Tubulin proteins are fundamental constituents of the cytoskeleton. Despite this, the function of the lesser-known subtypes, delta (TUBD1) and epsilon (TUBE1) tubulin, remain poorly understood in living mammalian systems. Our previous work has shown that TUBD1 and TUBE1 are highly enriched in the testis, particularly during meiosis and haploid germ cell development, and suggested that TUBD1 and TUBE1 are targeted by the microtubule severing enzyme, KATNAL2, to promote timely microtubule-dependent sperm head shaping [1]. Herein, we examined the function of TUBD1 and TUBE1 at complex microtubule structures during male germ cell development to identify their individual contributions to the microtubule cytoskeleton and advance our understanding of microtubule-dependent sperm head shaping in the mouse.
Using Stra8-Cre to generate pre-meiotic conditional germ cell knockouts for each of Tubd1 and Tube1, we reveal that both exotic tubulins are essential for male fertility and their loss results in a >99% reduction in functional sperm produced. At a cellular level, we show that TUBD1 and TUBE1 are required for the maintenance of spindle polarity, chromosome segregation and cytokinesis, and in the case of TUBD1, for stabilization of the meiotic kinetochore between metaphase and anaphase. We also reveal that TUBD1 and TUBE1 function to shape the sperm head at the microtubule-based nuclear shaping apparatus, the manchette, as evidenced by the formation of over-constricted sperm nuclei in their absence. Finally, we show that TUBD1 and TUBE1 are targets of key katanin microtubule severing enzyme action at the manchette and put forward an updated mechanistic model of manchette regulation. Overall, our results demonstrate that TUBD1 and TUBE1 are indispensable for spermatogenesis as they play key roles at complex microtubule structures during male germ cell development.