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Endocrinology, doi:10.1210/en.2003-1078
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Endocrinology Vol. 145, No. 2 959-966
Copyright © 2004 by The Endocrine Society

Regulation of Oocyte Microvilli Development in the Baboon Fetal Ovary by Estrogen

Nicholas C. Zachos, Reinhart B. Billiar, Eugene D. Albrecht and Gerald J. Pepe

Department of Physiological Sciences (N.C.Z., R.B.B., G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501; and Departments of Obstetrics, Gynecology, and Reproductive Sciences and Physiology (E.D.A.), The Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Gerald J. Pepe, Ph.D., Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. E-mail: pepegj{at}evms.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently showed that the number of primordial follicles was reduced by 50% in ovaries of near-term fetal baboons deprived of estrogen in utero and restored to normal in animals supplemented with estrogen. Oocytes are avascular and rely on surrounding granulosa cells for nutrients, a process facilitated by microvilli on the oocyte surface. However, our understanding of oocyte microvillus development in the primate fetal ovary is incomplete. Thus, we determined whether estrogen regulates formation of oocyte microvilli in utero. Fetal ovaries were obtained on d 165 gestation (term = d 184) from baboons untreated (n = 3) or treated on d 100–165 with aromatase inhibitor CGS 20267 (estrogen suppressed by 95%; n = 5) or CGS 20267 and estradiol (n = 4). Follicles with intact (homogeneous cytoplasm) or nonintact (cytoplasm vacuolated) oocytes were quantified and the number/height of oocyte microvilli determined by electron microscopy. In untreated baboons, the mean (±SE) number of follicles/0.08 mm2 with an intact oocyte (11.5 ± 0.5) was decreased (P < 0.05) by 70% in fetal ovaries of estrogen-suppressed baboons (3.4 ± 0.2) and restored (P < 0.05) by CGS 20267 and estradiol (11.2 ± 1.2). In estrogen-deprived fetuses, the number of microvilli/intact oocyte (23 ± 3) was 56% lower (P < 0.01) than normal (52 ± 5) and restored by CGS 20267 and estrogen (62 ± 4). Moreover, in intact oocytes of estrogen-suppressed baboons, height (nm) of microvilli (105 ± 11) was 54–62% lower (P < 0.01) than in intact oocytes of fetal ovaries of untreated (228 ± 13) or estrogen-treated (274 ± 17) baboons. In estrogen-replete baboons, the number of microvilli in intact oocytes was 2-fold greater (P < 0.01) than in nonintact oocytes. However, in estrogen-deprived baboons, no microvilli were detected in nonintact oocytes and the number of microvilli in intact oocytes was similar to that in nonintact oocytes of untreated fetuses. We conclude that development of microvilli in oocytes of primordial follicles in the primate fetal ovary is regulated by estrogen. Collectively, these results and those of our previous studies indicate that estrogen regulates fetal ovarian folliculogenesis and development of follicles with oocytes composed of microvilli critical for nutrient uptake and presumably long-term survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE RECENTLY SHOWED that the baboon fetal ovary expresses estrogen receptors {alpha} and ß (ER{alpha} and ERß) (1) and that the development of primordial follicles was significantly reduced in baboon fetuses in which estrogen production was suppressed throughout the second half of gestation by treatment with an aromatase inhibitor (2). Because ovarian maturation and folliculogenesis were restored to normal in animals concomitantly treated with aromatase inhibitor and estradiol benzoate, we proposed that estrogen regulates fetal ovarian maturation and thus development of the pool of follicles available for reproductive function in adulthood (2).

Oocytes of primordial follicles formed in utero must remain healthy for many years until recruited for growth, maturation, and ultimately ovulation. However, oocytes are avascular and dependent upon surrounding granulosa cells for uptake and exchange of metabolic products and thus survival (3, 4, 5). In epithelial cells of the kidney and small intestine, nutrient uptake from the systemic circulation is accomplished primarily by microvilli, and defects in the microvillus brush border typically result in various pathologies (see Ref.6 for review). Microvilli are present on oocytes of primordial follicles in the human fetal (7, 8) and adult (9) ovary, and in the adult rat ovary, oocytes undergoing atresia exhibit retraction of their microvilli as well as other morphological changes including cytoplasmic vacuolization (10). Thus, it appears that microvilli are essential for oocyte health and survival. Despite their physiological importance, however, our understanding of oocyte microvillus formation in the primate fetal ovary is incomplete.

Estrogen has previously been shown to increase the number and length of microvilli in rat pituitary cells (11) and in ER-positive MCF-7 breast cancer cells (12). Moreover, microvilli are markedly disrupted/depleted in ovarian primordial follicles of women undergoing chemotherapy, an effect partially prevented by pretreatment with estrogen-containing oral contraceptives (9). Therefore, in the current study we determined whether estrogen regulates the formation and/or maintenance of oocyte microvilli in the primate fetal ovary and whether the development of microvilli by the oocyte is associated with oocyte structural integrity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All fetal ovaries used in this study were from a contemporaneous group of baboons previously used to study other aspects of fetal ovarian development (1, 2). Fetal ovaries were obtained on d 100 (n = 3) and 165–170 (n = 6) gestation from untreated baboons (Papio anubis) and on d 165–170 gestation (term = d 184) from animals treated with the highly specific aromatase inhibitor CGS 20267 [Letrozole; 4,4-(1,2,3-triazol-1yl-methylene) bis-benzonitrite; Novartis Pharm AG, Basel, Switzerland] administered sc [115 µg/kg body weight (BW)·d·0.05 ml sesame oil; n = 6] to the mother on d 100–165 gestation essentially as described previously (13). Additional animals were injected with CGS 20267 (115 µg/d·kg BW) plus estradiol benzoate (50–175 µg/kg BW·d·0.1 ml sesame oil; n = 5) on d 100–165 gestation. Blood samples (3–5 ml) were obtained at 1- to 4-d intervals between d 85 and 164 of gestation via a maternal saphenous vein after sedation with an im injection of ketamine-HCl (10 mg/kg BW; Parke-Davis, Detroit, MI). Using this experimental protocol, we previously demonstrated (13) that the incidence of spontaneous abortion in untreated baboons (<5%) was increased in baboons treated with CGS 20267 (25%) and prevented in animals concomitantly treated with CGS 20267 and estrogen (<5%). On d 100 and 165 gestation, baboons were sedated with ketamine and anesthetized with isoflurane, and after obtaining maternal and umbilical venous and arterial blood samples, the placenta and the fetus were delivered by cesarean section and the fetus euthanized with an overdose of sodium pentobarbital. Maternal and umbilical venous serum samples were stored at -20 C and assayed for estradiol as described previously (2, 13). Fetal ovaries were excised, trimmed of fat, and weighed, and one ovary was stored in liquid nitrogen. A section from the middle region of the other ovary was fixed in Zamboni’s fixative (Newcomer Supply, Middleton, WI) for 24 h at room temperature, processed, and embedded in epon for electron microscopic analysis. The remaining portion of the fetal ovary was placed in 10% buffered formalin and paraffin embedded for histology. Ovaries were also available from three adult baboons euthanized with sodium pentobarbital 8 d or 48 h before or 6 d after anticipated ovulation as judged by menstrual cycle history and daily examination of perineal turgescence (14). Baboons were cared for and used strictly in accordance with United States Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication 85–23, 1985). The Institutional Animal Care and Use Committee of the Eastern Virginia Medical School approved the experimental protocol used in this study.

Histology and image analysis
Representative sections (4 µm) of paraffin-embedded fetal ovaries were stained with hematoxylin to determine the number of follicles/0.082 mm2 in which the oocyte was intact or vacuolized. Briefly, a minimum of 10 cortical areas (0.082 mm2) of 20–40 randomly selected sections of the fetal ovary were examined using an Optiphot-2 microscope attached to a video-based Image-1 analysis system, and parameters were established to ensure that follicles were counted only once. The number of intact or nonintact follicles was calculated for each animal, and data were expressed as an overall mean (±SE).

Electron microscopy
Zamboni-fixed ovarian sections were cut into 3-mm blocks, washed in PBS (Sigma Chemical Co., St. Louis, MO), and placed in 1% buffered osmium tetroxide (Aldrich Chemical Corp., Philadelphia, PA) at room temperature for 1 h. After three washes in PBS, ovarian blocks were dehydrated in 30, 50, 70, 95, and 100% ethanol and washed twice in propylene oxide (Sigma Chemical Co.) for 15 min. Samples were incubated in propylene oxide/PolyBed 812 (1:1) for 1 h, in propylene oxide/PolyBed 812 (1:2) for 2 h, and in 100% PolyBed 812 (Sigma Chemical Co.) overnight. Ovaries were embedded in Beam capsules (Fisher Scientific Corp., Philadelphia, PA) previously polymerized after incubation at 60 C for 24–36 h. Epon-embedded fetal ovaries were sectioned (1 µm), stained with Richard’s stain (Fisher Scientific Corp.), and examined using an Olympus BX50 upright microscope equipped with a spot slider digital camera. Ultrathin (90-nm) sections were prepared from three representative sections (cortex and medulla visible and encompassed by an intact capsule) per ovary, stained with uranyl acetate and lead citrate and mounted onto nickel grids, and imaged using a JEOL 1200 EX II (Peabody, MA) transmission electron microscope. The number and height of oocyte microvilli in two-dimensional sections through each oocyte were quantified in 30–40 follicles from three or four grids of randomly selected ultrathin sections from each fetal ovary obtained on d 100 and 165 gestation from untreated baboons (n = 3) and on d 165 from baboons treated with CGS 20267 (n = 5) or CGS 20267 and estrogen (n = 4). The number of microvilli on the surface of oocytes and contiguous with the plasma membrane were visualized at x10,000 and manually counted, and data were recorded as number of microvilli per intact or nonintact oocyte and as the number of microvilli per square micrometer oocyte surface area. To ensure that an oocyte was evaluated only once, the number of square areas comprising each grid covering a fetal ovarian section was recorded and areas within each grid analyzed sequentially and row by row. Thus, the entire area encompassing the fetal ovarian section was accounted for. In addition, to ensure that the entire circumference of the oocyte could be accurately measured, only oocytes that were not visually impaired by the grid overlay were analyzed. Microvillus height was measured at x25,000 using a calibration bar imprinted on the transmission electron microscope image screen and determined on microvilli that were evenly dissected and extended perpendicular to the plasma membrane. Data were imported to Excel files and analyzed, and results were calculated for each animal and expressed as an overall mean (±SE). The presence or absence of cytoplasmic vacuoles in oocytes as well as any alteration in morphology of surrounding granulosa cells (e.g. mitochondrial shape/structure) at various magnifications (x6,000–25,000) were also notated.

Statistics
Data (mean ± SE) were analyzed by ANOVA with post hoc comparisons of the means by Student-Newman-Keuls multiple comparison tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum estradiol concentrations
As demonstrated previously (2, 13), maternal serum estradiol levels rose from approximately 1 ng/ml on d 85–120 gestation to 2.5–3.0 ng/ml by d 165. Within 48–72 h of the onset of CGS 20267 treatment on d 100, maternal serum estradiol levels decreased to and remained at approximately 0.1 ng/ml. The pattern of serum estradiol in baboons treated with CGS 20267 plus estradiol benzoate was similar to that in controls; however, absolute levels were slightly greater than normal. Estradiol levels in umbilical venous serum on the day of delivery in untreated baboons (0.59 ± 0.13 ng/ml) were also decreased (P < 0.01) by administration of CGS 20267 (0.04 ± 0.01 ng/ml) and restored (0.19 ± 0.08 ng/ml) to 30% of normal in baboons treated with CGS 20267 and estradiol benzoate.

Effects of estrogen on oocyte/follicle development
In untreated baboons at midgestation (d 100), the fetal ovary was composed primarily of pregranulosa cells and oocytes in germ cell cords and very few primordial follicles (Refs. 1 and 2 and data not shown). As shown in Fig. 1Go and Table 1Go, in fetal ovaries of untreated baboons on d 165 gestation, the mean number of follicles/0.08 mm2 with an intact oocyte (11.5 ± 0.5) was decreased (P < 0.05) by approximately 70% in ovaries of baboons in which estrogen production was suppressed (3.4 ± 0.2) and restored (P < 0.05) to normal in animals treated with CGS 20267 and estradiol (11.2 ± 1.2). The mean number of follicles in which the cytoplasm of the oocyte was vacuolated (i.e. nonintact) was similar in fetal ovaries of baboons untreated (8.5 ± 1.2) or treated with CGS 20267 (7.6 ± 0.5) or CGS 20267 and estrogen (8.2 ± 0.8). Therefore, because the total number of primordial follicles was reduced by approximately 50% in estrogen-suppressed animals, the overall percentage of follicles with a vacuolated oocyte in estrogen-deprived baboons (68 ± 2%) was 2-fold greater (P < 0.05) than in ovaries of baboon fetuses untreated (35 ± 5%) or treated with CGS 20267 and estrogen (38 ± 3%). In contrast, the overall percentage of intact follicles in estrogen-suppressed baboons was only 50% of that in estrogen-replete animals.



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  		FIG. 1. A, Representative histology of intact and nonintact (vacuolized) follicles in near-term baboon fetal ovary; B–D, photomicrographs of intact (I) and vacuolized (V; nonintact) follicles in sections through the inner cortex of the fetal ovary on d 165 gestation in baboons untreated (B) or treated with CGS 20267 (C) or CGS 20267 and estradiol benzoate (D) as described in the footnote to Table 1Go. Paraffin sections (4 µm) were stained with hematoxylin, and follicles with oocyte cytoplasm that was homogenous (intact) or vacuolized (nonintact) were examined by light microscopy. Original magnifications: A, x600; B–D, x400. Bar, 20 µm.

 

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  		TABLE 1. Effect of estrogen on folliculogenesis in the near-term baboon fetal ovary

 
Effects of estrogen on oocyte microvilli development
In the adult baboon ovary, primordial follicles exhibited microvilli on the surface of oocytes as can be seen by transmission electron microscopy in Fig. 2AGo. In the small number of follicles that developed in the fetal ovary of untreated baboons at midgestation, oocytes did not exhibit microvilli; rather, oocytes appeared to contact surrounding granulosa cells via desmosomes (Fig. 2BGo). By late gestation, well- developed microvilli were detected on the surface of the oocyte of all primordial follicles examined (Fig. 2CGo). Moreover, granulosa cells appeared intact with well-defined nuclei and cytoplasm with electron-dense mitochondria (Fig. 2CGo). In contrast, in animals treated with CGS 20267, oocyte microvilli were absent or severely depleted in virtually all primordial follicles (Fig. 2DGo). However, despite alterations in oocyte microvilli, granulosa cells still appeared normal (Fig. 2DGo). It was also apparent that in fetal ovaries of CGS 20267-treated baboons, but not estrogen-replete animals, in many instances oocytes exhibited contact with granulosa cells via desmosomes, as observed at midgestation. In fetal ovaries of baboons treated with CGS 20267 and estradiol benzoate, oocyte microvilli formation was restored to normal (Fig. 2EGo). Additional analyses showed that in fetal ovaries of animals untreated or treated with CGS 20267 and estrogen, oocytes with intact cytoplasm contained mitochondria that appeared normal as judged by detection of an electron-dense matrix and abundant cristae (Fig. 3Go, A and C). In contrast, in fetal ovaries of estrogen-suppressed baboons in which oocytes exhibited a marked degree of cytoplasmic vacuolization, mitochondria appeared larger, less electron dense, and with fewer cristae (Fig 3BGo).



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  		FIG. 2. Representative photomicrographs of oocyte microvilli in primordial follicles of adult baboon ovary (A) and of fetal ovary at mid (B) and late (C) gestation in untreated baboons and at late gestation in animals treated with CGS 20267 (D) or CGS 20267 plus estradiol benzoate (E) as described in the footnote to Table 1Go. Epon-embedded ovaries were sectioned (1 µm), and ultrathin preparations (90 nm) from three representative sections (cortex and medulla visible and encompassed by a contiguous capsule) per ovary were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy. Original magnifications: A, x10,000; B and D, x17,000; C and E, x24,000. Bars: A, 1 µm; B–E, 500 nm.

 


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  		FIG. 3. Representative photomicrographs of oocyte mitochondria in primordial follicles of baboon fetal ovary at late gestation in animals untreated (A) or treated with CGS 20267 (B) or CGS 20267 plus estradiol benzoate (C) as described in the footnote to Table 1Go. Epon-embedded ovaries were examined by transmission electron microscopy as described in the legend to Fig. 2Go. Original magnifications, x13,000; bar, 500 nm. Insets in A–C are photomicrographs depicting cristae in oocyte mitochondria (original magnifications, x25,000).

 
The effects of estrogen on oocyte microvilli development were quantified using image analysis. Thus, as shown in Fig. 4AGo, the mean (±SE) number of microvilli/intact oocyte in estrogen-suppressed fetuses (23 ± 3) was 56% lower (P < 0.01) than normal (52 ± 5) and restored by CGS 20267 and estrogen (62 ± 4). Similar results were noted when the number of microvilli was expressed per square micrometer oocyte surface area (Fig. 4BGo). Moreover, as seen in Fig. 4CGo, average height of microvilli on intact oocytes of CGS 20267-treated baboons (105 ± 11 nm) was 54% lower (P < 0.01) than in baboons untreated (228 ± 13 nm) or treated with CGS 20267 plus estrogen (274 ± 17 nm).



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  		FIG. 4. Mean (±SE) number and height of oocyte microvilli in primordial follicles in fetal ovaries obtained on d 165 gestation from baboons untreated or treated with CGS 20267 or CGS 20267 and estradiol benzoate as described in the footnote to Table 1Go. Approximately 30–40 follicles per animal were examined by transmission electron microscopy as outlined in the legend to Fig. 2Go, and the number of microvilli per oocyte (A) or per square micrometer oocyte surface area (B) as well as microvillus height (C) were calculated for intact follicles. The number of oocyte microvilli in intact follicles compared with that in nonintact follicles are shown in B. Values with different letter superscripts differ from each other at P < 0.01 (ANOVA and Newman-Keuls multiple statistic).

 
Figure 4BGo compares the number of microvilli/µm2 oocyte surface area in follicles with oocytes intact or vacuolated. In untreated baboons, the number of microvilli/µm2 oocyte surface area in follicles with an intact oocyte (2045 ± 351) was 2-fold greater (P < 0.01) than in follicles with a nonintact oocyte (989 ± 144). However, in estrogen-deprived baboons, no microvilli were detected in nonintact oocytes and the number of microvilli in intact oocytes (1157 ± 135) was similar to that in nonintact oocytes of untreated fetuses. In fetal ovaries of baboons treated with CGS 20267 and estrogen the number of microvilli/µm2 oocyte surface area of intact (2498 ± 185) and nonintact (798 ± 43) oocytes were each restored (P < 0.01) to normal (Fig. 4BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the current study are the first to demonstrate that development of a microvillus brush border by the oocyte of primordial follicles is regulated in utero in the primate fetal ovary by estrogen. Thus, by late gestation, at which time the baboon fetal ovary was composed almost exclusively of primordial follicles, oocytes within these follicles contained an abundant number of microvilli on their plasma membrane. Moreover, both the number and size of microvilli were markedly decreased in oocytes of fetal ovaries from baboons deprived of estrogen and restored to normal in animals concomitantly treated with aromatase inhibitor and estrogen. Although the mechanism(s) by which estrogen regulates oocyte microvillus formation and growth remains to be determined, estrogen has previously been shown to increase the number and length of microvilli in rat pituitary lactotrophs (11) and enhance microvilli formation and the number of microvillus channels at the luminal surface of alveolar type I cells in the fetal bovine lung in utero (15). Estrogen also increased the number and height of microvilli in uterine luminal epithelial cells in ovariectomized rats in vivo (16) and in ER-positive MCF-7 cells in vitro (12). Moreover, the height of microvilli in epithelial cells of efferent ductules was markedly reduced in ER{alpha} knockout mice (17). Therefore, considering the results of the present study and because ER{alpha} and ERß proteins are expressed in pregranulosa (ER{alpha} and ERß) as well as granulosa (ERß) cells and perhaps oocytes (ERß) of the baboon fetal ovary during the second half of gestation (1), we suggest that estrogen regulates fetal ovarian microvillus development via receptor-mediated autocrine/paracrine mechanism(s).

The results of the current study further showed that in estrogen-suppressed animals, the number of follicles in which the oocyte cytoplasm was intact and contiguous with granulosa cells was markedly reduced and a significantly greater proportion of the remaining follicles contained an oocyte that exhibited cytoplasmic vacuolization. Moreover, these changes in oocyte structure were correlated with oocyte microvillus development. Thus, in estrogen-deprived fetal baboons, the size and number of microvilli in oocytes with an intact cytoplasm was decreased by approximately 50%, and no microvilli were detected in oocytes in which the cytoplasm was vacuolated. In the adult rat, oocyte cytoplasmic vacuolization and retraction of oocyte microvilli have been shown to be associated with oocyte demise (10). Therefore, it is possible that in estrogen-deprived fetal baboons, not only is there a reduction in the number/pool of primordial follicles available for adult reproductive function, but of the follicles that are available, some are also composed of oocytes that have either undergone (no microvilli) or are at risk of undergoing (reduced number of microvilli) demise. In support of this suggestion, in the majority of nonintact follicles examined in estrogen-suppressed baboon fetuses, oocyte mitochondrial size appeared to be increased and electron-density/cristae decreased, although these alterations were not quantified in the current study.

Whether the reduction/loss of the microvillus brush border was the cause or the consequence of oocyte vacuolization in the fetal ovary remains to be determined. However, in the current study, it was surprising to note that despite the qualitative status of the oocyte and number of microvilli formed, the surrounding granulosa cells in virtually all follicles examined appeared structurally intact and did not exhibit signs of impending demise. In the human fetal ovary, Vaskivuo et al. (18) demonstrated that apoptosis (i.e. DNA fragmentation) was detectable in oocytes and that the incidence markedly decreased with advancing gestation in association with the rise in fetal estrogen levels (19). However, apoptosis was never detected in pregranulosa at early- to mid-pregnancy or in follicular granulosa cells in late gestation, leading these authors to suggest that oocyte demise may be an oocyte-initiated phenomenon and not preceded by or the result of apoptosis in the granulosa cell (18). The results of the current study are consistent with this conclusion. Therefore, because microvilli exponentially increase the surface area of cells and contain many integral membrane proteins to ensure homeostasis, we speculate that the reduction and/or loss of oocyte microvilli in estrogen-suppressed fetal ovaries is not the result of but rather may be one of the factors that leads to oocyte demise. This suggestion, however, does not rule out alternative possibilities, e.g. that estrogen is acting on the granulosa cells to produce a factor(s) that then acts in a paracrine manner to maintain oocyte microvilli.

In the adult ovary, estrogen has been shown to play an important role in the protection of granulosa cells from apoptosis by regulating, via receptor-mediated mechanisms, the expression of components of the apoptotic machinery (20, 21, 22). Thus, it would appear these well-documented effects of estrogen on granulosa cell survival may not be manifest in the primate fetal ovary, despite the fact that these cells as well as oocytes express ERß (1). However, as described previously, the number of primordial follicles formed in estrogen-suppressed baboon fetuses was reduced by approximately 50%. Whether this loss resulted from removal of follicles and/or failure of oocytes to become encapsulated by granulosa cells (23) remains to be ascertained. However, in either case, it is apparent that estrogen is essential for granulosa cell development and follicle formation.

The biochemical mechanisms by which estrogen directly or indirectly regulates oocyte microvilli development in the baboon fetal ovary remain to be elucidated. It is well established that microvilli are composed of core actin filaments and several actin-binding proteins, including ezrin, radixin, and/or moesin, i.e. the ERM family of cytoskeletal proteins (24, 25, 26). Because microvillus structures are completely lost after treatment of cultured cells with antisense oligonucleotides that block ERM protein formation, these proteins have been proposed to play a key role in microvilli formation in general (27, 28). Preliminary studies in our laboratories indicate that ezrin is expressed in the oocyte of primordial follicles in the baboon fetal ovary and that cellular localization may be modified in oocytes of fetal ovaries from estrogen-suppressed baboons (Pepe, G. J., N. C. Zachos, and E. D. Albrecht, unpublished data). Kondo et al. (29) have also demonstrated that breakdown of microvilli resulted from disruption of the scaffolding proteins (e.g. ezrin) and was a very early event that preceded and resulted in apoptosis. Thus, Fas-ligand-induced apoptosis in mouse L929 fibroblast cells resulted first in disruption of the microvillus scaffolding proteins, which was then followed 3–4 h later by the initiation of DNA fragmentation. Based on these and an additional series of elegant experiments, Kondo et al. (29) proposed that ERM protein interaction with the actin cytoskeleton and plasma membrane provides a protective mechanism against physiological signals to cell death and that the destruction of the microvillus brush border is required for apoptotic changes in the nucleus (i.e. DNA fragmentation). These provocative findings are consistent with and thus further strengthen our hypothesis that estrogen promotes formation of healthy follicles by regulating development of oocyte microvilli.

In summary, we have shown that oocytes within primordial follicles of the fetal baboon ovary in late gestation contained an abundant number of microvilli on their plasma membrane. Moreover, both the number and size of the microvilli were markedly decreased in oocytes of fetal ovaries from baboons deprived of estrogen and restored to normal in animals concomitantly treated with aromatase inhibitor and estrogen. We conclude that development of a microvillus brush border on the oocyte of primordial follicles occurs in utero and is regulated by estrogen. Collectively, these results and those of our previous studies indicate that estrogen regulates fetal ovarian folliculogenesis and thus development of follicles with oocytes composed of a microvillus brush border critical for nutrient uptake and presumably long-term survival.


   Acknowledgments
 
We greatly appreciate the supply of CGS 20267 generously provided by Norvartis Pharma AG (Basel, Switzerland). We thank Sandra Huband for secretarial assistance with this manuscript and preparation of the figures and Jeff Dupree, Ph.D. (Department of Pathology and Anatomy, Eastern Virginia Medical School), for assistance in the conduct and interpretation of the electron microscopy studies.


   Footnotes
 
This work was supported by NIH Grant U54 36207 as part of the National Institute of Child Health and Human Development Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: BW, Body weight; ER, estrogen receptor; ERM, ezrin, radixin, and/or moesin.

Received August 18, 2003.

Accepted for publication October 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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