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a Department of Animal & Range Sciences,
b and Cell Biology Center, Biotechnology Institute, North Dakota State University, Fargo, North Dakota 58105
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ovariectomized (OVX), steroid-treated animal is an experimental paradigm that has been used extensively to evaluate the uterotropic effects of the ovarian steroids. Treatment of OVX mammals (including rodents, rabbits, guinea pigs, and primates) with estradiol-17ß (E2) stimulates uterine growth, cell proliferation, and hyperemia [4,7, 12–16]. Similarly, E2 stimulates increased uterine blood flow in OVX cows, ewes, and sows [6, 17, 18].
We recently have shown that treatment of OVX ewes with E2 for 48 h causes a 2- to 3-fold increase in uterine fresh and dry weights and a dramatic increase in the rate of uterine cell proliferation [19]. The purpose of the present study was to determine the time-course of the uterine growth response, and the associated endometrial microvascular development, in OVX, E2-treated ewes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty-two ewes of mixed breeding were ovariectomized (OVX) on Days 10–12 after estrus and allowed to recover for at least 30 days before steroid treatments were begun. Ovariectomized ewes either received no hormone (controls; n = 3 ewes) or received Silastic implants (Dow Corning, Midland, MI; 3.35-mm i.d. x 4.65-mm o.d. x 15-mm length; implanted s.c.) containing 50 mg of E2 (Sigma, St. Louis, MO; 2 implants per ewe), as we have described and validated previously [19]. Ewes were then slaughtered at 0 h (controls) or at 2, 4, 8, 24, 48, or 72 h after receiving the E2 implant (n = 4 to 5 ewes per group).
An i.v. injection of (+)-5-bromo-2'-deoxyuridine (BrdU; Aldrich, Milwaukee, WI; 5 mg/kg BW), a thymidine analogue, was given 1 h before slaughter to evaluate the relative rate of cell proliferation, as we have described and validated previously [8, 19, 20]. Because BrdU is a carcinogen and teratogen, carcasses were disposed of by incineration. At slaughter, the uterus was separated from the adnexa, and total uterine fresh weight (both uterine horns + uterine body) was determined as described previously [8,19–21]. For each ewe, portions of the whole uterus, endometrium, and myometrium were reweighed and freeze-dried for determination of the dry weight:fresh weight ratio [8, 19–21]. Samples (~1 g each) of whole uterus, endometrium, and myometrium also were snap-frozen in liquid nitrogen for later homogenization and determination of DNA, RNA, and protein concentrations. In addition, cross sections (~0.5 cm wide) from the mid-portion of one uterine horn were fixed in Carnoy's solution for immunohistochemical evaluation of cell proliferation (nuclear BrdU incorporation) and histochemical evaluation of microvascular development [8, 19–21].
DNA, RNA, and Protein Concentrations and Contents
To evaluate cellular growth of uterine tissues, concentrations of DNA, RNA, and protein of whole uterus, endometrium, and myometrium were determined as described previously [8, 19–21]. Uterine DNA content was calculated by multiplying uterine weight by DNA concentration of whole uterine samples. Uterine DNA content was used as an index of hyperplasia, and uterine RNA:DNA and protein:DNA ratios were used as indexes of hypertrophy [8,19–21]. Although endometrial and myometrial weights were not determined, and thus their total DNA contents could not be evaluated, their RNA:DNA and protein:DNA ratios were determined.
Endometrial and Myometrial Cell Proliferation
To evaluate the relative rate of cell proliferation, BrdU was immunolocalized in paraffin-embedded uterine cross sections (6 µm), as previously described [8, 19, 20], by using a mouse anti-BrdU monoclonal antibody (Boehringer Mannheim, Indianapolis, IN; 1:100 [1 µg/ml] in blocking buffer) and the avidin-biotinylated peroxidase complex (ABC) system (Vectastain; Vector, Burlingame, CA). Control sections were incubated with normal horse serum (Vector) or mouse ascites fluid (ICN Biochemicals, Costa Mesa, CA) in place of the primary antibody. Tissue sections were counterstained briefly with Harris' hematoxylin, and the relative rate of nuclear incorporation of BrdU, which provides an index of the rate of cell proliferation [8, 19, 20], was evaluated subjectively for luminal epithelial, luminal stromal, glandular epithelial, and myometrial tissue compartments across one entire section. Each tissue compartment was evaluated by two individuals who had no knowledge of the treatment groups and who assigned one of three relative levels of staining: (-) = little or no BrdU labeling (< 1% of cells); (+) = slight to moderate labeling (intermediate between little or no labeling and a relatively large proportion of labeling); or (++) = a relatively large proportion of cells labeling with BrdU (~4–5% of luminal epithelial, 2–5% of luminal stromal, 10–20% of glandular epithelial, and 2–5% of myometrial cells, based on previous studies [19]). The data are reported as the consensus of these two individuals. We chose to evaluate the BrdU labeling subjectively because we already had quantified the response of the various uterine tissue compartments to 48 h of E2 treatment in OVX ewes [19], and for the present study we were interested primarily in the time of onset of cell proliferation after E2 treatment. In addition, we evaluated only luminal endometrial tissue compartments (i.e., those within 0–360 µm, which is approximately 20 cell diameters, of the uterine lumen) because we previously had shown that these are the primary endometrial compartments that proliferate when the uterus grows during the estrous cycle or early pregnancy, or after E2 treatment [8, 19, 20].
Endometrial Microvascular Development
To determine the endometrial vascular response to E2, endometrial vascularity was objectively evaluated by using the morphometric method of point-counting, using the same procedures we have previously described and validated [21, 22]. Briefly, for each uterine horn, a Carnoy's-fixed paraffin-embedded section (6 µm) was stained with Harris' hematoxylin and periodic acid-Schiff reagent (PAS), which has been used extensively by us and others as a histochemical marker of microvascular basement membranes to determine the microvascular density of tissues [21–23]. In addition, we have found close agreement between values obtained using PAS histochemistry and those obtained using other markers of microvascular endothelial cells, such as Factor VIII or lectins ([21, 22]; unpublished observations). As in previous studies, we did not differentiate among arterioles, venules, and capillaries, and therefore the entire microvascular bed was evaluated.
For each uterine horn, caruncular and intercaruncular tissues of the endometrium were evaluated in two regions: within 0–360 µm (~20 cell diameters) of the uterine lumen (luminal), and 360–1080 µm (~20–60 cell diameters) away from the uterine lumen (deep). For each tissue type (caruncular or intercaruncular endometrium) and region (luminal or deep) within each uterine horn, the total area that was counted, as determined by using a stage micrometer, was 66 900 µm2 (6690 µm2 x 2 sites x 5 micrographs). For each tissue type and region within each uterine horn, the microvascular volume density was calculated, on the basis of stereological principles [21, 24], as the percentage of the grid points that were in contact with a microvessel.
Because the microvascular volume density represents the percentage of the tissue volume occupied by microvessels [21, 24], and thus is not a measure of total microvascular volume, we also estimated the volume of the endometrium and used that estimate to determine total microvascular volume as follows. Entire cross sections of each uterine horn that had been stained with hematoxylin and PAS were placed under a stereomicroscope that was equipped with a color video camera (model VI-470; Optronics, Goleta, CA). Each section was magnified at x10 or x15 total magnification so that the entire cross section was in view and then was digitized by using an image analysis system (Kontron KS 400; Roche Image Analysis Systems, Elon College, NC). For each digitized section, a polygon was drawn around the entire cross section, around the myometrial-endometrial border, and around the uterine lumen. The image analysis system was then used to calculate the total uterine area (largest polygon minus uterine luminal polygon), as well as the area of the myometrium and endometrium in mm2, as we have described and validated previously for areas of ovarian follicular tissue compartments [25]. The percentage of the total uterine area occupied by the endometrium was then calculated, and this value was multiplied by the total uterine weight for that ewe to obtain an estimate of the endometrial volume. This estimate is consistent with established stereological principles [21, 24] and is based on the assumption that 1 g of tissue weight ~ 1 ml of tissue volume. To determine total microvascular volume of the endometrium, the endometrial volume (ml) was multiplied by the microvascular volume density (percentage), averaged across all of the endometrial tissue compartments.
Statistical Analysis
Data were analyzed statistically by using least-squares (General Linear Models) ANOVA with treatment group and, when appropriate, tissue type, region, and all possible interactions included in the model [26]. When tissue type and(or) region and the interactions were included in the model, the data were analyzed as a split-plot design, and ewe within treatment was used as the error term for treatment [8, 19, 20]. When an F-test was significant, differences between specific means were evaluated by using Bonferroni's t-test [27]. In addition, linear and nonlinear regression procedures were used to evaluate responses over time [26]. Data are reported as means with their respective pooled SE, unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, the responses of uterine fresh weight, dry weight, and dry weight:fresh weight were best described by using an exponential growth model of the form:
y = ae (b1 - b2 · t) t,
where y = weight in grams, a = weight at Time 0, b1 = initial growth rate in % per hour, b2 = change in growth rate in % per hour, and t = time in hours after estradiol treatment, as described previously [11, 28, 29]. These exponential growth equations were all highly significant (p < 0.001), as follows:
Fresh wt = 22.71758e (0.038019-0.000323t)t, r2 = 0.87, CV = 4.7%;
Dry wt = 4.40795e (0.024130-0.000174t)t, r2 = 0.81, CV = 8.5%; and
Dry wt:Fresh wt = 0.19556e (-0.014383+0.000154t)t, r2 = 0.82, CV = 3.0%.
The cross-sectional areas of the uterine lumen, endometrium, and myometrium all increased (p < 0.01) from 0 to 72 h (Table 2). In addition, across all groups the cross-sectional area of the endometrium was less (p < 0.01) than that of the myometrium (Table 2). The endometrium and myometrium remained constant proportions (p > 0.10) of the total uterine (endometrial + myometrial) tissue area from 0 to 72 after E2 treatment, averaging 26.2 ± 1% and 73.8 ± 1%, respectively (Table 2). Thus, both the endometrium and myometrium also remained constant proportions of uterine volume [24].
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Total uterine DNA content, which provides an index of the number of cells [8, 19–21, 30], exhibited a linear increase (p < 0.01) from 0 to 24 h and then remained constant (Table 3). In contrast, the uterine ratios of RNA:DNA and protein:DNA, which provide indexes of cell size [8, 19–21, 30], did not increase until 24 h (p < 0.01), and then increased further (p < 0.01) at 48 h (Table 3). In addition, the 1.5- to 2-fold increase in uterine size that occurred by 24 h after E2 treatment (Table 1) was due approximately equally to hyperplasia and hypertrophy, since uterine DNA content and the RNA:DNA ratio had both increased (p < 0.01) by approximately 75% at 24 h (Table 3). The further dramatic increase in uterine size that occurred between 24 h and 48 h (Table 1), however, was due primarily to increased cell size, since both the RNA:DNA and protein:DNA ratios increased (p < 0.01), whereas uterine DNA content remained constant (Table 3). In addition, the cellular hypertrophy was reflected by cellular morphology, with an increase in nuclear and cellular volume throughout all of the uterine tissue compartments by 24–48 h after E2 treatment (data not shown).
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For endometrium and myometrium, the dry weight:fresh weight ratio exhibited patterns similar to that of the whole uterus, decreasing about 20–25% (p < 0.01) from 0 to 24 h (0.17 vs. 0.13 and 0.20 vs. 0.16 for endometrium and myometrium, respectively, at 0 vs. 24 h; SE = 0.01) and then remaining constant. Similarly, endometrial and myometrial RNA:DNA and protein:DNA ratios exhibited patterns similar to those of whole uterus, increasing dramatically (p < 0.01) between 8 h and 24 h after E2 treatment (RNA:DNA ratio: 0.30 vs. 0.51 and 0.33 vs. 0.58, protein:DNA ratio: 7.3 vs. 11.0 and 7.0 vs. 13.6, for endometrium and myometrium, respectively, at 8 vs. 24 h; SE = 0.03 for RNA:DNA and 1.4 for protein:DNA) and increasing further through 72 h (RNA:DNA ratio: 0.89 and 1.16, protein:DNA ratio: 16.4 and 21.5, for endometrium and myometrium, respectively, at 72 h). In addition, the ratios of dry weight:wet weight, RNA:DNA, and protein:DNA all were less (p < 0.01) for endometrium compared with myometrium (15.4 vs. 17.2, 0.50 vs. 0.60, and 9.7 vs. 12.9, for endometrium vs. myometrium, respectively).
The relative rate of uterine cell proliferation (nuclear incorporation of BrdU) was low through 4 h, with a slight increase in labeling in the luminal epithelium at 8 h (Table 4). By 24 h, however, moderate (glandular epithelium) to extensive (luminal epithelium and stroma) BrdU incorporation was observed in the endometrium; BrdU labeling in the myometrium, however, was still low (Table 4). At 48 h and 72 h, a relatively large proportion of the cells exhibited nuclear incorporation of BrdU, indicating a relatively high rate of cell proliferation, in all of the uterine compartments including the myometrium (Table 4). As observed in our previous studies [8, 19, 20], BrdU labeling was exclusively nuclear, and endometrial labeling occurred mostly in the luminal compartments (data not shown).
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Endometrial microvascular volume increased dramatically from 0 to 24 h after E2 treatment; histologically, this was manifest as a dramatic increase in the density of microvessels (Fig. 1). Across all of the endometrial tissue compartments, volume density of the microvasculature increased linearly (p < 0.01) between 0 h and 24 h (4.7 vs. 8.4%; SE = 1.2%) and then remained constant (Fig. 2). In addition, across all times the volume density of microvessels was similar for caruncular and intercaruncular tissues (6.8 vs. 6.1%; SE = 0.4%) but was greater (p < 0.01) for luminal than for deep regions (6.9 vs. 5.8%; SE = 0.4%).
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Because the endometrium remained a constant proportion of uterine volume from 0 to 72 h, its mass increased proportionately with the increase in uterine fresh weight. Thus, from 0 to 24 h the increase in tissue mass (Table 1) combined with the increase in microvascular volume density (Fig. 2) resulted in a 5-fold increase in total microvascular volume of the endometrium (0.21 vs. 1.01 ml; SE = 0.01 ml). In addition, although the proportion of the tissue occupied by microvessels remained relatively constant from 24 to 72 h (Fig. 2), the increase in tissue mass (Table 1) resulted in a further increase in total microvascular volume of the endometrium by 72 h (1.37 ml).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For example, the doubling time, which is a key measure of growth rate [25, 30], was about 20.0 h for uterine fresh weight from 0 to 24 h and about 27.9 h for uterine fresh weight from 0 to 48 h after E2. This dramatic growth rate is equal to or greater than that of the fastest-growing tissues known [30, 34]. However, in addition to the rapidity of the uterine growth response to E2, we have also shown that it results from both cell proliferation and increased cell size. This pattern of growth is similar to that of most tissues, especially during the postnatal period [30, 35, 36]. Thus, the uterine growth in response to E2 should make a good model not only for study of uterine function but also of tissue growth in general.
In the present study, the uterine growth response was due primarily to tissue growth (hyperplasia and hypertrophy) rather than to a change in the ratio of tissue dry weight:fresh weight. In addition, tissue growth was due approximately equally to hyperplasia and hypertrophy during the initial 24 h after E2 treatment. The initial uterine growth response differed from that observed for immature or ovariectomized rats, in which uterine growth at 6–12 h after E2 is due primarily to a change in the tissue dry weight:fresh weight ratio [37, 38]. However, by 24 h after E2 treatment, the growth of the rodent uterus was reflected by uterine hyperplasia and hypertrophy [7, 14, 15], which is similar to our observations for the sheep uterus. In addition, the endometrium and myometrium remained a constant proportion of the uterus in OVX, E2-treated mice, similar to our observations in the present study.
The pattern of cell proliferation and cell growth also differed somewhat in sheep compared with rodents. In the present study, we observed a dramatic increase in the rate of cell proliferation, beginning in the luminal epithelium at 8 h and culminating in extensive cell proliferation in all of the uterine compartments, including the myometrium, by 24–48 h. Similarly, in OVX mice, E2 stimulated a dramatic increase in the rate of cell proliferation in the luminal epithelium, glands, and stroma within the first 24 h, as reflected by increased numbers of cells in these uterine compartments [39]. In the mouse myometrium, however, nuclear incorporation of [3H]thymidine increased only slightly, and numbers of cells did not change, even though myometrial volume increased dramatically [39]. Thus, in OVX, E2-treated mice, the response of the myometrium was due almost entirely to increased cell size, whereas in sheep, both increased cell proliferation and cell size contributed to myometrial growth.
In the present study, cell number (DNA content) of the uterus did not change from 24 to 72 h after E2 treatment, even though the rate of cell proliferation remained high in all of the uterine tissue compartments. This observation suggests that cell death must have increased dramatically after 24 h and is consistent with a dramatic increase in cell death observed at 16–40 h after E2 treatment in OVX mice [39]. In mice, however, the onset of cell death was accompanied by a decrease in uterine weight and tissue volume, whereas in sheep we did not observe a decrease in uterine weight nor in the volume of the uterine tissue compartments.
Whereas uterine fresh and dry weights increased 2.5- to 3-fold, total microvascular volume increased 6.5-fold by 72 h after E2 treatment. Thus, the uterine growth response was accompanied by an even more dramatic increase in microvascular volume. Microvascular volume density (the proportion of the tissue occupied by microvessels) normally remains constant during growth of uterine as well as other tissues, since microvascular growth normally keeps pace with tissue growth [10, 23, 40]. However, in the present study, microvascular volume density increased nearly 2-fold from 0 to 24 h. Thus, our data indicate that vasodilation probably accounts for a significant portion of the uterine microvascular response to E2, which is consistent with its well-known role as a powerful stimulator of uterine blood flow [16]. It is highly unlikely, however, that the uterine microvascular response was due solely to vasodilation, because many of the microvessels that we quantified were capillaries, which do not possess vascular smooth muscle and are therefore incapable of dilation [23]. The dramatic increase in total microvascular volume, therefore, most likely involved both microvascular vasodilation and growth.
The volume density of the uterine microvasculature also increased approximately 2-fold during early pregnancy, resulting in a 3-fold increase in total uterine microvasculature by 24–30 days after mating in ewes [11, 21]. In addition, uterine microvascular growth continues throughout pregnancy in sheep [11]. We have hypothesized that uterine microvascular growth during pregnancy is regulated by the conceptus [11, 21]. Data from the present study, in which the uterine microvascular response resembled that previously observed during early pregnancy, suggest that E2 may, at least in part, mediate the effects of the conceptus on the uterine microvasculature. This suggestion is consistent with the observation that chronic infusion of E2 into the uterine lumen was able to maintain a 10-fold increase in uterine blood flow, and thus mimicked the increase in uterine blood flow observed during early pregnancy [41, 42].
It also is clear from the present data that microvascular growth cannot account for the early and substantial increase in uterine blood flow that occurs within 30–60 min after estrogen treatment in ovariectomized mammals and that is probably due exclusively to vasodilation of the uterine vascular bed [16]. Nevertheless, the sustained increase in uterine blood flow that occurs throughout pregnancy is probably due primarily to growth of the entire uterine vascular bed, including the uterine arteries [11, 43]. In addition, a portion of the angiogenic response of the uterus, including the up-regulation of endometrial angiogenic factor expression [44], may be caused by the early increase in uterine blood flow, since sheer-stress resulting from increased flow has been shown to induce angiogenesis in a variety of tissues [23, 45].
In conclusion, we have shown that the uterine growth in OVX, E2-treated sheep is due to hyperplasia and hypertrophy, with only a relatively small change in the tissue dry weight:fresh weight ratio. The most dramatic uterine growth occurred between 8 and 48 h after E2 treatment and was associated with increased cell proliferation in all of the uterine tissue compartments. In addition, the uterine growth response was associated with an even more dramatic increase in the volume of endometrial microvasculature. These studies will provide a foundation for further elucidation of the mechanisms responsible for uterine growth and microvascular development in response to steroids. For example, in a companion paper [44], we have investigated endometrial expression of two major angiogenic factors, namely basic fibroblast growth factor and vascular endothelial growth factor, and related the expression of these factors to the microvascular response observed in the present study.
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FOOTNOTES |
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2 Correspondence. FAX: (701) 231–7590; lreynold{at}prairie.nodak.edu
Accepted: April 21, 1998.
Received: January 7, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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