![]() Western blot analysis confirmed RSPH6A expression in the testis ( Fig. 1C). To examine RSPH6A protein expression levels, we generated an antibody that recognized amino acid residues 145-163. As mentioned previously, Rsph1 KO mice are male infertile ( Tokuhiro et al., 2008) but our RT-PCR results show strong expression of Rsph1 in the lung, ovary, brain and testis ( Fig. 1B). Compared with other RS head genes, Rsph6a is one that is strongly enriched in the testis. This analysis revealed that while Rsph6a is abundant in the testis, it also has minimal expression in the lungs and thymus, in contrast with the human expression pattern. To determine the expression pattern in mice, RT-PCR of Rsph6a as well as the other RS head genes in multi-tissues from adult mice was conducted ( Fig. 1B). Quantitative RT-PCR reveals that Rsph6a is testis-specific in humans ( Kott et al., 2013). In this KO mouse line, spermatid formation was abnormal, as shown by deformed heads and stunted flagellum formation ( Tokuhiro et al., 2008). However, there is currently only one RS head KO mouse model – Rsph1 KO – that has been reported infertile. In addition to ciliary motility defects, PCD patients can be infertile because of abnormal flagella. Loss-of-function mutations in RSPH1 showed a similar phenotype, with abnormal axoneme structures such as defects in the RS and central pair of microtubules ( Kott et al., 2013 Knowles et al., 2014). Mouse knockout (KO) models confirm that RSPH4A is essential for normal ciliary motility ( Shinohara et al., 2015). Human patients with mutations in RSPH4A and RSPH9 had PCD due to abnormalities in the RS and central microtubular pair ( Castleman et al., 2009). PCD is a condition that shows abnormal cilia movement, often leading to chronic respiratory tract infections and abnormally positioned internal organs. In mammals, mutations in RS head proteins have been linked to primary ciliary dyskinesia (PCD) ( Frommer et al., 2015). There are five RS head proteins found in Chlamydomonas (RSP1, -4, -6, -9 and -10) and all of these proteins are conserved in mice and humans (RSPH1, -4A, -6A, -9 and -10B, respectively). Using these mutation analyses, research related to RS head proteins and its impact on mammalian systems have been explored. The temperature-sensitive mutant pf-26ts lacks RSP6, which is also localized in the spoke head, and mutant Chlamydomonas was found to be paralyzed when introduced into a restrictive temperature. Chlamydomonas mutants pf-1 and pf-17, missing RSP4 and RSP9, respectively, were deficient of all RS head proteins but not stalk proteins, causing paralysis ( Huang et al., 1981). Additional mutant lines were found to further understand the role of specific protein components within the RSs. Chlamydomonas mutants that lack the entire RS become paralyzed and no longer possess the ability to propagate ( Witman et al., 1978). RSs regulate flagellar motility, as elucidated by Chlamydomonas mutant strains. Purification of the RS complex resulted in the identification of 23 Chlamydomonas flagellar RS proteins ( Piperno et al., 1981 Huang et al., 1981 Yang et al., 2006). Through experiments on Chlamydomonas reinhardtii, much about the RS proteins has been uncovered. This article has an associated First Person interview with the first author of the paper.įirst characterized in sea urchins ( Afzelius, 1959), the RS is a T-shaped protein complex that extends from the doublet microtubules towards the central pair of single microtubules, possessing an elongated ‘stalk’ that is bound to the doublet microtubules and terminates at a bulbous ‘head’. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. Manchette removal is also impaired in the KO testis. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. ![]()
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