Limits...
Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci.

Comazzetto S, Di Giacomo M, Rasmussen KD, Much C, Azzi C, Perlas E, Morgan M, O'Carroll D - PLoS Genet. (2014)

Bottom Line: We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis.We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice.Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory (EMBL), Mouse Biology Unit, Monterotondo Scalo, Italy.

ABSTRACT
Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility.

No MeSH data available.


Related in: MedlinePlus

miR-34bc/449 regulates a small cohort of genes in spermatocytes.(A) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc−/−;449−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log2 fold change of 0.5 (red) are shown. (B) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (C) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc−/−;449−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (D) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc−/−;449−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199480&req=5

pgen-1004597-g005: miR-34bc/449 regulates a small cohort of genes in spermatocytes.(A) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc−/−;449−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log2 fold change of 0.5 (red) are shown. (B) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (C) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc−/−;449−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (D) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc−/−;449−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.

Mentions: We next wanted to explore the mechanisms by which miR-34bc/449 supports spermatogenesis. The binding of miRNA:RISC to target mRNAs results in transcript destabilization, this facet of miRNA silencing has been used to reliably identify miRNA targets from the analysis of cellular transcriptomes with gain or loss of a specific miRNA function [19]–[21]. miRNAs exert a relatively small impact in the order of 1.5–3 fold change of target mRNAs, therefore the isolation of pure populations of cells from wild type and mutant mice is critical for comparative transcriptomic analysis and the identification of miRNA target genes. MiR-34bc/449 deficiency impacts both meiosis and the latter stages of spermiogenesis, FACS can be used to sort both of these populations, however in the case of elongating spermatids cells are damaged during the process losing both their tails and cytoplasm. We therefore decided to profile pachytene spermatocytes from control and miR-34bc−/−;449−/− mice. This population also has the added advantage in that it is the cell type where the onset of miR-34bc/449 expression is first observed and thus likely the most promising stage to define the primary impact of miR-34bc/449-deficiency. The comparison of wild type and miR-34bc−/−;449−/− pachytene cells revealed relatively minor changes in the transcriptome (Fig. 5A), setting a threshold of 1.4 fold (Log2>0.5 fold) and with significance value greater than 0.05, we found 22 genes upregulated and 2 genes downregulated in the mutant (Fig. 5A). In miRNA loss of function experiments, the expectation is the loss of repression and concomitant increase in target dosage. Strikingly, 13 of the 22 upregulated genes contained 3′UTR miR-34 ‘seed’ matches and were predicted targets of the miR-34 family (Fig. 5 A–B). Deregulation of 9 from the 13 predicted targets could be confirmed by qRT-PCR (Fig. 5C). We next employed Sylamer to search for significant enrichment of 7 nucleotide motifs corresponding to all known miRNA seed motifs across the 3′UTRs of all genes arranged from most upregulated to most downregulated in the mutant (Fig. 5D). This unbiased approach revealed a highly significant enrichment (p = 2.44×10−9) for the complementary seed match of miR-34 family (CACTGCC) in the cohort of most unregulated genes (Fig. 5D). Most importantly, no other significant miRNA seed matches were identified in this analysis. From the 9 validated miR-34 target genes identified, the forkhead transcription factor FoxJ2 merits special interest as it contains two highly conserved miR-34 binding sites and has been shown that transgenic levels of FoxJ2 overexpression are incompatible with male fertility [40]. Together, these analysis show that the loss of miR-34bc/449 has an intrinsic impact on the meiotic transcriptome and identifies a small cohort of likely direct miR-34bc/449 target genes.


Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci.

Comazzetto S, Di Giacomo M, Rasmussen KD, Much C, Azzi C, Perlas E, Morgan M, O'Carroll D - PLoS Genet. (2014)

miR-34bc/449 regulates a small cohort of genes in spermatocytes.(A) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc−/−;449−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log2 fold change of 0.5 (red) are shown. (B) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (C) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc−/−;449−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (D) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc−/−;449−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199480&req=5

pgen-1004597-g005: miR-34bc/449 regulates a small cohort of genes in spermatocytes.(A) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc−/−;449−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log2 fold change of 0.5 (red) are shown. (B) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (C) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc−/−;449−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (D) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc−/−;449−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.
Mentions: We next wanted to explore the mechanisms by which miR-34bc/449 supports spermatogenesis. The binding of miRNA:RISC to target mRNAs results in transcript destabilization, this facet of miRNA silencing has been used to reliably identify miRNA targets from the analysis of cellular transcriptomes with gain or loss of a specific miRNA function [19]–[21]. miRNAs exert a relatively small impact in the order of 1.5–3 fold change of target mRNAs, therefore the isolation of pure populations of cells from wild type and mutant mice is critical for comparative transcriptomic analysis and the identification of miRNA target genes. MiR-34bc/449 deficiency impacts both meiosis and the latter stages of spermiogenesis, FACS can be used to sort both of these populations, however in the case of elongating spermatids cells are damaged during the process losing both their tails and cytoplasm. We therefore decided to profile pachytene spermatocytes from control and miR-34bc−/−;449−/− mice. This population also has the added advantage in that it is the cell type where the onset of miR-34bc/449 expression is first observed and thus likely the most promising stage to define the primary impact of miR-34bc/449-deficiency. The comparison of wild type and miR-34bc−/−;449−/− pachytene cells revealed relatively minor changes in the transcriptome (Fig. 5A), setting a threshold of 1.4 fold (Log2>0.5 fold) and with significance value greater than 0.05, we found 22 genes upregulated and 2 genes downregulated in the mutant (Fig. 5A). In miRNA loss of function experiments, the expectation is the loss of repression and concomitant increase in target dosage. Strikingly, 13 of the 22 upregulated genes contained 3′UTR miR-34 ‘seed’ matches and were predicted targets of the miR-34 family (Fig. 5 A–B). Deregulation of 9 from the 13 predicted targets could be confirmed by qRT-PCR (Fig. 5C). We next employed Sylamer to search for significant enrichment of 7 nucleotide motifs corresponding to all known miRNA seed motifs across the 3′UTRs of all genes arranged from most upregulated to most downregulated in the mutant (Fig. 5D). This unbiased approach revealed a highly significant enrichment (p = 2.44×10−9) for the complementary seed match of miR-34 family (CACTGCC) in the cohort of most unregulated genes (Fig. 5D). Most importantly, no other significant miRNA seed matches were identified in this analysis. From the 9 validated miR-34 target genes identified, the forkhead transcription factor FoxJ2 merits special interest as it contains two highly conserved miR-34 binding sites and has been shown that transgenic levels of FoxJ2 overexpression are incompatible with male fertility [40]. Together, these analysis show that the loss of miR-34bc/449 has an intrinsic impact on the meiotic transcriptome and identifies a small cohort of likely direct miR-34bc/449 target genes.

Bottom Line: We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis.We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice.Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory (EMBL), Mouse Biology Unit, Monterotondo Scalo, Italy.

ABSTRACT
Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility.

No MeSH data available.


Related in: MedlinePlus