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BOR - Papers in Press, published online ahead of print July 11, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060152
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BIOLOGY OF REPRODUCTION 77, 872–879 (2007)
DOI: 10.1095/biolreprod.107.060152
© 2007 by the Society for the Study of Reproduction, Inc.

Insulin Signaling in Mouse Oocytes1

Nicole Acevedo 3 7, Jun Ding 3 6, and Gary D Smith 2 4 5 6 7

Departments of Molecular and Integrative Physiology,4 Urology,5 and Obstetrics and Gynecology,6 and the Reproductive Sciences Program,7 University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-ß. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 µg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 µg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.

chromatin remodeling, gamete biology, glycogen synthase kinase-3, insulin, kinases, meiosis, oocyte, oocyte development

INTRODUCTION

Mammalian oocyte or follicle growth in the presence of prolonged elevated insulin levels has a negative impact on oocyte developmental competence, defined as the ability of oocytes to support embryonic development to the blastocyst stage and/or establish normal pregnancies [1, 2]. Mouse oocyte/granulosa cell complexes (OGCC) cultured in insulin, with or without FSH, give rise to morphologically normal metaphase (M)II oocytes that have compromised embryo development following fertilization [3]. It is well recognized that oocyte growth and maturation are co-ordinated with follicular growth and differentiation [4–6]. Interaction of insulin with FSH promotes inappropriate differentiation of in vitro-cultured granulosa cells and differences in global protein expression compared to in vivo-derived granulosa cells [7, 8]. However, the mechanism by which prolonged elevated insulin, with or without FSH, ultimately reduces subsequent embryonic developmental competence is unknown.

In vivo, FSH can influence granulosa cell responses to insulin via the regulation of receptors to insulin and/or insulin-like growth factor 1 (IGF1) [9–12]. Insulin receptor transcripts have been described in human, bovine, and rat oocytes [13–15]. At the protein level, insulin receptors (INSR) are present in porcine [16] and human [17] oocytes, while the Insr transcript was reported to be absent from mouse oocytes [18]. The absence of an oocyte transcript does not necessarily signify a lack of the protein product, since some mRNAs are ‘masked' via deadenylation to maintain the stability of mRNA stored for long periods of time, whereas other transcripts are translated immediately during oocyte growth and then rapidly degraded [19, 20]. Considering the potential direct effects of prolonged elevated insulin on oocyte developmental competence, it is essential to determine whether oocytes contain INSR, and if receptor stimulation has consequences for oocyte competence via downstream signaling pathways.

Insulin binding to its receptor initiates a signal transduction cascade through phosphorylation of insulin receptor substrates, IRS1 and/or IRS2, and subsequent activation of phosphatidylinositol pathways [21]. Specifically, activated phosphoinositide-3 kinase signals through second messenger molecules to phosphorylate 3-phosphoinositide-dependent protein kinase-1 (PDPK1), which, in turn, phosphorylates its substrate thymoma viral proto-oncogene 1 (AKT1) [22, 23]. Activated AKT1 is the primary regulator of the terminal enzyme in insulin signaling, glycogen synthase kinase 3A/B (GSK3A/B), and AKT1-mediated phosphorylation of GSK3A at serine 21 or GSK3B at serine 9 results in GSK3 inactivation [24–28].

GSK3 is involved in a wide range of cellular processes far beyond the regulation of glycogen synthase [29]. Glycogen synthase kinase 3 is a key regulatory enzyme in the WNT/ß-catenin transduction pathway [30]. It has also been implicated in the regulation of other transcription factors, such as JUN, MYC, and CREB [31–33]. Oocytes of Xenopus [34], Caenorhabditis elegans [35], and the mouse [36] contain GSK3 mRNA and protein. It has long been recognized that GSK3 phosphorylates the inhibitory subunit (PPP1R2) of the protein phosphatase 1 (PPP1)-PPP1R2 complex, thereby activating cytoplasmic PPP1 [37, 38]. In addition, GSK3 regulates the phosphorylation of microtubule-associated proteins, thus influencing microtubule polymerization, stability, spindle formation, and function [39–42]. Lastly, pharmacologic inhibition of mouse oocyte GSK3 causes abnormal meiotic spindle configuration and function, chromatin organization, and bivalent chromatin segregation [19, 43].

The objectives of the present study were to determine the expression and function of receptors and enzymes in the insulin signaling cascade within mouse oocytes, to assess the effect of acute insulin exposure during oocyte maturation on meiotic progression, and to evaluate the effect of continuous insulin exposure during follicle/oocyte growth on GSK3 activity, meiotic progression, and meiotic metaphase chromatin configuration.

MATERIALS AND METHODS

All procedures involving animals were reviewed and approved by the University Committee on Use and Care of Animals at the University of Michigan and performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. All experiments were performed using follicles and oocytes collected from CF1 female mice (Harlan, Indianapolis, IN).

Oocyte Collection and In Vitro Maturation

For collection of growing oocytes, ovaries were isolated from 11-day-old, 15-day-old, and 19-day-old mice, and germinal vesicle-intact (GVI) oocytes were isolated by manual rupturing of the preantral follicles in Hepes-buffered Human Tubal Fluid medium (HTFH; Irvine Scientific, Santa Ana, CA) that was supplemented with 0.3% (w/v) BSA (Fisher Scientific, Pittsburgh, PA). Oocytes were completely denuded of surrounding granulosa and cumulus cells by manual pipetting. Cumulus-free oocytes were washed in HTFH plus 0.3% (wt/vol) polyvinylpropylene (PVP; Fisher Scientific), then frozen in liquid nitrogen and stored at –80°C for immunoblot analysis. For the collection of fully grown oocytes, ovaries were isolated from 19- to 21-day-old mice following injection of 10 IU eCG (Sigma Chemical Co., St. Louis, MO), and the oocytes were isolated as described above. To assess the effects of short-term insulin (Sigma) exposure on oocyte maturation, oocyte-cumulus cell complexes or denuded oocytes were cultured in HTF plus 0.3% BSA with or without 5 µg/ml insulin for 19 h at 37°C in 5% CO2 in air in a humidified incubator. The insulin used in this and subsequent experiments was from bovine pancreas, of HCLP potency, and was free of IGF1 contamination. A supraphysiologic dose of insulin was used based on experiments demonstrating that oocyte/granulosa cell complexes cultured in 5 mg/ml insulin, with or without FSH, give rise to oocytes with compromised embryonic developmental competence [1]. The current study focused on understanding the cellular/intracellular pathways by which oocyte exposure to insulin influences embryonic development. Cumulus-free oocytes were assessed for stage of development, washed in HTFH plus 0.3% PVP, and then prepared for either immunoblotting or chromatin analysis.

Follicle Collection and Culture

For follicle culture experiments, preantral follicles were collected from 11-day-old mice by microdissection of ovaries in HTFH that was supplemented with 10 µg/ml transferrin (Sigma), 50 µg/ml ascorbic acid (Sigma), 2 ng/ml selenium (Sigma), 50 µg/ml gentamicin, and 0.3% (w/v) BSA. Isolated secondary follicles, with intact basal lamina and surrounding theca/interstitial cells, were transferred to follicle culture media (FCM) that consisted of {alpha}-MEM (Gibco, Carlsbad, CA) supplemented with 10 µg/ml transferrin, 50 µg/ml ascorbic acid, 2 ng/ml selenium, 50 µg/ml gentamicin, 4 mM hypotaurine, 2 mM glutamine, and 5% fetal bovine serum (FBS; Gibco), as described previously [44]. To monitor follicle/oocyte growth, individual follicles were cultured in 50-µl drops of FCM overlaid with embryo-grade mineral oil (Irvine Scientific) at 37°C in 5% CO2 in air in a humidified incubator. Following overnight incubation, the follicles were placed into one of the following treatment groups: FCM alone, FCM supplemented with 10 ng/ml (0.1 IU) FSH (Serono, Rockland, MA), and FCM supplemented with 10 ng/ml FSH plus 5 µg/ml insulin. The medium was replaced every 2 days for up to 9 days by removal and replacement of 40 µl of the liquid. Follicle development was assessed at 200x magnification under an inverted microscope. Following 10 days of culture, follicles in each experimental group were treated with 100 mIU/ml hCG (Sigma) for 16 h. Oocytes were released from the follicles, denuded, and either frozen for immunoblot analysis or cultured for 7 h at 37°C in 5% CO2 in air in a humidified incubator, to assess maturation to MI and for chromatin analysis.

Immunoblot Analyses

Frozen cumulus cell-free GVI, MI, and MII oocytes were thawed in 2x SDS-PAGE sample buffer (80 mM Tris-HCl [pH 6.8], 20% [vol/vol] glycerol, 4% [wt/vol] SDS, 4% [vol/vol] 2-mercaptoethanol, 0.04% [wt/vol] bromophenol blue), vortexed, and placed on ice for 15 min. Following sonication on ice for 10 sec, samples were denatured at 90°C for 10 min and cooled on ice for 5 min. Entire cell lysates of a fixed number of oocytes were added per lane and separated by one-dimensional SDS-PAGE, as described previously [19]. Control lysates for the blots were generated from mouse brain (5 µg) and granulosa cells (GCs; 10 µg). The blots were probed with anti-INSR-ß (1:500; Upstate Biotechnology, Charlottesville, VA), anti-PDPK1 (1:200; Cell Signaling Technology, Beverly, MA), anti-AKT1 (1:200; Upstate), anti-GSK3A/B (1:1000; Upstate), and anti-phosphoserine-GSK3A/B (A S21, B S9, 1:1000; Cell Signaling) antibodies. This anti-phosphoserine-GSK3 antibody detects endogenous levels of GSK3 only when phosphorylation is present at S21 of GSK3A and S9 of GSK3B [45, 46]. Western blotting and antibody detection were performed in duplicate and visualized with ECL Plus chemiluminescence reagents (Amersham Life Sciences, Buckinghamshire, UK). Densitometry analyses of the immunoblots were performed using the Scion Image analysis software (Scion Corporation, Frederick, MD).

Chromatin Analysis

Oocyte chromatin was prepared as described previously [47]. Briefly, after 7 h of culture, MI oocytes were collected, washed once with 1x PBS (137 mM NaCl, 8.1 mM Na2HPO4-2H2O, 2.68 mM KCl, 1.47 mM KH2PO4 [pH = 7.4]), and then placed in 0.6% proteinase K solution (Promega, Madison, WI), to remove the zona pellucida. Following zona pellucida removal, the oocytes were washed in HTFH plus 0.3% BSA and briefly transferred to 1x PBS. For fixation, clean microscope slide were dipped in a solution of 1% paraformaldehyde (in distilled H2O that contained 0.15% Triton X-100 and 3 mM dithiothreitol [pH 9.2]). Oocytes were carefully pipetted along the length of each slide, and the slides were quickly transferred to humidified chambers (prewarmed to 37°C) and allowed to dry slowly overnight at room temperature. On the following day, the slides were air-dried and stained with Hoechst 33342 (5 µg/ml; Sigma). Chromatin condensation and configuration were assessed, as described previously [48]. The slides were coded and analyzed in a blinded manner with regard to treatment and visualized at 1000x magnification under a Leica DMR epifluorescence microscope (Leica Microsystems Inc., Bannockburn, IL).

Immunocytochemical Analysis

Localization of the INSR-ß protein was assessed in granulosa cells and oocytes of prepubertal and pubertal mice. Follicles from 11-day-old, 15-day-old, and 19-day-old mice were fixed at 37°C for 1 h in 2% paraformaldehyde that contained 0.04% Triton X-100 (Sigma) on poly-L-lysine-coated coverslips, as described previously [19]. Following fixation, the samples were washed and blocked overnight with 1x PBS plus 0.3% BSA (wt/vol) at 4°C. Follicles and enclosed oocytes were then incubated with anti-INRS antibody (10 µg/ml) for 1 h at 37°C in a humidified chamber. After washing, the samples were incubated with anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, Eugene, OR) for 1 h at 37°C. Slides were visualized and imaged using an Olympus FluoView 500 laser scanning confocal microscope (Olympus America Inc., Melville, NY).

GSK3 Activity Assay

To prepare oocyte extracts for GSK3 activity assays, 200 fully grown GVI oocytes per assay group were isolated from antral follicles, as described above. Oocytes were washed three times with cold PBS, and transferred to 200 µl cold lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM DTT, and EDTA-free protease inhibitor [Roche, Indianapolis, IN]). The oocytes were sonicated on ice and the cell lysates were cleared by centrifugation at 13 000 rpm for 15 min. Supernatants were collected for the kinase activity assay. For HeLa cell extracts, 2 x 107 HeLa cells were serum-starved for 16 h in Dulbecco modified Eagle medium (DMEM). HeLa cell lysates were prepared and collected for the kinase activity assay, as described above. Specific GSK3 activity was determined by measuring the transfer of 32P from [32P]-{gamma}-ATP to the GSK3-specific peptide substrate (GS-2; Upstate). The GSK3 specificity control peptide (GS-Ala; Upstate) was identical to GS-2 with the exception of a serine to alanine point mutation at the GSK3 phosphorylation site. The assay conditions were set as previously described [49]. Kinase activity was assayed in a total, volume of 25 µl of kinase buffer that contained 4 mg/ml of either GS-2, GS-Ala, or GS-2 plus 20 µM alsterpaullone, a selective GSK3 inhibitor [50], 400 µM of cold ATP, and 0.125 µCi of [32P]ATP. After an 8-min incubation at room temperature, 20 µl of the supernatant was spotted onto P81 phosphocellulose filter paper (Whatman Inc., Florham Park, NJ). The filters were washed in three changes of 0.75% phosphoric acid, rinsed in acetone, dried, and 32P incorporation was measured in a liquid scintillation counter. Kinase activity is expressed as counts per million (CPM) of incorporated 32P. The HeLa cell kinase assay was performed in duplicate and the values as shown as means ± SEM.

Statistics

Developmental and morphological analyses were performed in triplicate and the data were analyzed using Chi-square ({chi}2) analysis. Differences were considered significant at P < 0.01.

RESULTS

Insulin Receptor–ß Protein Is Expressed and Functional in Mouse Oocytes

The expression of IR-ß protein was assessed in oocytes of varying meiotic competence, to determine whether mouse oocytes have the ability to respond directly to insulin. GVI oocytes from 11-day-old CF1 mice are meiotically incompetent and begin to acquire meiotic competence within antral follicles on approximately Day 15. Full meiotic competence is acquired by the GVI oocytes contained in the antral follicles of 19-day-old mice. Both meiotically incompetent (d11) and meiotically competent (d19) oocytes showed expression of the 97 Mr x 10–3 INSR-ß protein (Fig. 1A). To address whether gonadotropins influence INSR-ß expression in oocytes, 19-day-old mice were stimulated with gonadotropin, and fully grown GVI oocytes were isolated on Day 21 and completely denuded of surrounding granulosa cells. Oocytes isolated after eCG stimulation were matured in vitro for 7 h (MI oocytes) and 16 h (MII oocytes). Western blot analysis revealed increased INSR-ß protein expression in oocytes derived from antral follicles that were previously stimulated with eCG, as compared to oocytes isolated from antral follicles without previous eCG treatment (Fig. 1A). Unexpectedly, the antibody recognized a single band at 97 Mr x 10–3 in granulosa cells and identified a doublet of immunoreactive bands in oocytes (Fig. 1A). Although we have not characterized the modification that distinguishes the INSR-ß isoforms, the doublet bands do not represent phosphotyrosine isoforms of the INSR because treatment of the extracts with tyrosine phosphatase did not influence the recognition of these bands (data not shown).


Figure 01
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  		FIG. 1 Expression of INSR-ß protein in meiotically incompetent (d11) and competent (d19, d21) oocytes at various developmental stages, with or without previous in vivo exposure to eCG. A) Cell extracts were analyzed by immunoblotting with an anti-INSR-ß antibody. Granulosa cell lysate (GC; 10 µg) was used as a positive control for INSR-ß (97 Mr x 10–3) expression. Total protein samples from 200 GVI (labeled GV-I in fig.), MI, or MII oocytes were loaded per lane. B) Immunocytochemical analysis of INSR-ß localization in growing oocytes (Oo) and granulosa cells (GC) of prepubertal mice (d11 and d15) and fully grown oocytes of pubertal mice (d19). N, nucleus.

Immunocytochemical analysis showed INSR-ß localization in granulosa cells, growing oocytes, and fully grown oocytes (Fig. 1B). In granulosa cells, INSR-ß was expressed in the cytoplasm, with intense localization at the juxtaposition of adjacent cells. In growing oocytes, INSR-ß was evenly dispersed throughout the cytoplasm. However, in fully grown GVI oocytes, INSR-ß was predominantly localized to a polar region of the oocyte, in juxtaposition to the plasma membrane. These results demonstrate that INSRs are present in the oocyte, that their expression is influenced by gonadotropin stimulation, and that receptor trafficking changes occur during oocyte growth.

Expression of Intermediate Enzymes and Endogenous Activity of Terminal Enzyme of Insulin Signaling in Oocytes

To determine whether insulin can propagate intracellular signaling within mammalian oocytes, we assessed the oocyte expression of intermediate enzymes in the insulin signaling cascade. Western blot analysis demonstrated the presence of both PDPK1 and AKT1 protein in fully grown oocytes (Fig. 2). The terminal enzyme of insulin signaling, GSK3A/B, has been previously identified by Western blot analysis in fully grown oocytes [19].


Figure 02
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  		FIG. 2 Expression of intermediate enzymes of the insulin regulatory pathway in fully grown GVI oocytes. Cell extracts were analyzed by immunoblotting with the anti-PDPK1 or anti-AKT1 antibody. Total protein samples from 200 GVI oocytes were loaded per lane. Mouse brain (5 µg) was used as a positive control for both PDPK1 (58–68 Mr x 10–3) and AKT1 (60 Mr x 10–3) expression in fully grown GVI oocytes.

To verify that endogenous GSK3 activity is present in fully grown oocytes, GSK3 specific activity was assayed by measuring the transfer of 32P from [32P]-{gamma}-ATP to the GSK3-specific peptide substrate GS-2 in the absence or presence of the selective GSK3 inhibitor, alsterpaullone. The GSK3 specificity control (GS-Ala) was used to measure the non-GSK3 kinase contribution to background phosphorylation. The kinase activities in HeLa cell extracts assayed with GS-2 (10906.5 ± 162.3; mean CPM ± SEM), GS-Ala (5080.7 ± 72.4), and GS-2 + alsterpaullone (5998.5 ± 451.05) demonstrated an 84% reduction in GSK3 activity following specific inhibition of GSK3 with alsterpaullone, and validated the assay for measurement of specific GSK3 activity in oocyte lysates. The kinase activities measured in oocyte extracts assayed with GS-2, GS-Ala, and GS-2 plus alsterpaullone (4301.3, 3006.4, and 2470.9 CPM, respectively) demonstrated a 59% reduction in GSK3 activity following specific inhibition of GSK3, which confirms that GSK3 is present and enzymatically active in mammalian oocytes. Collectively, these results confirm the presence in oocytes of an intermediate enzyme that is necessary for the regulation of GSK3, i.e., the terminal enzyme of insulin signaling.

Effect of Insulin Exposure During In Vitro Oocyte Maturation on Meiotic Progression

To assess whether insulin exposure during oocyte meiosis influences meiotic progression and chromatin segregation, cumulus cell-enclosed or denuded GVI oocytes were matured in vitro in the presence or absence of 5 µg/ml insulin. After 19 h of culture, the oocytes were assessed morphologically for GVBD and development to MII, as determined by extrusion of the first polar body. The oocytes that were classified morphologically as MII were assessed for normal chromatin condensation and formation of chromosome pairs [48]. Insulin exposure during meiosis did not significantly alter meiotic progression or MII chromosome condensation patterns in either cumulus-enclosed or denuded GVI oocytes, as compared to controls (Table 1).


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  		TABLE 1 Influence of insulin exposure on GVI oocytes during in vitro maturation (19 h).

To evaluate whether insulin exposure during meiosis regulates GSK3 protein expression or phosphorylation, denuded GVI oocytes were matured in vitro for 19 h in the presence or absence of 5 µg/ml insulin. Both the A and B isoforms of GSK3 were expressed within the control tissue (mouse brain), GVI, and MII oocytes, as described previously [19]. The expression of GSK3A was higher than that of GSK3B in MII oocytes, although insulin treatment during in vitro maturation did not alter the relative GSK3A/B expression levels, as compared to oocytes matured in the absence of insulin (Fig. 3). Interestingly, N-terminal serine phosphorylation was greater for GSK3B (S9) than for GSK3A (S21) in both fully grown GVI and in vitro-matured MII oocytes, although there was a marked increase in phosphorylation of GSK3A (S21) in MII oocytes compared to GVI oocytes. Insulin exposure during maturation did not influence GSK3A or GSK3B phosphorylation (Fig. 3). These results demonstrate that insulin exposure during oocyte meiotic progression does not influence oocyte development or alter the regulation of its terminal enzyme, GSK3.


Figure 03
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  		FIG. 3 Short-term insulin exposure does not affect GSK3 protein expression or phosphorylation during in vitro maturation of GVI oocytes. Fully grown GVI oocytes were matured in vitro in the absence (MII) or presence (MII +I) of 5 µg/ml insulin. Top panel: Cell extracts were analyzed by immunoblotting with an anti-GSK3A/B antibody. Mouse brain lysate (5 µg) was used as a positive control for GSK3A/B expression (51 Mr x 10–3 and 46 Mr x 10–3, respectively). Total protein samples from 200 GVI, MII, or MII plus I oocytes were loaded per lane. Bottom panel: Phosphorylation of GSK3A and GSK3B at serine 21 and serine 9, respectively.

Elevated Insulin Exposure of Follicles During Oocyte Growth Increases Oocyte GSK3 Phosphorylation and Meiotic Chromatin Remodeling Errors

To determine whether prolonged insulin exposure during oocyte growth influences intra-oocyte GSK3A/B activity, preantral follicles were cultured for 10 days in FCM alone (control), FCM media supplemented with 10 ng/ml FSH, or FCM supplemented with 10 ng/ml FSH and 5 µg/ml insulin. Previous studies have shown that continuous in vitro exposure of OGCC to a combination of 5 µg/ml insulin and FSH results in compromised oocyte developmental competence, even though the general morphology and competence to resume meiosis of the oocytes appear to be unaffected [1, 51]. Preantral follicles were manually isolated from 11-day-old females, so as to retain the surrounding basal lamina and thecal interstitial cells in the culture milieu (Fig. 4). After 10 days of culture, GVI oocytes were retrieved from antral follicles for Western blot analysis. Although the total expression levels of GSK3A and GSK3B total did not differ between the treatment groups, continuous treatment of follicles with insulin caused hyper-phosphorylation of GSK3B (S9) in GVI oocytes, as compared to oocytes from follicles cultured in the absence of insulin (Fig. 5).


Figure 04
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  		FIG. 4 Representative micrograph of in vitro culture of follicles and enclosed oocyte growth. For follicle culture experiments, preantral follicles were collected by microdissection from 11-day-old mice. Individual follicles were cultured for 10 days in one of the following: FCM alone; FCM plus 10 ng/ml FSH; FCM plus 10 ng /ml FSH plus 5 µg/ml insulin. On Day 3 of culture, granulosa cell (GC) layers start to expand, as compared to a follicle at day 0 (inset). On Day 6, GC expansion continues and interstitial cells (theca interna; TI) continue to proliferate. C–D) Antrum (A) formation surrounding the growing oocyte is evident by Day 8 and fully expanded by Day 10 of culture. O, oocyte; BM, basement membrane (basal lamina). Original magnification x200.


Figure 05
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  		FIG. 5 Continuous insulin exposure during follicle culture leads to decreased oocyte GSK3B activity. Preantral follicles were collected and cultured in either FCM alone (Control), FCM plus FSH (FSH) or FCM plus FSH plus insulin (FSH/Insulin). A) After 10 days of culture, follicles from each experimental group were treated with 100 mIU/ml hCG for 16 h, followed by oocyte dissociation from follicles for the immunoblot analysis. Total protein samples from 200 GVI oocytes per treatment group were immunoblotted with the anti-GSK3A/B and anti-phosphoserine-GSK3A/B (S21/S9) antibodies. B) Densitometry analysis of GSK3A/B immunoblot band densities. The upper bar graph compares the GSK3A and GSK3B total protein band intensities from oocytes cultured in Control, FSH or FSH/Insulin media. The lower bar graph compares the phospho-GSK3B (S9) band intensities from GVI oocytes cultured in each treatment group. Densitometry analysis was not performed on phospho-GSK3B (S21) because no quantifiable phosphorylation signal was detected.

To assess the effects of continuous insulin exposure during oocyte growth on meiotic chromatin remodeling, preantral follicles were cultured as described above, and after 10 days of culture, oocytes were retrieved from antral follicles and transferred to IVM media for an additional 7 h of culture. The oocytes were then assessed for normal development to MI, as judged by chromatin analyses of bivalent chromosomes and formation of tetrads. There was no significant difference between the number of oocytes that resumed meiotic progression to MI following 10 days of follicle culture in media with FSH or with FSH plus insulin (Table 2). However, there was a significant decrease in the incidence of normal chromatin condensation within MI oocytes following follicle culture in FSH plus insulin compared to follicle culture in FSH alone (Table 2; P < 0.01). Chromosome spreads of MI oocytes from follicle cultures treated with FSH revealed the formation of normally condensed bivalent chromosomes, whereas the MI chromosomes from follicle cultures treated with FSH plus insulin were poorly condensed (Fig. 6). These results demonstrate that prolonged insulin exposure during oocyte growth results in both increased phosphorylation (inactivation) of oocyte GSK3B at its N-terminal serine residue (S9) and impaired chromatin remodeling and condensation during meiosis.


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  		TABLE 2 Chromatin analysis of oocytes matured in vitro (7 h) following follicle culture in the presence or absence of insulin.


Figure 06
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  		FIG. 6 Representative micrographs of MI oocyte chromatin following 10 days of follicle culture in media that contained FSH alone (n = 21) or FSH plus insulin (n = 32). Preantral follicles were cultured and denuded GVI oocytes were collected. Denuded oocytes were cultured for 19 h in IVM medium and prepared for chromatin analysis. Original magnification x1000.

DISCUSSION

The present study demonstrates that insulin signaling is both present and responsive in mammalian oocytes, and that the effects of insulin signaling are mediated by gonadotropins. The INSR is composed of {alpha} and ß subunits, which form a mature dimeric receptor that translocates to the plasma membrane following proreceptor processing and dimerization in the endoplasmic reticulum [52, 53]. Insulin binding to the {alpha} subunit leads to autophosphorylation and conformational changes in the ß subunit, thereby increasing receptor tyrosine kinase activity and initiating signal transduction [54]. We have shown the presence of INSRs in mouse oocytes during oocyte growth and maturation, in contrast to a previous report that the insulin receptor transcript is absent [18]. A discrepancy between transcript and protein expression is not uncommon for oocytes, since some mRNAs undergo posttranscriptional modifications for long-term storage, while other transcripts undergo translation and rapid degradation during oocyte growth [19, 20]. Both growing and fully grown oocytes expressed a doublet form of INSR-ß, in contrast to the single band recognized in granulosa cells. The higher molecular mass band detected in oocytes does not appear to be a phosphorylated form of the insulin receptor. The unique lower molecular mass band may represent a degradation product or a gamete-specific isoform.

Mammalian oocytes also express Igf1r mRNA [55, 56]. Insulin and IGF1 receptors can form functional hybrids and insulin can bind to either the INSR or IGF1R to initiate a cellular response. Whether this doublet represents an INSR/IGF1R hybrid is currently unknown. It is worth recalling that insulin binds to its own receptor with high affinity and binds to the IGF1R with low affinity [57–59]. Therefore, at the doses of insulin used in the present study, activation of the INSR, IGF1R or a hybrid receptor is possible and requires further investigation.

Immunocytochemical analysis showed diffuse cytoplasmic expression of INSR-ß in growing oocytes of prepubertal females, whereas INSR-ß was polarized to a single region of the oocyte cytoplasm near the plasma membrane in fully grown oocytes from pubertal females. These results reveal temporal and spatial changes in INSR trafficking as oocytes grow and attain meiotic competence, and suggest that receptor polarization in fully grown oocytes reflects posttranslational modifications that mediate receptor translocation to the plasma membrane and receptivity to insulin signaling. Insulin receptor trafficking to and from the plasma membrane is mediated in part by a family of proteins called sorting nexins (SNX1, SNX2, SNX4, and SNX9), and insulin stimulation directs SNX translocation from the cytosol to the plasma membrane [60, 61]. Therefore, in the present study, exposure to insulin during follicle culturing may alter the insulin receptor trafficking patterns in growing oocytes, irrespective of meiotic competence.

We have also shown that gonadotropins increase INSR-ß expression in fully grown oocytes, although it is currently unknown whether this regulation of oocyte INSR-ß is due to the FSH-like and/or luteinizing hormone-like activities of eCG. FSH has been shown to influence granulosa cell responses to insulin through the regulation of INSR and/or IGF1R [9–11]. While the expression of FSH receptors has been recently described for human and porcine oocytes [62], it is currently unknown whether gonadotropins directly influence oocyte INSR-ß expression/function or signal through gap junctions of surrounding granulosa cells to alter oocyte responsiveness to insulin.

Key intermediate enzymes in insulin signaling are present and activated in fully grown oocytes. We identified the expression of PDPK1, which is the intermediate enzyme necessary for activation of AKT1. Activated AKT1, in turn, inactivates the terminal enzyme of the insulin signaling cascade, GSK3A/B, via phosphorylation of N-terminal serine residues [24]. We have previously reported the presence of GSK3A/B in mouse oocytes, and that inhibition of GSK3 by lithium chloride (LiCl) results in abnormal chromatin segregation and abnormal meiotic spindle formation [19]. Although LiCl is an ATP noncompetitive inhibitor of GSK3 activity that has been widely used to evaluate the effects of GSK3B, it is known to inhibit other targets, such as casein kinase 2, mitogen-activated protein kinase 14, mitogen-activated protein kinase 2, inositol polyphosphate phosphatase-like 1, and inositol (myo)-1(or 4)-monophosphatase 1 [63]. For this reason, we employed a more selective inhibitor of GSK3, alsterpaullone, to evaluate endogenous GSK3 activity in the oocyte. Alsterpaullone is a potent and selective inhibitor of both GSK3 and cyclin-dependent kinase (CDK) 1/2/5 activity in cell-free extracts [50, 64]. In the present study, we measured GSK3 specific activity and verified that endogenous GSK3 activity is present and regulatable in fully grown oocytes.

In a previous study, OGCC culture without basal lamina or interstitial cell layers in the presence of 5 µg/ml insulin resulted in oocytes with compromised developmental competence [1]. In the current study, we manually isolated preantral follicles with retained intact basal lamina and interstitial cell layers, to mimic a more physiologic cellular milieu during follicle culture. We tested the effects of long-term culture with 5 µg/ml insulin on oocyte insulin signal transduction. Fully grown oocytes isolated from preantral follicles cultured in the presence of insulin displayed increased levels of GSK3B (S9) phosphorylation in comparison to oocytes grown in the absence of insulin. This establishes that exposure to insulin during follicle culture initiates signal transduction within the follicle-contained oocytes, resulting in the inactivation of GSK3. Analysis of GVI oocytes matured to MI following follicle culture in the presence of insulin revealed severe chromatin remodeling and condensation errors in oocytes, as compared to oocytes grown in the absence of insulin, even though the MI oocytes in both treatment groups appeared normal under light microscopy. These results support previous findings that inhibition of GSK3 causes abnormal spindle formation and significantly increases the incidence of abnormal homologue segregation during mouse oocyte meiosis [36]. However, it is not clear whether the main insulin-mediated effect during oocyte meiosis is dysregulation of chromatin condensation and/or spindle function. Therefore, these results suggest that prolonged treatment of follicles with insulin causes increased phosphorylation of oocyte GSK3, leading to inactivation of GSK3 and chromatin condensation errors, which may cause misalignment of chromosomes on the meiotic spindle, resulting in congression failure.

In conclusion, we have demonstrated that the insulin signaling cascade is present and active in oocytes, and that gonadotropins influence oocyte insulin receptor expression during meiosis. We have also determined that exposure to insulin during oocyte growth has detrimental effects on meiotic chromatin remodeling and induces condensation errors, which may be mediated by abnormal regulation of GSK3 activity. These results begin to elucidate how prolonged exposure to elevated levels of insulin during oocyte growth may compromise subsequent embryonic developmental competence.

ACKNOWLEDGMENTS

We thank Dr. Matthew Wishart for a critical review of this manuscript.

FOOTNOTES

3These authors contributed equally to this work. Back

1Supported by NIH grant HD046768 (to G.D.S.). Support for N.A. was provided by NIH Training Grant in Reproductive Sciences T32-HD07048 (to D. Foster). This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, which is funded by NIH5P60 DK20572 from the National Institute of Diabetes and Digestive and Kidney Diseases. Back

Correspondence: 2Gary D. Smith, 6428 Medical Sciences I, 1301 E. Catherine St, Ann Arbor, MI 48109-0617. FAX: 734 936 8617; e-mail: smithgd{at}umich.edu

Received: 12 January 2007.

First decision: 7 February 2007.

Accepted: 11 July 2007.

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