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Ovary |
Department of Animal Science,4 University of Missouri, Columbia, Missouri 65211
Monsanto Company,5 St. Louis, Missouri 63198
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, follicle, gene regulation, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanisms underlying ovarian follicular development have been intensely studied. It is generally accepted that antral and preovulatory follicular development is primarily controlled by pituitary gonadotropins [1]. Early stages of follicular development (preantral and small antral stages) are controlled by a variety of locally produced hormones and growth factors [5, 6]. Despite continuing progress in the area of ovarian biology, the factors controlling some of the key events in follicular development, including the initiation of primordial follicle growth, antrum formation, follicular dominance, and follicular atresia, remain to be identified.
Large-scale sequencing of cDNA libraries is a rapid and efficient means of discovering new genes and providing both quantitative and qualitative information regarding gene expression within specific tissues or cell types [7–12]. We carried out large-scale sequencing of cDNA from the pig ovary to identify novel intraovarian factors and mechanisms involved in ovarian development. Here, we report a catalog of genes expressed in the porcine ovary (expressed sequence tags [ESTs]) as well as a preliminary view of their expression patterns throughout ovarian development, follicular growth, and ovulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovarian tissue (whole ovary, dissected follicles, or corpora lutea) (Table 1) was collected from cross-bred pigs (Sus scrofa domestica), frozen in liquid nitrogen shortly after collection, and stored at –80°C until RNA extraction. The tissues from several individual pigs were pooled for RNA extraction. The procedures used for tissue collection were approved by the Animal Care and Use Committee of the University of Missouri-Columbia.
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The procedures for constructing the Day 0 follicle (pd0fol), Day 5 ovary (pd5ov), Day 12 follicle (pd12fol), and Day 12 corpus luteum (pd12CL) cDNA libraries were reported previously [12]. The methods described herein were used for production of the 2-mm follicle (p2mm), 4-mm follicle (p4mm), 6-mm follicle (p6mm), 8-mm follicle (p8mm), fetal ovary (pfeto), neonatal ovary (pnatal), and prepubertal ovary (pputal) libraries. Total cellular RNA was extracted using the Trizol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Poly(A) RNA was isolated from total cellular RNA using the Oligotex mRNA kit (Qiagen, Chatsworth, CA). The integrity and purity of extracted RNA were verified by measuring the ratio of absorbance at 260 and 280 nm and by electrophoresis of an RNA aliquot on formaldehyde-agarose gels.
The cDNA libraries were constructed by using the SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) according to the manufacturer's instructions with a modified reverse transcription reaction [13]. The reverse transcription was performed by annealing 1 µg of poly(A) RNA with 10 µg of an oligo(dT) primer (GCTGCTCGCGGCCGC-tag-T18, where tag = library-specific five-base sequence identifier) at 42°C or by annealing 1 µg of poly(A) RNA with 4 µg of an anchored oligo(dT) primer (GCTGCTCGCGGCCGC-tag-T18VN, where V = A, G, or C and N = A, G, C, or T) at 50°C. An excessive amount of oligo(dT) primer or an anchored oligo(dT) primer was used to reduce the number of clones with long poly(A) tails in the cDNA libraries [13]. The cDNAs were ligated into either the pT7T3 vector (pd0fol, pd5ov, pd12fol, and pd12CL cDNA libraries) or the pSport1 vector (p2mm, p4mm, p6mm, p8mm, pfeto, pnatal, and pputal libraries; Life Technologies) at the NotI and SalI sites. Ligated vector-cDNAs were transformed into DH10B Escherichia coli-competent cells (Life Technologies) by electroporation. The complexity of the library was estimated according to the manufacturer's instructions (Life Technologies).
The quality of each cDNA library was assessed before large-scale cDNA sequencing was performed. Bacteria clones from each library were picked and individually grown overnight at 37°C in deep-well, 96-well plates containing 1.4 ml of Terrific Broth with ampicillin [14]. Plasmid extractions from bacterial cultures were performed by using the QIAprep 96 Turbo Miniprep kit (Qiagen) according to the manufacturer's instructions. The average cDNA insert size and the percentage of clones without an insert were determined by EcoRI and HindIII restriction-enzyme digestion of plasmid isolated from 96 randomly picked clones. Libraries with an average insert size of greater than 1 kilobase (kb) and more than 95% of clones having a cDNA insert were subjected to preliminary cDNA sequencing. Then, 288 randomly picked clones were sequenced. The ABI Prism Big Dye Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA) with a T3 (cDNA ligated into the pT7T3 vector) or Sp6 (cDNA ligated into the pSport1 vector) sequencing primer was used for the sequencing reactions. The sequencing reaction products (3' cDNA sequence) were analyzed on an ABI 377 Automated DNA sequencer (Applied Biosystems). The cDNA sequence data from the preliminary sequencing were evaluated. A cDNA library was subjected to large-scale sequencing if it met the following criteria: 1) More than 95% of the clones contained inserts, 2) the average insert size was greater than 1 kb, 3) less than 5% of the clones contained ribosomal RNA, 4) less than 10% of the clones contained long poly(A) tails (>40 adenines), and 5) the complexity of the library was at least 106 colony-forming units. The large-scale sequencing was performed as described for the preliminary sequence analysis above.
Sequence Clustering and Annotation
The cDNA sequences from the 11 libraries were clustered into contigs using the SeqMan II software system (DNASTAR, Inc., Madison, WI). The original trace files from the automated DNA sequencing were loaded into the software system. The trace files were evaluated for quality using the Trace Quality Evaluation system within SeqMan II. Poor-quality sequence, vector sequence, and poly(A) tails were trimmed from the sequence. The trimmed cDNA sequences were assembled into contigs using the assembly process within SeqMan II. A 90% minimum match percentage was used. The actual average match percentage for entry into the contig was 97.1%. A consensus sequence for each contig was exported from SeqMan II.
The consensus cDNA sequence for each cluster was compared with The Institute for Genomic Research (TIGR) Porcine Gene Index nucleotide sequence databases (Porcine Index, release 8.0; TIGR, Rockville, MD; http://www.tigr.org/tdb/tgi) using the BLAST program [15]. The contig consensus sequence was considered to be homologous to a TIGR Tentative Consensus (TC) sequence or mature transcript (ET) sequence when the BLAST score was greater than 200. Contigs that were homologous to the TIGR TC sequence were annotated with a gene name by using the TIGR EST Annotator.
Functional classifications from Gene Ontology (GO) Consortium [16] were assigned to each contig using the homologous TIGR TC number and the functional classifications assigned by TIGR. A set of higher-level parent classifications (GO Slim) [17] were used for the GO assignments. The GO Slim assignments were performed manually by visually inspecting the Tree view and identifying the appropriate GO Slim category. Genes whose component, process, or function fell into multiple GO Slim categories were given equal weighting across categories.
Electronic Northern Blot Analyses
Contigs were comprised of cDNA clones arising from the original cDNA libraries. An "electronic Northern" was performed by analyzing the frequency of library clones within each contig. Three collective libraries were selected for analyses. A collective follicle library (combined p2mm, p4mm, p6mm, and p8mm libraries) was compiled and analyzed, because the individual follicle libraries represented follicles at different stages of preovulatory development [18]. The pd12fol and pd12CL libraries were constructed from follicles and corpora lutea collected from the same ovaries on the same day of the estrous cycle. Therefore, a collective Day 12 ovary library (pd12fol and pd12CL) also was complied and analyzed for cDNAs that were differentially expressed in follicles and corpora lutea. Finally, a collective library representing developmental stages (fetal, neonatal, and prepubertal) was analyzed to identify cDNAs whose presence was specific to developmental stages of the ovary. Independence was examined using the chi-square test. The observed value was the number of clones arising from each library. Each library should contribute a proportional number of clones to the contig if the frequency of clones was independent of library. The expected value (assuming independence) therefore was the total number of clones in the contig multiplied by the proportion of accepted sequences in the subject library (relative to the total number of sequence in the collective libraries).
Comparison of mRNA Amount in Tissues with cDNA Frequency in Libraries
Ribonuclease protection (RP) and Northern blot assays were used to determine the mRNA expression levels of selected genes in ovaries, follicles, and corpora lutea. The expression levels of seven mRNAs were measured. These mRNAs were selected because their cDNAs appeared to be differentially represented within closely related libraries. The expressions of three mRNAs were compared for fetal, neonatal, and prepubertal tissues, and the expression of three mRNAs were compared for 2-, 4-, 6-, and 8-mm follicle tissues. The expression of one mRNA was compared between follicle and corpus luteum. Total cellular RNA was extracted from tissues as described above. Radiolabeled probes were synthesized from selected cDNA plasmids. The RP and Northern blot assays were performed as single experiments using previously described procedures [19, 20]. The mRNA expression level was subjectively compared to the number of cDNA isolated from each library.
Data and cDNA Clones Access
The sequence for each cDNA library clone reported herein has been deposited in GenBank. A list of cDNA library clones within each contig, an annotated list of the contigs (contig number, gene name, frequency of clones from each library, etc.), and the consensus sequence for each contig are available at http://www.swine.rnet.missouri.edu.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 22 176 cDNA were sequenced and subjected to the Trace Quality Evaluation system within SeqMan II. Seventy percent of the cDNA sequences (n = 15 613) contained sufficient cDNA sequences of acceptable quality for clustering (Table 2). The percentage of accepted sequences for individual libraries ranged from 23% (Day 5 ovary library) to 80% (neonatal ovary library). The average trimmed sequence length across all libraries was 348 ± 1 (mean ± SEM) base pairs (bp; range, 99 to 659 bp) (Fig. 1). Clustering of the cDNA sequences resulted in 8507 contigs (Table 2). Single-sequence contigs comprised 74% of the total, and 99% of the contigs had 20 or fewer members (Fig. 2).
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Annotation of cDNA and Contigs
Sixty-eight percent of the contigs (n = 5797) were defined as homologous (BLAST score, 
200) to existing TC or ET in the TIGR Porcine Gene Index (Fig. 3). The average BLAST score for homologous contigs was 601 ± 3. Forty-eight contigs has 20 or more members (Table 3). Nearly half of the largest contigs had consensus sequences that were homologous to genes involved in protein synthesis (initiation factors, elongation factors, and ribosomal proteins). Other large contigs had consensus sequences for enzymes involved in intracellular energy production (e.g., components of the electron-transport chain), for structural components of the cell, for proteins involved in protein turnover, or for proteins involved in transport.
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The consensus sequences for contigs were homologous to a number of genes that function in the endocrinology of ovarian cells (Table 4). The sequenced cDNA encoded insulin-like growth factor (IGF) system genes (including IGF-binding proteins), inhibin/activin system genes, relaxin and relaxin-like protein genes, and transforming growth factor ß family genes. The inhibin 
chain (contig 756) represented 0.29% of the sequenced cDNA. The cDNAs for three separate prostaglandin synthesis enzymes and four separate steroidogenic enzymes were sequenced. Prostaglandin F synthase 1 (contig 273) was one of the most abundant cDNAs and represented 0.30% of cDNA sequences. Steroid acute regulatory protein (StAR; contig 3043) represented 0.12% of the sequenced cDNAs. The cDNAs for hormone receptors and components of hormone second-messenger systems (cAMP, inositol triphosphate, phosphatidylinositol 3-kinase, mitogen-associated protein kinase, Janus kinase, and sma/mad) were also sequenced.
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The cDNAs were classified according to the GO Index (cellular component, molecular function, and biological process). We found that 797 contigs (3242 individual cDNA clones) had GO cellular component annotations in the TIGR Porcine Gene Index. Each contig contained one or more cDNA clones. Based on the number of cDNA clones (an indirect indicator of mRNA species within the cell), the majority of cellular mRNA encoded components of the ribosome, mitochondrion, and nucleus (Fig. 4). We found that 996 contigs (3370 individual cDNA clones) had a GO function annotation. The consensus sequences for most contigs were homologous to genes whose products were involved in binding or catalytic activity (enzymes). We found 949 contigs (2948 individual cDNA clones) with a GO process annotation. Nearly three-quarters of the cDNAs sequenced encoded genes involved in cellular metabolism and cell growth and/or maintenance.
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Frequency of cDNAs in Different Libraries (Electronic Northern Blot Analysis)
The frequency of library cDNA clones within the largest individual contigs is shown for follicle libraries (p2mm, p4mm, p6mm, and p8mm) (Table 5), Day 12 ovary libraries (p12fol and p12CL) (Table 6), and whole-ovary libraries (pfeto, pnatal, and pputal) (Table 7). Independence of cDNA distribution within the libraries was tested by chi-square (statistical significance inferred at minimum threshold of P < 0.10). A lack of independence (statistically significant result) indicated that within a contig, specific cDNAs were found in a higher percentage in some libraries relative to others.
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We found 18 large contigs (10 members) whose cDNA clones were not equally distributed across follicle libraries (Table 5). Nine contigs (contig 2 [elongation factor 1], contig 1 [cytochrome c oxidase], contig 160 [cytochrome b], contig 18 [identity unknown], contig 222 [actin ], contig 1711 [translation elongation factor eEF-2], contig 5141 [retinoic acid receptor-responder protein 1], contig 163 [ADP ATP carrier protein isoform T2], and contig 5152 [glutathione S-transferase]) had cDNA clones that were predominantly found in the collective p6mm and p8mm libraries. Three contigs (contig 288 [ferritin, heavy polypeptide], contig 273 [prostaglandin F synthase 1], and contig 756 [inhibin ]) had cDNA predominately found in the p6mm library. Four contigs (contigs 36 and 37 [glutathione S-transferase], contig 651 [cathepsin L], and contig 1448 [ATP synthase 
]) had cDNA clones predominately found in the p8mm library. Two contigs (contig 161 [cytochrome c oxidase III] and contig 370 [thymosin ß4]) had cDNA clones primarily found in p2mm and p4mm libraries. The remaining contigs with more than 10 members had cDNA equally distributed across follicle libraries.
Eight contigs had cDNA clones that were not equally distributed within the collective p12fol and p12cl libraries (contigs with four or more members) (Table 6). Eleven contigs had cDNAs that were found primarily in pd12CL, and seven contigs had cDNAs that were found primarily in pd12fol. The greatest difference in cDNA distribution toward pd12CL was found for contigs 1 (cytochrome c oxidase), 12 (prostate-secreted seminal plasma protein [PSP-94 protein]), 33 (ATP synthase A chain), 3043 (StAR), 5146 (tissue metalloproteinase inhibitor [TIMP]-1), and 5207 (CYP1B1). The cDNAs for a variety of proteins and enzymes involved in progesterone synthesis were found in higher proportions within pd12CL (contigs 3043 and 5325 [StAR], contig 2308 [CYPXIA1; side chain-cleavage enzyme], and contig 282 [3-ß-hydroxysteroid dehydrogenase]). The greatest difference in cDNA distribution toward pd12fol was found for contigs 278 (nexin-1), 18 (unknown), 368 (translationally controlled tumor protein [TCTP]), 7 (collagen 2[I] chain), 255 (unknown), and 2326 (alanine glyoxylate aminotransferase). The cDNAs within contigs 7, 18, and 368 (found in high abundance in pd12fol) were also abundant in the individual follicle libraries (Table 5).
Eleven contigs had cDNA clones that were not equally distributed within the collective pfeto, pnatal, and pputal libraries (contigs with six or more members) (Table 7). Three contigs (contig 33 [ATP synthase A chain], contig 161 [cytochrome c oxidase III], and contig 2285 [guanine nucleotide-binding protein ß subunit-like protein]) contained cDNAs that were predominately found in the pfeto library. Three contigs contained cDNAs that were predominately found in the pnatal library (contig 412 [collagen, type III, 1], contig 533 [eukaryotic translation initiation factor 4A], and contig 5141 [retinoic acid receptor-responder protein 1]). Five contigs contained cDNAs that were predominately found in the pputal library (contig 288 [ferritin, heavy polypeptide], contig 430 [monooxygenase], contig 64 [selenoprotein P-like protein], contig 756 [inhibin chain], and contig 34 [vimentin]). The five contigs whose cDNAs were predominately found in the pputal library (created from tissue-containing antral follicles) were among the largest contigs for the individual follicle libraries (Table 5).
Comparison of mRNA Amount in Tissues with cDNA Frequency in Libraries
Seven mRNAs were independently examined for expression level within ovarian tissues using RP assay (Fig. 5, A and B) or Northern blot analysis (Fig. 5C). In most cases, the level of mRNA expression within a tissue reflected the relative number of cDNA clones isolated from the respective libraries. For example, the RP assay did not detect the inhibin subunit mRNA in fetal or neonatal ovary, but a radiographic signal was readily detected for inhibin subunit in prepubertal ovary (Fig. 5A). The corresponding clone frequency within contig 756 (inhibin ) was one, zero, and seven for pfeto, pnatal, and pputal, respectively. Likewise, the mRNA expression level within fetal, neonatal, and prepubertal tissues for selenoprotein P (contig 64) and an unknown gene (contig 2542) (Fig. 5A) as well as the mRNA expression level for 2-, 4-, 6-, or 8-mm follicles for inhibin ß (contig 3428) and LH-receptor mRNA (contig 1894) (Fig. 5B) were visually correlated with the number of clones isolated from the respective libraries. The 17-hydroxylase mRNA was highest in 6-mm follicles (Fig. 5B), but the corresponding number of cDNA clones for p2mm, p4mm, p6mm, and p8mm was one, zero, four, and five, respectively. The apparent decrease in 17-hydroxylase mRNA in 8-mm follicles was not reflected in the cDNA frequency across libraries. The PSP-94 cDNA was found almost exclusively in the corpus luteum (one cDNA from pd12fol compared to 41 clones from pd12CL), and Northern blot analyses demonstrated expression of PSP-94 (
600 bp) in corpus luteum but not in the follicle or nonluteal ovary (Fig. 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As revealed by the cDNA frequency, cytochrome c oxidase cDNA was the most abundant cDNA, representing 1.42% of the 15 613 clones (Table 3). Cytochrome c oxidase is an enzyme in the mitochondrial electron-transport chain (intracellular energy production). Other highly expressed genes in porcine ovary were also involved in intracellular energy production (ATP synthase and cytochrome c oxidase III). Elongation factor 1 had the second-highest cDNA frequency (1.38% of the cDNA clones), and other genes involved in protein synthesis (including ribosomal proteins) were also highly expressed. More than 50% of the cDNA sequences encoded proteins involved in metabolism (GO Slim process) (Fig. 4), and nearly 50% of the genes encoded components of the ribosome or mitochondria (GO Slim component) (Fig. 4). Thus, the bulk of the mRNA within an ovarian cell encodes genes that are involved in the intracellular activities that are not unique to ovarian cells (metabolism, protein synthesis, etc.), and only a small percentage of the major cDNA species appeared to be specific to reproductive tissues. Reproductive genes with a high expression level (high frequency of sequenced cDNA) included inhibin and PSP-94. The abundance of cDNA for these two reproductive genes in ovary is unusual, because most other genes previously associated with ovarian function were found in relatively low numbers. For example, only a single copy of aromatase (contig 1576), FSH receptor (contig 7993), zona pellucida protein 3 (contig 7528), and IGF-I (contig 5255) were sequenced. Fewer than five cDNAs were sequenced for several other genes that play physiologically important roles within the ovary (IGF-II, IGF-binding protein-2, LH receptor) (Table 4). Tissue-specific ovarian genes therefore may be found only in contigs with a small number of members.
The cDNA libraries sequenced in the present study were not subtracted or normalized. The frequency of cDNA sequences therefore may approximate the expression level of the mRNA in the tissue from which the cDNA library was made [21–24]. For this reason, the frequency of each cDNA from each library was counted. Analysis of the cDNA frequency across different libraries suggested differential patterns of expression within different-size follicles (diameter, 2–8 mm) (Table 5), between follicles and corpora lutea (Table 6), and across developmental time-points (fetal, neonatal, and prepubertal ovary) (Table 7). The expression of selected genes was consistent with the frequencies of their respective cDNA in the individual libraries (analyzed by RP assay or Northern blot) (Fig. 5).
Distinct biochemical processes may underlie the morphological and physiological changes in follicles as they grow from 2 to 8 mm in diameter. Follicles of 2 to 4 mm in diameter either may continue growth or may undergo atresia [25]. Follicles of 6 mm in diameter are highly estrogenic, and in our previous analyses, follicles of 6 mm in diameter had more aromatase mRNA than those of 2, 4, or 8 mm in diameter [18]. Follicles of 8 mm in diameter had less aromatase, because they had been exposed to the LH surge and had undergone luteinization before ovulation. Eight contigs had cDNA clone frequency that was highly disproportionate (P <0.001) across follicle libraries (Table 5). Five contigs had cDNA clone frequencies that increased in the collective p6mm and p8mm libraries (contig 2 [elongation factor 1], contig 1 [cytochrome c oxidase], contig 160 [cytochrome b], contig 36 [glutathione S-transferase], and contig 18 [unknown]). The physiological basis for the observed changes in gene frequency is open to further scientific investigation. The increase in elongation factor 1 mRNA and cytochrome c oxidase mRNA may reflect an increase in protein synthesis and energy metabolism, respectively, in large follicles. The increase in glutathione S-transferase that we observed in the p8mm library is supported by previous research showing an increase in glutathione S-transferase in large follicles [26]. The increase in glutathione S-transferase is believed to support progesterone synthesis in preovulatory follicles that have been exposed to the LH surge and are undergoing luteinization [27]. The cytochrome b cDNA was increased in large follicles as well. Cytochrome b is a component of the oxidase system of neutrophils, and neutrophils are attracted to the follicle during ovulation [28]. The increase in cytochrome b therefore may reflect an infiltration of neutrophils into the preovulatory follicle. Contig 18 represents a cDNA that encodes an unknown protein. The cDNA sequence is found within the TIGR database but, nonetheless, cannot be tied to any known gene. The identity and function of this unknown protein that is increased in large porcine follicles is a potential subject for future investigations.
Contig 288 (ferritin, heavy polypeptide), contig 273 (prostaglandin F synthase 1), and contig 756 (inhibin ) were specifically increased in the p6mm library at P < 0.001. The high expression of inhibin is consistent with the role of inhibin as an endocrine factor secreted from large follicles [29]. Others have reported an increase in inhibin mRNA in midstage preovulatory follicles, with a subsequent decrease in inhibin mRNA is late-stage preovulatory follicles [30]. Ovarian follicles synthesize prostaglandins E, D, and F, as evidenced by our sequencing of synthase enzymes for the respective enzymes (Table 4) and previously published literature [31]. Prostaglandin synthesis increases in preovulatory follicles, particularly around the time of the LH surge [32], and an increase in prostaglandin synthesis is required for ovulation in pigs [33]. A clear increase in ferritin cDNA was noted within the p6mm library, and to our knowledge, changes in ferritin mRNA have not been previously reported in preovulatory follicles. Iron is required for coordinating the heme within P450 enzymes, which is a class of enzymes that includes those involved in steroidogenesis [34]. Ferritin sequesters and stores intracellular iron in a nontoxic form [35]. The increase in ferritin in preovulatory follicles may play an important role in iron homeostasis during the preovulatory period.
Twelve contigs had cDNA frequencies that differed at P < 0.05 across the pd12fol and pd12CL libraries (Table 6). Six of the contigs (contig 1 [cytochrome c oxidase], contig 12 [PSP-94], contig 33 [ATP synthase], contig 3043 [StAR], contig 5146 [TIMP-1], and contig 5207 [CYP1B1]) contained cDNAs that were more abundant in the pd12CL library. An increase in cytochrome c oxidase and ATP synthase activity in corpora lutea relative to follicles has been demonstrated in previous studies [36, 37]. Cytochrome c oxidase and ATP synthase are mitochondrial enzymes, and the tissue differences may simply reflect the greater density of mitochondria in corpus luteum relative to follicle. Greater StAR mRNA within corpora lutea relative to follicles has also been found in porcine ovary [38]. The difference in cDNA frequency may reflect the greater mitochondrial density as well as greater steroidogenic capacity of corpus luteum relative to follicle. Tissue inhibitors of metalloproteinases play a role in remodeling of the extracellular matrix [39]. The expression of TIMP-1 mRNA increases during late preovulatory development and increases further within the corpus luteum [40]. To our knowledge, specific expression of CYP1B1 (a P450 enzyme involved in steroid metabolism) [41] in corpus luteum has not been previously reported.
The abundance of prostate-secreted plasma protein (PSP-94 or ß-microseminoprotein) in corpus luteum relative to follicle was unexpected (Table 6). The PSP-94 protein is a major component of seminal fluid from a variety of species. Although PSP-94 has been used as a marker of prostatic development in males [42], a specific function for PSP-94 in prostate physiology has not been identified. The PSP-94 protein will bind immunoglobulin and may influence the activity of immune cells [43]. A recent study found an inhibitory effect of PSP-94 in prostate tumor growth [44]. Thus, PSP-94 may control cell growth and development either through an indirect effect on immune cells or through a direct effect on luteal cells themselves. We examined PSP-94 in cow and pig corpus luteum and found that the mRNA was only expressed in pig corpus luteum (unpublished observations). Furthermore, the mRNA was maximally expressed in midcycle corpora lutea. Thus, the porcine corpus luteum may be unique from the corpus luteum of other species in that it specifically expresses PSP-94 (Fig. 5). The highly specific expression of PSP-94 mRNA suggests a unique role for this protein in porcine corpus luteum.
Six contigs had cDNAs that were more abundant in pd12fol compared to pd12cl (P < 0.05; contig 278 [nexin-1], contig 18 [unknown], contig 368 [TCTP], contig 7 [collagen 2 (I) chain], contig 255 [unknown], and contig 2326 [alanine glyoxylate aminotransferase]) (Table 6). The cDNAs within contigs 7, 18, and 368 were also abundant in the individual follicle libraries (Table 5). The relative abundance of nexin-1 (a serine protease inhibitor) in follicles compared to corpus luteum confirms a recent report showing a high level of nexin-1 expression in preovulatory follicle [45]. Nexin-1 mRNA decreases after the LH surge. Thus, the decrease in a specific protease inhibitor may facilitate ovulation. Translationally controlled tumor protein was recently implicated as a protein that binds elongation factor eEF1A during the elongation step of protein synthesis [46]. The abundance of TCTP cDNA in follicle libraries (Tables 5 and 6) highlights the importance of protein synthesis during follicular development. The presence of collagen 2 (I) in pd12fol may reflect the relative abundance of specific collagen subunits in follicles versus corpora lutea [47]. The two contigs classified as unknown should be investigated further as genes controlling unique aspects of follicular development.
Several genes were differentially expressed when the pfetal, pnatal, and pputal libraries were examined. Ovarian follicular development in pigs is different from that in humans and other farm animals, because the neonatal ovary (sampled herein) is compact, with a dense population of primordial and primary follicles. Oocytes may still reside in egg nests at birth, and a period of 1–2 wk after birth may be required before follicular populations are fully established and primordial follicles enter the growth phase [48]. The ovary then undergoes a protracted period of follicular growth before puberty. Three genes had contigs that were highly represented in either fetal or neonatal ovary (P < 0.001; contig 33 [ATP synthase A chain], contig 161 [cytochrome c oxidase III], and contig 412 [collagen, type III, 1]) (Table 7). The fetal ovary is densely packed with oocytes, and oocytes have a large number of mitochondria. Thus, the high frequency of ATP synthase and cytochrome c oxidase in the pfeto libraries may reflect the preponderance of mitochondria in developing oocytes. Ovarian follicles change the collagen composition of their basal lamina when they commence growth [49]. The shift in expression toward collagen type III 1 within the pnatal library may be indicative of an active phase of follicular growth in the neonatal ovary. Additional cDNAs had a frequency that appeared to be greater in either the pfeto (contig 2285 [guanine nucleotide-binding protein ß subunit-like protein]) or pnatal (contig 5141 [retinoic acid-responder protein 1] and contig 533 [eukaryotic translation elongation factor 1]) libraries. Five contigs (contig 288 [ferritin, heavy polypeptide], contig 430 [monooxygenase], contig 64 [selenoprotein P-like protein], contig 756 [inhibin chain], and contig 34 [vimentin]) had cDNAs that were predominately derived from prepubertal ovaries (pputal library). The prepubertal ovaries used to construct the pputal library contained antral follicles. Each of the five contigs was among the largest contigs in the collective follicle libraries (Table 5). The observed frequencies of cDNA in the pputal library therefore supported the sequencing data from the follicle libraries (Table 5).
In summary, the present study has provided a catalog of 8507 contigs derived from 15 613 cDNA sequences obtained in porcine ovarian tissue. The list of ovarian cDNAs and their frequencies in each ovarian library will be a useful index of ovarian gene expression. The observed differences in cDNA frequency across libraries appeared to be logical given existing knowledge of ovarian biology. For most contigs, the frequency of the sequenced genes was too few to reliably study gene expression across tissues. Greater sensitivity for gene expression analysis will be achieved using solid-phase microarrays constructed from the cDNAs generated herein. This second phase of the current project is underway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Matthew C. Lucy, 164 Animal Science Research Center, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827; lucym{at}missouri.edu
3 Current address: Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA
Received: 10 May 2004.
First decision: 16 June 2004.
Accepted: 23 July 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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K.M. Whitworth, C. Agca, J.-G. Kim, R.V. Patel, G.K. Springer, N.J. Bivens, L.J. Forrester, N. Mathialagan, J.A. Green, and R.S. Prather Transcriptional Profiling of Pig Embryogenesis by Using a 15-K Member Unigene Set Specific for Pig Reproductive Tissues and Embryos Biol Reprod, June 1, 2005; 72(6): 1437 - 1451. [Abstract] [Full Text] [PDF]
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