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Gamete Biology |
The Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry,3 Temple University School of Medicine, Philadelphia, Pennsylvania 19140
The Jackson Laboratory,4 Bar Harbor, Maine 04609
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, follicle, gamete biology, granulosa cells, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth differentiation factor 9 (GDF9), an oocyte-specific member of the transforming growth factor-ß superfamily, is produced throughout oocyte development. Follicles fail to develop beyond the primary stage in Gdf9 null mice [5]. Because recombinant GDF9 promotes granulosa cell proliferation and cumulus expansion [6, 7], it is thought to participate in GC development throughout follicular development. In fact, both FGOs and GDF9 can promote the expression by mural GCs of hyaluronan synthase 2 (Has2), a gene normally expressed by cumulus cells and required for cumulus expansion [6, 8, 9]. The mural GCs of preovulatory follicles express luteinizing hormone/choriogonadotropin receptor (Lhcgr) mRNA, but mouse cumulus cells do not [10, 11]. Oocytes and recombinant GDF9 suppress the expression of Lhcgr mRNA in both cumulus and mural GCs in vitro [6, 11]. Because, in the absence of the oocyte, GCs from antral follicles generally express the mural GC phenotype, it is thought that the default pathway of GC development in the antral follicles of mammalian ovaries leads to the mural GC phenotype. Oocytes can abrogate this default program and promote the expression of the cumulus cell phenotype [3, 12]. However, it is not known whether any aspects of mural GC differentiation might also be influenced by the oocyte. This study used high-resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis to compare the effects of oocytes and recombinant GDF9 on the differentiation of GCs during the 2° to 3° follicle transition. The experimental strategy was to compare the pattern of proteins labeled in vitro with 35S-methionine in 2° GCs isolated from follicles of 12-day-old mice with the pattern of proteins synthesized in mural 3° GCs of 16-day- old mice. In addition, these different populations of GCs were cocultured with either GO or FGO isolated from 2° or 3° follicles, respectively, or with recombinant GDF9. Thus, the stage-specific effects of oocytes or GDF9 on the differentiation of GCs at different stages of their development were evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes and GCs were isolated from the ovaries of (C57BL/6J x SJL/ J)F1 mice. All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals. Preantral GCs (2° follicles) of 12-day-old mice (Fig. 1) were isolated by digestion of the ovary in medium containing 1 mg/ml collagenase and 0.02 mg/ml DNase. Removal of GOs was accomplished by aspirating the preantral granulosa cell-oocyte complexes repeatedly in and out of a borosilicate glass pipet having an internal diameter slightly smaller than the oocyte of 2° follicles. Mural GCs (3° follicles) of mice 16 days of age were isolated by puncturing the small 3° antral follicles with 30-gauge needles and collecting the clumps of mural cells released, avoiding any obviously darkened or degenerating cells. For each experiment, clumps of GCs from fifteen 12-day-old mice or twelve 16-day-old mice were pooled and washed manually by three serial passages using micropipets through dishes of fresh medium and distributed equally to control and experimental groups. The clumps were cultured in 48-well tissue-culture plates containing 150 µl medium per well. FGOs were obtained for coculture by puncturing 3° follicles of 20-day-old mice with 30-gauge needles, collecting all of the germinal vesicle-stage oocytes released, and stripping any remaining cumulus cells from the oocytes by aspirating them through a narrow-bore pipet. Collection of cells and overnight culture employed Medium 199 (Invitrogen, Carlsbad, CA), which was prepared as described previously [13] and supplemented with 3 mg/ml bovine serum albumin (BSA) and 10 µM milrinone (Sigma Chemical Co., St. Louis, MO) to maintain oocytes in meiotic arrest [14]. In coculture groups, GCs were cultured with either 1 FGO/µl or 2 GO/µl. These concentrations were chosen to be comparable on the basis of oocyte volume [15]. Some cultures of GCs were supplemented with either 25 or 100 µg/ml recombinant GDF9 generously provided by Dr. Martin M. Matzuk, Baylor College of Medicine, Houston, TX. All groups of GCs within an experimental replicate, regardless of age, coculture with oocytes, or treatment with GDF9, were cultured at the same time
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The GCs of all groups, including control 2° and 3° GCs, were labeled after 12 h in culture. Oocytes were removed from the coculture groups and the GCs were washed with BSA-free Whitten medium [16], then incubated with 1 mCi/ml 35S-methionine (1175 Ci/mmole; DuPont NEN Research Products, Boston, MA) in BSA-free Whitten medium [16] for 3 h. GCs were then lysed in 30 µl of hot dSDS solution and heated as described previously [17–19]. After treatment with DNase and RNase, the lysates were frozen, lyophilized, and redissolved in an equal volume of sample buffer as described previously [17–19].
High-Resolution Two-Dimensional Protein Gel Electrophoresis
Isoelectric focusing was performed using approximately 1–2 x 106 acid precipitable counts of radiolabeled protein, separated over a pH 3.5– 10 gradient. A mixture of 92.5 µl Resolyte 3.5–10 (British Drug House, Cater Chemicals, Bensenville, IL), 92.5 µl Ampholine 3.5–10 (Amersham- Pharmacia, Piscataway, NJ), 31 µl Servalyt 5–6 (Crescent Chemical, Hauppauge, NY), and 61 µl Ampholine 7–9 (Amersham-Pharmacia) was employed to enhance isoelectic focusing. Second dimension gel electrophoresis was performed on 12.5% polyacrylamide gels. Gels were then dried and imaged using a Fuji phosphorimager (Fujifilm, Tokyo, Japan). A total of 37 gels, comprising 2–5 gels representing independent samples for each stage and condition analyzed, were included in the study.
Previous studies have illustrated the value of two-dimensional protein gel image analysis for revealing global patterns of changes in gene expression [19, 20]. Although the identities of all individual protein spots may remain unknown, the ability to resolve and quantify the rates of synthesis of nearly 1000 gene products permits a powerful statistical comparison of samples and permits the creation of sets of valuable molecular markers of cell states and a perspective not readily achieved by alternative means [17, 21, 22].
Data Analysis
Gel images were processed and analyzed using the PD Quest software package (Bio-Rad Laboratories, Hercules, CA) using procedures employed previously [17, 22, 23]. Spots were detected and quantified using the Gaussian curve-fitting algorithm. Selected spots were matched manually to serve as landmarks for further automated matching. Automated matches were checked manually and inconsistent or partial matches resolved to the greatest extent possible. More than 900 fully matched spots of global quality 50 or greater (a measure of overall spot intensity, focus, and resolution) were successfully matched among the 37 gel images in the set. Phosphorimager values of individual spot intensities were summed for each gel. The individual spot intensities were then normalized to the total for all spots and expressed in units of parts per million (ppm). As with any method that provides for a global analysis of gene expression pattern (e.g., 2D-PAGE, microarrays), it is useful to characterize changes in gene expression by identifying sets of coordinately regulated gene products that change with developmental stage or treatment. To accomplish this, spots representing proteins that displayed statistically significant differences between stages or between treatment conditions were identified using a t- test. This yielded spot sets made of spots that increased or decreased significantly (P < 0.05) in rates of synthesis either during development or in response to a given treatment. Examination of the numerical degree of spot set overlaps provides one means of evaluating whether different treatments exert similar or distinct effects. Additionally, it is useful to compare effects on spot sets as a whole. To do this, it is necessary to obtain average expression profiles for the different spot sets, which can then be depicted as set graphs. To obtain average expression profiles for spot sets, the mean values of spots for each stage and treatment were determined and then expressed as the percentage of the maximum mean value over the entire range of stages and treatments. These normalized values were then averaged together to yield the average expression profile for each set. Normalizing individual spots to the percentage maximum value avoids disproportionate effects of the most intensely synthesized spots on the average expression profile. The statistical significance of effects of treatments and stage on spot sets was evaluated by applying the paired t-test to spots within the set, the results of which are indicated in the set graphs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Developmentally Regulated Proteins
As a first step toward evaluating the effects of oocyte coculture and GDF9 treatment on granulosa cell development, we sought to determine whether either treatment affected the expression of developmentally regulated proteins. We first compared 2° and 3° GCs (Fig. 2) that were cultured for 12 h without either oocytes or GDF9 to identify such proteins. This comparison revealed approximately 48 protein spots (5% of reliably scored spots, 3.3% displaying twofold or greater alterations) that were increased or decreased in intensity between these two stages of GC differentiation (Fig. 2; Fig. 3, sets A and F; and Fig. 4). These developmentally regulated sets of proteins provided a starting point for determining the degree to which oocyte coculture and GDF9 treatments could direct the transition from 2° GC to 3° GC phenotype in culture.
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Effects of Oocyte Coculture and GDF9 Treatment on Developmentally Regulated Proteins
As a set, those proteins for which rates of synthesis declined between 2° and 3° GCs (set A) showed significantly reduced rates of synthesis in 2° GCs cocultured with GOs or FGOs or treated with GDF9 (set A, Fig. 3). Synthesis of these proteins, particularly those proteins that were also affected in 2° GCs by coculture with GOs or 25 ng/ml GDF9 (A 
B, A D) ( denotes the intersection between sets), tended to increase in synthesis in 3° GCs upon treatment with 25 ng/ml GDF9, but not by the other treatments. A larger number of proteins in set A were affected by GOs and 25 ng/ml GDF9 than by FGOs or 100 ng/ml GDF9.
Those proteins that were synthesized at a greater rate in 3° GCs than in 2° GCs (set F) were significantly elevated in 2° GCs cocultured with either GOs or FGOs or treated with either concentration of GDF9 (Fig. 4). Within set F, some proteins were affected by only a single treatment, whereas others were affected by two or more treatments (Fig. 3). As with set A, a greater number of set F proteins were affected by GOs and 25 ng/ml GDF9 than were affected by FGOs or 100 ng/ml GDF9 (see set overlaps and graph, Fig. 3). Interestingly, synthesis of set F proteins was also slightly, but significantly, reduced in 3° GCs treated with GO or GDF9 compared with untreated 3° GCs, although this varied among the subsets defined on the basis of statistically significant differences observed with any given treatment (set F, Fig. 4).
To summarize the effects of treatments on developmentally regulated proteins, coculture with oocytes or treatment with GDF9 promoted a more mature phenotype in 2° GCs resembling the phenotype of mural GCs of 3° follicles. GOs exerted a greater effect than FGOs, and 25 ng/ml GDF9 was more effective than 100 ng/ml. Last, 2° GCs and 3° GCs displayed in some instances quantitatively opposing responses to some of these treatments (compare, e.g., increased rates of synthesis in treated 2° GCs versus untreated 2° GCs, as opposed to decreased rates of synthesis in treated 3° GCs versus untreated 3° GCs in set F, Fig. 4).
Broader Effects of Oocyte Coculture and GDF9 Treatment on Protein Synthesis
The data shown in Figures 3 and 4 indicated that not all developmentally regulated proteins were affected by either oocyte coculture or GDF9 treatment, and also that some spots were affected by these treatments but did not differ between untreated 2° and 3° GCs (i.e., some spots in sets B–F or G–J were not members of sets A or F). This indicated that oocyte coculture or GDF9 might normally support additional developmental transitions beyond those represented by sets A and F alone, and so could not be fully understood by examining only those proteins that differed between untreated 2° and 3° GCs. To explore more fully the effects of oocyte coculture and GDF9 treatment on GC phenotype, we examined the sets of affected proteins in three ways.
First, we examined effects of treatments on those proteins that displayed increased or decreased rates of synthesis in 2° GCs following treatments (sets B–E and sets G– J, Figs. 3 and 5). Those proteins that were increased in synthesis in 2° GCs by any one treatment were also significantly elevated by any of the other three treatments (sets G–J, Fig. 5). There was also a statistically significant reduction in synthesis in 3° GCs treated with 25 ng/ml GDF9, particularly in set J. Those proteins that were reduced in rates of synthesis in 2° GCs by coculture with GOs or treatment with 25 ng/ml GDF9 were likewise reduced by the other three treatments. Based on the sizes (i.e., numbers of members) of the spot sets, treatment with 25 ng/ml GDF9 produced the greatest effect, while FGOs once again produced the smallest effect, as observed for sets A and F above.
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Next, we created spot sets on the basis of significant differences within treatment categories but between stages (Fig. 6). Of the 42 proteins differing between 2° and 3° GCs cocultured with GO (Fig. 6, top two panels), 13 were also significantly different when comparing 2° and 3° GCs not cocultured with GOs. Of the 45 proteins differing between 2° and 3° GCs cocultured with FGOs, only 9 were also significantly different when comparing 2° and 3° GCs not cocultured with FGOs. This indicates that certain GC proteins differed between developmental stages when the GCs were cocultured with oocytes, but not in the absence of oocytes. Omitting those proteins that were different when comparing 2° and 3° GCs not cocultured with oocytes (i.e., subtracting sets A and F from the treated sets), it became apparent that coculture with either GOs or FGOs could exert opposite effects on 2° versus 3° GCs, see sets [(K)–(F)] and [(M)–(F)], Fig. 6; a similar trend was apparent in those proteins for which expression increased in the presence of oocytes, i.e., [(L)–(A)] and [(N)–(A)], but this did not reach the level of statistical significance. Interestingly, sets of proteins that differed between stage and within treatment were not seen for either 25 ng/ml or 100 ng/ml GDF9 treatment. This indicated that oocyte coculture may be permissive for some developmentally regulated changes in gene expression for which GDF9 is not permissive. It was also noted that only one protein spot was found shared between sets [(K)–(F)] and [(M)–(F)] and only one protein spot was found shared between sets [(L)–(A)] and [(N)–(A)]. Thus, the GOs and FGOs exerted distinct effects on the 2° and 3° GCs.
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Last, we examined the effects of each of the four treatments on 3° GCs (Fig. 7). The sizes of these spot sets were smaller than those observed in 2° GCs. Interestingly, coculture with FGOs affected more spots in 3° GCs than in 2° GCs (compare Figs. 3 and 7). There appeared to be a greater degree of independence in responses of 3° GCs to the different treatments, as indicated by the reduced frequency of proteins for which synthesis was affected by more than one treatment.
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Relationship Between Developmental Regulation and Response to Treatment
The examination of developmentally regulated proteins (sets A–J) indicated that each of the four treatments, and particularly coculture with GOs and 25 ng/ml GDF9, could exert opposite effects on the rates of synthesis of affected proteins in 2° GCs as compared with 3° GCs. To explore this relationship further, we examined those subsets of proteins that were significantly altered by each treatment when compared with untreated cultures of the same stage but that did not differ significantly between untreated 2° and 3° GCs (Fig. 8). The tendency for opposing effects was not apparent among these proteins. Thus, the opposing effects of treatments on 2° versus 3° GCs appear to be restricted to the developmentally regulated proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Coculture of 2° GCs with GOs or treatment with GDF9 promoted many of the changes exhibited during the transition to 3° GCs. Moreover, these changes were stable; i.e., the changes in 2° GCs promoted by coculture with GO or treatment with GDF9 were expressed by 3° GCs without oocytes or GDF9. Additionally, the effects of GOs on the transition from the 2° GCs to 3° GCs appear to be mediated, to a significant extent, by GDF9. Coculture of 2° GCs with FGOs did not promote the same changes in protein synthetic patterns as did coculture with GOs. Because the effects of FGOs on 2° GCs are reduced compared with those of GOs and GDF9, it can be inferred that FGOs and GOs either secrete different amounts of GDF9, with an accompanying difference in GC response, or that other factors that mitigate the effects of GDF9 are also secreted by FGOs.
The ability of GOs to induce 2° GCs to adopt characteristics of 3° GCs implicates the oocyte in an early step in the process of mural granulosa cell differentiation. It has been proposed that the default pathway of granulosa cell differentiation is that leading to the mural GCs, but that oocytes can abrogate the default pathway and promote differentiation to the cumulus cell phenotype [3, 12]. This hypothesis was based on evidence that the oocyte prevents the expression of Lhcg mRNA and can account for the observation that mural GCs, but not cumulus cells, of large antral follicles express this transcript [11, 24]. However, the results presented here reveal a more pervasive, previously unappreciated, effect of the oocyte on the development of the mural granulosa cell phenotype. These results indicate that some aspects of this phenotype are stably programmed by paracrine factors produced by the oocyte during the 2° to 3° transition. Moreover, one oocyte-derived paracrine factor contributing to this programming is GDF9.
It does not appear, however, that FGOs lose all ability to affect GC function, as the FGOs exerted a more pronounced effect on the 3° GC phenotype than did the GOs. This indicates that the array of signals emanating from the oocyte changes during the 2° to 3° transition, and that the mural GCs therefore continue to respond to oocyte-derived signals even at the 3° stage and despite the increased distance between the oocyte and mural GCs.
Just as the signals produced by the oocytes change between the 2° and 3° stages, so does the response of the GCs to those signals. This was evident not only in the greater response of 3° GCs than of 2° GCs to FGOs, but also in the opposing effects of GOs and of GDF9 on 2° versus 3° GCs. Based on these opposing effects, it is clear that the actions of oocytes and the responses of GCs to them are both developmentally regulated. This was also observed in the regulation of Kitl mRNA expression in GCs by oocytes and GDF9. GOs had a slightly positive effect on Kitl expression by 2° GCs. In contrast, FGOs strongly suppressed Kitl expression by both 2° and 3° GCs (both mural and cumulus GCs) [25, 26]. The effects of recombinant GDF9 on Kitl expression in 2° and 3° GCs were essentially the same as those of FGOs, but not of GOs [25, 26]. The differing activities of oocytes and responsiveness of GCs at different developmental stages are also illustrated by the mitogenic activity of GOs and FGOs. FGOs stimulate the proliferation of both 3° and 2° GCs, but GOs have little mitogenic effect on GCs of either developmental stage [27, 28].
Differing responses of GCs at different stages of their development could be explained by several different potential mechanisms, hypothesis of which, at this point, would be highly speculative. The differing actions of oocytes at different stages of development suggest either that they secrete multiple ligands or that they somehow modulate the effect of a single ligand. Although GDF9 appeared to have effects similar to those of GOs on protein synthetic patterns in GCs, they were not identical. Taken together, these considerations strongly suggest that multiple oocyte-derived paracrine factors and potential modulators promote different aspects of granulosa cell differentiation. It is also important to consider whether the denuded oocytes used in these experiments produce or secrete these factors similarly to oocytes in situ. GCs could modify these functions of oocytes via membrane contact or gap junction-mediated mechanisms, and some signals may be transmitted via gap junctions or direct cell contact.
Mouse oocytes transferred from 2° follicles to primordial follicles essentially doubled the rate of follicular development [29]. In addition to an increase in the rate of formation of antral follicles, this transfer promoted precocious differentiation of GCs exhibiting functional and molecular characteristics appropriate to both the cumulus and mural granulosa cell phenotypes [29]. The finding reported here that the oocyte exerts advance programming of mural granulosa cell differentiation at the 2° to 3° follicle transition, and thus regulates the developmental pathway of cells that no longer are in close association with the oocyte at a later stage, may help explain this precocious differentiation of the mural GCs. Moreover, the present findings reveal a heretofore unexpected complexity in the interactions between oocytes and the entire follicular complement of GCs during folliculogenesis and oogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 207 288 6073; jje{at}jax.org
Received: 2 October 2003.
First decision: 29 October 2003.
Accepted: 16 December 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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