Microtubules (MTs) are core cytoskeletal filaments that play a vital role in spermatogenesis, including in germ cell division, spermatid remodelling and flagella formation. How MTs achieve this incredible diversity of function however is poorly understood. Emerging data suggests that posttranslational modifications to the MT surface, the 'tubulin code', are essential for MT function acting as instructional signposts for MT regulatory enzymes including MT severing enzymes. Herein we aimed to define a key aspect of the tubulin code, MT glutamylation, in spermatogenesis and the role of a key MT tubulin glutamylase, TTLL1.
Using antibodies targeting different glutamylation modifications, we first defined the precise tubulin glutamylation pattern during male germ cell development, revealing tubulin glutamylation is predominantly distributed in the later steps of spermatogenesis, with a majority localised to the manchette and the sperm axoneme.
To explore how tubulin glutamylation is encoded and its functions during spermatogenesis, we then characterised a TTLL1 whole-body knockout mouse model. Ttll1 null male mice were sterile, and our analysis revealed this was due to defects in sperm morphology and number. Daily sperm production were reduced by 35%, and epididymal sperm were significantly decreased. Moreover, histological analyses revealed TTLL1 KO sperm had abnormal head shapes and shortened or entirely absent sperm flagella. Immunostaining on testis sections and Western blots revealed significantly decreased broad-, poly- and beta tubulin mono-glutamylation. Together, our data reveals that TTLL1-dependent tubulin glutamylation is required for microtubule dynamic changes during spermatogenesis by playing a critical role in spermatid reshaping, notably in sperm tail formation and head shaping.