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Biol Reprod. 2008 December; 79(6): 1160–1168.
Prepublished online 2008 August 20. doi:  10.1095/biolreprod.108.067900
PMCID: PMC2780470

Regulation of Expression of Microvillus Membrane Proteins by Estrogen in Baboon Fetal Ovarian Oocytes1

Abstract

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.

Keywords: baboon, estradiol, estrogen, fetal oocytes, follicular development, microvilli, oocyte development, ovary

INTRODUCTION

We previously showed that the number of primordial follicles developed in utero and available for reproductive function in adulthood was reduced by more than 50% in fetal baboons in which estrogen levels were depleted during the second half of gestation by treatment with an aromatase inhibitor [1]. Moreover, whereas most of the follicles formed in untreated (i.e., estrogen-replete) baboons contained oocytes with an intact cytoplasm and healthy appearance, in estrogen-depleted fetuses, the majority (>70%) of follicles that formed contained oocytes in which the cytoplasm exhibited vacuolization and was composed of swollen and less electron-dense mitochondria and a marked reduction in the number and/or depletion of microvilli [2]. Earlier studies by others [3, 4] have shown that oocyte growth and maturation and, thus, survival requires uptake of nutrients (e.g., glucose) from the surrounding granulosa cells, functions attributed to intercellular interaction between oocytes and granulosa cells and typically facilitated by microvilli [5]. It also has been shown that breakdown of the microvillus brush border in transformed renal epithelial cells facilitated cellular response to an apoptotic signal [6], and in the adult rodent ovary, oocytes undergoing demise also exhibited cytoplasmic vacuolization and concomitant retraction of their microvilli [7]. Based on these observations and our previous studies showing that follicle formation and oocyte cytoplasmic integrity and microvilli development were restored to normal in fetal baboons treated with aromatase inhibitor and estradiol [1, 2], we proposed that estrogen regulates development of follicles comproed of healthy oocytes by controlling the development of microvilli on the surface of oocytes [8].

It is well established that ezrin, a member of the ezrin-radixin-moesin-merlin (ERM) family of cytoskeletal proteins, plays an important role in the formation and maintenance of microvilli [912]. Thus, following phosphorylation, ezrin phosphate links f-actin filaments together to create a structure that serves as the frame/scaffold for the formation of microvilli [12], whereas Na+/H+ exchange regulatory factor 1 (solute carrier family 9 [sodium/hydrogen exchanger, member 3 regulator 1, NHERF1, now termed SLC9A3R1]), also termed ezrin-binding protein, anchors the ezrin-f-actin frame/scaffold directly to the plasma membrane. Other critical proteins, most notably α-actinin, which cross-links f-actin filaments, are required to complete the formation of microvilli and establishment of a microvillus brush border [13, 14]. However, despite the apparent importance of ERM proteins to formation of microvilli in epithelial cells [9], there have been no studies of the ERM family of proteins in the fetal ovary, and thus our understanding of the structural proteins that underpin microvilli formation in the fetal oocyte is incomplete. We propose that estrogen promotes development of the oocyte microvilli necessary for healthy follicle formation in the fetal ovary by regulating expression and localization of ERM cytoskeletal proteins. Therefore, the current study was designed to determine whether ezrin (total and phosphorylated), SLC9A3R1, and α-actinin were expressed in fetal oocytes and whether expression and localization were developmentally regulated by estrogen.

MATERIALS AND METHODS

Animals

Female baboons (Papio anubis) weighing 12–18 kg were housed individually in stainless steel cages in air-conditioned quarters and fed Purina monkey chow (Ralston Purina, St. Louis, MO) and fresh fruit and/or vegetables daily and water ad libitum. Females were paired with males for 5 days at the anticipated time of ovulation, and pregnancy was confirmed subsequently as described previously [15]. Peripheral saphenous blood samples were obtained at 1- to 4-day intervals during the study period and from the maternal saphenous vein and the umbilical vein at the time of placental-fetal delivery, and serum was stored at −20°C until assayed for estradiol levels as described previously [16]. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (86–23; 1985). The Animal Care and Use Committee of Eastern Virginia Medical School approved the experimental protocol used in this study.

Animal Treatment and Collection of Fetal Ovaries

Fetal ovaries were obtained on Days 98–105 (n = 5) and on Days 160–178 of gestation (term = Day 184) from baboons untreated (n = 9) or treated with the highly specific aromatase inhibitor CGS 20267 (letrozole; 4,4-[1,2,3-triazol-1yl-methylene]-bis-benzonitrite; 115 μg/kg body weight per day; n = 6; Novartis Pharm AG, Basel, Switzerland) or with CGS 20267 plus estradiol benzoate (each at 115 μg/kg; n = 6) administered s.c. daily to the mother beginning on Day 100 of gestation, as described previously [16]. After removal, one of the fetal ovaries was fixed in phosphate-buffered formalin and embedded in paraffin for immunocytochemistry (ICC) studies, and the other was frozen and stored in liquid nitrogen for Western blot experiments. Archived paraffin-embedded ovaries from two adult baboons also were used for ICC study.

Immunocytochemistry

The immunocytochemical detection of total ezrin, phosphorylated ezrin (T567), SLC9A3R1, and α-actinin was determined essentially as described previously [17, 18]. Briefly, paraffin-embedded sections (4 μm) of fetal ovaries were mounted onto Superfrost/Plus microscope slides (Erie Scientific, Portsmouth, NH), heat fixed and, for diaminobenzidine (DAB) detection, endogenous peroxidase blocked with 0.3% H2O2 (Sigma-Aldrich Corp., St. Louis, MO) in methanol (Fisher Scientific Co., Pittsburgh, PA). Sections (n = 5–10 per ovary per animal) were incubated overnight (4°C) with antibodies to human ezrin (goat polyclonal; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA; or mouse monoclonal; 1:5000; BD Transduction Laboratories, BD Biosciences, San Jose, CA), which recognize all ezrin isoforms (i.e., phosphorylated and nonphosphorylated), ezrin phosphate (rabbit polyclonal; 1:100; Cell Signaling Technology Inc., Danvers, MA), NHERF1/SLC9A3R1 (1:7000; rabbit polyclonal, generously supplied by Dr. Chris Yun, Emory University, Atlanta, GA), or α-actinin (1:1000; mouse monoclonal; Chemicon International, Temecula, CA) diluted in 5% normal horse or normal goat serum (NHS or NGS) in phosphate-buffered saline (PBS; Amresco, Solon, OH). Ovarian sections then were washed twice in PBS for 10 min and incubated with biotinylated horse anti-goat immunoglobulin G (IgG; ezrin; Santa Cruz Biotechnology), goat anti-mouse IgG (ezrin; α-actinin; BD Transduction Laboratories), or goat anti-rabbit IgG (ezrin phosphate; SLC9A3R1), and proteins were localized using DAB-imidazole-H2O2 (brown precipitant) or by immunofluorescence using streptavidin conjugated with Alexa Fluor 488 (Invitrogen Corp., Carlsbad, CA). Briefly, for detection with DAB, sections were treated with VectaStain Elite Kit (Vector Laboratories Inc., Burlingame, CA), rinsed, stained with DAB-imidazole-H2O2, counterstained with Harris hematoxylin, mounted in Cytoseal-XYL (Richard Allan Scientific, Kalamazoo, MI), and examined by light microscopy [17]. For immunofluorescence analysis, before blocking with NHS or NGS, sections were incubated with Image-iT signal enhancer (Molecular Probes) and blocked for endogenous biotin with the Streptavidin/Biotin Blocking kit (Vector Laboratories Inc., Center Valley, PA). After treatment with streptavidin-conjugated Alexa Fluor 488 diluted 1:500 with 5% NGS or NHS in PBS, sections were incubated with filtered 1% Sudan Black (Sigma-Aldrich) in 70% methanol to quench autofluorescence, rinsed, and treated with propidium iodide (1.0 μg/ml PBS) to stain nuclei red. Following application of mounting media, slides were sealed with nail polish and stored in the dark at 4°C until examination using an Olympus BX41 microscope (Olympus America Inc., Center Valley, PA) equipped with an Olympus DP70 digital camera with FITC and TRITC filter sets.

Western Blot Analysis of α-Actinin, Ezrin, SLC9A3R1, and GAPDH

Fetal ovaries were homogenized on ice and digested in sample buffer (PBS with 1% cholic acid, 0.1% SDS, and 1 mM EDTA [Sigma-Aldrich]) that included a protease inhibitor cocktail as described previously [2]. After determination of protein concentrations using the bicinchoninic acid procedure (Sigma-Aldrich), samples were treated with 5× Laemmli buffer, heated to 100°C for 5 min, cooled on ice for 2 min, and briefly centrifuged. Either 8 or 25 μg of each protein sample then was loaded onto SDS-polyacrylamide gels (8% α-actinin; total ezrin; ezrin phosphate; 12% SLC9A3R1), electrophoresed in a Bio-Rad Mini-Protean II electrophoresis chamber (Bio-Rad Laboratories, Richmond, CA) containing chilled 25 mM Tris, pH 8.3; 0.192 M glycine; and 0.1% SDS buffer, and proteins were wet transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA). Membranes were blocked (37°C) in solution I (50 mM Tris, pH 7.5, containing 150 mM NaCl and 0.1% Tween-20) with 3% BSA or normal sera (Sigma-Aldrich) and then incubated (1 h; room temperature) with primary antibody diluted (α-actinin 1:1000; ezrin 1:500; and SLC9A3R1 1:10 000) in solution II (50 mM Tris, pH 7.5, containing 150 mM NaCl, 0.1% Tween-20, and 0.1% IGEPAL [Sigma-Aldrich]) with 1.5% BSA or normal sera. In select experiments, ovarian samples also were incubated with rabbit polyclonal antibody to GAPDH (0.75 μg/ml; Abcam Inc., Cambridge, MA) to correct for loading/transfer. Membranes were washed and incubated with anti-mouse (α-actinin), anti-goat (ezrin), or anti-rabbit (SLC9A3R1, GAPDH) IgG horseradish peroxidase-conjugated secondary antibody (Vector Laboratories) diluted (1:20 000) in Solution II, washed, and proteins detected using enhanced chemiluminescence (Amersham Biosciences, Pittsburgh, PA), as described previously [18]. Specificity of the primary antibodies was determined by incubation of samples without primary antibody. Membranes then were exposed to Fugi Super RX medical x-ray film (Fugifilm Medical Systems USA Inc., Roselle, IL), developed, and band intensities quantified by densitometry using the MetaMorph Image Analysis system (Molecular Devices, Downingtown, PA) or Image J software (National Institutes of Health, Bethesda, MD).

Statistics

Data (mean ± SEM) were analyzed by ANOVA with posthoc comparisons of the means by Students-Newman-Keuls multiple comparison tests.

RESULTS

Serum Estradiol Concentrations

As described previously [1,16], in untreated baboons maternal serum estradiol levels gradually increased from approximately 1 ng/ml on Days 85–120 of gestation to approximately 3.0 ng/ml by Days 160–175. Within 72 h of the onset of CGS 20267 administration, maternal serum estradiol decreased to and was maintained at levels that ranged between 0.10 and 0.15 ng/ml. Mean (±SE) estradiol level in umbilical venous serum on the day of delivery in untreated baboons at midgestation (0.22 ± 0.08 ng/ml) was increased approximately 2-fold by late gestation (0.59 ± 0.13 ng/ml) and decreased at term by >95% in baboons treated with CGS 20267 (0.04 ± 0.01 ng/ml). In baboons treated with CGS 20267 and estradiol benzoate, maternal and umbilical venous serum estradiol levels were restored to 90%–120% and 10%–30% of normal, respectively.

Expression of α-Actinin, Ezrin Phosphate, Ezrin, and SLC9A3R1

Immunocytochemistry confirmed that α-actinin expression in oocytes and pregranulosa cells of the fetal ovary was minimal at midgestation (Fig. 1A) and markedly increased in and localized to oocytes but not granulosa cells of primordial follicles in late gestation (Fig. 1, B and B inset). In contrast, in fetal ovaries of animals in which estrogen production was suppressed and the number and height of oocyte microvilli reduced [2] by in vivo treatment with CGS 20267, expression of α-actinin by oocytes of primordial follicles was minimal (Fig. 1, C and C inset), and in some sections was not detectable. Significantly, treatment with CGS 20267 and estradiol, which restored oocyte microvilli [2], increased but did not totally restore expression of α-actinin in oocytes of virtually all primordial follicles (Fig. 1, D and D inset). Abundant expression of α-actinin also was detected in blood vessels (Fig. 1E), presumably vascular smooth muscle of the fetal (Fig. 1E, E,1–3)1–3) and adult (Fig. 1E4) ovary. However, in contrast to the estrogen-dependent developmental changes in protein expression in oocytes, abundant expression in vascular cells was detected at mid (Fig. 1E1) and late (Fig. 1E2) gestation in untreated baboons and at late gestation in ovaries of animals treated with CGS 20267 (Fig. 1E3) or CGS 20267 and estrogen (data not shown). Specificity of primary antibody was confirmed by absence of signal in sections of fetal term ovary incubated without primary antibody (Fig. 1F).

FIG. 1.
Representative photomicrographs of the immunocytochemical expression of α-actinin in oocytes and pregranulosa cells in the fetal ovary of untreated baboons on Day 100 of gestation (A) and in oocytes of primordial follicles on Day 165 (B ...

Western blot showed that α-actinin protein was expressed and detected in extracts of the entire baboon fetal ovary as a single band of approximately 105 kDa (Fig. 2). The baboon fetal ovary also expressed the 36-kDa protein GAPDH (Fig. 2), the levels (arbitrary units × 10−4/μg protein) of which in late gestation were similar in baboons untreated (302 ± 11) or treated with CGS 20267 (295 ± 9) or CGS 20267 and estrogen (298 ± 13), and were approximately 16% greater (P < 0.05) than at midgestation (250 ± 8). The levels of α-actinin expressed either as arbitrary units ×10−3/μg protein or as a ratio to GAPDH appeared higher at late (208 ± 23 and 0.70 ± 0.10, respectively) than at mid (124 ± 17 and 0.49 ± 0.05, respectively) gestation, and appeared lower in late gestation in baboons treated with CGS 20267 (154 ± 48 and 0.52 ± 0.14, respectively) or CGS 20267 plus estrogen (141 ± 30 and 0.47 ± 0.06, respectively). However, the levels of α-actinin were not statistically different, presumably reflecting the cell-specific effects of estrogen on expression of α-actinin in oocytes but not vascular cells as shown by ICC.

FIG. 2.
A) Western immunoblot of α-actinin (105 kDa) and GAPDH (36 kDa) proteins in extracts of whole fetal ovaries obtained on Days 100 (mid) and 165 (late) of gestation from baboons untreated and on Day 165 from animals treated with CGS 20267 or CGS ...

Ezrin phosphate was expressed abundantly in fetal ovaries at mid and late gestation. At mid gestation (Fig. 3A), the phosphorylated protein was localized primarily to the surface of oocytes and pregranulosa cells in germ cell nests, and in late gestation (Fig. 3B) it was detected on the surface of oocytes of primordial follicles, lightly expressed in oocytes and/or pregranulosa cells in interfollicular germ cell nests (data not shown), and only lightly expressed or in several instances not detected in granulosa cells of primordial follicles (Fig. 3, B and B inset). Although ezrin phosphate appeared to be abundantly expressed in oocytes of primordial follicles in baboons treated with CGS 20267 (Fig. 3C), in several follicles, particularly those in the inner cortex, ezrin phosphate was primarily localized to and detected on the surface of granulosa cells and not oocytes (Fig. 3, C and C inset). In contrast, in animals treated with CGS 20267 and estradiol (Fig. 3D), ezrin phosphate expression was primarily localized to the surface of oocytes in the majority of follicles, and was only lightly expressed in granulosa cells. In the adult ovary, ezrin phosphate was expressed and tightly aligned to the surface of oocytes of follicles at all stages of development (i.e., primordial to preovulatory) and also was detected in granulosa cells of preantral and antral follicles (Fig. 3E). Specificity of primary antibody was confirmed by absence of signal in sections of fetal term ovary incubated without primary antibody (Fig. 3F).

FIG. 3.
Representative photomicrographs of ezrin phosphate expression in fetal ovary on Days 100 (A) and 165 (B and inset of B) of gestation in baboons untreated and on Day 165 in animals treated with CGS 20267 (C) or CGS 20267 and estradiol benzoate (D), as ...

Total ezrin (i.e., nonphosphorylated ezrin and ezrin phosphate) was expressed abundantly in fetal ovaries at mid (Fig. 4A) and late (Fig. 4C) gestation and exhibited a pattern of localization that paralleled that of ezrin phosphate; that is, localized and tightly aligned to the surface of oocytes and pregranulosa cells in germ cell nests at mid gestation (Fig. 4A) and to the surface of oocytes of primordial follicles, particularly those in the inner but not the outer cortex, in late gestation (Fig. 4, C1 and C2). In contrast, in estrogen-suppressed baboons, although ezrin (but not ezrin phosphate) was expressed abundantly in oocytes of primordial follicles, expression appeared to be more diffuse and to extend into the oocyte cytoplasm (Fig. 4D). Moreover, in the majority of primordial follicles formed in estrogen-suppressed animals, particularly those in the inner cortex, total ezrin also was detected in granulosa cells (Fig. 4D1) as well as in oocytes and granulosa cells in interfollicular germ cell nests. In animals treated with CGS 20267 and estradiol (Fig. 4, E1 and E2), total ezrin expression again paralleled that of ezrin phosphate, and thus was primarily localized and tightly aligned to the surface of oocytes in the majority of follicles and only lightly expressed or in several instances not detected in the granulosa cells. In the adult ovary, total ezrin, as with ezrin phosphate, was expressed and tightly aligned to the surface of oocytes of follicles at all stages of development (i.e., primordial to preovulatory; Fig. 4B) and also was detected in granulosa cells of preantral and antral follicles (Fig. 4B) but not in any of the early-stage (e.g., primordial; primary) follicles (data not shown). Specificity of primary antibody was confirmed by the absence of signal in sections of fetal term ovary incubated without primary antibody (shown in Fig. 5F).

FIG. 4.
Representative photomicrographs of total ezrin (nonphosphorylated ezrin and ezrin phosphate) expression in fetal ovary on Days 100 (A) and 165 (C and inset of C) of gestation in baboons untreated and on Day 165 in animals treated with CGS 20267 (D) or ...
FIG. 5.
Representative photomicrographs of NHERF1 (now termed SLC9A3R1) expression in fetal ovary on Days 100 (A) and 165 (B) of gestation in baboons untreated and on Day 165 in animals treated with CGS 20267 (C) or CGS 20267 and estradiol benzoate (D) and in ...

SLC9A3R1 was expressed abundantly in fetal (Fig. 5, A–D) and adult (Fig. 5E) baboon ovaries and also exhibited a pattern of staining comparable to that noted for total and phosphorylated ezrin. Thus, like ezrin phosphate, SLC9A3R1 was localized to the surface of oocytes and pregranulosa cells within germ cell nests at mid gestation (Fig. 5A), and in late gestation localized primarily to the surface of oocytes and only lightly expressed or was not detected in granulosa cells of primordial follicles (Fig. 5B). Moreover, although SLC9A3R1 was localized to the oocytes of primordial follicles in estrogen-suppressed animals, in several but not all follicles, like total ezrin, expression appeared to be more diffuse (Fig. 5C) and not tightly aligned to the oocyte surface, as in estrogen-replete baboons; for example, animals untreated (Fig. 5B) or treated with CGS 20267 and estrogen (Fig. 5D). In the adult ovary (Fig. 5E), SLC9A3R1, like phosphorylated ezrin, was localized to the surface of oocytes at all stages of follicular development as well as granulosa cells of preantral and antral follicles, but not smaller (e.g., primary) follicles (data not shown), and also was detected in interstitial cells between larger follicles. Specificity of primary antibody was confirmed by the absence of signal in sections of fetal term ovary incubated without primary antibody (Fig. 5F).

Western blot analyses verified that the 80-kDa ezrin (Fig. 6A) and 50-kDa SLC9A3R1 (Fig. 6B) proteins were expressed and detected as single bands in fetal ovaries of untreated baboons at mid and late gestation, in late gestation in animals treated with CGS 20267 or CGS 20267 and estradiol, and in adult baboon ovary (Fig. 6). Moreover, the respective levels of expression of ezrin and SLC9A3R1 were not altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen (Fig. 7, A and B).

FIG. 6.
Western immunoblot of 80-kDa ezrin (A) and 50-kDa NHERF1 (now termed SLC9A3R1; B) in fetal ovary on Days (d) 100 and 165 of gestation in baboons untreated and at Day 165 in animals treated with CGS 20267 or CGS 20267 and estradiol (E2) and in adult ovary. ...
FIG. 7.
Western immunoblot of ezrin (A) and NHERF1 (now termed SLC9A3R1; C) in fetal ovary on Day 165 of gestation in baboons (n = 3–8 per group) untreated or treated with CGS 20267 or CGS 20267 and estradiol (E2). Mean (±SEM) concentrations (arbitrary ...

DISCUSSION

The results of the current study are the first to show that the oocyte of the baboon fetal ovary in late gestation expresses the microvillus scaffolding proteins ezrin, NHERF1 (now termed SLC9A3R1), and α-actinin, and that these proteins are localized almost exclusively to the oocyte surface, consistent with their well-established role in the development of microvilli in epithelial cells [9, 10, 13, 14]. The current study also showed that oocyte expression of α-actinin was negligible at mid gestation, markedly increased on the surface of oocytes of primordial follicles in late gestation, prevented in the ovaries of fetuses in which estrogen production was suppressed by administration of CGS 20267, and increased by concomitant treatment with CGS 20267 and estrogen. Therefore, we propose that the development of α-actinin expression in fetal oocytes is regulated by estrogen. Moreover, this effect of estrogen appears to be relatively specific, since α-actinin expression in vascular cells, presumably vascular smooth muscle, was comparable at mid and late gestation and not altered by estrogen. We showed previously that the height and number of microvilli on oocytes of primordial follicles were markedly increased between mid and late gestation and prevented in fetuses in which estrogen production was suppressed and restored to normal by concomitant restoration of estrogen [2]. Although the present study showed that α-actinin expression was not totally restored to normal in baboons treated with the aromatase inhibitor and estrogen, level of expression was apparently sufficient to support microvillus development. Therefore, we suggest that estrogen regulates cell-specific expression of α-actinin, a protein required to link f-actin filaments to complete formation of microvilli [14] on the oocyte surface of the developing primate fetal ovary.

It is well established that nonphosphorylated ezrin typically resides and thus is detected in the cytoplasm of cells in a “dormant” (i.e., closed conformation) form in which its N and C terminals are masked and unable to link to other proteins, notably f-actin and SLC9A3R1 [13, 19]. However, following binding to phosphatidylinositol 4,5 bisphosphate (PIP2) and phosphorylation of a conserved threonine (T567) in the C-terminal of ezrin [20], which weakens interaction with the N-terminal (i.e., ezrin is unmasked), ezrin phosphate binds to f-actin, which leads to appropriate orientation of active ezrin within the apical membrane and creation of a structure that serves as the frame/scaffold for the formation of microvilli [12, 2124]. In addition, the retention of phosphorylated ezrin to the apical membrane requires interaction of the phosphorylated protein with the C-terminal ERM domain of SLC9A3R1 [24, 25]. In intestinal epithelial cells of Slc9a3r1-null mice, the actin-rich region upon which the microvilli are anchored is markedly disrupted [26], and the number of microvilli is significantly reduced [26]. These observations and studies showing that SLC9A3R1 is typically coexpressed with ezrin in various epithelial cell lines has led to the proposal that these two scaffolding proteins are both integral to microvillus formation [27, 28]. In the current study, we showed that SLC9A3R1 and ezrin (total and phosphorylated) were abundantly expressed and localized primarily and tightly aligned along the surface of oocytes of primordial follicles in late gestation. The detection of ezrin at this site probably reflects the fact that the ezrin antibody employed did not distinguish between phosphorylated and nonphosphorylated ezrin, and therefore it is likely that all of the ezrin detected on the oocyte surface in estrogen-replete animals is phosphorylated. Importantly, the latter observations contrast with the diffuse and apparent cytoplasmic or non-membrane-associated pattern of ezrin and SLC9A3R1 expression detected in oocytes of ovaries of near-term animals in which estrogen production was suppressed by treatment with CGS 20267. This diffuse/cytoplasmic pattern of staining implies that ezrin was not phosphorylated, consistent with our finding that phosphorylated ezrin was not detected in oocytes of estrogen-suppressed fetuses. Moreover, these observations correlate with our previous results showing that there was a significant decrease in the height and number of microvilli on the surface of oocytes in late-gestation fetal ovaries from estrogen-suppressed baboons [2]. Therefore, we further propose that estrogen, in addition to regulating oocyte expression of α-actinin and linking of f-actin filaments to form the apical microvilli, also modulates signals that induce phosphorylation of ezrin and its interaction with SLC9A3R1 to form the frame upon which the microvilli are developed.

The mechanism by which estrogen acts to regulate ezrin phosphorylation and α-actinin expression, and thus development of oocyte microvilli, remains to be determined. However, we demonstrated recently that baboon fetal oocytes isolated by laser capture microdissection from fetal ovaries in late gestation express mRNA and protein for estrogen receptor ESR2 (estrogen receptor beta) but not ESR1 (estrogen receptor alpha) [29]. We propose, therefore, that estrogen exerts direct effects on oocyte development, either by classic genomic mechanisms (i.e., nuclear receptor) and/or membrane receptor-mediated events that typically are associated with protein phosphorylation. Although phosphorylated ezrin was detected in oocytes at mid gestation, expression of α-actinin was minimal. It is possible that the latter reflects differences in cellular expression of the numerous coactivators that influence estrogen action and specific gene expression [30] and/or cell sensitivity to estrogen levels, which are greater at term than at mid gestation, as well the presence/absence of membrane estrogen receptor that links to phosphorylation of proteins via cell kinases [31].

We showed previously that follicular development, namely the encapsulation/surrounding of oocytes by pregranulosa cells, increased dramatically between mid and late gestation and that the number of follicles developed in late gestation in estrogen-suppressed baboons was 50% lower than that in estrogen-replete animals [1]. In the current study, we demonstrated that ezrin phosphate and SLC9A3R1 were detected on the surface of pregranulosa cells as well as oocytes at mid gestation (i.e., prior to follicle formation). In contrast, in late gestation, whereas both ezrin phosphate and SLC9A3R1 were still expressed in the oocytes of primordial follicles, these two proteins were not expressed in the surrounding granulosa cells. This contrasts with the localization of SLC9A3R1 and ezrin phosphate in the follicles of estrogen-suppressed fetal baboon ovaries in which the surrounding granulosa expressed both ezrin phosphate and SLC9A3R1, whereas expression in oocytes was abolished. We suggest, therefore, that as follicle formation proceeds, the interaction-communication of the oocyte and pregranulosa cells, perhaps via the latter microvillar proteins, is changed and development of microvilli initiated in the oocyte. Furthermore, the findings that granulosa cells in the follicles of estrogen-suppressed fetuses still expressed ezrin phosphate and the oocyte did not express α-actinin suggest that communication between oocyte and granulosa cells is regulated by estrogen, although additional experiments are required to examine these possibilities.

In summary, the results of the current study showed that the oocyte of the baboon fetal ovary in late gestation expressed the microvillus scaffolding proteins ezrin, SLC9A3R1, and α-actinin, and that these proteins were localized almost exclusively to the oocyte surface, consistent with their well-established role in the development of microvilli in epithelial cells [9, 10, 13, 14]. The present study also showed that the increase in oocyte expression of α-actinin between mid and late gestation and the change in oocyte and granulosa cell ezrin phosphate and SLC9A3R1 localization in estrogen-deprived baboon fetuses were regulated by estrogen and correlated with the estrogen-dependent increase in height and number of microvilli on oocytes of primordial follicles, as demonstrated previously [2]. Therefore, we propose that development of microvilli on the oocyte surface requires expression of the f-actin linker protein α-actinin and that expression of this protein and alterations in ezrin phosphate and SLC9A3R1 localization are regulated by estrogen.

Acknowledgments

The authors sincerely appreciate the secretarial assistance of Ms. Sandra Huband with the manuscript and preparation of the photomicrographs. The authors greatly appreciate the generous provision of CGS 20267 by Novartis Pharma AG, Basel, Switzerland.

Footnotes

1Supported by the National Institute of Child Health and Human Development/National Institute of Health through cooperative agreement U54 HD 36207 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

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