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Regular Article |
a Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108
b School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea
ABSTRACT
Differential display reverse transcription polymerase chain reaction was used to isolate a novel cDNA clone (C47) that was initially shown to be downregulated in senescent chicken embryo fibroblast cells. In a tissue environment, C47 transcripts were only detected in gonadal tissue. The expression of the larger isoform (C47L) was essentially restricted to the ovary, and the smaller isoform (C47S) was predominately expressed in the testis. Although levels of the C47L mRNA were relatively high in both the small white and the developing larger follicles, there was very low expression in regressed and postovulated follicles. Nucleotide sequence analysis indicated that two different transcripts of the single-copy C47 gene were generated by differential polyadenylation in the 3' untranslated region. As a result of a single nucleotide deletion, the C47L mRNA produced a smaller 48-kDa protein, and the C47S mRNA generated a larger 57-kDa protein when both were translated in vitro. Both protein isoforms were shown to contain conserved C2H2 Zn finger motifs and nuclear localization signals suggestive of being putative transcription factors. These results suggest that the C47L and C47S isoforms might play an important role in the regulation and maintenance of ovarian and testicular functions, respectively, in the chicken.
follicle, follicular development, ovary, testes
INTRODUCTION
Differential changes in gene expression are major mediators of altered physiological properties both in vitro and in vivo. Therefore, the identification and characterization of genes differentially expressed in proliferating, senescing, and immortal cells would be an important initial step for understanding the mechanisms governing the cellular and organismic conversion of various growth states. In the present study, using differential display reverse transcription polymerase chain reaction (DD-RT-PCR), we have isolated a novel cDNA clone (C47). C47 has a long (C47L) and a short (C47S) isoform that were initially shown to be downregulated in the senescent chicken embryo fibroblast (CEF) cells but were later found to be predominately expressed in chicken gonadal tissues (e.g., the ovary and testis).
Follicular arrangement, especially in the context of the avian ovary, provides a unique model to study the changes associated with the discrete stages of ovarian follicular development, maturation, ovulation, senescence, and apoptosis. The ovary of a regularly laying hen contains a cluster of yolk-filled follicles arranged in a hierarchy according to size. The largest follicle (F1) is ovulated first, the second largest (F2) the following day, and so on until an anovulatory day interrupts the sequences of oviposition. The ovary in a laying hen also contains several million follicles of which several thousand are macroscopically visible. At peak production, the hen's ovary contains about 60 small yolky follicles between 1 and 8 mm in length [1].
The number of small yolky follicles tends to decrease due to atresia as the size of the follicle increases. Follicle atresia occurs almost exclusively in prehierarchal follicles [2]. Hen atretic follicles exhibit extensive oligonucleosome formation characteristic of apoptosis, and these formations occur almost exclusively within the granulosa layer [3, 4].
Recent findings showed that LH receptor and FSH receptor mRNA levels decrease in atretic follicles compared with normal follicles. Although the decrease in gonadotropin receptor mRNA levels might account for the loss of gonadotropin-induced steroidogenesis in atretic follicles [5–7], the molecular and cellular mechanisms governed by altered gene expression in follicular development, regression, and atresia and in ovarian senescence remain largely unknown.
MATERIALS AND METHODS
Cell Culture
All primary and immortal DF-1 CEF cells were grown in Dulbecco minimum essential high-glucose medium (DMEM) enriched with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, and 2 mM L-glutamine. All cell culture reagents were purchased from Life Technologies (Gaithersburg, MD). The population doubling (PD) and the senescence-associated ß-galactosidase activity [8] were examined to determine cell proliferation and senescence rates, respectively. Cell growth was arrested by standard serum starvation (0.2% FCS) or by contact-inhibiting methods and was analyzed using FACScan and Cell Quest software program (Becton Dickinson, San Jose, CA).
RNA Analysis
Total cellular RNA was isolated from cultured CEF cells and from follicles of various sizes using TRIzol reagent (Life Technologies) following the manufacturer's instructions. The various sizes and stages of follicles (F1, F5, small white, postovulated, and regressed) were collected from 1-yr-old single-comb white Leghorn laying hens (University of Minnesota, St. Paul, MN) as previously described [1]. Total cellular RNA from chicken tissues was provided from Drs. F. Abel Ponce de León and In-Jeong Kim (University of Minnesota, St. Paul, MN).
Using RNA isolated from primary young, senescent, and immortal CEF (DF-1) cells, C47 was identified by DD-RT-PCR following the procedure described previously [9]. One microgram of DNase I-treated total RNA was reverse transcribed in a 20-µl reaction volume using T12MA primers, where M represents degeneracy for G, A, and C. The DD-RT-PCR was performed in a reaction mixture (20 µl) containing 1:10 volume of the reverse transcription mixture, 2.5 µM of the appropriate anchored oligo dT primer, 0.5 µM 10-mer random primer (KitA-19; Operon Biotechnology, Alameda, CA), 1x PCR buffer, 2 µM dNTPs, 0.5 µM [
-35S]dATP (1200 Ci/mmol), 2 mM MgCl2, and 1 U AmpliTaq DNA polymerase (5 U/µl; Perkin-Elmer, Norwalk, CT), and the reaction mixture was covered with 30 µl of mineral oil. The DD-RT-PCR was carried out using the following parameters: denaturation at 95°C for 5 min followed by 40 cycles at 94°C for 30 sec, 40°C for 2 min, and 72°C for 30 sec and then a final extension at 72°C for 10 min. A second round screening process using DD-RT-PCR cDNA fragments was performed by reverse Northern dot-blot analysis to eliminate false-positive cDNA clones, as described previously [10].
Northern blot analysis using total cellular RNA (20 µg) prepared from either cultured cells or various tissue samples was performed by standard hybridization methods using an [-32P]dATP-labeled C47 cDNA fragment. Equal RNA loading was determined by staining the membrane with methylene blue after transfer.
Genomic DNA Analysis
Total genomic DNA from primary (passage 5) CEF cells was isolated using the Qiagen cell culture DNA kit (Qiagen, Valencia, CA). The DNA was digested with either EcoRI or HindIII and was resolved on a 0.8% agarose gel. The gel was blotted onto a positively charged nylon membrane and hybridized with the [-32P]dATP-labeled C47 cDNA fragment.
cDNA Library Screening and Sequence Analysis
An immortal chicken CEF cell line (DF-1) cDNA library was screened using an [-32P]dATP-labeled C47 cDNA fragment. Full-length open reading frames of C47L and C47S cDNA isoforms were obtained by several consecutive rounds of library screening. Both strands of the in vivo excised (Stratagene, La Jolla, CA) C47L and C47S cDNA clones were sequenced by automated DNA sequencing at the Advanced Genetic Analysis Center (University of Minnesota, St. Paul, MN).
In Vitro Translation Analysis
The pC47L and pC47S expression plasmids were constructed by ligating the full-length C47L and C47S cDNA isoforms into the expression vector pcDNA3.1 (Invitrogen, San Diego, CA). Coupled transcription and translation of pC47L and pC47S isoforms was carried out with the TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI) following the manufacturer's instructions.
Protein Motif Analysis
The PSORT program (http://www.yk.rim.or.jp) was used to analyze and predict the protein-sorting signal encoded in the putative amino acid sequences of the C47L and C47S isoforms. The protein motif search was conducted with the MOTIF program (http://www.yk.rim.or.jp).
Data Documentation and Statistical Analysis
Each experiment was conducted at least three times. All image data were processed by the Eagle Eye II still video system (Stratagene) and analyzed with the NIH Image software program. The normal probability of data was ensured as judged by Wilk-Shapiro rankit values. Statistical analysis was performed using the two-tailed Student t-test.
RESULTS
Analysis of Chicken C47 Isoforms in Cultured Cells
We used the DD-RT-PCR method to compare altered gene expression in primary young, senescent, and immortal CEF cells. Among the initial 108 DD-RT-PCR cDNA fragments, 56 fragments were differentially expressed in immortal and senescent CEF cells (threefold differences) compared with primary young CEF cells as determined by reverse Northern dot-blot analysis (data not shown), 55 fragments were shown to be upregulated in immortal CEF cells compared with primary young CEF cells (data not shown and the detailed characterization of these genes will be reported elsewhere), and 1 fragment (C47) was found to be downregulated in senescent CEF cells compared with primary young and immortal CEF cells.
The verification of senescence-specific downregulation of C47 was further confirmed by Northern blot analysis (Fig. 1A), and two different sizes (2.6 and 1.8 kilobases [kb]) of transcripts were found to be expressed (Fig. 1). Expression of the 2.6-kb transcript in primary CEF cells (passages 16 through 19 in which 
70–84% of CEF cells were shown to be senescence-associated ß-galactosidase positive [8]) was significantly reduced (P < 0.01) compared with primary young CEF passage 5 cells (Fig. 1B). To determine whether the downregulation of C47 mRNA was senescence-specific or merely related to decreased growth rates, we compared the steady-state levels of C47 mRNA from exponentially growing cells with that from cells whose growth was arrested by either contact inhibition or serum starvation. Expression of C47 mRNA did not decrease (and even increased, although not significantly; P > 0.05) in growth-arrested cells compared with exponentially growing cells (Fig. 1C). These results suggest that expression of C47 mRNA is specifically downregulated in senescent cells cultured in vitro.
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Chicken Gonad Tissue-Specific Expression of C47
To determine the expression patterns of C47 in tissue environments, Northern blot analysis was performed using total cellular RNA isolated from a variety of tissues, such as pituitary gland, hypothalamus, brain, muscle, gizzard, gut, kidney, lung, liver, heart, testis, follicle, stroma, thymus, bursa, and bone marrow (Fig. 2A). Under low-stringency (2x standard saline citrate [SSC], 45°C) or subsequent high-stringency (0.1x SSC, 68°C) washes, the only tissues that expressed C47 transcripts were gonadal in origin (Fig. 2A). However, low-stringency washes showed that there were four different tissues, such as hypothalamus, brain, thymus, and bursa, that expressed the C47 2.6-kb transcript, although the 2.6-kb signal from these tissues was barely detectable compared with those in ovarian follicle, ovarian stroma, and testis tissues (data not shown). The 1.8-kb transcript was observed in only the testis, and the 2.6-kb transcript was predominately observed in follicular and stromal tissues (a minor 1.8-kb band was also observed in the follicle). Analysis of another independent Northern blot confirmed that the ovary tissue predominantly expressed the larger 2.6-kb transcript, with a faint band at 1.8 kb, whereas the testis tissue produced almost exclusively the shorter 1.8-kb transcript (Fig. 2B). As normalized to the RNA content used, the expression levels of the 1.8-kb transcript in the testis were 2-fold lower than that of the 2.6-kb transcript in the ovary.
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Expression of C47 in Ovarian Follicles
The avian ovary contains discrete stages of developing, matured, and regressed/atretic follicles (which are analogous to senescent or apoptotic cells in culture). These follicles provide an excellent model to determine whether the downregulated C47 expression observed in senescent cells grown in vitro is correlated with the in vivo expression of regressed follicular tissue. We compared the expression pattern of C47 mRNA using Northern blot analysis for various stages of follicular development (small white, F5, and F1 follicles) and for follicles that were postovulated or regressed (Fig. 3). When normalized to 28S rRNA, the expression of the 2.6-kb transcript that hybridized to the C47 probe was high in small white and F5 follicles and slightly decreased in F1 follicles. Conversely, the postovulated and regressed follicles expressed significantly lower levels (P < 0.01) when compared with the small white, F5, and F1 follicles (Fig. 3). Therefore, downregulation of C47 expression in both senescent cells and postovulated or regressed follicles suggests that C47 expression might be associated with cellular and organismic senescence.
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Genomic Southern Analysis of C47
To determine whether multiple genes were generating the two different mRNA transcripts, chicken genomic DNA was digested with restriction enzymes and subjected to Southern blot analysis using an [-32P]dATP-labeled C47-specific probe. Figure 4 clearly shows that C47 is single copy by virtue of a single hybridizing band when genomic DNA was digested with either EcoRI or HindIII. Therefore, the two C47 transcripts are being transcribed from the same gene.
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Sequence Analysis of C47L and C47S
We obtained 11 larger C47L and 2 smaller C47S isoforms from four rounds of DF-1 CEF cDNA library screening and sequenced full-length cDNAs for both isoforms (Fig. 5). The two isoforms were identical in nucleotide sequence except for a single base deletion (base 972, bold asterisk) in the C47L mRNA isoform. This single base change resulted in a shorter open reading frame corresponding to a peptide of 374 amino acids (the stop codon is in bold and underlined, TGA). The C47S mRNA isoform included an additional base (base 972, bold A) that resulted in a longer open reading frame corresponding to a peptide of 404 amino acids (the stop codon is in bold and underlined, TAA). Altered mRNA processing can be modulated via differential recognition of two alternative poly(A) sites by the tissue-specific activity of basic polyadenylation factors or gene-specific regulators [11, 12]. Thus, the two putative alternative poly(A) sites found in C47 likely account for the differences in the transcript size of the two isoforms. The longer 404-amino acid peptide (generated from the shorter mRNA, C47S) has a polyadenylation signal at 1484–1489 base pairs (bp) (ATTAAA), and the 373-amino acid peptide (generated from the longer mRNA, C47L) has the signal at 2142–2147 bp (ATTAAA).
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To determine whether there was a deleted base (base 972, bold asterisk) in the longer mRNA C47L isoform or whether the shorter mRNA C47S isoform had an insertion (base 972, bold A), we amplified C47 from ovary or testis tissues by PCR using total cellular RNA from pooled tissue samples from each of five different commercial white Leghorn chickens and used control genomic DNA from CEF cells. As shown in Figure 6, the cDNA fragment of C47 from the ovary showed only two AA bases, whereas the cDNA fragment of C47 from the testis revealed three AAA bases. The genomic DNA also revealed three AAA bases at the same position as the C47 cDNA fragment from testis. Furthermore, the single base deletion in C47L mRNA was also observed in various CEF cells derived from the East Lansing Line 0 (ev-0; Avian Disease and Oncology Laboratory, East Lansing, MI), SPAFAS (Preston, CT), and HyVac (Gowrie, IA) chickens and in ovary and testis tissues obtained from HyVac chickens (data not shown). Because C47 is a single-copy gene, some type of transcriptional or posttranscriptional processing event might have caused a single A base deletion in C47L mRNA, resulting in a shorter 374-amino acid peptide. Although a molecular event similar to this processing has been documented in viruses, fungi, and protozoa as RNA editing [13], it is currently unclear whether the same RNA editing occurs in more complex organisms.
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In Vitro Translation Analysis of C47L and C47S
The predicted molecular mass of both C47L and C47S was shown to be 42 and 46 kDa, respectively, as determined by the Expert Protein Analysis system (http://www.expasy.proteome.org.au/tools/#pattern). However, in vitro translation analysis showed that two different proteins of 48 and 57 kDa were translated from the open reading frames of C47L and C47S, respectively (Fig. 7, lanes 1 and 2). Luciferase (61 kDa) was translated in vitro as a positive control (Fig. 7, lane 3). In the current study, we could not elucidate the obvious differences between the computer-predicted and the in vitro translated molecular masses. Posttranscriptional modifications might account for these differences (although glycosylation can be ruled out because there is no evidence for potential glycosylation sites in either protein). Therefore, further analysis should be performed to determine the exact molecular masses of each protein.
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Protein Motif Analysis of C47L and C47S
The nucleotide sequence of C47 did not show significant similarity to previously identified GenBank entries. However, a number of unidentified genes from various species were highly similar to C47 mRNA (score bits >96, identities >80%, E-value <9-17) as searched by dbEST (GenBank BLASTN-dbEST): chicken (AI980811, putative Zn finger protein in T-cells; AW239766, T-cells), human (AA506325, unknown; AA630405, HeLa cell; AA364410, pineal gland), mouse (AA874252, thymus; W59370, embryo; AI047179, embryonic stem cell), fish (AI407219, zebrafish), and frog (AW198790, Xenopus oocyte). The peptide sequences were searched using the PSORT and the MOTIF programs to identify possible protein sublocalization and motifs of interest. Figure 8A shows the structure of both C47L (shorter peptide) and C47S (longer peptide). Three areas of interest were identified from the motif search: 1) four C2H2 Zn finger motifs (amino acids 6–22, 69–85, 246–267, and 300–316), 2) a nuclear localization signal (amino acids 74–90), and 3) a coiled-coil domain (amino acids 79–208). The amino acid sequence of C47 also showed relatively higher similarities (52–64%) to a putative C2H2 Zn finger transcription factor from yeast, plants, and Caenorhabditis elegans (Fig. 8B). Furthermore, the PSORT program predicted that C47L and C47S proteins have a 95.7% and 78.3% probability of localizing in the nucleus, respectively (data not shown).
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DISCUSSION
In the present study, C47L expression was found to be prominent in actively growing follicles but decreased in postovulated and regressed follicles, suggesting that C47 may play a role in follicular development and in the proliferation of CEF cells. Furthermore, the reactivation of C47 expression in the immortal DF-1 CEF cells that bypassed senescent barriers and the inactivation of C47 expression in atretic follicles also suggest that maintenance of C47 levels might play an important role in cell survival. Because follicular development and growth appear to be regulated by various gonad-specific hormonal factors [14–20] and because the expression of C47 was predominately restricted in the gonadal tissues, C47 expression may be induced through gonadotropic and local growth factors. Therefore, the physiological function of C47 in relation to intraovarian factors during follicular development needs to be elucidated.
The inferred amino acid and structural motif search predicted C47L and C47S as putative Zn finger proteins. The Zn finger motif is one of many motifs involved in the DNA binding of proteins and represents the conserved Cys2-His2 residues [21] commonly found in a variety of transcription factors, including TFIIIA [22], Sp1 [23], and steroid receptors [24]. C47 isoforms also have four conserved Cys2-His2 finger motifs. If the C47 isoforms are DNA binding proteins, they should localize to the nucleus. In this regard, the conserved bipartite nuclear localization signal (KRFSTFNAYENHLKSKK) was found in both C47L and C47S proteins. The C47 protein isoforms also have a coiled-coil motif found in several leucine zipper containing DNA-binding proteins. This coiled-coil domain facilitates homo- or heterodimerization [25, 26]. Recently, elegant studies demonstrated that WT1, one of Zn finger proteins, functions as a transcriptional repressor of the inhibin- gene in the ovarian follicles [27, 28]. The C47 isoforms may be gonad-specific transcription factors. Therefore, the biological function of C47L and C47S in the ovary and testis should be further addressed to help us understand their critical roles in these reproductive tissues.
ACKNOWLEDGMENTS
We thank Drs. F. Abel Ponce de León and In-Jeong Kim (University of Minnesota) for providing total cellular RNA from various chicken tissues.
FOOTNOTES
First decision: 4 October 2000.
1 These studies were supported in part by USDA/NRICGP grant 9603280 and by a grant from American Home Products. 
2 Correspondence: Douglas N. Foster, 495 AnSci/VetMed, 1988 Fitch Ave., St. Paul, MN 55108. FAX: 612 625 2743; foste001{at}tc.umn.edu
3 These authors contributed equally in this work.
Accepted: December 26, 2000.
Received: September 7, 2000.
REFERENCES
gene promoter. Mol Endocrinol 1995; 9:1356–1366[Abstract]
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