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BOR - Papers in Press, published online ahead of print January 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.024992
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BIOLOGY OF REPRODUCTION 70, 1306–1316 (2004)
DOI: 10.1095/biolreprod.103.024992
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Diethylstilbestrol Versus Estradiol as Neonatal Disruptors of the Hamster (Mesocricetus auratus) Cervix1

William J. Hendry, III2,3, William S. Branham4, and Daniel M. Sheehan5

Department of Biological Sciences,3 Wichita State University, Wichita, Kansas 67260 Division of Genetic and Reproductive Toxicology,4 National Center for Toxicological Research,Jefferson, Arkansas 72079 Daniel M. Sheehan and Associates,5 Little Rock, Arkansas 72202


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The synthetic estrogen diethylstilbestrol (DES) is an established, estrogenic endocrine disruptor (ED). The Syrian golden hamster (Mesocricetus auratus) offers some unique advantages as an experimental system to investigate the perinatal ED action of DES and other estrogenic EDs. Previous analyses regarding the consequences of neonatal administration (100 µg) of DES versus estradiol-17ß (E2) showed that DES had a more potent disruptive effect on morphogenesis and gene expression in the uterus, oviduct, and ovary as well as in the testis and male accessory organs. The objectives of the present study were to describe the histopathological consequences of the two neonatal treatment regimens in the hamster cervix and to compare them with our previous observations in the hamster uterus. As previously found in the hamster uterus, DES was more potent than E2 as a neonatal disruptor of the hamster cervix in prepubertal animals and in ovarian-intact adult animals. However, the cervix-versus-uterus scenario diverged in animals that were ovariectomized prepubertally and then chronically stimulated with natural estrogen (E2). We confirmed previous observations that neonatal exposure to DES, but not to E2, permanently alters estrogen responsiveness in the adult hamster uterus, but neither neonatal treatment regimen affected estrogen responsiveness in the adult hamster cervix. These results suggest that an unidentified ovarian factor influences the extent of neonatal DES-induced disruption of the cervix, but not of the uterus, in hamsters.

cervix, developmental biology, estradiol, female reproductive tract, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The synthetic estrogen diethylstilbestrol (DES) is an established perinatal endocrine disruptor (ED) in both humans and experimental animals [1, 2]. Of the rodent species used to evaluate the potency and mechanism of action of DES and other putative EDs, the Syrian golden hamster (Mesocricetus auratus) offers some unique advantages. These advantages have been reviewed recently [3] and include 1) a very short and predictable gestation period (exactly 16 days); 2) a serum alpha-fetoprotein (AFP) moiety that does not bind the natural estrogen, estradiol-17ß (E2); 3) an estrous cycle that is very regular (exactly 4 days) and easy to monitor; and 4) cheek pouches that are immunologically privileged and convenient sites (amenable to repetitive inspection and manipulation) for tissue transplantation studies.

Our initial efforts in the hamster consisted of treating newborn animals with DES and then assessing reproductive tract morphology in mature (postpubertal) females. The reproductive tract region that first attracted our attention was the uterus, because 100% of the DES-exposed organs developed endometrial hyperplasia and a large proportion (~40%) progressed to neoplasia (endometrial adenocarcinoma) [4]. Subsequent analysis regarding the early stage of the phenomenon revealed that neonatal DES exposure induced both acute and persistent changes in mitotic activity, organization, and dimensions of individual tissue compartments in the immature (prepubertal) uterus [5]. To investigate the later stage of the phenomenon, we used mature animals that had been ovariectomized and then chronically stimulated with E2, and we observed endometrial hyperplasia/adenocarcinoma in the uteri exposed to DES neonatally, but not in the control uteri [4, 6, 7]. Those observations, when considered from the context of the two-step model for carcinogenesis, seemed to be consistent with the hypothesis that 1) neonatal DES treatment directly and permanently alters the developing hamster uterus (initiating event) such that 2) the uterus responds abnormally later in life to stimulation with E2 (promotion event).

Strong support for the two-stage hypothesis came from a study in which immature uteri from control and neonatally DES-treated donors were cross-transplanted and allowed to develop in the cheek pouches of control and neonatally DES-exposed hosts that had also been ovariectomized and then chronically stimulated with E2 [6]. Closer examination of the histopathological lesions that developed in the hamster uterus following neonatal DES exposure and later stimulation with E2 revealed that the hyperplastic endometrial epithelium also displayed intense apoptosis according to morphological (apoptotic bodies), biochemical (internucleosomal DNA fragmentation), and histochemical (in situ labeling of free 3' DNA ends) evidence [6, 7]. According to Northern blot and immunohistochemical analyses, those neonatal DES-induced morphological responses in the uterus were accompanied by the altered expression of several proto-oncogenes that are implicated in the regulation of both cell proliferation (c-jun, c-fos, c-myc) and apoptosis (bax, bcl-2, bcl-x) [8].

To provide a resource that would allow us to more fully define the phenomenon of neonatal DES-induced endocrine disruption throughout the female hamster reproductive tract and probe its mechanism at the cellular and molecular level, we have generated a comprehensive archive of formalin- fixed and paraffin-embedded tissues. This archive consists of various reproductive tract regions harvested at several time points from 1) prepubertal animals; 2) intact, mature animals; and 3) mature animals that were ovariectomized just before puberty and then exposed chronically to E2. Because few direct, in vivo comparisons of perinatal endocrine-disruptive potency have been made between the synthetic estrogen, DES, and the primary natural estrogen, E2, the archive includes tissues from groups of hamsters that were treated neonatally with each of the two estrogens. We have already used this archive for an extensive histomorphological analysis in the uterus and a more preliminary analysis in the ovary. A key finding in both reproductive tract regions was that, at the same dose level, DES was much more potent than E2 as a neonatal ED [3, 9]. Evaluation of a similar archive of reproductive tract tissues from the male hamster demonstrated that DES was also more potent than E2 as a neonatal ED of the testis and seminal vesicle [3, 10].

The present study represents a logical extension of the use of our tissue archive to investigate the endocrine-disruptive action of DES in the hamster cervix. It shows that 1) neonatal DES treatment does disrupt morphogenesis of the hamster cervix; 2) as previously found in the hamster uterus and ovary, DES was more potent than E2 as a neonatal disruptor of the hamster cervix; and 3) the cervical disruption phenomenon did not completely mimic the DES- initiation/E2-promotion profile observed in the hamster uterus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatment

Treatment of neonates, ovariectomy, chronic E2 stimulation, anesthesia, and killing by decapitation were performed according to established protocols [4, 5, 11] in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility according to the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Timed pregnant Syrian golden hamsters (M. auratus) from Charles River Breeding Laboratories (Wilmington, MA) or Harlan Sprague Dawley, Inc. (Indianapolis, IN), were caged singly under a 14L:10D photoperiod with food and water provided ad libitum. Within 6 h of birth (Day 0), litter size was adjusted to eight neonates/litter by eliminating males, and all littermates received a single s.c. injection of 50 µl of corn oil vehicle either alone (control) or containing 100 µg (~33 mg/kg body wt) of either E2 or DES. Those dose levels are high, but they are not unreasonable considering that DES ingestion by pregnant women is as much as 150 mg daily and 18.2 g in total during pregnancy [12]. Ovariectomy and placement of E2 pellets were performed at 21 days of age under pentobarbital-induced anesthesia. The pellets were inserted s.c. between the shoulder blades and consisted of a plugged Silastic (Dow Corning Corp., Midland, MI) tube (open lumen length, 1.0 cm; inner diameter, 1.57 mm; outer diameter, 2.41 mm) filled with crystalline E2. According to previous determinations [4, 6, 13], this procedure maintains serum E2 levels at approximately 200 pg/ml for at least 5 mo. At all the ages studied, at least three animals from each treatment group were anesthetized with CO2, weighed, killed, and immediately processed as described below.

Tissue Harvesting, Processing, and Histological Analysis

The bodies of prepubertal animals were eviscerated, and the lower torsos were immersed in ice-cold fixative for at least 24 h before reproductive tracts were excised, trimmed of adhering fat and mesentery under a dissecting microscope, and placed back in fixative. The fixative consisted of 4% paraformaldehyde in Dulbecco PBS (pH 7.4). For the adult animals, reproductive tracts were immediately excised and then also placed in fixative for at least 24 h before trimming and placement back in fixative. The harvested tracts were ultimately divided into cervix, uterine horns, and oviduct/ovarian (when present) regions and then embedded in paraffin. For the present study, 4- to 5-µm midfrontal sections from the cervical region of the tracts were processed for light microscopy using standard hematoxylin-and-eosin staining. At least four sections cut from each of the cervices harvested from all of the animals per time point and treatment group were analyzed. Digital images were captured with a Kodak MDS 290 system (Eastman Kodak Co., Rochester, NY). The photomicrographs shown are representative of the histomorphological conditions in the triplicate specimens from the three treatment groups at every observation point. Using photomicrographs, cervical diameters were determined by comparing manual measurements of tissue section dimensions (at the uterine/endocervical junction) to those of an objective micrometer.

Statistics

For the data regarding animal weight, cervical diameter, and normalized cervical diameter, an SAS statistical package [14] was used to calculate the mean, the SEM, and the significance levels of differences among the three neonatal treatment groups at all the time points. Factorial ANOVA was followed by the Tukey honestly significant difference test for multiple comparisons [15], and means were considered to be significantly different at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Prepubertal Animals

Effects of neonatal DES versus E2 treatment on body weight, absolute size (diameter) of the cervix, and size of the cervix normalized to body weight in prepubertal animals are shown in Figure 1. The data in Figure 1A show that neither of the two neonatal treatment regimens had a significant effect on animal growth from Days 1 to 21. Consequently, neonatal treatment effects on cervical growth were comparable whether values were expressed on an absolute (Fig. 1B) or a normalized (Fig. 1C) basis. In both cases, cervical growth was significantly enhanced by Day 3 in the neonatal E2 and DES treatment groups. Thereafter, the enhanced cervical growth response was more dramatic in the neonatally DES-exposed groups than in the neonatally E2-exposed groups (all DES vs. E2 group differences, except for the Day-21 values, were statistically significant).



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  		FIG. 1. Effects of neonatal DES versus E2 treatment on body weight, absolute size of the cervix, and normalized size of the cervix in prepubertal animals. Following injection on the day of birth (Day 0) with vehicle alone (control [C]) or 100 µg of either E2 (E) or DES (D), female animals at the indicated days of age were weighed (A), and their reproductive tracts were fixed, separated into various regions, and processed for standard paraffin embedding, sectioning, and hematoxylin-and-eosin staining. Cervical diameters were measured at the uterine/endocervical junction of midfrontal tissue sections (see Fig. 2) and were expressed both on an absolute basis (B) and after being normalized to the animal's body weight (C). Values represent the mean ± SEM (n = 3; errors bars are not shown where the variability was so small as to be masked by the data point), and means that are significantly different (P < 0.05) from each other at each time point are indicated by different lowercase letters

To further illustrate the differential treatment effects on prepubertal growth of the cervix, low-magnification views are shown of representative tissue sections from control animals (Fig. 2) and from animals that were treated neonatally with E2 (Fig. 3) or with DES (Fig. 4). They provide visual confirmation that both neonatal treatment regimens enhanced cervical growth as early as Day 3, but then (Day 5 to Day 21) that response was more dramatic in the neonatally DES-exposed animals than in the neonatally E2- exposed animals. Another anatomical feature that can be appreciated in Figures 2–4 (and in subsequent figures that contain low-magnification micrographs) is that the hamster has a true duplex uterus with two separate cervical canals, each with their own external os. Still another finding evident even at low magnification was that early cytodifferentiation of the luminal epithelium in the ectocervical region was altered to a different extent by neonatal DES treatment than by neonatal E2 treatment. For instance, as early as Day 5, precocious cornification of the stratified squamous epithelium was obvious in the DES-exposed cervix (Fig. 4). That phenomenon is illustrated at higher magnification in Figure 5 and is contrasted to what occurs in the columnar epithelium that lines the lumen in the uterine region of the hamster reproductive tract. In the ectocervical region of the 5-day-old tracts (Fig. 5, left), stratification of the luminal epithelium was 1) incipient in control animals; 2) multilayered, but noncornified, in neonatally E2-treated animals; and 3) extensively cornified in neonatally DES- treated animals (indicated by an asterisk in Fig. 5). In the uterine-adjacent region of the same sections, histology of the luminal epithelium was distinct from that observed in the ectocervical regions but mimicked what we had found previously in sections taken from the midregion of the uterus in 5-day-old hamsters [9]. Specifically, in the lower uterine region adjacent to the cervix (Fig. 5, right), the columnar epithelium was low cuboidal in control and neonatally E2-treated animals but taller and pseudostratified in neonatally DES-treated animals.



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  		FIG. 2. Cervical histology in control, prepubertal hamsters. Low-magnification micrographs of midfrontal cervical sections positioned with their cranial (uterine-adjacent) aspect to the right and caudal (vaginal-adjacent) aspect to the left. The tissues are from hamsters that had been injected on the day of birth (Day 0) with vehicle alone (control [CON]) and are representative of the histology observed in the cervices from three separate animals at each age.



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  		FIG. 3. Cervical histology in prepubertal hamsters following neonatal E2 treatment. Low-magnification micrographs of midfrontal cervical sections positioned with their cranial (uterine-adjacent) aspect to the right and caudal (vaginal-adjacent) aspect to the left. The tissues are from animals injected on the day of birth with vehicle containing 100 µg of E2



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  		FIG. 4. Cervical histology in prepubertal hamsters following neonatal DES treatment. Low-magnification micrographs of midfrontal cervical sections positioned with their cranial (uterine-adjacent) aspect to the right and caudal (vaginal-adjacent) aspect to the left. The tissues are from animals injected on the day of birth with vehicle containing 100 µg of DES



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  		FIG. 5. Effects of neonatal DES versus E2 exposure on the luminal epithelium of the uterine and ectocervical regions of reproductive tracts from 5-day-old hamsters. Using the same representative sections shown for Day 5 in Figures 2 (control [CON]), 3 (E2), and 4 (DES), separate high-magnification micrographs were taken from the cranial (cr) or uterine-adjacent region and from the caudal (ca) or vaginal-adjacent region. Each micrograph shows the cellular organization of a length of luminal epithelium (E) and a portion of its underlying mesenchymal or stromal tissue (S). The asterisk marks a region where the stratified squamous epithelium was cornified (flattened, keratinized cells to the right of the asterisk and desquamated sheets of cells to the left)

Adult, Intact Animals

Effects of neonatal DES versus E2 treatment on body weight, absolute size (diameter) of the cervix, and size of the cervix normalized to body weight in 1- to 5-mo-old adult animals are shown in Figure 6. Body weight tended to be reduced by both neonatal treatment regimens, but the difference was statistically significant only for the DES- exposed versus the control group at the 3-mo time point. Consequently, body weight differences cannot account for the following treatment-induced differences in cervical size. At the earliest time point, cervical size in both absolute (Fig. 6B) and normalized terms (Fig. 6C) was enhanced in the neonatally DES-exposed group, but that effect was not statistically significant. Thereafter, cervical size was enhanced in both neonatal treatment groups. The enhancement in absolute cervical size was statistically significant at the 2- to 4-mo time points in the neonatally DES-exposed group and at the 3-mo time point in the neonatally E2- exposed group. The same was true for the enhancement of normalized cervical size, except that it was also statistically significant at the 5-mo time point in the neonatally DES- exposed group.



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  		FIG. 6. Effects of neonatal DES versus E2 treatment on body weight, absolute size of the cervix, and normalized size of the cervix in adult, intact hamsters. Following injection on the day of birth (Day 0) with vehicle alone (control [C]) or 100 µg of either E2 (E) or DES (D), female animals at the indicated days of age were weighed (A), and their reproductive tracts were fixed, separated into various regions, and processed for standard paraffin embedding, sectioning, and hematoxylin and eosin staining. Cervical diameters were measured at the uterine/endocervix junction of mid-frontal tissue sections (see Fig. 2) and were expressed both on an absolute basis (B) and after being normalized to the animal's body weight (C). Values represent the mean ± SEM (n = 3; errors bars not shown where variability was so small as to be masked by the data point), and means that are significantly different (P < 0.05) from each other at each time point are indicated by different lowercase letters

The differential effect of neonatal DES vs. E2 treatment on cervical size was modest and somewhat variable (Fig. 6, A and B), but the differential effect of the two treatments on histomorphology of the adult cervix was striking and very consistent from the 2-mo time point onward. For instance, the low-magnification micrographs in Figure 7 confirm the rank order of cervical size shown for the three groups of animals at the 2-mo time point shown in Figure 6 (DES > E2 > control). However, inspection of the same sections at higher magnification revealed that luminal epithelial histology in both the ectocervix (Fig. 8, left) and uterine-adjacent regions (Fig. 8, right) was quite different between the two neonatal treatment groups. In the ectocervical region, the stratified squamous epithelium was multilayered, but noncornified, in both the control and neonatally E2-treated animals, but it was extensively cornified in the neonatally DES-treated animals (indicated by an asterisk in Fig. 8). In the uterine-adjacent region of the same sections, epithelial histology again was distinct from that observed in the ectocervical regions and again mimicked what we had found previously in sections taken from the midregion of adult uteri [9]. Specifically, the luminal epithelium in the lower uterine region adjacent to the cervix (Fig. 8, right) was 1) low cuboidal in control animals, 2) slightly taller and pseudostratified in E2-treated animals, and 3) extremely tall in neonatally DES-exposed animals. In addition, the cell's basal aspects were difficult to distinguish from the underlying stromal tissue compartment in neonatally DES-treated animals.



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  		FIG. 7. Cervical histology in adult, intact hamsters. Shown are low-magnification micrographs of midfrontal sections of the cervical regions positioned with their cranial (uterine-adjacent) aspect to the right and caudal (vaginal-adjacent) aspect to the left. The tissues are from 2-mo-old hamsters that had been injected on the day of birth with vehicle alone (control [CON]) or containing 100 µg of either E2 or DES and are representative of the histology observed in the cervices from three separate animals per treatment group



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  		FIG. 8. Effects of neonatal DES versus E2 exposure on the luminal epithelium of the uterine and ectocervical regions of reproductive tracts from adult, intact hamsters. Using the same representative sections shown and described in Figure 7, separate high-magnification micrographs were taken from the cranial (cr) or uterine-adjacent region and from the caudal (ca) or vaginal-adjacent region. Each micrograph shows the cellular organization of a length of luminal epithelium (E) and a portion of its underlying mesenchymal or stromal tissue (S). The asterisk marks a region where the stratified squamous epithelium was cornified

Adult, Ovariectomized, and Estrogen-Replaced Animals

Previously, we used the strategy of prepubertal ovariectomy plus sustained E2 replacement (O+E2) to demonstrate that neonatal DES, but not neonatal E2, treatment directly and permanently disrupts estrogen responsiveness in the adult hamster uterus [3, 4, 6–9]. Under the same endocrine-manipulation conditions, the scenario in the hamster cervix was somewhat different. Figure 9 shows the influence of neonatal DES versus E2 treatment on body weight, absolute size (diameter) of the cervix, and size of the cervix normalized to body weight in animals that were ovariectomized on Day 21 of age and then chronically stimulated with E2 until 1 or 2 mo of age. In contrast to the trend noted above in the intact adult animals, body weight in the O+E2 animals (Fig. 9A) was slightly increased (not statistically significant) in the neonatally E2-treated group but slightly decreased in the neonatally DES-treated group (statistically significant only at 2 mo). These changes in absolute size of the cervix induced by neonatal treatment were not statistically significant (Fig. 9B), but they did follow the same pattern as that noted above for body weight. Consequently, normalized cervical size (Fig. 9C) was not affected by either neonatal treatment regimen in the O+E2 hamsters. Figure 9 also shows that, between the two time points studied, body weight increased, but absolute size of the cervix did not change. Thus, the normalized size of the cervix decreased in all three groups.



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  		FIG. 9. Effects of neonatal DES versus E2 treatment on body weight, absolute size of the cervix, and normalized size of the cervix in ovariectomized and estrogen-replaced adult hamsters. Following injection on the day of birth (Day 0) with vehicle alone (control [C]) or 100 µg of either E2 (E) or DES (D), female animals at 21 days of age underwent ovariectomy plus implantation with an E2-releasing pellet. Then at the indicated days of age, they were weighed (A), and their reproductive tracts were fixed, separated into various regions, and processed for standard paraffin embedding, sectioning, and hematoxylin and eosin staining. Cervical diameters were measured at the uterine/endocervix junction of mid- frontal tissue sections (see Fig. 2) and were expressed both on an absolute basis (B) and after being normalized to the animal's body weight (C). Values represent the mean ± SEM (n = 3; errors bars not shown where variability was so small as to be masked by the data point), and means that are significantly different (P < 0.05) from each other at each time point are indicated by different lowercase letters

Cervical histomorphology was another characteristic that did not change between 1 and 2 mo of age within any of the three groups of O+E2 animals. Consequently, only examples from the 2-mo time point are shown. The low-magnification micrographs of the representative tissue sections in Figure 10 confirm that neither neonatal treatment regimen altered cervical diameter to a significant extent. However, inspection of the same sections at higher magnification revealed that the O+E2 condition induced cornification of the squamous epithelium in the ectocervical region of all three groups (Fig. 11, left). That result contrasts with what was observed in the uterine-adjacent regions of the same sections (Fig. 11, right) and was found previously in sections taken from the miduterine region of adult, O+E2 hamsters [9]. Specifically, the luminal epithelium in the lower uterine region adjacent to the cervix was moderately tall and pseudostratified in both the control and the neonatally E2-treated group. In the neonatally DES-treated group, it was extremely tall and disorganized, riddled with cavities that often contained degenerating cells, and again, the cell's basal aspects were difficult to distinguish from the underlying stromal tissue compartment. Together, these results indicate that neonatal E2 versus DES treatment also has differential disruptive effects on the hamster cervix, but the phenomenon does not completely mimic that observed in the hamster uterus.



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  		FIG. 10. Cervical histology in ovariectomized and estrogen-replaced adult hamsters. Shown are low-magnification micrographs of midfrontal sections of the cervical regions positioned with their cranial (uterine-adjacent) aspect to the right and caudal (vaginal-adjacent) aspect to the left. The tissues are from hamsters that had been injected on the day of birth with vehicle alone (control [CON]) or containing 100 µg of either E2 or DES and then had undergone ovariectomy plus implantation of an E2- releasing pellet at 21 days of age, and they are representative of the histology observed in the cervices from three separate animals at each age



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  		FIG. 11. Effects of neonatal DES versus E2 exposure on the luminal epithelium of the uterine and ectocervical regions of reproductive tracts from ovariectomized and estrogen-replaced adult hamsters. Using the same representative sections shown and described in Figure 10, separate high-magnification micrographs were taken from the cranial (cr) or uterine-adjacent region and from the caudal (ca) or vaginal-adjacent region. Each micrograph shows the cellular organization of a length of luminal epithelium (E) and a portion of its underlying mesenchymal or stromal tissue (S). The asterisks mark regions where the stratified squamous epithelium was cornified.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The tissue archive used for the present study documents the disruption phenomenon that develops throughout the female reproductive tract of hamsters following neonatal exposure to either DES or E2. It includes both the prepubertal and mature stages in intact animals plus what happens in mature animals after they are ovariectomized prepubertally and then chronically stimulated with E2. Our objectives were to describe the histopathological consequences of each treatment in the cervical region and to compare them with our previous observations in the hamster uterus.

The ability of perinatal DES exposure to disrupt morphogenesis of the cervix has previously been reported in the hamster [16] and in the more commonly used experimental species of mice [17–23] and rats [24–26]. However, few direct comparisons have been made of the ED activity of DES versus E2. Those that have been performed either suggested that E2 was less effective than DES as a perinatal ED agent [19, 27, 28] or led to differing conclusions about whether the perinatal ED action of DES involves factors other than its relative estrogenicity [29, 30].

Consideration of such topics inevitably leads to a discussion of the biology of AFP. This plasma glycoprotein is produced and circulates at very high levels during fetal/ neonatal life but then declines drastically as mammals mature [31]. Because AFP in most rodents is an effective binder of E2 but not of DES [31, 32], reduced bioavailability of perinatally administered E2 would seem to be a reasonable explanation for its lower potency as a perinatal ED agent compared to DES. However, that cannot be the case for the differential ED potency of E2 versus DES in the hamster, because hamster AFP is like that in humans, which does not bind E2 [33, 34].

An alternative explanation stems from various in vitro and in vivo assays showing that DES is inherently more estrogenic than E2 [35, 36]. Of course, in a whole animal, the relative potency of such agents is influenced by a web of complex biological and pharmacokinetic dynamics, such as the method of agent administration (e.g., s.c. injection compared to the more "natural," oral route), developmental stage, metabolic activation/clearance, and influence of serum-binding proteins other than AFP (e.g., albumin, sex hormone-binding globulin). For instance, a finding relevant to the latter topic is that albumin does bind DES preferentially to E2 in rats and hamsters [37, 38]. Also, preliminary observations (unpublished results) indicate that the conjugated estrogen, E2-benzoate, more closely mimics the neonatal ED action of DES in the male and female hamster than does the natural ovarian estrogen, E2. On the other hand, a recent report that focused on a variety of putative ED agents [39] raises the possibility that potency as a perinatal ED agent at the whole animal level may rely on characteristics other than a given agent's relative estrogenicity as assessed by simple in vitro assays of binding affinity to the estrogen-receptor (ER) protein and/or transactivation of ER-responsive target gene constructs. Because such unresolved questions directly relate to the mechanisms and putative consequences of endocrine disruption, they deserve continuing attention.

Most studies of the perinatal ED action of DES have assessed the later consequences in adult animals. In the hamster system, we have also assessed the early consequences in prepubertal animals. In the present study, we show that, beginning immediately following neonatal injection and progressing through the prepubertal period, overall growth of the hamster cervix was accelerated to a much greater extent by DES than by E2 treatment. A similar DES-selective effect on overall growth of the early, developing cervix was reported in the guinea pig following a prenatal treatment regimen with DES versus E2 [27]. However, something we observed in the immature hamster cervix that was not reported in the immature guinea pig study was that DES, but not E2, treatment caused precocious cornification of the squamous epithelium lining the ectocervical lumen. Other precocious events occur in the prepubertal hamster uterus following neonatal DES, but not E2, treatment. One such event, reported previously [9] and confirmed in the present study, is the development of pseudostratification in the columnar epithelium of the endometrial lumen. Another is the development of invaginating endometrial glands [5, 9]. More recently, we also detected precocious induction of the progesterone-receptor protein in the prepubertal hamster uterus following neonatal DES exposure [40]. Although those events are considered to be abnormal when they occur before puberty, they are some of the most well-established responses to estrogen stimulation of the normal, adult reproductive tract [41, 42]. The obvious implication is that the ultimate disruptive consequences of neonatal DES exposure are mediated by a functional ER system in the hamster's early, developing reproductive tract. Indeed, similar levels of ER{alpha} protein were detected in control and neonatally DES-exposed uteri from 5-day-old hamsters [43]. So, the inherent estrogenicity of DES may not be the sole determinant of its action as a perinatal ED agent, but it is a necessary one. This conclusion is supported by the extensive clinical and experimental evidence that perinatal DES-induced teratogenesis and neoplasia are confined to estrogen target tissues [1, 2].

In mature animals with ovaries, neonatal DES, but not E2, treatment resulted in hypertrophic cervical regions and a squamous epithelium in the ectocervix that was extensively cornified. In guinea pigs at a similar stage of maturity, DES, but not E2, treatment also resulted in hypertrophy of the cervical region, but cornification of the luminal epithelium was not reported [28]. Furthermore, postnatal DES treatment had no discernable effect on the overall morphology or histology of the female reproductive tract in adult guinea pigs [28]. Such observations contribute to the evidence from numerous studies that there exist species differences and critical periods of exposure for the perinatal ED action of DES. As shown previously [9] and confirmed in the present study, neonatal DES treatment is also more potent than neonatal E2 treatment in terms of inducing overall hypertrophy of the adult uterus plus hypertrophy/ hyperplasia and pseudostratification of its endometrial epithelium. Such changes in organ size and histology are considered to be classical signs of hyperestrogenism and warrant comparison to what is observed in adult mice following prenatal or neonatal DES treatment. The uteri and cervices of the prenatally DES-exposed mice exhibited the same hypertrophic/hyperplastic/hyperestrogenic qualities that we observed in the neonatally DES-exposed hamsters, whereas those of the neonatally DES-exposed mice were hypotrophic [19, 21]. Thus, we conclude that 1) in addition to the differential effects observed in the prepubertal cervix, neonatal DES, but not E2, treatment induces long-term hyperestrogenic changes in the cervix of adult, intact hamsters and 2) treatment of hamsters once on the day of birth with DES approximates the situation in which mice are treated repeatedly with DES during the mid to late stages of gestation.

In adult hamsters under the O+E2 conditions, we were surprised in the present study to find that their cervical regions were of similar size and histology in all three treatment groups, whereas in both the present and a previous study [9], their uteri were hypertrophic and hyperplastic/ dysplastic in the DES-treatment group only. In other words, neonatal DES, but not E2, treatment directly elicits a hyperestrogenic effect in the early, developing (prepubertal) cervix and uterus, but it has differential effects on the two regions when the adult tract is chronically stimulated with estrogen. That is, it neither inhibits nor enhances the change in size or histology (epithelial cornification) of the cervix, but it results in progressive histopathological responses in the uterus. Why neonatal DES exposure permanently alters estrogen responsiveness in the uterus, but not in the cervix, of adult hamsters is not yet known. A related question is what drives the DES-specific difference in size and histology of cervical regions from the adult hamsters with ovaries (see Figs. 7 and 8). The responses observed in intact versus ovariectomized and estrogen-replaced animals seem to indicate a differential effect of the two neonatal treatment regimens on ovarian factors. In fact, preliminary results indicate that neonatal DES treatment does disrupt morphogenesis of the hamster ovary to a greater extent than does neonatal E2 treatment [3, 44]. Whether neonatal DES treatment disrupts morphogenesis of the hamster ovary by a direct or indirect mechanism and how its follicular development and steroidogenic function may be disrupted are currently under investigation.

In conclusion, both the early and long-term development of the cervix in intact hamsters was differentially disrupted by neonatal exposure to the same high dose of DES versus E2. The extent of this phenomenon in adult animals may be caused, at least in part, by differing degrees of altered ovarian function between the two neonatal treatment groups. However, the mechanisms and ovarian agents responsible for differential disruption in the postpubertal cervix are not yet known. Relevant to this topic, and in contrast to what was found in the hamster uterus, the response of the adult hamster cervix to chronic estrogen stimulation did not differ between the two neonatal treatment groups or between the two neonatal treatment groups and the control group. Future efforts in this experimental system should use a range of lower, environmentally relevant doses of DES, E2, plus other suspected EDs and should continue to probe for differences in molecular dynamics among specific reproductive tract regions (cervix, uterus, oviduct, and ovary).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent processing of histology samples by Pathology Associates International (Jefferson, AR). We also thank Dr. Karen L. Brown-Sullivan (Department of Biological Sciences, Wichita State University, Wichita, KS) for valuable guidance in performing statistical analyses.


   FOOTNOTES
 
1 Supported by U.S. Public Health Service grants CA60250 and P20 RR16475 (BRIN program of the National Center for Research Resources), the Flossie E. West Memorial Foundation, and the U.S. Food and Drug Administration. Back

2 Correspondence: William J. Hendry III, Department of Biological Sciences, Wichita State University, 1845 Fairmount, Wichita, KS 67260- 0026. FAX: 316 978 3772; william.hendry{at}wichita.edu Back

Received: 31 October 2003.

First decision: 21 November 2003.

Accepted: 23 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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