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A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome.

Bhattacharya R, Devi MS, Dhople VM, Jesudasan RA - BMC Cell Biol. (2013)

Bottom Line: Novel proteins are still being reported from acrosome.MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome.The role of Y chromosome in the regulation of this complex is however not clear from the current study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. rachel@ccmb.res.in.

ABSTRACT

Background: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.

Results: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.

Conclusions: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

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Comparative analyses of normal and subfertile mice using MAST antibody. (A) Comparative Western blotting with testes and sperm lysates from normal (XY) and subfertile (XYq-del) mice probed with MAST antibody shows presence of a 20 kDa protein in comparable quantities in the testis lysates of both. MAST was absent in the subfertile sperm proteome. Tubulin antibody served as the loading control. (B) Shows cytoplasmic localization of the protein in the entire cell types of normal (XY) testis. In the subfertile (Yq-del) testis there is a substantial reduction in signal, indicating either reduced expression or alternatively lack of localization to the correct locus. Pre-immune serum was used as negative control and PI was the counterstain. Magnification - 100× and scale bar = 25 μm. (C) Comparative immunolocalization in sperms: Immunolocalization with antibody to MAST protein showed that in normal (XY) sperms the protein localizes to the acrosome and sperm tail. In sperms from subfertile (XYq-del) mice a very faint signal is observed only in the midpiece of sperm tail. DAPI was used as the counterstain for nucleus and pseudo color red was used for representation. Magnification - 63×; scale bar = 10 μm.
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Figure 5: Comparative analyses of normal and subfertile mice using MAST antibody. (A) Comparative Western blotting with testes and sperm lysates from normal (XY) and subfertile (XYq-del) mice probed with MAST antibody shows presence of a 20 kDa protein in comparable quantities in the testis lysates of both. MAST was absent in the subfertile sperm proteome. Tubulin antibody served as the loading control. (B) Shows cytoplasmic localization of the protein in the entire cell types of normal (XY) testis. In the subfertile (Yq-del) testis there is a substantial reduction in signal, indicating either reduced expression or alternatively lack of localization to the correct locus. Pre-immune serum was used as negative control and PI was the counterstain. Magnification - 100× and scale bar = 25 μm. (C) Comparative immunolocalization in sperms: Immunolocalization with antibody to MAST protein showed that in normal (XY) sperms the protein localizes to the acrosome and sperm tail. In sperms from subfertile (XYq-del) mice a very faint signal is observed only in the midpiece of sperm tail. DAPI was used as the counterstain for nucleus and pseudo color red was used for representation. Magnification - 63×; scale bar = 10 μm.

Mentions: A strain of mutant mouse wherein there is a 2/3rd deletion of the heterochromatic long arm of the Y chromosome is deleted shows sperm abnormalities and subfertility [12]. Comparative immunoblotting using testis and sperm lysates from normal and Yq-del mice showed that MAST was present in testes of normal and Yq-del mice in equal quantities; MAST was present in sperm lysate from wild type mice, but absent in sperms from Yq-del mice (Figure 5A). Comparative immunolocalization studies on testes sections of wild type and Yq-del mice showed that in Yq–del testes signal was barely present in cells of the basal lamina and round spermatids and was substantially reduced in elongated spermatids (Figure 5B). Yq-del sperms showed only a faint signal restricted to midpiece (Figure 5C). Thus MAST was found to be absent in Yq-del sperms by western blot analysis and immunolocalization. Western blot using normal and Yq-del testes lysates showed equal expression of MAST, but immunolocalization onto Yq-del testis sections showed a drastic reduction in signal in comparison to that of the wild type. As there was no difference in the intensity of the signal between the normal and Yq-del mice by western blot analysis, these results indicate that there is no reduction in expression of MAST in Yq-del testis; the reduction in intensity in immunolocalization studies might reflect failure of localization to the correct locus rather than a quantitative difference in expression.


A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome.

Bhattacharya R, Devi MS, Dhople VM, Jesudasan RA - BMC Cell Biol. (2013)

Comparative analyses of normal and subfertile mice using MAST antibody. (A) Comparative Western blotting with testes and sperm lysates from normal (XY) and subfertile (XYq-del) mice probed with MAST antibody shows presence of a 20 kDa protein in comparable quantities in the testis lysates of both. MAST was absent in the subfertile sperm proteome. Tubulin antibody served as the loading control. (B) Shows cytoplasmic localization of the protein in the entire cell types of normal (XY) testis. In the subfertile (Yq-del) testis there is a substantial reduction in signal, indicating either reduced expression or alternatively lack of localization to the correct locus. Pre-immune serum was used as negative control and PI was the counterstain. Magnification - 100× and scale bar = 25 μm. (C) Comparative immunolocalization in sperms: Immunolocalization with antibody to MAST protein showed that in normal (XY) sperms the protein localizes to the acrosome and sperm tail. In sperms from subfertile (XYq-del) mice a very faint signal is observed only in the midpiece of sperm tail. DAPI was used as the counterstain for nucleus and pseudo color red was used for representation. Magnification - 63×; scale bar = 10 μm.
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Figure 5: Comparative analyses of normal and subfertile mice using MAST antibody. (A) Comparative Western blotting with testes and sperm lysates from normal (XY) and subfertile (XYq-del) mice probed with MAST antibody shows presence of a 20 kDa protein in comparable quantities in the testis lysates of both. MAST was absent in the subfertile sperm proteome. Tubulin antibody served as the loading control. (B) Shows cytoplasmic localization of the protein in the entire cell types of normal (XY) testis. In the subfertile (Yq-del) testis there is a substantial reduction in signal, indicating either reduced expression or alternatively lack of localization to the correct locus. Pre-immune serum was used as negative control and PI was the counterstain. Magnification - 100× and scale bar = 25 μm. (C) Comparative immunolocalization in sperms: Immunolocalization with antibody to MAST protein showed that in normal (XY) sperms the protein localizes to the acrosome and sperm tail. In sperms from subfertile (XYq-del) mice a very faint signal is observed only in the midpiece of sperm tail. DAPI was used as the counterstain for nucleus and pseudo color red was used for representation. Magnification - 63×; scale bar = 10 μm.
Mentions: A strain of mutant mouse wherein there is a 2/3rd deletion of the heterochromatic long arm of the Y chromosome is deleted shows sperm abnormalities and subfertility [12]. Comparative immunoblotting using testis and sperm lysates from normal and Yq-del mice showed that MAST was present in testes of normal and Yq-del mice in equal quantities; MAST was present in sperm lysate from wild type mice, but absent in sperms from Yq-del mice (Figure 5A). Comparative immunolocalization studies on testes sections of wild type and Yq-del mice showed that in Yq–del testes signal was barely present in cells of the basal lamina and round spermatids and was substantially reduced in elongated spermatids (Figure 5B). Yq-del sperms showed only a faint signal restricted to midpiece (Figure 5C). Thus MAST was found to be absent in Yq-del sperms by western blot analysis and immunolocalization. Western blot using normal and Yq-del testes lysates showed equal expression of MAST, but immunolocalization onto Yq-del testis sections showed a drastic reduction in signal in comparison to that of the wild type. As there was no difference in the intensity of the signal between the normal and Yq-del mice by western blot analysis, these results indicate that there is no reduction in expression of MAST in Yq-del testis; the reduction in intensity in immunolocalization studies might reflect failure of localization to the correct locus rather than a quantitative difference in expression.

Bottom Line: Novel proteins are still being reported from acrosome.MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome.The role of Y chromosome in the regulation of this complex is however not clear from the current study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. rachel@ccmb.res.in.

ABSTRACT

Background: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.

Results: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.

Conclusions: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

Show MeSH
Related in: MedlinePlus