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LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Liu Y, DeBoer K, de Kretser DM, O'Donnell L, O'Connor AE, Merriner DJ, Okuda H, Whittle B, Jans DA, Efthymiadis A, McLachlan RI, Ormandy CJ, Goodnow CC, Jamsai D, O'Bryan MK - PLoS Genet. (2015)

Bottom Line: Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette.Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2.Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Australia.

ABSTRACT
Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

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LRGUK-1 is highly expressed in the testis and the localizes to the acrosome-acroplaxoneme-manchette-tail network.(A) Quantitative PCR analysis of Lrguk-1 mRNA expression in various tissues (B) and during the establishment of the first wave of spermatogenesis n = 3. Expression was normalised against Ppia expression and shown as mean±SEM. (C) The localization of LRGUK (red) in purified round spermatids, scale bar = 2μm. Acrosomes were visualised by PNA (green). (D) The localization of LRGUK-1 (red) in purified elongating spermatids, co-labelled with α-tubulin (green) to mark the manchette (scale bar = 2 μm). The red arrowhead indicates LRGUK localization at the leading edge of the acrosome (E) The localization of LRGUK (red) within caudal epididymal sperm, co-labelled with acetylated tubulin (Ac, green) to mark the axoneme (upper panel) and sperm basal body (upper and lower panel). Red arrowheads denote LRGUK localization in the sperm basal body. Scale bar = 4 μm. In all images, nuclei were labelled with DAPI (blue). Please see S3 Fig. for negative control images.
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pgen.1005090.g003: LRGUK-1 is highly expressed in the testis and the localizes to the acrosome-acroplaxoneme-manchette-tail network.(A) Quantitative PCR analysis of Lrguk-1 mRNA expression in various tissues (B) and during the establishment of the first wave of spermatogenesis n = 3. Expression was normalised against Ppia expression and shown as mean±SEM. (C) The localization of LRGUK (red) in purified round spermatids, scale bar = 2μm. Acrosomes were visualised by PNA (green). (D) The localization of LRGUK-1 (red) in purified elongating spermatids, co-labelled with α-tubulin (green) to mark the manchette (scale bar = 2 μm). The red arrowhead indicates LRGUK localization at the leading edge of the acrosome (E) The localization of LRGUK (red) within caudal epididymal sperm, co-labelled with acetylated tubulin (Ac, green) to mark the axoneme (upper panel) and sperm basal body (upper and lower panel). Red arrowheads denote LRGUK localization in the sperm basal body. Scale bar = 4 μm. In all images, nuclei were labelled with DAPI (blue). Please see S3 Fig. for negative control images.

Mentions: qPCR analysis revealed that Lrguk-1 mRNA was highly enriched in the testis compared to other adult tissues (Fig. 3A). An analysis of mouse testes taken at defined periods during the establishment of the first wave of spermatogenesis also revealed that Lrguk-1 mRNA was detectable at low levels from birth, up-regulated at day 14 coincident with the appearance of pachytene spermatocytes, then maximal from day 18 coincident with the appearance of haploid germ cells (Fig. 3B). This result was suggestive of Lrguk-1 being predominantly expressed in haploid germ cells. During spermatid development, and using an antibody that should detect all of LRGUK1–3, LRGUK protein was initially localised to a supra-nuclear region of round spermatids, and was particularly evident at the leading edge of the developing acrosome and acroplaxome (Fig. 3C). As maturation proceeded and nuclear elongation initiated, LRGUK moved distally to ultimately reside on the microtubules of the manchette (Fig. 3D and S2 Fig.). LRGUK was also evident in the sperm basal body and the sperm tail (Fig. 3E). These data, and the abnormal sperm head and tail morphology in LrgukKaos/Kaos germ cells, suggested that LRGUK-1 has a role in acrosome and tail biogenesis. We note that LRGUK protein was not detectable in our hands in spermatocytes. At present it is unknown if this was due to a lack of abundance or a translational delay as is often seen in spermiogenic genes [23].


LRGUK-1 is required for basal body and manchette function during spermatogenesis and male fertility.

Liu Y, DeBoer K, de Kretser DM, O'Donnell L, O'Connor AE, Merriner DJ, Okuda H, Whittle B, Jans DA, Efthymiadis A, McLachlan RI, Ormandy CJ, Goodnow CC, Jamsai D, O'Bryan MK - PLoS Genet. (2015)

LRGUK-1 is highly expressed in the testis and the localizes to the acrosome-acroplaxoneme-manchette-tail network.(A) Quantitative PCR analysis of Lrguk-1 mRNA expression in various tissues (B) and during the establishment of the first wave of spermatogenesis n = 3. Expression was normalised against Ppia expression and shown as mean±SEM. (C) The localization of LRGUK (red) in purified round spermatids, scale bar = 2μm. Acrosomes were visualised by PNA (green). (D) The localization of LRGUK-1 (red) in purified elongating spermatids, co-labelled with α-tubulin (green) to mark the manchette (scale bar = 2 μm). The red arrowhead indicates LRGUK localization at the leading edge of the acrosome (E) The localization of LRGUK (red) within caudal epididymal sperm, co-labelled with acetylated tubulin (Ac, green) to mark the axoneme (upper panel) and sperm basal body (upper and lower panel). Red arrowheads denote LRGUK localization in the sperm basal body. Scale bar = 4 μm. In all images, nuclei were labelled with DAPI (blue). Please see S3 Fig. for negative control images.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363142&req=5

pgen.1005090.g003: LRGUK-1 is highly expressed in the testis and the localizes to the acrosome-acroplaxoneme-manchette-tail network.(A) Quantitative PCR analysis of Lrguk-1 mRNA expression in various tissues (B) and during the establishment of the first wave of spermatogenesis n = 3. Expression was normalised against Ppia expression and shown as mean±SEM. (C) The localization of LRGUK (red) in purified round spermatids, scale bar = 2μm. Acrosomes were visualised by PNA (green). (D) The localization of LRGUK-1 (red) in purified elongating spermatids, co-labelled with α-tubulin (green) to mark the manchette (scale bar = 2 μm). The red arrowhead indicates LRGUK localization at the leading edge of the acrosome (E) The localization of LRGUK (red) within caudal epididymal sperm, co-labelled with acetylated tubulin (Ac, green) to mark the axoneme (upper panel) and sperm basal body (upper and lower panel). Red arrowheads denote LRGUK localization in the sperm basal body. Scale bar = 4 μm. In all images, nuclei were labelled with DAPI (blue). Please see S3 Fig. for negative control images.
Mentions: qPCR analysis revealed that Lrguk-1 mRNA was highly enriched in the testis compared to other adult tissues (Fig. 3A). An analysis of mouse testes taken at defined periods during the establishment of the first wave of spermatogenesis also revealed that Lrguk-1 mRNA was detectable at low levels from birth, up-regulated at day 14 coincident with the appearance of pachytene spermatocytes, then maximal from day 18 coincident with the appearance of haploid germ cells (Fig. 3B). This result was suggestive of Lrguk-1 being predominantly expressed in haploid germ cells. During spermatid development, and using an antibody that should detect all of LRGUK1–3, LRGUK protein was initially localised to a supra-nuclear region of round spermatids, and was particularly evident at the leading edge of the developing acrosome and acroplaxome (Fig. 3C). As maturation proceeded and nuclear elongation initiated, LRGUK moved distally to ultimately reside on the microtubules of the manchette (Fig. 3D and S2 Fig.). LRGUK was also evident in the sperm basal body and the sperm tail (Fig. 3E). These data, and the abnormal sperm head and tail morphology in LrgukKaos/Kaos germ cells, suggested that LRGUK-1 has a role in acrosome and tail biogenesis. We note that LRGUK protein was not detectable in our hands in spermatocytes. At present it is unknown if this was due to a lack of abundance or a translational delay as is often seen in spermiogenic genes [23].

Bottom Line: Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette.Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2.Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Australia.

ABSTRACT
Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

Show MeSH
Related in: MedlinePlus