Mol. Hum. Reprod. Advance Access originally published online on February 6, 2008
Molecular Human Reproduction 2008 14(3):137-142; doi:10.1093/molehr/gan002
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Identification and characterization of novel mammalian spermatogenic genes conserved from fly to human
1Divison of Reproductive Biology Research, Department of Obstetrics and Gynecology, Center for Genetic Medicine, Northwestern University, Feinberg School of Medicine, Lurie 7-117, Chicago, IL 60611, USA 2Department of Health Sciences, UAMI, 09340 Mexico City, Mexico
3 Correspondence address. Tel: +1-312-5030481; Fax: +1-312-9033074; E-mail: e-xu{at}northwestern.edu
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Abstract |
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Spermatogenesis is a complex and highly regulated developmental process by which round spermatogonial stem cells undergo mitotic proliferation and meiosis, followed by extraordinary differentiation into highly specialized elongated mature sperm. Extensive differences in terms of sperm production such as testicular structure and organization, hormonal regulation are reported between humans and insects, yet it is not known to what extent components of the process could be conserved and furthermore to what extent the underlying genetic regulators could be shared from insects to mammals. We hence take a genomic approach to identify genes which are expressed in the testes of both fly and mouse through in silico analysis and are phylogenetically conserved across metazoans. Fifty eight testis-enriched, phylogenetically conserved from fly to mouse genes were identified. Among them, 12 genes are novel. Detailed characterization of their murine and human homologs indicate most of them are testis-restricted or enriched and developmentally regulated, thus suggesting that they are important regulators of sperm development in mammals and potential human fertility factors. Our results reveal the existence of spermatogenic homologs with similar testicular expression across a large evolutionary distance, further functional study will be needed to explore the functional conservation among those spermatogenic orthologs.
Key words: spermatogenesis/male fertility/germline development/testis/conserved genes
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary dataFundingAcknowledgementsReferences |
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Infertility resulting from a severe defect in sperm production affects 5% of men worldwide (de Kretser, 1997; Cram et al., 2004). Although it can be caused by many different factors, genetics undoubtedly plays a role in this disorder (Reijo et al., 1995, 1996; Carrell et al., 2006). It is reported that genes expressed during spermatogenesis are involved in the pathogenesis of male infertility, e.g. SYCP3, DAZ, AURKC, Protamines 1–2, Ubiquitin specific protease—USP9Y (Reijo et al., 1995, 1996; Sun et al., 1999; Miyamoto et al., 2003; Christensen et al., 2005; Paduch et al., 2005; Oliva, 2006; Dieterich et al., 2007).
However, the formation of a mature sperm is a very complex process involving many genes. Current estimates suggest that the interplay of some 2000 different genes are involved in testicular development, germ cell differentiation, meiosis and the successive stages of spermiogenesis (Bhasin et al., 2000). Defects in any one of these genes could result in impaired sperm or no sperm at all. It is currently believed that idiopathic infertility is mainly of genetic origin. Thus, the study of genes that are involved in the control of spermatogenesis may help to distinguish between intrinsic and acquired male infertility (Kleiman et al., 2003).
The network of genes and the pathways regulating spermatogenesis in mammals is not well understood (Cooke and Saunders, 2002). A lot more is known about the genetic regulators for germ cell development, spermatogenesis in particular, in other more manipulatable biological systems such as in flies and worms (Castrillon et al., 1993; Fuller 1998; Chu et al., 2006). As in mammals, spermatogenesis in flies takes place inside a separate organ—the testis. Spermatogenesis consists of spermatogonial stem cells at the apical tip of the testis, proliferating spermatogonial cells, spermatocytes, meiotic cells and elongating spermatids. Mature sperm is released into a sperm storage place called the seminal vesicle. The major cell types and stages of spermatogenesis are also present in human and mice. The striking parallel in spermatogenesis between the human and the fly raised a question as to whether the underlying genetic regulators are conserved through evolution. Identification of those highly conserved spermatogenic regulators might help us to uncover key regulators of human spermatogenesis. An example of a key regulator gene of spermatogenesis is BOULE, whose Drosophila homolog is the known meiotic regulator boule (Eberhart et al., 1996). Loss of boule function in Drosophila results in azoospermia due to meiotic arrest during sperm development. Since human BOULE can rescue the meiotic defect in fly boule mutants, it suggests that BOULE may encode a key conserved switch that regulates progression of germ cells through meiosis in men (Xu et al., 2003). Although a few other homologs of fly germ cell regulators are also found to be important for mammalian spermatogenesis such as Nanos, Piwi, Mvh (Tanaka et al., 2000; Deng and Lin, 2002; Tsuda et al., 2003), it remains unknown to what extent genetic regulators are conserved across such a large evolutionary distance.
We reason that if we could identify testicular genes, which meet the three following criteria: (i) they are expressed specifically in testis; (ii) are phylogenetically conserved across metazoans and (iii) are developmentally regulated; we are likely to discover important regulators of spermatogenesis. In this study, we have used in silico and in vitro approaches in order to identify genes meeting these criteria, and identified 12 candidate fertility factors in mammals.
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Materials and Methods |
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Identification of conserved testis genes in mouse and human
Two in silico approaches were used. In the first approach, we analyzed transcription profiles of Drosophila (fruitfly) germline development from our fly microarray data to identify testis-enriched genes in the fly. Nine pairs of fly testes were dissected and RNA was extracted and linear RNA amplification was performed essentially as described (Klebes et al., 2002). Microarrays were produced as described (Xu et al., 2003). The common reference samples to which we compare all the wild-type testis samples with was generated by pooling amplified RNA from adult males and females. There are over 1000 genes highly expressed in testis versus reference simples (Xu et al., unpublished). In order to determine to what extent fly testicular genes with enriched expression in the testis could be conserved in mammals, we decided to randomly choose 50 genes and set out to find if any homologs could be identified in mouse and whether they are also highly expressed in the mouse testis.
Starting with this subset of fly testis-enriched genes, we then identified mouse homologs showing high protein sequence similarity and testis-enriched expression using Unigene and BLAST databases at NCBI (available on the World Wide Web at www.ncbi.nlm.nih.gov). A gene was considered as testis-enriched when there were only testicular EST(s) of a given gene or when the number of testicular ESTs was much higher than that of non-testicular ESTs. In the second approach, Unigene was used to identify testis-enriched genes in the mouse. Out of this preliminary list, we then selected those genes with homology to Drosophila genes. The human orthologs for the selected genes were identified using MGI database. MGI GO database (available at http://www.informatics.jax.org/searches/GO_form.shtml) was used for gene ontology.
Reverse transcriptase–polymerase chain reaction
Total RNA was extracted from various tissues from mice using TRIzol (GibcoBRL, Gaithersburg, MD, USA) according to the manufacturer's recommendation. The first-strand synthesis was performed using 5 µg RNA, random primers and reverse transcriptase Superscript II (GibcoBRL) at 42°C for 1 h.
For human expression pattern analysis, first-strand cDNA samples were purchased from Origene (Rockville, MD, USA).
To determine the tissue distribution of novel gene expression, PCR experiments were performed using cDNA from the multiple organs of mice and human. To investigate whether the novel genes are expressed specifically in germ cells of testis, RT–PCR was performed using total RNA isolated from the germ cell-less testes of WBB6F1/J-KitW/KitW-v mutant mice (The Jackson Laboratory). To test whether transcripts are expressed at particular stages of spermatogenesis, prepubertal and adult male mice (ages 3, 9, 18 and 23 days) were sacrificed, and total RNA isolated from their testes was used for reverse transcription. PCR was performed for 30 cycles of 94°C for 45 s, 65°C for 45 s and 72°C for 1 min. Gene-specific primers were designed using Primer3 program (MIT, available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers used in this study to amplify mouse and human transcripts are listed in Table I.
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Northern blot analyses
Total RNAs were isolated from the various organs of mice and quantified by optical density measurement. Ten microgram of RNA were separated on a 1.0% agarose gel with 1X MOPS buffer and 0.66 M formaldehyde (final concentrations) and transferred onto Hybond N+membrane (Amersham Pharmacia Biotech) in a 20X SSC buffer. The filter was pre-hybridized for 5 h and then hybridized for 17 h in a solution of 5xSSC, 5xDenhardt's solution, 0.1% SDS and 5% dextran sulfate at 42°C with an [32P]dCTP-labelled cDNA probe. After hybridization, the filter was washed one time 20 min in 2% SSC at room temperature followed by one time 20 min in 2XSSC, 0.1% SDS at 65°C. The hybridization signal was visualized by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary dataFundingAcknowledgementsReferences |
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In order to identify human conserved fertility factors, we decided to look for spermatogenic genes conserved between fly and mouse first. We reasoned that the evolutionary distance between human and mice is relatively insignificant in comparison with that of human and fly, and genes conserved between human and fly should also be conserved in mice, and vice versa. Hence we decided to start by comparing mouse and fly genes to identify conserved homologs first, and then we would characterize novel murine spermatogenic genes and determine if they are developmentally regulated during development. Finally, we would identify candidate human fertility factors based on mouse–human synteny map and sequence homology.
Two in silico approaches were used in this study in order to identify some putative fertility factors in the mouse (Supplementary Figure). In the first approach, we analyzed transcription profiles of Drosophila germline development from our fly microarray data to identify testis-enriched genes in the fly (Xu et al., unpublished). Starting with a subset of 50 fly testis-enriched genes, we identified 12 mouse homologs showing high protein sequence similarity and restricted or highly enriched expression in testes based on EST information from Unigene and BLAST databases (Supplementary Table 1). The tissue specificity and/or the developmental expression of nine of these genes had already been carried out. In this study, we focused on the three novel genes DnaicI, Spata20 and Wdr63. In the second approach, we searched Unigene for testis-enriched genes in the mouse, and then we used Unigene and BLAST to identify those genes with homology to Drosophila genes. Forty-seven genes were selected by using this approach (Supplementary Table 2), 10 of them were novel genes showing expression restricted to or highly enriched in the testis and were selected in this study for further analyses.
A total of 12 highly conserved candidate germline regulators were identified using both approaches [one gene was identified by both approaches (Spata 20)]. The list of the 12 candidate genes and their fly and human homologs is shown in Table II. Gene ontology information obtained from MGI-GO database revealed diverse putative regulatory roles for most of these genes, as well as for the rest 46 previously characterized, testis-enriched, conserved from fly to mouse genes (Table II and supplementary Tables 1 and 2). These 58 genes represent a first glimpse of highly conserved core germ cell machinery. They were categorized based on their known or putative molecular function (Fig. 1). Many of them are transcription factors, post-transcriptional regulators, protein binding and modification factors, and coordinators of multi-protein complexes, thus implicating a relevant role in spermatogenesis.
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The tissue-specificity of the 12 selected genes was then analyzed by RT–PCR on several adult mouse tissues (Fig. 2). All of them were shown to be testis-enriched. A mouse mutant—steel mice from Jackson lab which lack germ cells but maintain somatic cells inside the mutant testis was used to determine whether these genes are specifically expressed in germ cells in the testis. Ten of these 12 genes are found to be germ-cell-specific, and two of them may be expressed also in testis somatic cells (Slc25a2 and Mael).
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We further analyzed the developmental expression pattern of the selected genes by RT–PCR from testes of different developmental stages (Fig. 3). It was observed that seven genes are only expressed during the meiotic and post-meiotic phases of spermatogenesis (day 18 onwards). Three other genes (Arpm1, 1700012H05 and 4933400A11) are expressed only in the post-meiotic phase of spermatogenesis (days 23 and 90), and another two genes (Slc25a2 and Mael) are expressed (at low levels for Slc25a2) during the mitotic phase of spermatogenesis (days 3 and 9), and continue to be expressed during the meiotic and post-meiotic phases.
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Northern blot analyses were then carried out to analyze the transcript abundance of the candidate genes (Fig. 4). Consistent with our RT–PCR results, all the genes showed testis-enriched expression and presented at the expected sizes. No splicing variants were observed. The analysis of gene expression by in situ hybridization has been accomplished for genes 1700012G21 and 1700012H05. Supporting the RT–PCR analysis of these genes during the development, it was found that these genes are expressed exclusively during the post-meiotic phase of spermatogenesis (data not shown).
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The human orthologs to five of these genes have been previously characterized, and there is evidence that three of them are key fertility factors. Here we analyzed the tissue specificity of the other seven human orthologs (Fig. 5). It was found that six of them (ANKRD20A, CASKIN1, SLC25A2, SPATA20, WDR16 and WDR63) are specifically and/or abundantly expressed in testis, thus suggesting possible roles of these genes in spermatogenesis. The only human homolog that is not detected in human testis is CAZ1. It is possible that our primers fail to pick up the CAZ1 transcript efficiently. Alternatively other homologs of CAZ1 might have taken over the testicular function of CAZ1 and CAZ1 is not specifically expressed in human testis. Functional analysis of those identified conserved genes could be performed either with mouse knockout mutations or through RNAi coupled with in vitro germ cell differentiation from embryonic stem cell culture.
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Our results demonstrate the feasibility of identifying important human fertility factors through such comparative genomics comparison between mice and fly, providing further support for the conservation of genetic regulators for spermatogenesis from insects to human.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary dataFundingAcknowledgementsReferences |
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Although frequently suggested, evidence of a genetic etiology of impaired spermatogenesis is scarce (Turek and Pera, 2002). Identifying the full complement of key genes that regulate this process will undoubtedly lay a solid base for our further understanding and better manipulation of spermatogenesis.
Several studies have identified genes expressed at a specific stage or in a particular cell type during spermatogenesis using differential display, cDNA/oligonucleotide array or substractive hybridization (Fujii et al., 2002; Schultz et al., 2003; Almstrup et al., 2004; Rossi et al., 2004). Hong et al. (2005) identified 28 novel candidate regulators of male germ cell development by using in silico and in vitro approaches based mainly on two criteria: (i) these genes are specifically expressed in testis and (ii) they are developmentally regulated. Since several studies have shown that regulators of spermatogenesis may also be phylogenetically conserved (Reijo et al., 1995; Dai et al., 1998; Tanaka et al., 2000; Tsuda et al., 2003; Xu et al., 2003); in this study, we have used two in silico and in vitro approaches in order to identify genes meeting these three criteria.
From a group of genes highly expressed in fly testis or in mouse testis, we identified 58 homologous genes with conserved testicular expression in both fly and mouse. They include previously reported spermatogenic or germ-cell-specific genes such as Odf2, Zfp29, Nasp, Gapds, Mili, Dnahc8, Catsper4, Adam1a, Ddx4 and Dazl. Twelve novel highly conserved, testis-enriched genes were identified by using in silico and comparative genomics approaches (Table II). All of them are testis-restricted or enriched, developmentally regulated and phylogenetically conserved from the fruitfly to humans, and some of them have been reported as essential for spermatogenesis and male fertility, thus validating our approach in identifying candidate genes with key roles in spermatogenesis (Welch et al., 1990; Denny and Ashworth, 1991; Petersen et al., 1999; Tanaka et al., 2000; Samant et al., 2002; Kuramochi-Miyagawa et al., 2004; Miki et al., 2004; Lin and Page, 2005).
A detailed molecular characterization was then carried out in order to determine whether the selected candidates are genuine novel testis-enriched genes and whether they are expressed in developmentally regulated and stage-specific patterns. First, we examined the tissue-specificity of the candidate genes by RT–PCR expression analysis on different adult mouse tissues. All these genes were considered as testis-enriched, since they were specifically and/or abundantly expressed in testis, validating our approach in identifying testis-enriched genes. We next asked if these genes are specifically expressed in germ cells or in somatic cells in the testis. Ten of these 12 genes are found to be germ-cell-specific, since we were not able to detect their mRNA in mutant—steel mice testes, which lack germ cells. Another hallmark of key regulator genes is that they are developmentally regulated, so they can be responsible for the sequence of spermatogenesis. It was found that the 12 candidate genes in this study are expressed in developmentally regulated patterns. The Northern blot analysis revealed the expression level of the candidates and confirmed the testis-specificity of these genes. The transcripts showed the expected size, and no splicing variants were observed. Examination of gene expression profile in microarray gene expression database-SymAtlas reveals 11 out of 12 genes show either testis-restricted or enriched expression pattern, further confirming the enriched testicular expression of the identified novel spermatogenic genes (http://symatlas.gnf.org/SymAtlas/). Gene ontology information obtained from MGI-GO database revealed diverse putative regulator roles for most of these genes, again validating our approach at identifying highly conserved spermatogenic genes.
There is no overlapping between the 28 candidate to key regulators of spermatogenesis reported by Hong et al. (2005) and the candidate genes identified in our study. This is probably due to (i) the criteria applied in the in silico assays, (ii) the search of premeiotic, meiotic and post-meiotic genes in our study, while Hong et al. focused on post-meiotic genes, and (iii) the high number of testis specific genes, which limited both studies to the analysis of only a subset of the preliminary selected genes.
The corresponding human homologs to the 12 candidate genes were identified using MGI and Unigene databases. There is strong evidence supporting three of them (HGCL, hnRNP G-T and DNAI1) as important fertility factors: it has been reported that the expression of HGCL is affected in parallel with the severity of testicular impairment found in azoospermic men with severe germinal cell impairment (Kleiman et al., 2003). The human gene hnRNP G-T has been shown to be associated with impaired spermatogenesis in man (Westerveld et al., 2004), and DNAI1, which encodes a dynein intermediate chain, is involved in primary ciliary dyskinesia, an autosomal disease characterized by sinusitis and bronchiectasis, and usually associated with male infertility, due to the absence of dynein arms in respiratory cilia and sperm flagella (Pennarun et al., 1999). The tissue-specificity analysis of the seven uncharacterized human homologs revealed that six of them are specifically expressed in testis (ANKRD20A, SLC25A2, CASKIN1, SPATA20, WDR16 and WDR63), thus suggesting a pivotal role of these genes in human spermatogenesis. The ongoing RNA inhibition assays on those novel candidates using mouse in vitro germ cell differentiation system from ES cells will provide valuable evidence for their roles as important spermatogenic regulators. It will be interesting to determine if defects in these genes are associated to male infertility in humans, an extraordinarily common problem affecting 1 in 20 normal men (Cram et al., 2004).
The 12 candidate fertility factors identified in this study, along with the 46 testis enriched, phylogenetically conserved genes (Supplementary Table 1), belong to a very interesting group of genes that have been conserved from fly to humans and represent a first glimpse of highly conserved core germ cell machinery. Here we can identify, mainly, transcription and post-transcriptional regulators, protein binding and modification factors, and coordinators of multi-protein complexes (Fig. 1). The importance of these proteins has been demonstrated in other biological processes not only by their critical roles in many essential biological functions ranging from signal transduction, transcription regulation, to apoptosis, but is also recognized by their association with several human diseases. Defining the function of these proteins during germ cell differentiation is the current challenge.
In conclusion, we have identified 12 testis-enriched, developmentally regulated and highly conserved genes that are strong candidates as primary regulators of germ cell development in mammals. However, identification of the full complement of key genes that regulate spermatogenesis is far from being completed. Further studies are necessary to identify candidate genes with essential roles in all of the phases of spermatogenesis, and furthermore to investigate their biological roles in this precisely orchestrated developmental process. Investigating such roles will require functional studies such as identification of partners and/or targeted invalidation.
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Supplementary data |
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Supplementary data are available at http://molehr.oxfordjournals.org/
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Funding |
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The work was supported by NIH grant U01 HD045871 and by EAM grant from the State of Illinois Department of Public Aid and Northwestern Memorial Hospital.
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Acknowledgements |
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The authors are grateful to Drs Renee Reijo Pera, Ansgar Klebes and Tom Kornberg for their helpful suggestions and discussion. E.B. is a visiting fellow of MEXUS-Conacyt program.
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References |
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Submitted on August 3, 2007; resubmitted on December 19, 2007; accepted on January 7, 2008.
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