Limits...
Yeast model identifies ENTPD6 as a potential non-obstructive azoospermia pathogenic gene.

Wang Q, Liu C, Tang C, Guo H, Liu Y, Wang L, Zhao H, Shang Y, Wen Y, Lin Y, Zhou T, Zhou Z, Dong W, Hu Z, Guo X, Sha J, Li W - Sci Rep (2015)

Bottom Line: Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity.After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene.These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
Approximately ten percent of male infertility is caused by non-obstructive azoospermia (NOA), but the etiologies of many NOA remain elusive. Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity. However, those variants that lack genome-wide significance might still be essential for fertility. Functional analysis of genes surrounding these variants in Drosophila identified some spermatogenesis-essential genes. As a complementary method of Drosophila screening, SK1 background Saccharomvces cerevisiae was used as a model to screen meiosis-related genes from the NOA GWAS data in this study. After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene. The deletion of GDA1 resulted in the failure of yeast sporulation. Further investigations showed that Gda1p was important for pre-meiotic S phase entry. Interestingly, the meiotic role of Gda1p was dependent on its guanosine diphosphatase activity, but not it's cytoplasmic, transmembrane or stem domains. These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA.

No MeSH data available.


Related in: MedlinePlus

Identification of potential non-obstructive azoospermia pathogenic genes by functional genomic screening in yeast.(a) Flow chart of the screening strategy. The selection criteria for candidate genes in Saccharomvces cerevisiae included a tSNP with an association P-value < 10−5 and a P-value ≥ 5*10−8 after multiple validations, and human genes flanking the tSNPs within 100 kb, homology type (one to many or one to one) and orthology identity >20% were considered. After eliminating the well-studied genes in meiosis and lethal genes, the candidate genes were screened for their sporulation efficiency after deletion. (b) The sporulation efficiency of the yeast in which candidate genes were deleted. Wild type and candidate gene deletion stains were incubated in sporulation medium for 24 hrs. Sporulation efficiency was the percentage of cells induced to sporulate that became dyads and tetrads by staining with DAPI. (c) The gda1Δ strain showed a decrease in sporulation efficiency compared with the WT strain. A sporulation time course indicated the percentage of cells/asci with dyads and tetrads in the gda1Δ and WT strains. Diploid yeast cells were deprived of nutrients, induced to enter sporulation synchronously, and stained with DAPI at different times post-induction. (d) WT and gda1Δ spores were stained with DAPI to show the decrease of sporulation efficiency.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495445&req=5

f1: Identification of potential non-obstructive azoospermia pathogenic genes by functional genomic screening in yeast.(a) Flow chart of the screening strategy. The selection criteria for candidate genes in Saccharomvces cerevisiae included a tSNP with an association P-value < 10−5 and a P-value ≥ 5*10−8 after multiple validations, and human genes flanking the tSNPs within 100 kb, homology type (one to many or one to one) and orthology identity >20% were considered. After eliminating the well-studied genes in meiosis and lethal genes, the candidate genes were screened for their sporulation efficiency after deletion. (b) The sporulation efficiency of the yeast in which candidate genes were deleted. Wild type and candidate gene deletion stains were incubated in sporulation medium for 24 hrs. Sporulation efficiency was the percentage of cells induced to sporulate that became dyads and tetrads by staining with DAPI. (c) The gda1Δ strain showed a decrease in sporulation efficiency compared with the WT strain. A sporulation time course indicated the percentage of cells/asci with dyads and tetrads in the gda1Δ and WT strains. Diploid yeast cells were deprived of nutrients, induced to enter sporulation synchronously, and stained with DAPI at different times post-induction. (d) WT and gda1Δ spores were stained with DAPI to show the decrease of sporulation efficiency.

Mentions: A recent study showed that approximately 32% of GWAS SNPs are located in deoxyribonuclease I hypersensitive sites (DHSs), which are markers of regulatory DNA that can regulate genes within 100 kb30. Thus, we considered genes flanking the tSNPs (Tag Single Nucleotide Polymorphisms) within 100 kb in this study. Our recent work in Drosophila has demonstrated that this approach is effective in identifying genes that are essential for male fertility based on SNPs without genome-wide significant associations with human NOA26. However, Drosophila spermatogenesis does not involve chromosome recombination27, which is an important event for human spermatocyte meiosis. Because yeast is the most powerful model to study meiosis, we used yeast in the present study to screen meiosis-related genes from the NOA GWAS data and used the strategy described in Fig. 1a. In summary, 9 candidate orthologous yeast genes were obtained, corresponding to 11 human genes and 7 susceptible tSNPs (Table S1). Among these, MPP10, RFC5, RPC19 and SLD5 were found to be essential to yeast survival, thus prohibiting our further screening by their deletion. MSH5 was found to be involved in meiosis in yeast2829. Finally, 4 genes, CKB2, GDA1, GPH1, and PMC1, were selected and underwent functional analysis in the SK1 background yeast strain, which sporulates faster and more synchronously than other strains and is commonly used for the study of sporulation or meiosis31. After deleting these genes by homologous recombination32, wild type (WT) and candidate gene deletion stains were deprived of nitrogen and incubated in sporulation medium for 24 hrs, and the sporulation efficiency was detected by staining with 4’,6-diamidino-2-phenylindole (DAPI). We found that the sporulation efficiency of the gda1Δ strain showed a significant decrease compared with that of the WT strain (Fig. 1b–d), which is similar to some NOAs of humans. ENTPD6 is the orthologous human gene of GDA1, and it is a member of the ENTPD family and localizes in the Golgi apparatus33. Thus, the functional genomic screening of NOA GWAS data in yeast resulted in the identification of ENTPD6 as a potential pathogenic gene of NOA.


Yeast model identifies ENTPD6 as a potential non-obstructive azoospermia pathogenic gene.

Wang Q, Liu C, Tang C, Guo H, Liu Y, Wang L, Zhao H, Shang Y, Wen Y, Lin Y, Zhou T, Zhou Z, Dong W, Hu Z, Guo X, Sha J, Li W - Sci Rep (2015)

Identification of potential non-obstructive azoospermia pathogenic genes by functional genomic screening in yeast.(a) Flow chart of the screening strategy. The selection criteria for candidate genes in Saccharomvces cerevisiae included a tSNP with an association P-value < 10−5 and a P-value ≥ 5*10−8 after multiple validations, and human genes flanking the tSNPs within 100 kb, homology type (one to many or one to one) and orthology identity >20% were considered. After eliminating the well-studied genes in meiosis and lethal genes, the candidate genes were screened for their sporulation efficiency after deletion. (b) The sporulation efficiency of the yeast in which candidate genes were deleted. Wild type and candidate gene deletion stains were incubated in sporulation medium for 24 hrs. Sporulation efficiency was the percentage of cells induced to sporulate that became dyads and tetrads by staining with DAPI. (c) The gda1Δ strain showed a decrease in sporulation efficiency compared with the WT strain. A sporulation time course indicated the percentage of cells/asci with dyads and tetrads in the gda1Δ and WT strains. Diploid yeast cells were deprived of nutrients, induced to enter sporulation synchronously, and stained with DAPI at different times post-induction. (d) WT and gda1Δ spores were stained with DAPI to show the decrease of sporulation efficiency.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495445&req=5

f1: Identification of potential non-obstructive azoospermia pathogenic genes by functional genomic screening in yeast.(a) Flow chart of the screening strategy. The selection criteria for candidate genes in Saccharomvces cerevisiae included a tSNP with an association P-value < 10−5 and a P-value ≥ 5*10−8 after multiple validations, and human genes flanking the tSNPs within 100 kb, homology type (one to many or one to one) and orthology identity >20% were considered. After eliminating the well-studied genes in meiosis and lethal genes, the candidate genes were screened for their sporulation efficiency after deletion. (b) The sporulation efficiency of the yeast in which candidate genes were deleted. Wild type and candidate gene deletion stains were incubated in sporulation medium for 24 hrs. Sporulation efficiency was the percentage of cells induced to sporulate that became dyads and tetrads by staining with DAPI. (c) The gda1Δ strain showed a decrease in sporulation efficiency compared with the WT strain. A sporulation time course indicated the percentage of cells/asci with dyads and tetrads in the gda1Δ and WT strains. Diploid yeast cells were deprived of nutrients, induced to enter sporulation synchronously, and stained with DAPI at different times post-induction. (d) WT and gda1Δ spores were stained with DAPI to show the decrease of sporulation efficiency.
Mentions: A recent study showed that approximately 32% of GWAS SNPs are located in deoxyribonuclease I hypersensitive sites (DHSs), which are markers of regulatory DNA that can regulate genes within 100 kb30. Thus, we considered genes flanking the tSNPs (Tag Single Nucleotide Polymorphisms) within 100 kb in this study. Our recent work in Drosophila has demonstrated that this approach is effective in identifying genes that are essential for male fertility based on SNPs without genome-wide significant associations with human NOA26. However, Drosophila spermatogenesis does not involve chromosome recombination27, which is an important event for human spermatocyte meiosis. Because yeast is the most powerful model to study meiosis, we used yeast in the present study to screen meiosis-related genes from the NOA GWAS data and used the strategy described in Fig. 1a. In summary, 9 candidate orthologous yeast genes were obtained, corresponding to 11 human genes and 7 susceptible tSNPs (Table S1). Among these, MPP10, RFC5, RPC19 and SLD5 were found to be essential to yeast survival, thus prohibiting our further screening by their deletion. MSH5 was found to be involved in meiosis in yeast2829. Finally, 4 genes, CKB2, GDA1, GPH1, and PMC1, were selected and underwent functional analysis in the SK1 background yeast strain, which sporulates faster and more synchronously than other strains and is commonly used for the study of sporulation or meiosis31. After deleting these genes by homologous recombination32, wild type (WT) and candidate gene deletion stains were deprived of nitrogen and incubated in sporulation medium for 24 hrs, and the sporulation efficiency was detected by staining with 4’,6-diamidino-2-phenylindole (DAPI). We found that the sporulation efficiency of the gda1Δ strain showed a significant decrease compared with that of the WT strain (Fig. 1b–d), which is similar to some NOAs of humans. ENTPD6 is the orthologous human gene of GDA1, and it is a member of the ENTPD family and localizes in the Golgi apparatus33. Thus, the functional genomic screening of NOA GWAS data in yeast resulted in the identification of ENTPD6 as a potential pathogenic gene of NOA.

Bottom Line: Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity.After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene.These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
Approximately ten percent of male infertility is caused by non-obstructive azoospermia (NOA), but the etiologies of many NOA remain elusive. Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity. However, those variants that lack genome-wide significance might still be essential for fertility. Functional analysis of genes surrounding these variants in Drosophila identified some spermatogenesis-essential genes. As a complementary method of Drosophila screening, SK1 background Saccharomvces cerevisiae was used as a model to screen meiosis-related genes from the NOA GWAS data in this study. After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene. The deletion of GDA1 resulted in the failure of yeast sporulation. Further investigations showed that Gda1p was important for pre-meiotic S phase entry. Interestingly, the meiotic role of Gda1p was dependent on its guanosine diphosphatase activity, but not it's cytoplasmic, transmembrane or stem domains. These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA.

No MeSH data available.


Related in: MedlinePlus