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Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Tirmarche S, Kimura S, Sapey-Triomphe L, Sullivan W, Landmann F, Loppin B - G3 (Bethesda) (2014)

Bottom Line: During spermiogenesis, histones are massively replaced with protamines.A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile.Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.

View Article: PubMed Central - PubMed

Affiliation: Centre de Génétique et de Physiologie Moléculaire et Cellulaire - CNRS UMR 5534 - Université Claude Bernard Lyon1, 69100 Villeurbanne, France.

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Related in: MedlinePlus

Knocking-out Mst35Ba/b genes by homologous recombination. (A) Representation of the Mst35b locus before (wild-type; WT) and after (ΔMst35B, w+) “ends-out” targeting using the pW25 vector (Gong and Golic, 2003, 2004; details are available upon request). The ΔMst35B allele was obtained after Cre-mediated excision of the white cassette. The tandem protamine genes Mst35Ba and Mst35Bb are represented with their coding regions in pale blue. The dark blue hexagons representing termination codons in the six reading frames and the Lox sites (black triangles) are from the pW25 ends-out targeting vector. Arrowheads indicate the position of the primer pair used in polymerase chain reaction analyses. (B) Sequence alignment of Mst35Ba and Mst35Bb proteins. The peptide used to generate the rabbit antipeptide “NGI” polyclonal antibody is highlighted in green and other identical residues are shaded in gray. (C) Immunofluorescence staining of WT or ΔMst35B spermatid nuclei with the antipeptide “NGI” antibody. The antibody stains WT spermatid nuclei in late canoe stage but not more advanced nuclei (asterisk), which are too compacted for antibody penetration (Bonnefoy et al. 2007). No staining was observed in mutant spermatid nuclei at the late canoe stage. Bar: 10 µm. (D) Western blot analysis of testicular protein extracts using a rabbit antibody directed against a recombinant full-length Mst35Bb protein. Mst35Ba/b-specific bands are detected in WT but not in ΔMst35B extracts. The anti-α-Tubulin antibody was used as loading control.
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fig1: Knocking-out Mst35Ba/b genes by homologous recombination. (A) Representation of the Mst35b locus before (wild-type; WT) and after (ΔMst35B, w+) “ends-out” targeting using the pW25 vector (Gong and Golic, 2003, 2004; details are available upon request). The ΔMst35B allele was obtained after Cre-mediated excision of the white cassette. The tandem protamine genes Mst35Ba and Mst35Bb are represented with their coding regions in pale blue. The dark blue hexagons representing termination codons in the six reading frames and the Lox sites (black triangles) are from the pW25 ends-out targeting vector. Arrowheads indicate the position of the primer pair used in polymerase chain reaction analyses. (B) Sequence alignment of Mst35Ba and Mst35Bb proteins. The peptide used to generate the rabbit antipeptide “NGI” polyclonal antibody is highlighted in green and other identical residues are shaded in gray. (C) Immunofluorescence staining of WT or ΔMst35B spermatid nuclei with the antipeptide “NGI” antibody. The antibody stains WT spermatid nuclei in late canoe stage but not more advanced nuclei (asterisk), which are too compacted for antibody penetration (Bonnefoy et al. 2007). No staining was observed in mutant spermatid nuclei at the late canoe stage. Bar: 10 µm. (D) Western blot analysis of testicular protein extracts using a rabbit antibody directed against a recombinant full-length Mst35Bb protein. Mst35Ba/b-specific bands are detected in WT but not in ΔMst35B extracts. The anti-α-Tubulin antibody was used as loading control.

Mentions: In most animal species, the mature sperm nucleus is characterized by an extreme level of DNA compaction achieved after the massive replacement of somatic-type histones with sperm-specific nuclear basic proteins (SNBPs) (Lewis et al. 2003; Miller et al. 2010; Ward 2010; Kanippayoor et al. 2013). In mammals, the bulk of sperm chromatin is organized with two small protamines (Protamine 1 and 2) highly enriched in arginine residues (Balhorn 2007). Drosophila comprises at least three SNBPs: two paralogous protamine-like proteins, Mst35Ba and Mst35Bb, which are conserved among drosophilids, and the HILS1-related protein Mst77F (Russell and Kaiser 1993; Jayaramaiah Raja and Renkawitz-Pohl 2005; Alvi et al. 2013; Rathke et al. 2014). The almost identical Mst35Ba and Mst35Bb proteins are larger than mammalian protamines and are enriched in lysine residues. Mst35Ba/b proteins are incorporated in elongating spermatid nuclei at the late canoe stage and remain associated with mature sperm nuclei until fertilization (Jayaramaiah Raja and Renkawitz-Pohl 2005). Although the functions of Drosophila SNBPs remain poorly understood, Rathke et al. (2010) reported the surprising observation that Drosophila males homozygous for a genomic deficiency covering the Mst35B locus were fertile. This result was indeed unexpected when considering for instance the haploinsufficiency of mouse protamine genes for male fertility (Cho et al. 2001). However, according to Flybase (Flybase.org), the deficiency generated by Rathke et al. (2010) (named protΔ) is a 73.6-kb deletion that not only uncovers Mst35Ba and Mst35Bb but also removes four additional protein encoding genes (CG42682, CG15279, CG4480, CG15278) as well as three noncoding RNAs (CR43805, CR45727, CR45302). Furthermore, all these genes and noncoding RNAs are expressed in the adult testis or accessory glands, with the exception of CG15279, and transcripts of three of these genes (CG33309, CG4480, and CG15278) were detected in early spermatids (Flybase; Rathke et al. 2010). Because the simultaneous deletion of these other genetic elements could potentially interfere with a detailed functional analysis of Mst35B genes, we generated a precise deletion of the Mst35B locus by homologous recombination using the “Ends-Out” targeting technique (Gong and Golic 2003, 2004) (Figure 1A). The resulting allele, named ΔMst35B, eliminates a 5-kb genomic DNA fragment that only contains the Mst35Ba and Mst35Bb genes. To validate the elimination of these genes in the new deletion allele, we raised an antiserum against a peptide common to Mst35Ba and Mst35Bb proteins (Figure 1B). This antibody specifically stained late canoe stage spermatid nuclei of wild-type males but not those of ΔMst35B homozygous males (Figure 1C). At later stages of spermiogenesis, the highly compacted chromatin of spermatids is no longer accessible to antibodies (Bonnefoy et al. 2007), thus explaining the absence of staining beyond the canoe stage in wild-type testes. In addition, another anti-Mst35B antibody raised against the whole Mst35Bb recombinant protein allowed us to confirm the absence of Mst35B proteins from ΔMst35B testicular protein extracts (Figure 1D).


Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Tirmarche S, Kimura S, Sapey-Triomphe L, Sullivan W, Landmann F, Loppin B - G3 (Bethesda) (2014)

Knocking-out Mst35Ba/b genes by homologous recombination. (A) Representation of the Mst35b locus before (wild-type; WT) and after (ΔMst35B, w+) “ends-out” targeting using the pW25 vector (Gong and Golic, 2003, 2004; details are available upon request). The ΔMst35B allele was obtained after Cre-mediated excision of the white cassette. The tandem protamine genes Mst35Ba and Mst35Bb are represented with their coding regions in pale blue. The dark blue hexagons representing termination codons in the six reading frames and the Lox sites (black triangles) are from the pW25 ends-out targeting vector. Arrowheads indicate the position of the primer pair used in polymerase chain reaction analyses. (B) Sequence alignment of Mst35Ba and Mst35Bb proteins. The peptide used to generate the rabbit antipeptide “NGI” polyclonal antibody is highlighted in green and other identical residues are shaded in gray. (C) Immunofluorescence staining of WT or ΔMst35B spermatid nuclei with the antipeptide “NGI” antibody. The antibody stains WT spermatid nuclei in late canoe stage but not more advanced nuclei (asterisk), which are too compacted for antibody penetration (Bonnefoy et al. 2007). No staining was observed in mutant spermatid nuclei at the late canoe stage. Bar: 10 µm. (D) Western blot analysis of testicular protein extracts using a rabbit antibody directed against a recombinant full-length Mst35Bb protein. Mst35Ba/b-specific bands are detected in WT but not in ΔMst35B extracts. The anti-α-Tubulin antibody was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232549&req=5

fig1: Knocking-out Mst35Ba/b genes by homologous recombination. (A) Representation of the Mst35b locus before (wild-type; WT) and after (ΔMst35B, w+) “ends-out” targeting using the pW25 vector (Gong and Golic, 2003, 2004; details are available upon request). The ΔMst35B allele was obtained after Cre-mediated excision of the white cassette. The tandem protamine genes Mst35Ba and Mst35Bb are represented with their coding regions in pale blue. The dark blue hexagons representing termination codons in the six reading frames and the Lox sites (black triangles) are from the pW25 ends-out targeting vector. Arrowheads indicate the position of the primer pair used in polymerase chain reaction analyses. (B) Sequence alignment of Mst35Ba and Mst35Bb proteins. The peptide used to generate the rabbit antipeptide “NGI” polyclonal antibody is highlighted in green and other identical residues are shaded in gray. (C) Immunofluorescence staining of WT or ΔMst35B spermatid nuclei with the antipeptide “NGI” antibody. The antibody stains WT spermatid nuclei in late canoe stage but not more advanced nuclei (asterisk), which are too compacted for antibody penetration (Bonnefoy et al. 2007). No staining was observed in mutant spermatid nuclei at the late canoe stage. Bar: 10 µm. (D) Western blot analysis of testicular protein extracts using a rabbit antibody directed against a recombinant full-length Mst35Bb protein. Mst35Ba/b-specific bands are detected in WT but not in ΔMst35B extracts. The anti-α-Tubulin antibody was used as loading control.
Mentions: In most animal species, the mature sperm nucleus is characterized by an extreme level of DNA compaction achieved after the massive replacement of somatic-type histones with sperm-specific nuclear basic proteins (SNBPs) (Lewis et al. 2003; Miller et al. 2010; Ward 2010; Kanippayoor et al. 2013). In mammals, the bulk of sperm chromatin is organized with two small protamines (Protamine 1 and 2) highly enriched in arginine residues (Balhorn 2007). Drosophila comprises at least three SNBPs: two paralogous protamine-like proteins, Mst35Ba and Mst35Bb, which are conserved among drosophilids, and the HILS1-related protein Mst77F (Russell and Kaiser 1993; Jayaramaiah Raja and Renkawitz-Pohl 2005; Alvi et al. 2013; Rathke et al. 2014). The almost identical Mst35Ba and Mst35Bb proteins are larger than mammalian protamines and are enriched in lysine residues. Mst35Ba/b proteins are incorporated in elongating spermatid nuclei at the late canoe stage and remain associated with mature sperm nuclei until fertilization (Jayaramaiah Raja and Renkawitz-Pohl 2005). Although the functions of Drosophila SNBPs remain poorly understood, Rathke et al. (2010) reported the surprising observation that Drosophila males homozygous for a genomic deficiency covering the Mst35B locus were fertile. This result was indeed unexpected when considering for instance the haploinsufficiency of mouse protamine genes for male fertility (Cho et al. 2001). However, according to Flybase (Flybase.org), the deficiency generated by Rathke et al. (2010) (named protΔ) is a 73.6-kb deletion that not only uncovers Mst35Ba and Mst35Bb but also removes four additional protein encoding genes (CG42682, CG15279, CG4480, CG15278) as well as three noncoding RNAs (CR43805, CR45727, CR45302). Furthermore, all these genes and noncoding RNAs are expressed in the adult testis or accessory glands, with the exception of CG15279, and transcripts of three of these genes (CG33309, CG4480, and CG15278) were detected in early spermatids (Flybase; Rathke et al. 2010). Because the simultaneous deletion of these other genetic elements could potentially interfere with a detailed functional analysis of Mst35B genes, we generated a precise deletion of the Mst35B locus by homologous recombination using the “Ends-Out” targeting technique (Gong and Golic 2003, 2004) (Figure 1A). The resulting allele, named ΔMst35B, eliminates a 5-kb genomic DNA fragment that only contains the Mst35Ba and Mst35Bb genes. To validate the elimination of these genes in the new deletion allele, we raised an antiserum against a peptide common to Mst35Ba and Mst35Bb proteins (Figure 1B). This antibody specifically stained late canoe stage spermatid nuclei of wild-type males but not those of ΔMst35B homozygous males (Figure 1C). At later stages of spermiogenesis, the highly compacted chromatin of spermatids is no longer accessible to antibodies (Bonnefoy et al. 2007), thus explaining the absence of staining beyond the canoe stage in wild-type testes. In addition, another anti-Mst35B antibody raised against the whole Mst35Bb recombinant protein allowed us to confirm the absence of Mst35B proteins from ΔMst35B testicular protein extracts (Figure 1D).

Bottom Line: During spermiogenesis, histones are massively replaced with protamines.A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile.Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.

View Article: PubMed Central - PubMed

Affiliation: Centre de Génétique et de Physiologie Moléculaire et Cellulaire - CNRS UMR 5534 - Université Claude Bernard Lyon1, 69100 Villeurbanne, France.

Show MeSH
Related in: MedlinePlus