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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

GDA1 is required for pre-meiotic S phase entry.The expression of Ime1p was only slightly delayed in the gda1Δ strain during sporulation. WT or gda1Δ strains expressing the IME1-3×myc allele were incubated in sporulation medium and samples were collected at different times. The expression of Ime1p-3×myc over time was analyzed by immunoblotting with anti-Myc antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5. (b) Real-time PCR analysis of the IME1 expression level in WT and GDA1 deletion strains. (c) The pre-meiotic DNA replication was inhibited in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). (d) The stabilization of Sic1p in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. The expression of Sic1p over time was analyzed by immunoblotting with specific anti-Sic1 antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5.
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f2: GDA1 is required for pre-meiotic S phase entry.The expression of Ime1p was only slightly delayed in the gda1Δ strain during sporulation. WT or gda1Δ strains expressing the IME1-3×myc allele were incubated in sporulation medium and samples were collected at different times. The expression of Ime1p-3×myc over time was analyzed by immunoblotting with anti-Myc antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5. (b) Real-time PCR analysis of the IME1 expression level in WT and GDA1 deletion strains. (c) The pre-meiotic DNA replication was inhibited in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). (d) The stabilization of Sic1p in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. The expression of Sic1p over time was analyzed by immunoblotting with specific anti-Sic1 antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5.

Mentions: Because the deletion of GDA1 inhibited sporulation, we next determined which phases of sporulation were affected in the GDA1 deleted strain. Meiosis in yeast is initiated by the expression of Ime1p, which serves as the master regulatory switch for meiosis3435. First, we detected the expression of Ime1p in the GDA1 deletion strain by generating a 3×Myc tag on the C-terminus of IME1. During the sporulation processes, we found that the expression of Ime1p in the GDA1 deletion strain was only delayed by approximately 1 hr compared with the WT strain (Fig. 2a). Real-time PCR analysis of IME1 mRNA in WT and the GDA1 deletion strain showed similar results to the protein level (Fig. 2b). These results suggest that GDA1 is not the major regulator of meiosis initiation, even though it is involved in this process to some extent. We next detected the pre-meiotic DNA synthesis by flow cytometry analysis to test whether the GDA1- mutant influenced pre-meiotic DNA replication. We found that the pre-meiotic DNA replication was repressed in the GDA1 deletion strain (Fig. 2c).


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)

GDA1 is required for pre-meiotic S phase entry.The expression of Ime1p was only slightly delayed in the gda1Δ strain during sporulation. WT or gda1Δ strains expressing the IME1-3×myc allele were incubated in sporulation medium and samples were collected at different times. The expression of Ime1p-3×myc over time was analyzed by immunoblotting with anti-Myc antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5. (b) Real-time PCR analysis of the IME1 expression level in WT and GDA1 deletion strains. (c) The pre-meiotic DNA replication was inhibited in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). (d) The stabilization of Sic1p in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. The expression of Sic1p over time was analyzed by immunoblotting with specific anti-Sic1 antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5.
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Related In: Results  -  Collection

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

f2: GDA1 is required for pre-meiotic S phase entry.The expression of Ime1p was only slightly delayed in the gda1Δ strain during sporulation. WT or gda1Δ strains expressing the IME1-3×myc allele were incubated in sporulation medium and samples were collected at different times. The expression of Ime1p-3×myc over time was analyzed by immunoblotting with anti-Myc antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5. (b) Real-time PCR analysis of the IME1 expression level in WT and GDA1 deletion strains. (c) The pre-meiotic DNA replication was inhibited in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. DNA content was analyzed by flow cytometry to detect pre-meiotic DNA replication (2C to 4C transition). (d) The stabilization of Sic1p in the gda1Δ strain during sporulation. WT or gda1Δ strains were incubated in sporulation medium and samples were collected at different times after induction. The expression of Sic1p over time was analyzed by immunoblotting with specific anti-Sic1 antibody. Pgk1p served as a loading control. Full-length blots/gels are presented in Supplementary Figure 5.
Mentions: Because the deletion of GDA1 inhibited sporulation, we next determined which phases of sporulation were affected in the GDA1 deleted strain. Meiosis in yeast is initiated by the expression of Ime1p, which serves as the master regulatory switch for meiosis3435. First, we detected the expression of Ime1p in the GDA1 deletion strain by generating a 3×Myc tag on the C-terminus of IME1. During the sporulation processes, we found that the expression of Ime1p in the GDA1 deletion strain was only delayed by approximately 1 hr compared with the WT strain (Fig. 2a). Real-time PCR analysis of IME1 mRNA in WT and the GDA1 deletion strain showed similar results to the protein level (Fig. 2b). These results suggest that GDA1 is not the major regulator of meiosis initiation, even though it is involved in this process to some extent. We next detected the pre-meiotic DNA synthesis by flow cytometry analysis to test whether the GDA1- mutant influenced pre-meiotic DNA replication. We found that the pre-meiotic DNA replication was repressed in the GDA1 deletion strain (Fig. 2c).

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