The naturally-occurring t haplotypes is an ideal model for studying activation and hyperactivation, two critical processes for successful fertilization. When homozygous, t haplotypes cause male sterility due in large part to the expression of a set of linked mutant alleles that impact negatively on flagellar function. This study is designed to compare four candidate protein function and localization in sperm based on predictions made from thorough computational analyses of relevant protein sequences.
Dnahc8, the first candidate for "curlicue" to be mapped, isolated, and characterized. DNAHC8 was shown to demonstrate restriction to the sperm flagellar principal piece. This finding made DNAHC8t an ideal candidate for the development of the chronic negative principal piece bend, also introduced a novel paradigm of outer dynein arm organization in mammalian sperm tails;
Tsga2, initially believed to be confined to the testis in male mice. Two major sperm isoforms were localized differentially in the sperm tail periaxonemal structures and in the anterior acrosome. In addition, bioinformatic analysis of the t-isoform strongly suggested that the highly conserved acid-rich regions (ARRs) in wild-type orthologs might govern Ca2+-induced conformational changes in the intervening MORN repeats, thus regulating TSGA2 isoform localization/function.
Tctex5, encoding an inhibitor of the testis/sperm-restricted serine/threonine protein phosphatase. PP1cγ2. TCTEX5t was shown to contain mutations in highly conserved residues that could potentially affect its ability to be phosphorylated, bind to PP1cγ2, and/or its stability. GST-pull down experiments suggested that TCTEX5t binds to PP1cγ2 as avidly as TCTEX5+, but western blot analysis demonstrated that TCTEX5t was significantly more unstable than TCTEX5 + without benzamidine.
Btbd9, encoding a BTB/POZ domain-carrying protein of unknown function. BTBD9 was shown as a 50 kDa in +/+ and t/t sperm, suggesting that BTBD9 played a role in axonemal organization and/or sperm motility. However, no differences between BTBD9+ and BTBD9t suggested that BTBD9t might not play a role in the "curlicue" phenotype.
In summary, the investigation of four linked t complex factors localizing to regions of the mouse sperm tail related to motility-associated signaling pathways has permitted their hypothetical integration at various points along these pathways, thus improving the current regulatory model of sperm motility.