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Department of Biology, One Trinity Place, Trinity University, San Antonio, Texas 78212, USA1 Department of Obstetrics and Gynecology, St Marianna University School of Medicine, Kawasaki 216-8511, Japan2 Department of Obstetrics and Gynecology, Kyoto University School of Medicine, Kyoto 606- 8507, Japan3 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
(Correspondence should be addressed to L L Espey; Email: lespey{at}trinity.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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27% (i.e. 22/82 clones) of the transcripts were fragments of paralogs of known pseudogenes. Out of the 22 clones reported here, 12 have high sequence similarity to the cytochrome P450 pseudogene Cyp21a1-ps, and 5 have high sequence similarity to both the Cyp21a1-ps and the aldo-keto reductase gene Akr1c6. The remaining five clones were paralogs of the endogenous retrovirus SC1 that has heavily infested the rat genome. Northern analyses reveal that peak expression of all the 22 paralogs occurs at 4–8 h into the ovulatory process. In situ hybridization shows that expression of these pseudogenes is primarily in the granulosa layer of ovulatory follicles. In summary, the results reveal that ovarian expression of Cyp21a1-ps- and SC1-like pseudogenes occurs concurrently with the ovulatory process. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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The present study focuses on multiple paralogs of several ovarian pseudogenes that are expressed during ovulation (by definition, ‘paralogs’ are homologous sequences that underwent evolutionary separation from a common ancestor following a gene duplication event (Fitch 2000)). In particular, this study examines a number of paralogs of cytochrome P45021a1 pseudogenes (Cyp21a1-ps) that are transcribed by multiple chromosomes during the ovulatory process. In addition, this study assesses the ovulation-related expression of several pseudogenes that are similar to segments of Cyp21a1-ps, but have distinct sequence similarity to a functional aldo-keto reductase (Akr1c6). Also, this study presents data on the ovulation-related expression of multiple forms of the endogenous retrovirus (ERV) SC1 in the rat ovary. Collectively, these genes represent several varieties of pseudogenes.
The ovulation-related expression of Cyp21a1-ps and SC1 paralogs was detected during RT-PCR differential display (DD) experiments conducted during the past 12 years. Routinely, the DD procedure detects only random gene expression in an experimental tissue. Therefore, the pseudogenes reported here are not based on any predetermined interest or bias. Out of a total of 82 different ovulation-related genes that were discovered, 22 of the sequences were identified as pseudogenes of the above types. None of the remaining 60 ovulation-related genes were of the pseudogene category. Thus, the unique expression of ovarian Cyp21a1-ps and SC1 merits closer examination because their transcription is a common characteristic of ovulation in the rat.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Immature Wistar rats were induced to superovulate by injections of equine chorionic gonadotropin (eCG) and hCG as described previously (Espey et al. 2000b). Ovarian RNA was extracted at the periovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. These extracts of nucleic acid were used for DD, and differentially expressed cDNA bands were used for northern analyses, in situ hybridization, and sequence analyses. The ovulation rate in the experimental animals was determined by a procedure that also has been described previously (Espey et al. 2000b). The rats were killed by exposure to CO2 in order to extract ovarian RNA and to determine ovulation rate. The number of ova in the oviducts of ten of the rats at 24 h after hCG was 70.6±3.0. This work was conducted in accordance with the accepted standards of humane animal care, and the experimental procedures conformed with The UFAW Handbook on the Care and Management of Laboratory Animals and the approval of the institutional committee on animal care, i.e. the Animal Research Committee of Trinity University.
RNA extraction
Total RNA was extracted from whole ovaries that were extirpated at 0, 2, 4, 8, 12, and 24 h after the ovulatory process had been initiated by a dose of 10 IU of hCG. The 0-h control group did not receive any hCG. At each of the six designated intervals, the ovaries from seven to ten rats were immediately frozen on blocks of dry ice and then pooled together to provide a total of 0.5 g ovarian tissue for RNA extraction. The RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure that has been described previously (Espey et al. 2000b). Different RNA extractions for DD RT-PCR and northern blotting were performed on numerous occasions over the course of this 12-year study of ovarian gene expression. The 12 Cyp21a1-ps clones were obtained from ten different RNA extractions, while the five Akr1c6 and the five SC1 clones were obtained from ten other RNA extractions.
DD protocols that led to detection of pseudogenes
The steps of the DD procedure were carried out as described previously (Espey et al. 2000b). In brief, RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure. RT-PCR was performed by primers from a number of different RNAimage kits (GenHunter Corporation, Nashville, TN, USA). The specific primer sets that yielded differentially expressed homologs of clones for Cyp21a1-ps, Akr1c6, and SC1 were quite diverse (Table 1). After extraction and reamplification of the differentially expressed cDNAs, standard northern analyses were performed to confirm the ovulation-related expression of the parent mRNAs for Cyp21a1-ps, Akr1c6, and SC1. Subsequently, the unique cDNA fragments were individually cloned using a PCR-TRAP Cloning System (P404, GenHunter), and cloning colonies containing each of the different cDNAs were identified by secondary northern analyses. The cDNAs were sequenced either manually or commercially (SeqWright, Houston, TX, USA). Each sequence has been entered into the NCBI database and the accession numbers are available (Table 1). In situ hybridization was carried out as described previously (Espey et al. 2000b).
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The nucleotide sequences reported in this paper have been submitted to GenBank under the accession numbers AF159099.1, DQ255905–DQ255907, DQ255909–DQ255911, DQ266363, DQ266365, DQ266370, DQ266372, and EF189906–EF189916.
Statistical analysis
The density of the signals from the northern blots was analyzed by the NIH Image program http://rsb.info.nih.gov/nih-image/using the download ‘NIH-image 162 fat.hqx.’ The raw densitometric numbers at each of the six time points during the ovulatory process were used to tabulate the means±S.E.M. at each of the six time points during the ovulatory process. The 0-h value, i.e., the pre-hCG value, was designated as the control group, while the 2-, 4-, 8-, 12-, and 24-h values were the experimental groups. The significance of the differences among the means was determined by Duncan's multiple range tests after a completely randomized one-way ANOVA of the means of the groups. The probability value used as the cutoff between ‘significant’ and ‘not significant’ was P=0.05. After completion of the statistical analysis, the mean of the time point that exhibited the highest signal density was arbitrarily set as 100%, and the remaining means and S.E.M. were expressed as proportions of 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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The northern analyses for cDNAs that had sequences similar to Cyp21a1-ps, Akr1c6, or SC1 showed comparable patterns of expression during the six different stages of the ovulatory process – with maximum expression occurring at 4–8 h after the animals were stimulated with an ovulatory dose of hCG (Figs 1–3
). In addition, all three sets of clones hybridized with variable lengths of RNA on the northern blots, indicating the presence of multiple forms of the extracted mRNAs that generated the differentially displayed cDNAs.
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In situ hybridization of ovarian tissues taken at 0, 8, and 24 h after hCG yielded signals that spatially localized the ovarian expression of mRNA for Cyp21a1-ps, Akr1c6-ps, and SC1 paralogs in a temporal pattern that corresponded with the DD autoradiographs and the northern analyses. Probes prepared from clones of the gene fragments of all three classes of pseudogenes consistently showed minimal signal from the in situ preparations of control tissue staged at 0 h, i.e. of ovaries from animals that had not been injected with hCG (Figs 4–6). By contrast, there were substantial signals radiating from the tissue preparations at 8 h after hCG, which was the approximate time when there was maximum signal from the DDs and northern blots. Most of the expression of pseudogene mRNA was localized in the stratum granulosum of the mature follicles, but there was also diffuse signal from the interstitial tissues of the ovaries. Later, as the ovarian follicles began to transform into luteal tissue at 24 h after hCG, the in situ evidence showed that mRNA expression for all of the pseudogenes declined.
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Based on BLASTN searches using the NCBI database, 12 clones were initially identified as having the highest sequence similarity to Cyp21a1-ps (Fig. 7), and 5 clones were initially identified as having the highest similarity to the Akr1c6 gene (Fig. 8). Subsequently, in more recent years, it has become evident from a closer analysis of the NCBI database that all five of the Akr1c6-like clones also possess 99–100% similarity to segments of the Cyp21a1-ps reference gene (NCBI accession number NG_004071). For this reason, sequences of the Akr1c6-like clones were also compared with the Cyp21a1-ps reference gene (Fig. 8). However, the sequences of the five Akr1c6-like clones were most similar to the Cyp21a1-ps reference gene at regions upstream and downstream from the locus for the 12 principal Cyp21a1-ps-like clones. Therefore, the Akr1c6-like clones were grouped separately from the Cyp21a1-ps-like clones (Fig. 8). By contrast, the five clones for SC1 were considered as a completely separate group because they had no sequence similarity to either the Cyp21a1-ps-like clones or the Akr1c6-like clones (Fig. 9).
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The NCBI database was used to identify the various chromosomal loci that shared the greatest similarity to each of the clones in this study. In each instance where a locus was established, the database indicated there was 99–100% similarity between the given clone and the genomic sequence at the locus identified (Table 2). For the Cyp21a1-ps-like fragments, clones 1, 2, and 13 had the greatest similarity to sequence in the rat genome at locus 1q22. Clones 3–6 were highly similar to sequence at locus 3q41. Clones 7–10 were most similar to sequence at locus 11p12. Clones 11 and 14–16 were very similar to sequence at locus 14p22. Clones 12 and 17 were essentially identical to sequences in the rat genome at loci Xq12 and Xq22 respectively (Table 2). By contrast, the five paralogs of SC1 were most similar to rat genomic sequences found at 1q21, 5q33, 13q21, 19q12, and Xq22 respectively (Table 2). Also, in this effort to identify the corresponding position of each clone within the rat genome, the specific genes that flanked each paralog locus at its 5' and 3' ends were tabulated (Table 2). Since many of the loci were on different chromosomes, the flanking genes varied substantially from paralog to paralog.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Pseudogenes are categorized into two fundamentally different groups, based on the nature of their origin. The ‘duplicated’ pseudogenes arise from tandem duplication of segments of chromosomes or from unequal crossing over, and they usually exhibit retention of the exon–intron pattern of their paralogous parental genes (Mighell et al. 2000, Zhang & Gerstein 2004, Svensson et al. 2006). By contrast, the ‘retrotransposed’ pseudogenes arise when freshly translated mRNAs undergo reverse transcription into cDNAs, which are subsequently reintegrated into the genome at new loci (Mighell et al. 2000, Zhang & Gerstein 2004, Svensson et al. 2006).
Cytochrome P450 genes make up one of the largest members of a multigene family that originated from a common ancestral gene over 3 billion years ago (Danielson 2002). The paralogous pseudogene Cyp21a1-ps presumably arose as a tandem repeat of the functional Cyp21a1 gene (involved primarily in steroidogenesis) (Kawaguchi et al. 1992, Riepe et al. 2005), and therefore it belongs to the category of ‘duplicated’ pseudogenes. The scientific literature contains conflicting information about whether pseudogenes such as Cyp21a1-ps can, or cannot, produce transcripts (Mighell et al. 2000, Zhang & Gerstein 2004, Prudhomme et al. 2005). Clearly, the present data from DD RT-PCR, northern analyses, and in situ hybridization, collectively, make it evident that a number of paralogs of Cyp21a1-ps are transcribed in the ovary at the time of ovulation in a cell-specific manner. Apparently, in some cases, pseudogenes retain or acquire functional promoter sequence that allows them to be transcribed (Mighell et al. 2000, Zhang & Gerstein 2004). In other instances, pseudogene transcription can be driven by a nearby promoter that is present in an unrelated, upstream sequence (Mighell et al. 2000). However, the upstream genes that flank each of the transcribed Cyp21a1-ps-like clones in this study are not known to be ovulation related (Table 2).
The specific function(s) of gonadally expressed Cyp21-type pseudogenes is uncertain. It has been suggested that the mass of pseudogenes that now clutter the mammalian genome could have a somewhat indirect function, namely, they could serve as a ‘pool’ of DNA sequence that has the potential of generating new genes that spontaneously gain functionality during the course of evolution (Zhang & Gerstein 2004). In other words, pseudogenes might serve as a reservoir of potentially functional genes or ‘potogenes’ (Balakirev & Ayala 2003).
The sequence similarity between Cyp21a1-ps and its normal Cyp21a1 counterpart has resulted in the loading of sequence databases with inappropriate ‘protein-coding’ transcripts (Zhang & Gerstein 2004, Claverie 2005, van Baren & Brent 2006). For example, several years ago, we reported that 3
-hydroxysteroid dehydrogenase (3-HSD) mRNA was transcribed in the rat ovary in response to an ovulatory dose of gonadotropin (Espey et al. 2001). (Note that, under present nomenclature, 3-HSD is now abbreviated as Akr1c6). However, based on the growing knowledge of pseudogenes and on the completion of the rat genome project, it is now evident that our previously reported sequence for ovarian 3-HSD is actually a Cyp21a1-ps-like pseudogene that is identical to clone 15 in the present paper (Fig. 8). That is to say, one of the paralogs of Cyp21a1-ps in the present study was reported previously as 3-HSD (i.e., as Akr1c6).
Over the course of millions of years, there has been a huge proliferation of genomic elements that are derived from only a few initial germ line invasions by exogenous retroviruses such as SC1 that become endogenous (Anway et al. 2001, Belshaw et al. 2004). These ERVs now appear to exist as rather permanent ‘symbiotic’ or ‘parasitic’ DNA sequences that use their hosts' replicative machinery for vertical transmission of their DNA along the evolutionary tree over the course of time (Prudhomme et al. 2005). Thus, ERVs can persist within any given individual as ‘proviruses’ that possess the same genetic machinery as a viable virus (Anway et al. 2001, Belshaw et al. 2004, Prudhomme et al. 2005). During the course of their replication (by retrotransposition or transposition) within a given individual, they retain transcription initiation and termination elements in their long-terminal repeat segments, and there is evidence that a significant number of these pseudogenes actually are transcribed (Prudhomme et al. 2005, Svensson et al. 2006). For example, the retrovirus SC1 that is clearly expressed during the ovulatory process contains CAAT, TATA, and GATA ‘box promoter elements and many putative transcription factor binding site regulatory elements’ ((Anway et al. 2001) or see NCBI accession number AY009092).
The first significant report of SC1 expression in mammalian ovarian tissue was published in 2001 (Anway et al. 2001). This study found that maximum transcription of SC1 in immature rat ovaries occurred at 48 h after the animals were primed with 10 units of eCG – a time point that corresponds with what most investigators considered to be 0 h into the ovulatory process in the immature rat model. Furthermore, this earlier study found that retroviral transcription in the rat ovary was relatively low at 14 h after the rats had been injected with hCG to initiate the ovulatory process. These earlier findings are quite different from the results of the present study. Clearly, the results in Fig. 3 shows that all five of the different clones for SC1 transcripts were at the lowest expression levels at 0 h into the ovulatory process (i.e. at 48 h after the administration of eCG to prime the immature animals), while the greatest expression was at 4–8 h into the ovulatory process. The reason(s) for these distinctly opposite findings in the present study are unclear.
It has been suggested that replication-competent ERVs that are comparable with SC1 can undergo persistent reinfection of a host organism (Belshaw et al. 2004). Such a chronic condition can result in ‘extremely long periods of smoldering infection’ (Belshaw et al. 2004). Therefore, since there is ample evidence that an ovulatory surge in gonadotropic hormones induces an acute inflammatory reaction in mammalian ovaries (Espey 1980, 1994, 2001, Hernandez-Gonzalez et al. 2006), it is difficult not to consider the possibility that the hormonally induced expression of SC1 in the rat ovary might contribute in some way to the inflammatory degradation and rupture of the follicle wall. In any event, the present data confirm that expression of transcripts of SC1 is associated with a specific inflammatory reaction in the ovary. Since recent studies also document that ovulating follicles express genes associated with innate immune responses that detect ‘self’ from ‘non-self’, or ‘altered-self’, it is possible that the SC1 mRNAs may impact these signaling pathways as well (Shimada et al. 2006). In future studies on the molecular events of ovulation, it would be helpful to learn whether SC1 elements induce the expression of proinflammatory cytokines or vice versa. If the former scenario is the case, then it would also be interesting to know whether antibodies to epitopes on the gag, pol, and/or env proteins from SC1 can affect the efficiency of mammalian ovulation.
In conclusion, it is evident that transcripts of pseudogenes of the types Cyp21a1-ps and retrovirus SC1 are a simultaneous event in the ovulatory process of immature rats. While there is no conspicuous role for the Cyp21a1-ps-like elements in the mechanism of ovulation, it is possible that translated products of endogenous SC1, such as the envelop protein for SC1, might contribute in some significant way to the ovarian inflammatory reaction that is a fundamental component of the ovulatory process.
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Declaration of Interest |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Funding |
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Received in final form 24 April 2008
Accepted 1 May 2008
Made available online as an Accepted Preprint 2 May 2008
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0.001) at 2 h into the ovulatory process. Asterisks indicate mean values that are statistically greater than the 0-h control. The actual northern pattern for one radiolabeled Cyp21a1-ps-like clone is displayed below the linear graph. The multiple banding on the northern is the consequence of expression of numerous mRNA paralogs of Cyp21a1-ps that are of variable lengths.
