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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.Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes.

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

Expression and function of Dicer in adult spermatogenesis.(A) Domain structure of the Dicer protein is shown. The organization of the 5′ portion of Dicer locus is depicted. The targeting vector used for introduction of FlagHA2 into the Dicer locus and the schematic map of the targeted Dicer gene before and after Cre mediated-recombination are shown. Triangles represent loxP sites as indicated. Rectangles indicate the position of Neomycin (Neo) and Diptheria toxin A (DTA) selection marker genes. The SacI restriction sites are indicated as well as the respective Southern fragments detected by the 3′probe. A schematic diagram of the resulting FlagHA2-Dicer protein is shown. (B) Southern blot of tail derived SacI-digested DNA from wild-type and Dcr+/FH-Neo mice is shown with the 3′ probe indicated in A. (C) Western blot using anti-HA and anti-SMC1 antibodies on extracts from adult wild type and Dcr+/FH testis is shown. (D) Immunofluorescence using anti-HA and anti-γH2AX antibodies on Dcr+/FH testis germ cells from adult testis sections is shown. Scale bar = 10 µm. (E) Hematoxylin and eosin stained testis section from adult DcrCtl and DcrC-KO mice with representative tubules shown. Scale bars = 50 µm and 20 µm in the upper and lower panel, respectively. (F) Increased apoptosis in DcrC-KO testis. A TUNEL assay counterstained with DAPI is shown on testis sections from adult DcrCtl and DcrC-KO mice. The apoptotic cells stain in green. Scale bars = 50 µm and 10 µm in the upper and lower panel, respectively. Abbreviations: P, pachytene and RS, round spermatid. Representative images are shown from at least 3 mice analyzed in panels D–F.
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pgen-1004597-g001: Expression and function of Dicer in adult spermatogenesis.(A) Domain structure of the Dicer protein is shown. The organization of the 5′ portion of Dicer locus is depicted. The targeting vector used for introduction of FlagHA2 into the Dicer locus and the schematic map of the targeted Dicer gene before and after Cre mediated-recombination are shown. Triangles represent loxP sites as indicated. Rectangles indicate the position of Neomycin (Neo) and Diptheria toxin A (DTA) selection marker genes. The SacI restriction sites are indicated as well as the respective Southern fragments detected by the 3′probe. A schematic diagram of the resulting FlagHA2-Dicer protein is shown. (B) Southern blot of tail derived SacI-digested DNA from wild-type and Dcr+/FH-Neo mice is shown with the 3′ probe indicated in A. (C) Western blot using anti-HA and anti-SMC1 antibodies on extracts from adult wild type and Dcr+/FH testis is shown. (D) Immunofluorescence using anti-HA and anti-γH2AX antibodies on Dcr+/FH testis germ cells from adult testis sections is shown. Scale bar = 10 µm. (E) Hematoxylin and eosin stained testis section from adult DcrCtl and DcrC-KO mice with representative tubules shown. Scale bars = 50 µm and 20 µm in the upper and lower panel, respectively. (F) Increased apoptosis in DcrC-KO testis. A TUNEL assay counterstained with DAPI is shown on testis sections from adult DcrCtl and DcrC-KO mice. The apoptotic cells stain in green. Scale bars = 50 µm and 10 µm in the upper and lower panel, respectively. Abbreviations: P, pachytene and RS, round spermatid. Representative images are shown from at least 3 mice analyzed in panels D–F.

Mentions: The importance of post-transcriptional regulation of gene expression in spermatogenesis prompted us to examine the contribution of the miRNA pathway to this process. The RNase III Dicer catalyzes the last step of canonical miRNA biogenesis and thus its expression levels within testicular germ cell populations would be indicative of where this pathway or the biogenesis of miRNAs for current or later use would be important. Since antibodies against mouse Dicer that function for tissue immunofluorescence are lacking, we therefore generated a knock-in allele in mice that carry an N-terminal Flag-HA2 tagged Dicer (DcrFH) (Fig. 1A–C). The Flag-HA2 tag did not adversely impact on the function of Dicer as mice homozygous for the DcrFH allele are viable and fertile. Visualization in adult testis sections of Flag-HA2-Dicer with anti-HA antibodies revealed abundant expression of Dicer in the mitotic spermatogonia and the early meiotic stages of pre-leptotene and leptotene. Thereafter Dicer was up regulated in zygotene reaching a maximum expression in early pachytene spermatocytes (Fig. 1D). From mid-pachytene onwards Dicer was downregulated but still detected in the later stages of spermiogenesis (Fig. 1D). The expression pattern of Dicer would suggest a critical function for the miRNA pathway in meiosis as well as during haploid germ cell development. While non-canonical miRNA biogenesis pathways do exist, only a single miRNA (miR-451) has been shown to be Dicer independent [22]–[24]. In addition to miRNAs, the other Dicer products, the endogenous siRNAs, have thus far only been found in oocytes and ESCs [25]–[27]. While the failure to detect siRNAs in the male germ cells cannot formally exclude their presence therein, the loss of Dicer can more than likely be used to explore the function of the miRNA pathway in post-mitotic spermatogenesis. The importance of Dicer in early germ cell development was shown through its conditional ablation during early embryogenesis in primordial germ cells (PGCs) using the TNAP-Cre [28]. This loss of Dicer results in proliferative defects in PGCs with either absent or retarded spermatogenesis in adult seminiferous tubules [28]. To understand whether Dicer is required during meiosis, we combined the Dicer LoxP (DcrFl) allele with the Stra8Cre transgene that deletes in differentiating spermatogonia to generate meiotic Dicer conditional knockouts (DicerC-KO) [29]–[31]. Fertility was lost in some of these animals; genotyping of pups sired by fertile DicerC-KO mice revealed the presence of the undeleted DcrFl allele, indicating the incomplete deletion in these animals. Histological examination of DicerC-KO testis sections revealed the presence of highly abnormal seminiferous tubules with a high apoptotic index (Fig. 1E–F). Thus the impairment of Dicer function has major impact on post-mitotic male germ cell development.


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)

Expression and function of Dicer in adult spermatogenesis.(A) Domain structure of the Dicer protein is shown. The organization of the 5′ portion of Dicer locus is depicted. The targeting vector used for introduction of FlagHA2 into the Dicer locus and the schematic map of the targeted Dicer gene before and after Cre mediated-recombination are shown. Triangles represent loxP sites as indicated. Rectangles indicate the position of Neomycin (Neo) and Diptheria toxin A (DTA) selection marker genes. The SacI restriction sites are indicated as well as the respective Southern fragments detected by the 3′probe. A schematic diagram of the resulting FlagHA2-Dicer protein is shown. (B) Southern blot of tail derived SacI-digested DNA from wild-type and Dcr+/FH-Neo mice is shown with the 3′ probe indicated in A. (C) Western blot using anti-HA and anti-SMC1 antibodies on extracts from adult wild type and Dcr+/FH testis is shown. (D) Immunofluorescence using anti-HA and anti-γH2AX antibodies on Dcr+/FH testis germ cells from adult testis sections is shown. Scale bar = 10 µm. (E) Hematoxylin and eosin stained testis section from adult DcrCtl and DcrC-KO mice with representative tubules shown. Scale bars = 50 µm and 20 µm in the upper and lower panel, respectively. (F) Increased apoptosis in DcrC-KO testis. A TUNEL assay counterstained with DAPI is shown on testis sections from adult DcrCtl and DcrC-KO mice. The apoptotic cells stain in green. Scale bars = 50 µm and 10 µm in the upper and lower panel, respectively. Abbreviations: P, pachytene and RS, round spermatid. Representative images are shown from at least 3 mice analyzed in panels D–F.
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Related In: Results  -  Collection

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pgen-1004597-g001: Expression and function of Dicer in adult spermatogenesis.(A) Domain structure of the Dicer protein is shown. The organization of the 5′ portion of Dicer locus is depicted. The targeting vector used for introduction of FlagHA2 into the Dicer locus and the schematic map of the targeted Dicer gene before and after Cre mediated-recombination are shown. Triangles represent loxP sites as indicated. Rectangles indicate the position of Neomycin (Neo) and Diptheria toxin A (DTA) selection marker genes. The SacI restriction sites are indicated as well as the respective Southern fragments detected by the 3′probe. A schematic diagram of the resulting FlagHA2-Dicer protein is shown. (B) Southern blot of tail derived SacI-digested DNA from wild-type and Dcr+/FH-Neo mice is shown with the 3′ probe indicated in A. (C) Western blot using anti-HA and anti-SMC1 antibodies on extracts from adult wild type and Dcr+/FH testis is shown. (D) Immunofluorescence using anti-HA and anti-γH2AX antibodies on Dcr+/FH testis germ cells from adult testis sections is shown. Scale bar = 10 µm. (E) Hematoxylin and eosin stained testis section from adult DcrCtl and DcrC-KO mice with representative tubules shown. Scale bars = 50 µm and 20 µm in the upper and lower panel, respectively. (F) Increased apoptosis in DcrC-KO testis. A TUNEL assay counterstained with DAPI is shown on testis sections from adult DcrCtl and DcrC-KO mice. The apoptotic cells stain in green. Scale bars = 50 µm and 10 µm in the upper and lower panel, respectively. Abbreviations: P, pachytene and RS, round spermatid. Representative images are shown from at least 3 mice analyzed in panels D–F.
Mentions: The importance of post-transcriptional regulation of gene expression in spermatogenesis prompted us to examine the contribution of the miRNA pathway to this process. The RNase III Dicer catalyzes the last step of canonical miRNA biogenesis and thus its expression levels within testicular germ cell populations would be indicative of where this pathway or the biogenesis of miRNAs for current or later use would be important. Since antibodies against mouse Dicer that function for tissue immunofluorescence are lacking, we therefore generated a knock-in allele in mice that carry an N-terminal Flag-HA2 tagged Dicer (DcrFH) (Fig. 1A–C). The Flag-HA2 tag did not adversely impact on the function of Dicer as mice homozygous for the DcrFH allele are viable and fertile. Visualization in adult testis sections of Flag-HA2-Dicer with anti-HA antibodies revealed abundant expression of Dicer in the mitotic spermatogonia and the early meiotic stages of pre-leptotene and leptotene. Thereafter Dicer was up regulated in zygotene reaching a maximum expression in early pachytene spermatocytes (Fig. 1D). From mid-pachytene onwards Dicer was downregulated but still detected in the later stages of spermiogenesis (Fig. 1D). The expression pattern of Dicer would suggest a critical function for the miRNA pathway in meiosis as well as during haploid germ cell development. While non-canonical miRNA biogenesis pathways do exist, only a single miRNA (miR-451) has been shown to be Dicer independent [22]–[24]. In addition to miRNAs, the other Dicer products, the endogenous siRNAs, have thus far only been found in oocytes and ESCs [25]–[27]. While the failure to detect siRNAs in the male germ cells cannot formally exclude their presence therein, the loss of Dicer can more than likely be used to explore the function of the miRNA pathway in post-mitotic spermatogenesis. The importance of Dicer in early germ cell development was shown through its conditional ablation during early embryogenesis in primordial germ cells (PGCs) using the TNAP-Cre [28]. This loss of Dicer results in proliferative defects in PGCs with either absent or retarded spermatogenesis in adult seminiferous tubules [28]. To understand whether Dicer is required during meiosis, we combined the Dicer LoxP (DcrFl) allele with the Stra8Cre transgene that deletes in differentiating spermatogonia to generate meiotic Dicer conditional knockouts (DicerC-KO) [29]–[31]. Fertility was lost in some of these animals; genotyping of pups sired by fertile DicerC-KO mice revealed the presence of the undeleted DcrFl allele, indicating the incomplete deletion in these animals. Histological examination of DicerC-KO testis sections revealed the presence of highly abnormal seminiferous tubules with a high apoptotic index (Fig. 1E–F). Thus the impairment of Dicer function has major impact on post-mitotic male germ cell development.

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.Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes.

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