Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2007; 9 (4): 522-527 |
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This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Review - Identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling Daniel S. Johnston1, Terry T. Turner2,3, Joshua N. Finger1, Tracy L. Owtscharuk1, Gregory S. Kopf1, Scott A. Jelinsky4 1Contraception, Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, PA 19426, USA
Abstract As part of our efforts to identify novel contraceptive targets in the epididymis we performed transcriptional profiling on each of the 10 and 19 segments of the mouse and rat epididymidis, respectively, using Affymetrix whole genome microarrays. A total of 17 096 and 16 360 probe sets representing transcripts were identified as being expressed in the segmented mouse and rat epididymal transcriptomes, respectively. Comparison of the expressed murine transcripts against a mouse transcriptional profiling database derived from 22 other mouse tissues identified 77 transcripts that were expressed uniquely in the epididymis. The expression of these genes was further evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis of RNA from 21 mouse tissues. RT-PCR analysis confirmed epididymis-specific expression of Defensin Beta 13 and identified two additional genes with expression restricted only to the epididymis and testis. Comparison of the 16 360 expressed transcripts in the rat epididymis with data of 21 other tissues from a rat transcriptional profiling database identified 110 transcripts specific for the epididymis. Sixty-two of these transcripts were further investigated by qPCR analysis. Only Defensin 22 (E3 epididymal protein) was shown to be completely specific for the epididymis. In addition, 14 transcripts showed more than 100-fold selective expression in the epididymis. The products of these genes might play important roles in epididymal and/or sperm function and further investigation and validation as contraceptive targets are warranted. The results of the studies described in this report are available at the Mammalian Reproductive Genetics (MRG) Database (http://mrg.genetics.washington.edu/). (Asian J Androl 2007 July; 9: 522_527) Keywords: mouse; rat; epididymis; transcriptional profiling; contraception; microarray; epididymis-specific; epididymis-selective Correspondence to: Dr Daniel S. Johnston, Contraception, Women's Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Rd,
N2312 Collegeville, PA 19426, USA.
DOI: 10.1111/j.1745-7262.2007.00317.x 1 The epididymis as a target for contraception Non-hormonal approaches to achieving contraception represent an innovative strategy for the development of novel contraceptive therapeutics. The strategy is designed to identify specific gene products associated with the reproductive tissues that mediate physiological processes required for the production of a mature gamete. In men, the goal is to inhibit successful sperm production or maturation. The concept of post-testicular maturation of spermatozoa as a target for male contraception is well established [1_3]. Sperm acquire progressive motility and the ability to undergo capacitation during transit through the epididymis. These changes are a result of, in part, changes in the composition of the epididymal luminal fluid microenvironment. Inhibition of these post-testicular maturational events in sperm by modulating the function of specific proteins associated with the epididymis represents an attractive option of contraception intervention [4, 5]. An advantage of targeting the epididymis through a non-hormonal approach is that normal male endocrine function is unlikely to be affected. This would preclude targeting the process of spermatogenesis, thus avoiding potential issues with aneuploidy. Moreover, reversibility of the contraceptive effect would be rapid owing to the lack of a latency period seen with strategies targeting the processes of spermatogenesis. As contraceptives are frequently given to healthy people for long periods of time, it is also essential that contraceptive drugs do not have significant side-effects. One way to minimize any potential off-target, drug profile is to focus on targets that are either unique to, or highly selective for, the reproductive tract. To this end, a large-scale segmental transcriptional profiling of both mouse and rat epididymides using commercial whole genome microarrays was performed. The data were compared to those from a large in-house database of other tissues derived from the same platform and generated with exactly the same protocol. Those putative epididymis-specific transcripts were investigated further using more sensitive amplification-based technologies. 2 Segmental profiling of the rat and murine epididymal transcriptomes To date, numerous strategies for identifying novel epididymis-specific transcripts or proteins have been used, including micro-arrays [6_8], proteomics [9] and signal sequence traps (Johnston, unpublished data). As part of our effort to identify novel epididymis-based contraceptive targets, we undertook a strategy to catalogue the mouse and rat epididymis transcriptomes at the highest possible resolution in a manner that would allow us to rapidly compare the expressed transcripts with large multi-tissue databases in order to identify novel epididymis-specific transcripts. Most of the published reports on transcriptional profiling of the epididymis have used epididymal tissue dissected into conventional caput, corpus, cauda regions [8] or slightly more refined regions, such as the initial segment (in the rat) [6]. In describing the localization of epididymal gene expression or gene products, these regions have also been described in greater detail (e.g. proximal-, mid- or distal-caput). At the time we were initiating our studies, one of us (Terry T. Turner), had begun investigating the biological role of epididymal segmentation in the mouse [10] and had demonstrated the ability to micro-dissect the caput region reproducibly. As it had become increasingly apparent from the literature that many gene expression and protein localizations within these regions are restricted to one or more segments, we hypothesized that it would be possible and beneficial to micro-dissect out all of the segments of the mouse epididymis and subject each of these segments to transcriptional profiling using genome-wide arrays. It was felt that this strategy would provide a high resolution cataloging of the mouse epididymal transcriptome and assist in the identification and characterization of putative targets. Complete segmentation of C57BL/6 mouse epididymidis reproducibly identified 10 distinct epididymal segments (Figure 1A) [11]. The expression profiles and several analyses of these data have been reported [11]. After completing the study in the mouse, several factors prompted us to use this strategy to investigate the segmented transcriptome of the Sprague-Dawley rat epididymis. First, this approach would help to identify additional contraceptive targets. Second, the rat is the most commonly used animal model for studies of the epididymis. Third, this species is a frequently used model within the pharmaceutical industry for efficacy and toxicology studies and we recognized that data from these studies would be important for planning and interpreting such studies. Fourth, we had established a large-scale transcriptional profiling effort of the entire male rat reproductive tract and we were confident that having the same high-resolution view of the rat epididymal transcriptome would ultimately be beneficial to our future efforts. Complete microdissection of Sprague-Dawley epididymides reproducibly identified 19 distinct epididymal segments (Figure 1B). Analysis of these data have been reported [12]. Importantly, the two profiling efforts described above have considerable importance beyond the identification of novel targets. The data from these studies provide a high resolution, physiologically relevant index of gene expression along the full length of mouse and rat epididymides, elucidate the epididymal expression of individual genes [11, 12], gene families [11, 12] and genes that comprise biological pathways, and allow comparison of gene expression between the two species [12]. Interested readers are encouraged to view these primary references and to utilize the data available on the public database (Mammalian Reproductive Genetics, MRG). The present report will provide novel data about epididymis-specific genes identified from our profiling efforts and will characterize the expression profiles of those transcripts. 3 Identification of epididymis-specific transcripts in the mouse The 17 096 transcripts expressed in the mouse epididymis were compared to an internal database of 22 mouse transcriptional profiling data to identify transcripts that were detected only in the samples of RNA from the mouse epididymis. The criteria to be considered "epididymis specific" are described in Johnston et al. [11]. Briefly, the transcript had to be called "present" in at least 67% of the samples in at least one epididymal segment with a mean expression of > 50 signal units and the transcript had never to be called "present" in any of the samples from the other 22 tissues. The data for each tissue were comprised of between 3 and 12 replicates. RNA from the following tissues were used for comparison: adrenal, bladder, brain, colon, embryo, eye, heart, kidney, liver, lung, lymph node, ovary, pituitary, prostate, salivary gland, small intestine, spleen, stomach, testis, thymus, uterus and vagina. Seventy-seven transcripts met the criteria for epididymal specificity, including a number of genes historically studied in the epididymis, such as lipocalin 5, glutathione peroxidase 5 and Adam 7. Interestingly, a large number of transcripts corresponding to β-defensin genes were also identified, including β-defensins 2, 9, 11, 13 and 15. The analysis also identified transcripts of several uncharacterized or hypothetical genes, including Hypothetical Protein A230091H23, RIKEN cDNA 9230116B18 Gene and cDNA Sequence AJ554213. A list of 49 epididymis-specific probe sets is provided in Johnston et al. [11]. The identity of the remaining 28 probe sets, so far as they were known, is provided in Table 1. The identity of all 77 probe sets can be found on the MRG database by selecting "Download Database" and selecting the link for "Datasets from Wyeth Research". We wished to confirm and characterize further the expression of transcripts identified as being epididymis-specific from the above analysis. In addition, we decided to increase the number of probe sets under evaluation by investigating both epididymis-specific probe sets (described above) and epididymis-selective probe sets. The criteria for epididymal-selective probe sets is described in Johnston et al. [11]. Briefly, for a transcript to be epididymal-selective, it had to present in 66% of the samples in at least one segment with a mean expression of > 50 signal units and it had to have a signal value > 3-fold higher than all of the other 21 normal tissues. As these criteria were less stringent, all of the 77 epididymis-specific transcripts met the criteria for being epididymis-selective. This resulted in the identification of 307 murine epididymis-selective transcripts. The identity of the epididymis-selective transcripts is available on the MRG database by selecting "Download Database" and selecting the link for "Datasets from Wyeth Research". The tissue expression profile of each of the 307 transcripts was visually inspected to identify those transcripts that had high signal values in other tissues and these probe sets were excluded. Approximately 130 appeared to show high selectivity in the epididymis and were selected for further analysis. The tissue distribution of these transcripts was analyzed by RT-PCR analysis of RNA from a total of 21 normal tissues, including the epididymis. The tissues used in these analyses were ovary, epididymis, testis, prostate, embryo, muscle, kidney, liver, placenta, heart, intestine, lung, brain, pancreas, stomach, hypothalamus, spleen, thymus, uterus, pituitary and colon. This analysis showed that only one transcript corresponding to Defensin β13 was expressed solely in the epididymis (Figure 2). Two additional transcripts corresponding to Eppin and RIKEN cDNA 9230105I15 gene each had strong expression in the epididymal samples and weak expression in the testis (data not shown). 4 Identification of epididymal-specific transcripts in the rat After completing the analysis of epididymis-specific transcripts in the mouse, a similar strategy was used to identify additional epididymis-specific transcripts in the rat. To this end, the 16 360 transcripts expressed in the rat epididymis were compared with a Wyeth Research database of rat profiling data from 22 distinct normal tissues. The tissues used for comparison were similar to those used for the mouse and are listed in the Materials and Methods of Jelinsky et al. [12]. The criterion for epididymal specificity was effectively identical to that used for the mouse and 110 transcripts met the criteria for epididymal specificity. Among the 110 transcripts were known "epididymis specific" genes, such as Cystatin 11, Adam7 and LCN5. Many of the identified transcripts have not been reported to be epididymis-specific, including Adora1, Gdf15 and Kcng3, and their function in the epididymis is not known. The complete list of probe sets has been provided [12] and has been placed on the MRG. We characterized the expression of the 110 epididymis-selective genes in 11 additional types of normal rat tissue, including bone, bone marrow, cerebellum, hippocampus, hypothalamus, cartilage, esophagus, white fat, meniscus, muscle and pancreas. We found that 48 transcripts were also expressed in at least one of these additional 11 tissues. The tissue expression of the remaining 62 transcripts was characterized by qPCR. Using PrimerExpress (Applied Biosystems, Foster City, CA, USA) transcript-specific primer/probe sets were identified and cross-reactivity of each primer probe set sequence was evaluated using the BLAST algorithm and determined to be specific for each qualifier. Twelve point standard curves were generated for caput, corpus, and cauda epididymidal total RNA and used to validate all 62 probe sets. One primer probe set failed to be validated and was therefore eliminated from further analysis. A panel of 28 tissues was evaluated, including 19 non-reproductive tissues and 9 male reproductive tissues. The 19 non-reproductive tissues were adipose tissue, adrenal gland, bone, cerebellum, cerebrum, colon, heart, small intestine, kidney, liver, lung, meniscus, pancreas, salivary gland, skeletal muscle, spleen, stomach, synovium and thymus. The nine male reproductive tissues were bulbourethral gland, prostate, seminal vesicle, testis, vas deferens, caput epididymidis, corpus epididymidis, cauda epididymidis and whole epididymis. All tissues were dissected in triplicate from littermates and frozen in liquid nitrogen. Total RNA was isolated using CsCl2 centrifugation followed by DNAase treatment on QIAgen RNAeasy columns and expression of each of the 61 transcripts in the 28 tissues was determined as previously described [12]. In total, 14 transcripts were found to have >100-fold higher expression in epididymal tissue than any other non-reproductive tissue and 5 were > 10 000-fold higher in the epididymis tissue than any other non-reproductive tissue. These 5 transcripts included ERABP (> 30 000), Defβ22 (> 28 000), Spag 11 (> 20 000), Transmembrane Epididymal Protein 1 (> 16 000) and cystatin 8 (> 11 000). These genes that are so highly enriched may be critical for epididymal function. Davies et al. [13] reported that disruption of mouse HE6 (Transmembrane Epididymal Protein 1) downregulates genes specific to the caput epididymidis and results in male infertility, indicating that this approach is valid. Other epididymis-specific genes, such as beta-galactosidase-like protein, ribonuclease 12 and indoleamine-2,3-dioxygenase have not been implicated in male reproduction, but might provide novel putative targets for male contraception. A representative electronic distribution and qPCR analysis for transmembrane epididymal protein 1 is shown in Figure 3. The identities of all 14 genes and the qPCR data have been placed on the MRG database and can be found as described above. 5 Conclusion The epididymis-selective genes described in this manuscript were identified using sensitive technologies that are amenable to high volume screening. These data should be viewed in the context of the sensitivity of the microarray technology and amplification procedures used to determine their expression and the panel of tissues used for analysis. Evaluation of these transcripts using additional tissues and/or more sensitive assays could alter their designation as epididymis selective/specific. This analysis provides an important dataset of genes and gene products to be further evaluated in order to understand the role they might play in epididymal function. It is the authors' hope that this resource will assist interested investigators to develop novel hypothesis about the cellular and molecular biology of the epididymis and, ultimately, lead to new insights into epididymal function. References 1 Turner TT, Johnston DS, Jelinsky SA. Epididymal genomics and the search for a male contraceptive. Mol Cell Endocrinol 2006; 250: 178_83. 2 Gottwald U, Davies B, Fritsch M, Habenicht UF. New approaches for male fertility control: HE6 as an example of a putative target. Mol Cell Endocrinol 2006; 250: 49_57. 3 O'Rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, et al. Reversible immunocontraception in male monkeys immunized with Eppin. Science 2004; 306: 1189_90. 4 Lyttle CR, Kopf GS. Status and future direction of male contraceptive development. Curr Opin Pharmacol 2003; 3: 667_71. 5 Johnston DS, Bai Y, Kopf GS. Utilization of human genome databases in strategies for contraceptive development. In: Turner TT, Hinton BT, editors. Epididymis III: The Third International Conference on the Epididymis. Charlottesville: The Van Doren Company; 2002: 256_70. 6 Hsia N, Cornwall GA. DNA microarray analysis of region-specific gene expression in the mouse epididymis. Biol Reprod 2004; 70: 448_57. 7 Jervis KM, Robaire B. Dynamic changes in gene expression along the rat epididymis. Biol Reprod 2001; 65: 696_703. 8 Chauvin TR, Griswold MD. Androgen-regulated genes in the murine epididymis. Biol Reprod 2004; 71: 560_9. 9 Dacheux JL, Belghazi M, Lanson Y, Dacheux F. Human epididymal secretome and proteome. Mol Cell Endocrinol 2006; 250: 36_42. 10 Turner TT, Bomgardner D, Jacobs JP, Nguyen QA. Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice. Reproduction 2003; 125: 871_8. 11 Johnston DS, Jelinsky SA, Bang HJ, Dicandeloro P, Wilson E, Kopf GS, et al. The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis. Biol Reprod 2005; 73: 404_13. 12 Jelinsky SA, Turner TT, Bang HJ, Finger JN, Solarz MK, Wilson E, et al. The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides. Biol Reprod 2007; 76: 561_70. 13 Davies B, Behnen M, Cappallo-Obermann H, Spiess AN, Theuring F, Kirchhoff C. Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption. Mol Reprod Develop 2007; 74: 539_53. |
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