| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Western Australia, 6907 Australia
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present work was carried out as part of a program to investigate the physiological significance of maternal estradiol-17ß (E2) concentrations throughout pregnancy in the rat. The objectives were, first, to define periods of sensitivity of rat embryos to estradiol administration at levels likely to reflect upper limits of the physiological range; and second, to investigate possible mechanisms contributing to deleterious effects. Of particular interest was the possibility that estradiol would act through depression of progesterone concentrations (estrogen can be luteolytic in the rat [16]) or through an imbalance of progesterone/estrogen ratios. The possibility that the primary effect of estradiol is on structural changes to the placenta and that this in turn affects embryonic and fetal development [17] was also considered.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Four experiments were conducted. The first examined effects of repeated daily administration of estradiol benzoate (EB) to determine broad ranges of sensitivity over specific periods of gestation. The second examined single injections of EB over Days 9–14 to more closely define periods of its lethal effect. The third assessed the onset of deleterious effects by examining fetuses one or two days after EB administration. The fourth used miniosmotic pumps to administer low doses of E2, rather than EB, to assess the lower range of maternal E2 blood concentrations likely to cause embryo mortality. Effects of EB on placental histology and maternal plasma progesterone concentrations were examined in some experiments, as was the effect of progesterone administration together with the EB.
Animals and Treatments
Nulliparous albino Wistar rats, 3–5 mo old and with a mean weight at mating of 225 ± 3 g (mean ± SEM; n = 201), were used. All rats were kept in an environmentally controlled room (temperature 19°C, range 18–21°C; relative humidity 64%, range 50–75%; lights-on from 0700 to 2100 h). Food and water were freely available. Rats were mated overnight, and the morning that spermatozoa were found in a vaginal smear was called Day 1 of gestation; rats from this colony normally litter on the morning of Day 23.
Treatments included s.c. injections of EB (Intervet; Organon, Durham, NC) in peanut oil (5 or 12.5 µg/ml), progesterone in peanut oil (50 mg/ml), and peanut oil alone (controls). The general methods used for these injections and for demonstration that peanut oil was free of estrogenic action have been described previously [1].
Miniosmotic pumps (Alzet, Palo Alto, CA; model 2001; 1.0 ± 0.01 µl/h delivery rate) were used to infuse E2 for the fourth experiment. Pumps were filled with E2 (Sigma, St. Louis, MO) at a concentration of 15 µg/ml, 30 µg/ml, or 40 µg/ml, in a carrier of 0.5% ethanol:99.5% propylene glycol. Control animals were given infusions of the carrier only. Rats were anesthetized lightly with ether, and the pumps were implanted s.c. in the subscapular region on the morning of the first day of treatment. This procedure took approximately 10 min.
Pregnancy Assessment
Animals were killed on Day 12, 13, 14, or 22 of gestation with an overdose of pentobarbitone sodium or ether, and the number, position and condition of each conceptus were recorded. The conceptus was classed as live if heartbeats or fetal movements could be elicited. Live 22-day fetuses and their placentas were weighed to the nearest milligram, and sex of the fetus was determined from observation of the internal and external genitalia. Some placentas were immersed in 10% formalin for later histological examination.
Placental Histology
Placentas were fixed in 10% formalin for at least 1 mo before embedding in paraffin. Central cross-sections (7 µm) were taken perpendicular to the longest axis of the placenta. Comparisons were made of cross-sectional areas occupied by the labyrinth (gas exchange region), basal zone (involved in hormone synthesis) [18], and maternal decidua as measured by an image analyzer.
RIAs
Plasma E2 concentrations were measured by a double-antibody RIA (KE2D; Diagnostic Products Corporation, Los Angeles, CA) as described previously [19], with minor modifications. Samples (250 µl) were extracted in 3.75 ml of ether; the solvent fraction was defatted with 250 µl of 0.02 molar NaOH, dried down under nitrogen, and reconstituted in 250 µl of 0 pg calibrator. Duplicate 100-µl aliquots were incubated with 50 µl of antiserum for 2 h and then with 50 µl of 125I-labeled E2 for 1 h. Precipitating solution (500 µl) was added; after 10 min the tubes were centrifuged and decanted, and radioactivity was measured for 1 min. All incubations were carried out at room temperature. Recovery of [3H]E2 was 85 ± 3% (mean ± SD, n = 11). Sensitivity of the assay was 1.9 pg/ml, and the intra- and interassay coefficients of variation were 10.2% and 9.6%, respectively. Concentrations of progesterone in plasma were measured as described previously [20].
Analysis of Results
Results were analyzed by one-way ANOVA, and, when the F test was significant, differences between each treated group and the control group were compared by the least-significant-difference (LSD) statistic [21]. Proportional data were transformed (arc-sine) and analyzed similarly: the results presented are retransformed means together with 95% confidence limits.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The method of EB administration was validated on conscious, Day 14, pregnant rats (n = 3) that had previously been fitted with an aortic cannula for collection of blood samples. After injection of 1 µg EB, plasma E2 rose from 43 ± 12 pg/ml at time zero, to 82 ± 5 pg/ml at 2 h, 112 ± 4 at 4 h, and 182 ± 19 pg/ml at 8 h. It then declined to 101 ± 4 pg/ml at 24 h. The results revealed no unexpected peaks of E2, and maximum values were within 5-fold control values.
Rats were treated with EB over Days 6–13, 8–13, 11–13, or 14–21 and examined on Day 22. Treatment had no apparent effect on overall health of the animals other than on reproductive status. Controls, given injections of 0.2 ml of peanut oil alone over Days 6–13 (n = 6), 8–13 (n = 4), or 14–21 (n = 11), revealed no significant differences between the three sub-groups, so their results were pooled.
Embryo and fetal mortality Treatment with 1 µg EB/day over Days 6–13 (n = 6) or 8–13 (n = 11) killed all fetuses except for a single survivor. Treatment over Days 11–13 reduced survival to 22% (Table 1). However, EB administered over Days 14–21 had no effect on survival at a dose of 1 µg/day and only a minor effect at 2.5 µg/day.
|
Fetal and placental growth Fetal weights were lighter in all groups treated with EB, and this was accompanied by similar reductions in placental weight. The growth-retarding effects were most pronounced in rats treated over Days 11–13. Only one treated fetus was malformed (within the expected rate for untreated rats). The percentage of male fetuses per litter was not affected, and there were no inconsistencies in the assessment of sex on the basis of examination of internal and external genitalia.
Placental histology The cross-sectional composition of placentas did not differ between controls (n = 7) and a combined group of treated rats (1 µg EB/day, Days 11–13, n = 3; 2.5 µg EB/day, Days 14–21, n = 3). The actual percentage values for the control and treated groups, respectively, were as follows: labyrinth, 70% and 72%; basal zone, 16% and 14%; and decidua, 14% and 14%.
Administration of progesterone together with EB treatment Eleven rats received injections of 1 µg EB and 15 mg progesterone/day over Days 11–13 to determine whether progesterone supplementation would protect embryos against the lethal effects of EB. By Day 22, however, fetal survival had fallen to 21% (95% confidence limits; range 6–41%), which was similar to the rate in rats treated with EB alone over Days 11–13 (22%; range 5–46%; Table 1).
Experiment 2: Effects of a Single Injection of EB on Fetal Survival, and Fetal and Placental Growth
To define the temporal sensitivity of embryos to EB, single injections were administered on Day 9, 10, 12, 13, or 14 of gestation, and effects on fetal development were examined on Day 22 (Fig. 1). Fetal survival was significantly reduced in rats treated on Days 9, 10, and 12 but was not affected when treatment was delayed to Day 13. Both fetal and placental weights were lighter than those of controls in all treated groups, but this effect was significant only for fetal weight in rats treated with 1 µg EB on Day 12 (3.89 ± 0.28 g; p < 0.01) and 2.5 µg EB on Day 14 (4.13 ± 0.14 g; p < 0.05). There were no malformed fetuses, and there was no discrepancy in sex ratios or in the assessment of sex based on an examination of the internal and external genitalia of each fetus.
|
Experiment 3: Onset of Fetal Death after EB Treatment over Mid-Gestation
Fetal survival in rats treated on Day 11 alone was reduced to 67% within two days (Fig. 2). In rats treated over Days 11–13, fetal survival fell to 51% by Day 14 and continued to fall to 22% by Day 22. EB did not appear to affect plasma progesterone concentrations. Control values on Day 13 were 74 ± 10 ng/ml (n = 6). This was comparable to values from rats treated on Day 11 and examined on Day 12 (65 ± 7 ng/ml, n = 5), treated on Day 11 and examined Day 13 (47 ± 6 ng/ml, n = 7), and treated over Days 11–13 and examined on Day 14 (71 ± 20 ng/ml, n = 6).
|
Experiment 4: Effects of E2 Administration by Miniosmotic Pump on Plasma E2 Concentrations and Fetal Survival
The effect of E2 infusion on plasma E2 concentrations was examined at the highest dose rate used (40 ng/h) to assess maximum likely elevations in maternal E2 concentrations. Rats were anesthetized on Day 10, and an aortic cannula was exteriorized over the subscapular region to provide blood samples. On Day 13, an initial blood sample (1.2 ml) was taken at 0900 h, and 1 h later a miniosmotic pump was inserted. Further blood samples were taken over the next 72 h, with blood from a pool of diestrus donor rats used to maintain blood volume.
Infusion of E2 produced a stable, 2-fold elevation in E2 plasma concentrations (Fig. 3). This rise was evident within 30 min of pump insertion, and no significant peaks were identified.
|
Administration of E2 over Days 10–13 Rats were treated with E2 (15 ng/h or 30 ng/h) or the carrier alone and examined on the last day of treatment or near term, Day 22 (Fig. 4). Fetal survival was significantly reduced at both rates of E2 infusion. This effect was significant by Day 13 at the higher rate (30 ng/h) and by Day 22 at the lower rate (15 ng/h). Infusion at 15 ng/h only slightly increased E2 concentrations by Day 13 (controls, 26 ± 1 pg/ml, n = 7; treated, 30 ± 2 pg/ml, n = 5; not significant).
|
Administration of E2 over Days 13–16 Rats were given infusions of E2 at a single rate of 40 ng/h or with the carrier alone. They were examined on the last day of treatment, Day 16, or near term, Day 22. E2 had no effect on fetal survival; values for the two treated and matched control groups (n = 6–8) all exceeded 90%.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The cause of the estrogen-induced mortality is unknown, but the present work clearly shows that it is only the early embryo and fetus that is susceptible and that death can occur rapidly. Over Days 9–13, the embryo undergoes major organogenesis. It would seem that some aspect of organ development is critically and directly affected by elevated E2, which leads to rapid death of the embryo or to a malformation that becomes incompatible with fetal life at a later stage. Estradiol crosses the placental barrier [24], and the early embryo and fetus express E2 receptor activity, at least in their reproductive organs [25]. To date, however, there has been no report on E2 receptor activity in organs critical to fetal survival, and it is possible that one or more such organs are peculiarly sensitive to E2. The failure to find structural malformations in the present work suggests that if critical organ malformation is the cause, then it must be at a fine structural or functional level beyond that detectable through the routine inspection of fetuses that was carried out. Extensive histological examination of cultured rat embryos subjected to extremely high levels of E2 also failed to reveal organ malformation other than to the central nervous system, which would be unlikely to fully account for the E2 toxicity [26]. The only specific change noted in the present study was an apparent increased vascularity of the decidual area of the placenta, observed in both dead and live fetuses of rats treated over Days 11–13 and examined on Day 14. If this change induced or reflected changes in the placental circulation, it may have precipitated fetal death. It is pertinent that estrogen administration, which is also lethal to the mouse and rabbit embryo, elicits hemorrhage around gestational sacs of the mouse [13, 15] and rabbit [27], and decreases placental blood flow in the latter species. Furthermore, vascularization of the decidua has been shown to be estrogen-dependent in the rat [28].
The possibility that the primary effect of EB was reduction of endogenous progesterone secretion and, in turn, of plasma progesterone concentrations was considered because E2 can be luteolytic [16] as well as luteotrophic in the rat [29, 30]. But in experiment 3 of the present work, there was clear evidence of fetal death despite no change in progesterone concentrations. Furthermore, in rats that received progesterone supplementation together with EB over Days 11–13, embryonic mortality was just as great as in those that received EB alone. This would suggest that E2 itself is the lethal factor rather than an altered estrogen/progesterone ratio, since the amount of progesterone added was designed to raise plasma concentrations by around 5-fold [31]. It was also considered that sudden reduction of E2 at the end of treatment, rather than elevated E2 as such, could have been lethal. But the finding that rats given one injection of EB only on Days 13 or 14 were not affected shows that the time of administration rather than withdrawal of EB was the important factor. Furthermore, rats given injections over Days 11–13 and killed the following day had already lost almost half of their fetuses before any withdrawal effect could be expected.
With regard to placental function, EB administration retarded placental growth, particularly within groups with the highest embryonic mortality or subjected to repeated EB administration. However, although EB reduced placental weight, it did not appear to affect any specific zone of the placenta. The relative proportions of the three major components—the labyrinth, basal zone, and decidua—were unchanged, thus maintaining parity of the gas exchange and the endocrine areas of the placenta. Fetal growth was also retarded in most EB-treated groups, and it is noteworthy that a single injection of 2.5 µg EB on Day 14 had an effect similar to repeated injections of 1 µg EB/day over Days 14–21. This result further emphasizes that the major period of fetal sensitivity to estradiol is early in gestation extending up to the beginning of the last third of pregnancy. The mechanism of the growth-retarding effect of estradiol may be different from that promoting fetal death. Estrogen supplementation has been reported to inhibit the accelerated fetal and placental growth seen in ovariectomized, progesterone-supplemented rabbits [11] and rats [8]. In both studies it was postulated that estradiol may have a specific growth-retarding effect on the placenta that secondarily limits fetal growth, and the present results accord with this view. It is relevant here that E2 also inhibits placental production of androstenedione and testosterone, essential precursors for its own synthesis by the ovary [32]. Collectively, these findings raise the intriguing possibility of an intricate feedback loop between the ovary and the placenta in the control of maternal E2 concentrations and fetal growth.
In overview, the present study shows that even minor elevations of maternal E2 can be lethal to the embryo and retard fetal and placental growth. Given this level of sensitivity, endogenous control of E2 secretion must be carefully regulated to maintain optimum conditions for fetal development. It remains to be seen whether aberrations in E2 secretion contribute to unexplained embryonic mortality or retarded fetal growth in otherwise normal rats.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 61 08 9380 1051; nbruce{at}anhb.uwa.edu.au
Accepted: February 15, 1999.
Received: December 8, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
reductase type 1 caused by estrogen excess. Mol Endocrinol 1997; 11:917–927. in Norwegian semi-domestic reindeer (Rangifer tarandus tarandus) during their first reproductive season. J Reprod Fertil 1995; 105:307–314.[Abstract]This article has been cited by other articles:
|
|
 |
|
  R. L. Kelley, K. L. Kind, M. Lane, R. L. Robker, J. G. Thompson, and L. J. Edwards Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E761 - E770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Wolf, G. A. LeBlanc, and L. E. Gray Jr. Interactive Effects of Vinclozolin and Testosterone Propionate on Pregnancy and Sexual Differentiation of the Male and Female SD Rat Toxicol. Sci., March 1, 2004; 78(1): 135 - 143. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
