crucial aspects of the experimental design in the present study were the measurement of birthweight and the process of cross-fostering. The reductions in birthweight, independent of litter size and sex ratio, observed in those groups in which fetal glucocorticoid exposure was increased confirm the efficacy of the treatments because similar birthweight reductions were previously observed after carbenoxolone (7) or dexamethasone (5) treatment. Conversely, birthweight was elevated after metyrapone treatment, similar to previously reported effects of metyrapone on fetal growth (19). The inclusion of cross-fostering in the experimental design ensured that all treatment effects were restricted to the prenatal period of life, with theoretically no difference in maternal postnatal behavior or lactation between experimental and control groups. In addition, no differences were observed between fostered and nonfostered control groups in all measures of postnatal development, indicating no adverse effects on development created by cross-fostering alone.
The observation that delayed puberty was more readily detected in females compared with males may reflect either a greater responsivity of females to glucocorticoids, or simply a more precise method of puberty detection in this sex (i.e. vaginal opening vs. preputial separation). With regard to the first possibility, prenatal glucocorticoid therapy has been shown to promote lung development more effectively in female compared with male fetuses (27) and so a similar effect could account for the more pronounced effect of prenatal glucocorticoid treatment on the timing of female puberty. Alternatively, the statistically significant sex-treatment interaction with respect to puberty onset may reflect differences in the accuracy of the puberty-detection method used in the two sexes. Thus, the overall pattern of puberty onset in male offspring paralleled that observed in females, but within-group variation was clearly greater. Although preputial separation is considered the most reliable method for determining male puberty onset from external genitalia (21), it requires some manipulation and thus subjective interpretation. In contrast, puberty onset in females is likely to be more accurately timed because vaginal opening is a more definitive developmental marker.
Several potential mechanisms may account for the effects of fetal glucocorticoid exposure on the timing of puberty onset in subsequent postnatal life. Importantly, a simple link to body weight can be ruled out because catch-up growth occurred in all growth-retarded newborns early in postnatal development. Consequently, weight at puberty onset was higher in female offspring from dexamethasone and carbenoxolone treated groups. There were also no changes evident in the pattern of plasma leptin across the peripubertal period, even though leptin provides a key signal in the timing of puberty onset in normal development (14, 15). Interestingly, however, it remains possible that responsivity to leptin may be compromised in puberty-delayed groups; indeed, high glucocorticoid levels have been associated with leptin resistance in adult rats, and several models of obesity are known to be adrenal dependent (16, 17, 18). Our data also show that sexual dimorphism in plasma leptin levels only became evident after puberty, presumably reflecting the greater body mass of males, and similar results were recently reported by Landt et al. (28). Plasma leptin was also elevated in male offspring of carbenoxolone-treated adrenalectomized mothers (pre- and postpubertally), and in male offspring of metyrapone-treated mothers (postpubertal only). Although unexpected, these elevated plasma leptin levels are consistent with the body weights of these offspring, and possibly reflect altered patterns of fetal glucocorticoid secretion (due to metyrapone) and fetal 11ß-HSD activity (due to carbenoxolone) in these groups. Further studies are required to elucidate the precise mechanisms involved in this apparent disturbance to adult body composition.
Fetal glucocorticoid exposure could also influence the timing of puberty via direct actions on the hypothalamic-pituitary-gonadal (HPG) axis, possibly acting at all three levels. Such reprogramming of hormonal axes has previously been observed in relation to the HPA axis (29) and glucose homeostasis (7). In the rat, GnRH neurons develop and migrate during the fetal period (30) so that altered glucocorticoid exposure at this time may program their number and/or normal function; indeed, glucocorticoids have been shown to induce neuronal apoptosis (31) and thus may influence the final number of GnRH neurons present in the adult. Pituitary gonadotrophs (32) and the ovary (33) and testis (34) are all glucocorticoid targets in the adult, and so may also be programmed by fetal glucocorticoids. Interestingly, peripubertal plasma LH was elevated in male offspring of metyrapone-treated mothers, consistent with their advanced puberty, but in puberty-delayed groups plasma LH appeared unaffected. While full pulsatile analysis of LH secretion would be required to confirm these LH patterns, the similarity in plasma LH among control and puberty-delayed groups raises the possibility that delayed puberty following excess fetal glucocorticoid exposure may involve gonadal hyporesponsiveness to LH. It is also noteworthy that estrous cycle length appeared normal after delayed puberty, whereas cycles appeared to be lengthened slightly in offspring of metyrapone-treated mothers (after normal puberty onset). Further studies are required to ascertain the mechanisms involved in these changes, and whether fertility was compromised in any of the offspring.
The effects of fetal glucocorticoid exposure on puberty onset provides an explanation for the previous observation of delayed puberty following maternal stress in pregnancy (9). Thus, because the placental glucocorticoid barrier provided by 11ß-HSD is clearly not impermeable (11), elevated maternal glucocorticoids associated with stress would be expected to reach the fetus. Indeed, normal variation in placental 11ß-HSD activity is positively correlated with fetal weight in the rat (5), which further suggests that the timing of puberty could also be programmed by normal variation in placental 11ß-HSD. Maternal undernutrition also limits fetal growth and predisposes to later disease (35, 36), an effect possibly mediated by increased glucocorticoid exposure via reduced placental 11ß-HSD activity (37). Potentially, therefore, maternal undernutrition could also delay the timing of puberty onset in offspring. Finally, our data further suggest that therapeutic administration of synthetic glucocorticoids for prevention of fetal respiratory distress syndrome (38) may delay the timing of puberty onset, particularly when repeated glucocorticoid doses are administered (39).
In conclusion, this study shows that excess glucocorticoid exposure during fetal development retards fetal growth and delays the subsequent onset of puberty in postnatal life. Importantly, the alterations in the timing of puberty onset following carbenoxolone and metyrapone treatments indicate that concentrations of maternal glucocorticoids within the normal physiological range impact upon the subsequent timing of puberty in offspring.
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Acknowledgments |
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Received November 12, 1999.
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