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Ovary |
Department of Physiological Sciences,3 Eastern Virginia Medical School, Norfolk, Virginia 23501
Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, The Center for Studies in Reproduction,4 University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin, but not activin ßA and ßB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.
activin, estradiol, follicular development, ovary, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and one of two ß subunits (ßA, inhibin A; ßB, inhibin B). Activins are homodimers or heterodimers of either of the two ß subunits (ßA:ßA, activin A; ßB:ßB, activin B; or ßA:ßB, activin AB) [3, 4]. Activins interact at the cell membrane with serine/threonine kinase receptors classed as type I or type II. Because a specific receptor(s) for inhibin remains to be characterized [5], inhibins act either by binding ß subunits to produce an inactive dimer, i.e. inhibin, and/or by binding to the activin receptor II, thereby preventing the action of activin [3, 4]. We recently demonstrated that the subunit of inhibin [6], which was not expressed in the baboon fetal ovary throughout the second half of gestation, was markedly up-regulated in pregranulosa and granulosa cells of fetal ovaries in which the number of follicles was significantly reduced by estrogen deprivation [7]. In contrast, estrogen deprivation had no effect on the expression of activin ßA in oocytes and activin ßA and ßB and the activin receptors in pregranulosa and granulosa cells [6]. We have proposed, therefore, that the primate fetal ovary does not produce inhibin and that estrogen regulates envelopment of oocytes by pregranulosa cells (i.e., folliculogenesis) by controlling -inhibin expression, and thus the fetal ovarian activin:inhibin ratio. Upon binding activin, the type II receptor kinase phosphorylates and thus activates the type I receptor, which then phosphorylates and activates a class of intracellular substrates collectively known as Smads [8, 9]. It appears that Smads 2 and 3 are phosphorylated by activin-activin receptor interaction [10] and then bind to a common mediator Smad 4, which translocates to the nucleus to activate target genes [11–13]. Although Smads 2 and 4 have been detected in human adult ovarian follicles at preantral stages [14], there are no studies of the ontogeny or developmental expression of the activin-dependent signaling substrates in the primate fetal ovary. Therefore, as a prelude to conducting experiments to test the hypothesis that estrogen regulates fetal ovarian follicular development by controlling the activin:inhibin ratio, it was important to determine whether the primate fetal ovary expressed Smads 2/3 and 4 during the second half of gestation, and whether expression of these signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed and fetal ovarian follicular development was markedly altered [7, 15].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetal ovaries used in previous experiments [6, 7] were obtained on Days 100 (n = 3) and 165–170 (n = 6) of gestation from untreated baboons (Papio anubis) and on Days 165–170 from animals treated daily beginning on Day 100 of gestation (term = Day 184) with the aromatase inhibitor CGS 20267 (Letrozole; 4,4-[1,2,3-triazol-1yl-methylene] bis-benzonitrite, Norvartis Pharm AG, Basel, Switzerland) administered s.c. (115 µg kg-1 body weight day-1 0.05 ml-1 sesame oil, n = 6) to the mother or treated with CGS 20267 (115 µg kg-1 body weight day-1) plus estradiol benzoate at doses (175 µg kg-1 body weight day-1 0.1 ml-1 sesame oil, n = 5) designed to replicate the normal pattern of serum estradiol essentially as described previously [7]. Briefly, baboons were sedated with ketamine and anesthetized with isoflurane. The placenta and the fetus were delivered by cesarean section, and the fetus was killed with an overdose of sodium pentobarbital. Fetal ovaries were excised, trimmed of fat, weighed, and one ovary was fixed in 10% buffered formalin and paraffin-embedded for subsequent histologic/immunocytochemical analyses as previously described [6, 15]. The other ovary was stored in liquid nitrogen for Western blot studies. Blood samples (3–5 ml) were also obtained at 1–4-day intervals between Days 85 and 165 of gestation via a maternal saphenous vein after sedation with an i.m. injection of ketamine-HCl (10 mg/kg body weight; Parke-Davis, Detroit, MI) and from the umbilical vein prior to delivery of the fetus on Days 165–170 of gestation. Maternal and umbilical venous serum estradiol levels were reported previously [7]. Ovaries were also available from three adult baboons and used previously to examine immunocytochemical localization of estrogen receptors and ß [15]. Baboons 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 (Publication 85-23, 1985). The experimental protocol utilized in this study was approved by the Institutional Animal Care and Use Committee of the Eastern Virginia Medical School.
Immunocytochemistry
The expression of Smads 2/3 and 4 in fetal (n = 3–6/group) and adult (n = 3) ovaries was determined essentially as described previously [6, 15]. Briefly, representative sections (4 µm) of paraffin-embedded fetal and adult ovaries were mounted onto Superfrost microscope slides (Fisher Scientific Co., Arlington, VA); heat fixed; and endogenous peroxidase blocked with 0.4% H2O2 in methanol. Sections were microwaved in 10 mM sodium citrate buffer, pH 6 (Sigma, St. Louis, MO), at power level setting 9 (General Electric Model JE 1540 oven, maximum power 900 W; General Electic Co., Fairfield, CT) for 20–25 min and then cooled for 30 min prior to analysis. All sections were washed in PBS and preblocked with 5% normal goat serum (Smad 4) or normal horse serum (Smad 2/3) in PBS for 30 min at room temperature. Sections (n = 5–10 per ovary per animal) were then incubated (4°C) for 48 h with goat polyclonal antibody to Smad 2/3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or mouse monoclonal antibody to Smad 4 (Santa Cruz) diluted in 5% normal horse serum or 5% normal goat serum, respectively, and used at concentrations of 0.5–4 µg/ml. Ovarian sections were then washed twice in PBS (10 min), incubated (40 min) with biotinylated horse anti-goat or goat-anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA), and then with the VectaStain Elite Kit (Vector). After rinsing in PBS, all sections were incubated for 6 min with diaminobenzidine (DAB)-imidazole-H2O2, counterstained with Gill hematoxylin diluted 1:5, mounted in Bio mount, and examined by light microscopy. Studies were also performed without primary antibody (Smad 4) or with primary antibody (Smad 2/3) preabsorbed with immunizing peptide (Santa Cruz).
Because the distribution of DAB in oocyte cytoplasm made it difficult to examine nuclear localization, immunofluorescence was employed to determine whether Smad 4 was localized to the nucleus. Briefly, sections of fetal ovaries were incubated with mouse monoclonal antibody to Smad 4 (1 µg/ml) and treated with biotinylated antimouse IgG as described above. Sections were then incubated for 30 min in the dark with streptavidin conjugated with Alexa Fluor 488 (Molecular Probes, Inc., Eugene, OR) diluted 1:200 with 5% normal goat serum. After incubation with Sudan Black (Sigma; 1% in 70% methanol) to quench autofluorescence, slides were rinsed and treated with Vectashield mounting medium containing propidium iodide (Vector) to stain nuclei red. Slides were cover-slipped and stored in the dark at 4°C until examined and photographed using an Olympus BX50 microscope (Optical Elements Corp., Melville, NY) equipped with a Spot Slider digital camera and FITC filter set (Diagnostic Instruments, Inc., Sterling Heights, MI). Under these conditions, Smad 4 protein localized to cytoplasm was detected as green and Smad 4 colocalized with nuclei was detected as yellow.
Western Immunoblot
Western blot analyses of Smads 2/3 and 4 were performed essentially as described previously [6, 16]. Briefly, samples of frozen fetal and adult ovaries were suspended in 1% cholic acid (Sigma), 0.1% SDS (Sigma), and 1 mM EDTA in PBS (pH 7.4) containing proteases and homogenized on ice. Samples were heated (100°C for 5 min); cooled; and then loaded (25 µg protein/lane) onto discontinuous 12% SDS-polyacrylamide minigels in electrophoresis chambers containing chilled 0.025 M Tris, 0.192 M glycine (Bio-Rad Laboratories, Hercules, CA), and 0.1% SDS buffer (pH 8.3); electrophoresed; and wet transferred to Immobilon P (Millipore Corp., Bedford, MA). Membranes were blocked and incubated with antibodies to Smad 2/3 or Smad 4 diluted to 0.2 µg/ml or 0.5 µg/ml, respectively, in 50 mM Tris, pH 7.5 buffer, washed, and then incubated with donkey anti-goat IgG or anti-mouse IgG horseradish peroxidase-conjugated second antibody (Amersham Life Sciences, Inc., Arlington Heights, IL). After washing and application of enhanced chemiluminescent reagent (ECL, Amersham), membranes were placed in x-ray film cassettes containing Fugi Medical x-ray film (Fugi Medical Systems, Stamford, CT) and exposed in a dark room for 30–120 sec. Samples were quantified by one-dimensional densitometry using an Image 1 analysis system (Universal Imaging Corp., Reamstown, PA), and results were expressed as arbitrary densitometric units per 25 µg protein. Smads 2/3 and 4 were each examined on two membranes with two of the same fetal ovarian samples on both membranes to normalize quantification. Moreover, each membrane was composed of at least one fetal ovarian sample from each of the experimental groups. The second antibody contributed no nonspecific bands at the concentrations employed.
Statistical Analyses
Data were expressed as mean ± SEM, and statistical differences were determined by ANOVA with post hoc comparison of the means by Newman-Keuls multiple comparison test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously demonstrated [7, 16] that maternal serum estradiol levels increased from approximately 1 ng/ml on Days 85–120 of gestation to 2.5–3.0 ng/ml by Day 165, and that within 48–72 h of the onset of CGS 20267 treatment on Day 100 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 the control, although absolute levels were slightly greater than normal. Mean (±SEM) estradiol levels in umbilical serum on Day 165 of gestation in untreated baboons (0.59 ± 0.13 ng/ml) were also reduced (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 (P < 0.05) in baboons treated with CGS 20267 and estrogen.
Fetal Ovarian Development
A major feature of primate fetal ovarian development is the ontogenesis of follicle development between mid and late gestation. Thus as previously demonstrated [15], on Day 100 of gestation the baboon fetal ovary was composed primarily of oocytes and pregranulosa cells in germ cell cords and very few primordial follicles. This contrasts with the situation at term in which primordial follicles abound and the number of oocytes and pregranulosa cells between follicles (i.e., interfollicular nests) was minimal.
Expression of Smads 2 and 3
Western blot analyses demonstrated that 59 kDa Smad 2 and 54 kDa Smad 3 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation, in late gestation in animals treated with CGS 20267 or CGS 20267 and estrogen (Fig. 1A), and in the adult baboon ovary (data not shown). Moreover, the level of expression of Smads 2 (Fig. 1B) and 3 (Fig. 1C) was similar (P > 0.5) at mid and late gestation and not altered (P > 0.5) in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry confirmed that Smads 2 and 3 were expressed in fetal (Fig. 2, A–C) and adult (Fig. 2E) baboon ovaries and localized in the fetus to oocytes and pregranulosa cells at midgestation (Fig. 2A) and to oocytes and granulosa cells of primordial follicles in late gestation (Fig. 2B). In late gestation, Smads 2/3 were also localized to interfollicular nests composed of pregranulosa and oocytes not enveloped to form a follicle and which were most prominent in fetal ovaries of baboons treated with CGS 20267 (Fig. 2C). Moreover, consistent with Western blot, the apparent level of Smad 2/3 immunocytochemical expression was not modified in fetal ovaries of baboons deprived of estrogen (Fig. 2C). Regardless of site of localization or treatment in vivo, expression of Smads 2/3 appeared to be predominantly cytoplasmic and only occasionally detected in the nucleus of some flattened granulosa cells but not in oocyte nuclei. In the adult ovary, Smads 2/3 were localized by immunocytochemistry to cytoplasm of the oocyte and granulosa cells of preantral (Fig. 2E) and antral follicles (data not shown). Specificity of the primary antibody was confirmed by the absence of signals in Western blots (not shown) and in sections of fetal ovaries incubated with primary antibody preabsorbed with immunizing peptide (Fig. 2D).
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Expression of Smad 4
The 64 kDa Smad 4 protein was detected by Western blot and thus expressed in fetal ovaries of untreated baboons at both mid and late gestation, in late gestation in animals treated with CGS 20267 or CGS 20267 and estrogen (Fig. 3A), and in the adult ovary (data not shown). Moreover, the apparent level of expression of Smad 4 was not significantly (P > 0.5) altered by development or in vivo treatment with CGS 20267 or CGS 20267 and estrogen (Fig. 3B). Immunocytochemistry also showed that Smad 4 was expressed in fetal (Fig. 4, A–C) and adult (Fig. 4E) baboon ovaries. In the fetus, Smad 4 was localized to oocytes and pregranulosa cells at midgestation (Fig. 4A) and oocytes and granulosa cells of primordial follicles, and interfollicular nests of oocytes and pregranulosa in late gestation (Fig. 4B). As seen in Figure 4C, consistent with Western blot, the apparent level of Smad 4 immunocytochemical expression was not modified in fetal ovaries of baboons deprived of estrogen. In the adult ovary, Smad 4 was localized by immunocytochemistry to cytoplasm of the oocyte and granulosa cells and theca cells of preantral (Fig. 4E) and antral (data not shown) follicles. Specificity was confirmed by the absence of signal in Western blots (data not shown) or sections of fetal ovary incubated without primary antibody (Fig. 4D).
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In the late gestation fetal ovary of untreated baboons, Smad 4 colocalized with propidium iodide and thus was detected in the nuclei of granulosa cells of several primordial follicles (Fig. 5B). Assuming equal labeling with propidium iodide, it would appear that in fetal ovaries of estrogen-suppressed baboons, expression of Smad 4 in granulosa cell nuclei was not as marked (Fig. 5C). In fetal ovaries of both untreated and estrogen-suppressed animals at late gestation, although oocytes did not stain intensely with propidium iodide, Smad 4 expression in the oocytes appeared to be primarily cytoplasmic and was only occasionally detected in oocyte nuclei (Fig. 5B). In the fetal ovary at midgestation, expression of Smad 4 in oocytes and pregranulosa cells was predominantly cytoplasmic (Fig. 5A). Specificity was confirmed by the absence of green (Fig. 5D) or yellow signal (Fig. 5E) in sections of a late gestation fetal ovary incubated with streptavidin-conjugated Alexa Fluor 488 and propidium iodide, but without primary antibody.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the current study also indicate that the level of expression of the Smads was not significantly altered in late gestation in fetal ovaries of baboons deprived of estrogen in utero. Because the expression of the activin receptors as well as activin subunits were also not altered by estrogen deprivation in utero [6], we suggest that estrogen is not required for the development of the activin receptors and the signaling cascade in the primate fetal ovary. However, we previously showed that fetal ovarian follicular development was markedly altered in estrogen-suppressed baboons (i.e., the number of primordial follicles was approximately 50% lower in fetal ovaries of baboons deprived of estrogen than in animals untreated [estrogen replete] or in which estrogen production was restored). Therefore, we further suggest that the decrease in the number of follicles developed in the ovaries of estrogen-suppressed animals does not reflect a change in the expression/availability of activin receptors or the intracellular Smad signaling system. Rather, as proposed previously [6], the upregulation of -inhibin in ovaries of estrogen-suppressed baboon fetuses could have depleted activin (i.e., by formation of inhibin) or interfered with activin signaling at the receptor level, although studies to directly address these possibilities remain to be conducted.
Activins have been shown to regulate several aspects of adult ovarian function. For example, activin enhanced oocyte maturation in the cow [18, 19] and monkey [20]; promoted the in vitro development of theca-free granulosa-oocyte complexes isolated from immature rat ovaries [21]; stimulated growth of rat preantral follicles and oocyte maturation [22]; and induced proliferation of human luteinized granulosa cells [23]. Although activin gene disruption in mice resulted in females with impaired reproductive ability [4], the role or sites of action of activin in the primate fetal ovary remain to be elucidated. It is generally believed [24], however, that follicle formation, which is initiated at midgestation in human [25] and nonhuman primates [15, 17, 26], involves encapsulation of oocytes by surrounding pregranulosa, a process that results by late gestation in the formation of the pool of primordial follicles available for reproductive/ovarian function in adulthood. It is possible, therefore, that activin may play a role in regulating this process, although the latter also remains to be determined. However, the results of the current study and of our previously published studies [6, 7] are consistent with this hypothesis. Thus, the 50% decrease in the number of follicles in fetal ovaries of estrogen-deprived baboons [7] was associated with a comparable increase in the number of interfollicular nests (i.e., oocytes and pregranulosa cells) and an upregulation of the -subunit of inhibin in these nests. However, because there was no change in activin subunits, activin receptors, or the Smads, it is possible that the decrease in follicle formation may have resulted, in part, from increased formation of inhibin secondary to a loss of estrogen.
That the activin signaling system may play a role in fetal ovarian development is supported by the finding that Smad 4 protein was detected in nuclei of granulosa cells of primordial cells in late gestation in untreated (e.g., estrogen replete) baboons and that colocalization of Smad 4 to granulosa cell nuclei appeared to be reduced in fetal ovaries of estrogen-suppressed animals. In contrast, however, activin did not appear to be colocalized in the nuclei of pregranulosa or oocytes at midgestation. Thus, whether the activin signaling cascade is involved in oocyte envelopment by granulosa cells will require additional studies. Recently, Mayo et al. [27, 28] showed that overexpression of -inhibin in the mouse ovary, independent of pituitary FSH, resulted in ovarian pathologies. Although the site(s) and mechanisms of estrogen action in the baboon fetal ovary remain to be defined, this proposed action of estrogen is absolutely essential for normal fetal ovarian maturation.
Localization of Smad 4 to the nucleus may also reflect Smad 1, 5, and/or 8 activities in addition to Smad 2/3 [29]. Moreover, it is well established that Smads 2/3 and Smad 4 also mediate signaling of other transforming growth factor (TGF)-ß family members, including TGF-ß 1, 2, and 3 [8, 12, 29] and the bone morphogenetic protein-related ligands GDF9 and GDF11 [29–32], whereas bone morphogenetic proteins utilize Smad 1 [29]. In addition, it has recently been shown that inhibin antagonized the actions of bone morphogenetic protein in various cell types in vitro [33]. Therefore, it is possible that TGF-ß family members other than or in addition to activins modulate estrogen-dependent primate fetal ovarian folliculogenesis.
In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary. Thus, Smads 2/3 and Smad 4 were localized to oocytes and pregranulosa cells at midgestation and to oocytes and granulosa cells of primordial follicles in late gestation. Moreover, the results of the current study showing that expression of Smads was not altered by estrogen deprivation in utero coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and up-regulated/induced expression of inhibin lend further support to our suggestion that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gerald J. Pepe, Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501-1980. FAX: 757 624 2269; pepegj{at}evms.edu
Received: 22 April 2003.
First decision: 14 May 2003.
Accepted: 21 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis. Biol Reprod 2003 68:1989-1996 and ß in the baboon fetal ovary. Biol Reprod 2002 66:1054-1060-subunit gene. Endocrinology 2001 142:4994-5004-subunit. Endocrinology 2001 142:5005-5014This article has been cited by other articles:
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  P. Yang and S. K. Roy Transforming Growth Factor B1 Stimulated DNA Synthesis in the Granulosa Cells of Preantral Follicles: Negative Interaction with Epidermal Growth Factor Biol Reprod, July 1, 2006; 75(1): 140 - 148. [Abstract] [Full Text] [PDF]
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