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Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, gene regulation, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies using ovarian organ cultures or null mutant knockout mice have begun to elucidate the cell-cell interactions that coordinate primordial follicle assembly and the primordial to primary follicle transition. The process of follicular assembly begins at mid gestation in large monovulators such as humans [5] and at birth in rodents [6]. Follicular assembly is the apoptotic breakdown of groups of recently proliferating gametes called oocyte nests [7]. This apoptosis is coordinated by apoptotic factors such as tumor necrosis factor alpha (TNF
) [8]. High progesterone concentrations in utero repress oocyte apoptosis and follicular assembly in rodent pups until birth when the pups experience a dramatic decline in steroid concentrations [9].
The cell-cell interactions that occur during the primordial to primary follicle transition are better characterized than those of primordial follicle assembly. The primordial to primary follicle transition is coordinated by a combination of positive and negative regulatory factors. The epithelial granulosa cells produce kit ligand [10] and leukemia inhibitory factor [11] that act on the oocyte and the surrounding stroma to promote the primordial to primary follicle transition. Specifically, kit ligand is involved in the recruitment of mesenchymal theca cells to the follicle [12]. Basic fibroblast growth factor is produced by the oocyte and acts on the granulosa and theca to promote the primordial to primary follicle transition [13]. Nerve growth factor acts on the granulosa to promote the primordial to primary follicle transition [14]. Bone morphogenic protein 4 produced by the theca and stroma cells acts as a follicle survival factor [15]. Insulin acts as an endocrine factor to promote the primordial to primary follicle transition [16]. Müllerian inhibitory substance (MIS) produced by larger follicles represses the primordial to primary follicle transition in adjacent follicles [17].
The current model of cell-cell interactions in primordial follicle assembly and the primordial to primary follicle transition is based primarily on the investigation of growth factors that have been shown to be important in cell-cell interactions of the better characterized large antral follicles. A number of the growth factors known to coordinate antral follicle growth have been shown to be involved in primordial follicle development. Additional factors will also likely be involved in the development of primordial follicles. The current study is a gene discovery approach to identify new factors that may coordinate primordial follicle assembly and the primordial to primary follicle transition. This gene discovery project investigates the transcriptomes of ovaries that contain predominately unassembled, primordial, or primary follicles. Primordial and primary follicles are too small to dissect or isolate from the ovary, and it is not possible to isolate individual cell types from them. The current study uses rat ovaries from different developmental stages that have predominately one follicle population. Postnatal Day 0 ovaries contain only unassembled follicles. Postnatal Day 4 ovaries contain predominately primordial follicles. Postnatal Day 0 ovaries cultured for 1 wk contain predominately primary follicles [9]. Comparing the global gene expression profiles (transcriptomes) from ovaries at these developmental stages allows a comparison between unassembled, primordial, and primary follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from Postnatal Day 0 Sprague-Dawley rat pups were dissected for both immediate RNA extraction and organ culture. Postnatal Day 4 rat ovaries were dissected for immediate RNA extraction. Postnatal Day 0 rat ovaries were also cultured for 7 days. Whole ovaries were cultured as previously described [10] on floating filters (0.4 µm; Millicell-CM; Millipore, Bedford, MD) in 0.5 ml Dulbecco modified eagle medium (DMEM)–Ham F-12 medium (1:1, vol/vol) containing 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO); 0.1% albumax (Gibco BRL, Gaithersburg, MD); 2.75 µg/ml transferrin; 0.05 mg/ml L-ascorbic acid (Sigma); and 1 µg/ml insulin (bovine; Sigma) in a four-well culture plate (Nunc plate; Applied Scientific, South San Francisco, CA). Medium was supplemented with penicillin, streptomycin, and gentamicin to prevent bacterial contamination. RNA was extracted using the TRIZOL reagent (Invitrogen, Grand Island, NY) per the manufacturer's instructions. About 20 ovaries were used for each RNA extraction. All procedures were approved by the Washington State University (WSU) Animal Care and Use Committee.
Microarray Analysis
RNA was hybridized to the Affymetrix U34A 8799 gene chip (Affymetrix, Santa Clara, CA). The Genomics Core in the Center for Reproductive Biology at Washington State University performed the analysis as previously described [18, 19]. Briefly, RNA from whole ovaries was reverse transcribed into cDNA, and cDNA was transcribed into biotin-labeled RNA. Biotin-labeled RNA was then hybridized to the Affymetrix U34A 8799 gene chips. Each gene set was composed of 16 pairs of 24-oligomer oligonucleotides, with one sense strand specific for the gene and one antisense strand with single point mutations for use as a comparative negative control. The oligonucleotides spanned the gene, so 5' and 3' regions contributed to the final signal obtained. Biotinylated RNA was then visualized by labeling with phycoerythrin-coupled avidin. The microarray was scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA). Two microarray chips from two different RNA samples were analyzed for each of the predominate unassembled, primordial, and primary follicle ovary preparations.
Bioinformatics and Microarray Statistics
Microarray output was examined visually for excessive background noise and physical anomalies. The default Microarray Suite (Affymetrix, Santa Clara, CA) statistical values were used for all analyses. An absolute analysis using Microarray Suite was performed to assess the relative abundance of the transcripts based on signal and detection (present, absent, or marginal) for the 16 different oligonucleotides per gene and comparison for analysis. The absolute analysis from Microarray Suite was imported into GeneSpring 5.1 software (Silicon Genetics, Redwood City, CA). The developmental time course data were normalized within GeneSpring using the default/recommended normalization methods. These included the setting of signal values below 0.01 to 0.01, total chip normalization to the 50th percentile, and normalization of each gene to the median. These normalizations allowed for the visualization of data based on relative abundance at any given time point, rather than compared with a specific control value. Data restrictions and analytical tools in GeneSpring were applied to isolate noteworthy and possibly important patterns of gene expression during the course of testicular development and spermatogenesis. Transcripts expressed differentially at a statistically significant level were determined using a one-way ANOVA parametric test with variances not assumed equal and a P-value cutoff of 0.05. This was applied to all three developmental stage samples and considered all transcripts represented on the arrays. Two repeats for each developmental stage was performed and allowed a 2 x 2 factorial comparison in the experiment. Subsequently, expression restrictions were applied to the transcripts expressed in a significant manner. These restrictions were designed so that the remaining transcripts met the following requirements in addition to being expressed in a significant manner: 1) each transcript must have a signal value of at least 100 in at least one of the three developmental stages, and 2) had an average fold change of two or greater in signal intensity between developmental stages. The resulting transcripts were screened using Excel (Microsoft, Redmond, WA) for redundant UniGene entries. Transcripts that passed these restrictions were considered for further analysis. Cluster analysis and patterns of gene expression were identified using unsupervised cluster analysis within the set of differentially expressed transcripts that met the requirements detailed previously. Clustering algorithms allow for the separation of distinct patterns of expression based on the similarity of expression profiles between different genes [20]. In this analysis, a hierarchical clustering algorithm using a smooth correlation with the default parameters was used to isolate distinct, nonrepetitive patterns of expression within the time course. A nonphylogenetic gene tree that illustrates the major expressional patterns within the differentially expressed transcripts (determined through statistical analysis) in a continuous fashion was produced from this analysis. Pathway Assist (Stratagene Inc., La Jolla, CA) software was used for detailed pathway analysis and gene associations. Previous studies have shown that microarray data correlates well with real-time quantitative PCR and Northern analysis [18, 21]. Therefore, microarray data does not need to be confirmed as previously suggested [19]. However, a selected gene (i.e., vascular endothelial growth factor) was used in a real-time quantitative PCR procedure as previously described [22] to help confirm the microarray procedure.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RNA from two different preparations of each ovarian stage and follicle type were analyzed separately using the Affymetrix U34A rat gene chips. This gene chip has 14 000 genes for analysis. The global gene expression profile comparison between unassembled, primordial, and primary follicles is shown in Figure 3. The ovarian transcriptome analyzed is a combination of somatic cell and oocyte contributions. The Venn diagram demonstrates 2332 genes expressed in all three developmental stages with 146 genes specific to unassembled follicles, 94 genes specific to primordial follicles, and 151 genes specific to primary follicles (Fig. 3). Similar numbers of genes were also in common between two different developmental stages in the analysis (Fig. 3). The principle genes of interest were those that change expression levels between the developmental stages. Specific genes were categorized using a relative hybridization signal limit minimum of 100 and a minimum change greater than twofold. The cutoff 100 for signal and greater than twofold change demonstrated that over 5000 genes did not change expression levels and/or had very low levels of expression. Analysis of primordial follicle assembly demonstrated that 80 genes are up-regulated and 44 genes are down-regulated between unassembled and primordial follicles. Analysis of the primordial to primary follicle transition demonstrated that 148 genes are up-regulated and 50 genes are down-regulated between primordial and primary follicles (Table 1).
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Genes were grouped by expression pattern into six gene clusters (Fig. 4). Half of these clusters displayed dramatic changes in gene expression (Fig. 4, B, D, and F). The transcriptomes of each developmental point were unique. The other three clusters displayed smaller changes in gene expression (Fig. 4, A, C, and E). The first displayed up-regulation at primordial and primary stage follicles (Fig. 4A). The second displayed up-regulation at the primary follicle stage (Fig. 4C). The third displayed up-regulation at the unassembled and primary follicle stage (Fig. 4E). A dendrogram cluster analysis for all of the genes changing greater than twofold between the developmental stages is shown in Figure 5. The genes with increased (red) and decreased (blue) expression between the stages are grouped and show unique clusters at each developmental stage. Specific examples of functional gene clusters of metabolic, signal transduction, and hormones/growth factors/cytokines are also shown in Figure 5. Each developmental stage has gene clusters increasing and decreasing between the developmental stages. The specific genes examined are listed by change in signal in Tables 2–5.
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Of the 80 genes up-regulated during primordial follicle assembly (Tables 1, 2, and 6), a number were steroidogenetic enzymes. Also up-regulated was the hormonal factor inhibin and the known repressor of the primordial to primary follicle transition MIS. All three zona pellucida genes, which code for the thick protein coat of the oocyte, were up-regulated. Members of the neu differentiation factor family were also up-regulated (Tables 2 and 6).
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Of the 44 genes down-regulated during follicular assembly (Tables 3 and 6), two were synaptonemal complex family genes involved in meiosis. A growth factor strongly down-regulated between the unassembled and primordial follicle stage was insulin-like growth factor II (IGFII), as was an IGF binding protein–2 (IGFBP2).
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Of the 148 genes up-regulated during the primordial to primary follicle transition (Tables 1, 4, and 7), 17 were immune or inflammatory response–related genes such as cytokines. In addition, 10 proteases (e.g., cathepsins) were up-regulated. Twenty-three of the genes were metabolic enzymes. Also up-regulated were the IGFII growth factor and the IGFBP2 (Fig. 6C). Interestingly, vascular endothelial growth factor (VEGF) was also found to be up-regulated (Fig. 6C). To confirm the microarray analysis, the VEGF gene expression change was also assessed with a real-time quantitative PCR procedure and found to give the same relative increase in expression (data not shown). Absent from this list are growth factors known to promote the primordial to primary follicle transition—such as kit ligand (data not shown)—primarily due to low levels (i.e., <100 signal) of expression. In addition to the known genes, two genes of unknown function were dramatically up-regulated during the primordial to primary follicle transition. The kidney-specific androgen-regulated protein (KAP) and the uterus ovary–specific transmembrane protein (UOSTP) were both up-regulated (28- and 42-fold, respectively; Fig. 6A).
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Of the 50 genes down-regulated during the primordial to primary follicle transition (Tables 1, 5, and 7), three where steroidogenic enzymes. The endocrine factors inhibin and MIS were also down-regulated in a situation reciprocal to the change between the unassembled and primordial follicles (Fig. 6B). Three globin genes were strongly down-regulated between the primordial and primary follicle stages (Tables 5 and 7).
Further analysis of the data with Pathway Assist software identified groups of genes in specific cellular pathways. The relationship of the steroidogenic genes and other interacting proteins is shown in Figure 7. Critical steroidogenic genes such as CYP19A1 and STAR have interactions with a large number of proteins. Some regulate activity (Fig. 7, dashed lines) and some regulate expression (Fig. 7, solid lines). All of the genes identified as changing during primordial follicle development are linked except Scd1 and DCHR7 (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in gene expression are anticipated as the primordial follicles leave developmental arrest and the somatic cells proliferate. The gene expression changes reflect contributions of both the somatic cells and developing oocyte. Of the 148 genes that are up-regulated, 23 are metabolic enzymes suggesting the follicles are generally more metabolically active. This increase in metabolic state in the primary stage follicles is anticipated [26]. The increase in expression of the zona pellucida genes during follicular assembly validated the experimental methodology and design. The zona pellucida is the protein coat of the oocyte that is produced as the follicle assembles [27]. The rise in zona pellucida gene expression was anticipated as the primordial follicles assembled [27, 28].
The transcriptional profile of MIS is shown in Figure 6B. MIS is known to inhibit the primordial to primary follicle transition [17]. MIS was up-regulated during primordial follicle assembly and down-regulated during the primordial to primary follicle transition, consistent with the inhibitory role of MIS. The accompanying change in inhibin expression was unexpected (Fig. 6B) [29]. Although MIS and inhibin production are known to be stimulated by estrogen in the antral follicle [30], these early stages of folliculogenesis have negligible steroidogenesis. In addition, the pituitary/gonadal axis is not active at this stage of development, and these follicle stages are hormone-independent. These data suggest that low levels of inhibin operating within the ovary may have a function in maintaining the primordial follicles in their developmentally arrested state. Characterization of this potential unique function for inhibin requires further investigation.
A number of steroidogenic enzymes were up-regulated in primordial follicles and then down-regulated in primary follicles. Although a number of steroidogenic genes have increased expression levels (Fig. 7), critical genes in the steroidogenic pathway (e.g., CYP11A) were not changed. Therefore, steroid production may be minimal. Two important genes, STAR and CYP19A1, were altered (Fig. 7). Previous studies have suggested steroids may be produced and be involved in primordial follicle development [31]. This unique role of steroids in early primordial follicle development requires further investigation.
The transcriptional profile of IGFII and IGFBP2 displayed a pattern inverse to that of MIS and inhibin (Fig. 6C). They were down-regulated during primordial follicle assembly and up-regulated at the primordial to primary follicle transition. Although IGFII is known to have important functions in the antral follicle and corpus luteum [32], it has no known function in the early developing primordial follicle. The microarray data suggests a potential role for IGFII at this stage of development.
Three neu differentiation factors were up-regulated at the time of follicular assembly. This large gene family has been shown to be involved in the differentiation and growth of mesenchymal and neuronal cells [33]. This may be related to the innervation of the ovary by neural cells. Previous studies have suggested ovarian innervation may be critical for the normal timing of early folliculogenesis [34]. Because the neu differentiation factor family is such a large and diverse group of factors, it is possible that these factors may act directly on primordial follicles themselves. Therefore, the neu differentiation factors are candidates for potential coordinators of primordial follicle assembly.
Interestingly, several growth factors known to promote the primordial to primary follicle transition such as kit ligand, leukemia inhibitory factor, and basic fibroblast growth factor [10–12] had no change in gene expression between the developmental stages (data not shown). This was in large part due to the low levels of gene expression of these factors in the whole ovary. These known facilitators of the primordial to primary follicle transition may be expected to be up-regulated during the primordial to primary follicle transition. Alteration of gene expression may not be required if translational control and/or the cells responsiveness to the growth factors (e.g., receptor expression) are more critical. Although the experimental approach identifies potential candidate regulatory factors, it is important to note that gene expression changes are not always essential. Further analysis at an individual gene level is required to determine the specific function of a given gene product.
VEGF (Fig. 6C) gene expression is significantly up-regulated during the primordial to primary follicle transition. This observation was confirmed with a quantitative polymerase chain reaction (PCR) procedure that helped validate the microarray procedure. VEGF effects endothelial cell migration and proliferation to influence angiogenesis of the ovary [35–37]. No function of VEGF that affects early stages of folliculogenesis has been identified, but VEGF does influence preantral follicle development [38]. The microarray data associates this growth factor with the primordial to primary follicle transition. An obvious function of VEGF in the primordial to primary follicle transition would be in the angiogenesis of the ovary and follicles. The actual role of VEGF in the primordial to primary follicle transition will require further characterization.
Immune response genes, such as cytokines, were also found to be up-regulated during the primordial to primary follicle transition. Interleukin 1-ß, for example, was up-regulated sevenfold. Interleukins have been shown to have activities in the ovary, but not at this early stage of development [39]. The relationship and functions of these genes in primordial follicle development remains to be elucidated. Microarray observations suggest these factors might also play a role in the coordination of the primordial to primary follicle transition.
In addition to genes with a characterized known function, the microarray analysis identified two genes of unknown function that are up-regulated during the primordial to primary follicle transition (Fig. 6A). These are the kidney-specific androgen-regulated protein KAP (Genebank reference NM_052802) [40] and the uterus ovary–specific transmembrane protein (Genebank reference AF022147) [41]. Both of these genes are known only by screening of cDNA libraries from androgen-treated kidney from the first case and from estrogen-treated uterus in the second. Nothing can be deduced about the function or localization of these proteins except that they are highly expressed at the time of primordial to primary follicle transition. These two genes are good candidates for regulators of the primordial to primary follicle transition and require further characterization of their localization and function.
In conclusion, a gene discovery project was undertaken to identify new coordinators of primordial follicle assembly and the primordial to primary follicle transition. The Affymetrix rat U34A 8799 gene chip was used to analyze the transcriptomes of unassembled, primordial, and primary follicles. This gene chip contains 14 000 genes, and therefore does not reflect the entire genome. The majority of these genes did not change expression during primordial follicle development or were expressed at low levels below the cutoff used in the current study. Further investigations are needed to assess a genome-wide transcriptome of the ovary. The unassembled and primordial follicle stages used in vivo tissue, whereas the primary stage used cultured ovaries. Although we have shown no effect of culture on organ or follicle viability [10–12], changes potentially induced during culture need to be considered in any data interpretation. A summary of the potential factors involved in the coordination of primordial follicle development are summarized below. The neu differentiation factors are among the genes identified as being up-regulated at the time of follicular assembly. The neu factors are candidates for promoters of follicular differentiation. MIS was found to be highly expressed at the primordial follicle, consistent with its proposed function as an inhibitor of the primordial to primary follicle transition. Its site of synthesis and action remain to be determined. Unexpectedly, the steroidogenic apparatus and inhibin were also highly expressed at the primordial follicle stage. This suggests that potentially both inhibin and steroids may have novel functions in maintaining the primordial follicle in its developmentally arrested state. Estrogen receptor activation has been shown to regulate MIS expression [42]. The speculation is that steroids may be performing a similar function in the neonatal ovary. Further studies are needed to characterize the role of MIS, steroids, and inhibin in the coordination of early folliculogenesis. IGFII was down-regulated in the primordial follicle stage, and therefore may influence follicle assembly. The growth factor VEGF was up-regulated in the primary follicle, making the factor a candidate for a facilitator of the primordial to primary follicle transition. Many cytokines such as interleukin 1-ß were also up-regulated in the primary follicle. These factors are also candidates for coordinators of the primordial to primary follicle transition. In addition, two new genes of unknown function, KAP and the UOSTP, were up-regulated and are also candidates for coordinators of the primordial to primary follicle transition. Therefore, the microarray approach identified a number of factors potentially involved in primordial follicle development.
Primordial follicle assembly and the rate of primordial to primary follicle transition influences the size of the primordial follicle pool [43]. The size of the primordial follicle pool sets the number and availability of follicles in the female. Although it has recently been speculated that new follicles can be recruited into the primordial follicle pool in adult rodents from germ-line stem cells [2], further investigation is required to confirm this possibility. When the primordial follicle pool is depleted, reproduction and steroidogenesis ends and menopause begins. Dysfunction in primordial follicle assembly and the primordial to primary follicle transition compromises the primordial follicle pool and may lead to an early menopause and/or the condition of premature ovarian failure. Further analysis of the factors involved in these processes of primordial follicle assembly and the primordial to primary follicle transition will improve our understanding of such pathological conditions as premature ovarian failure. The genes identified in the current study provide candidates for further analysis and will help elucidate this critical biological process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michael K. Skinner, Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4231. FAX: 509 335 2176; Skinner{at}mail.wsu.edu
Received: 12 May 2004.
First decision: 2 June 2004.
Accepted: 30 August 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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