A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope via microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the “zygotene chromosomal bouquet”. Here, we identified the “zygotene cilium” in oocytes. This cilium provides a cable system for the bouquet machinery, extending throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies, and suggests that cilia can control chromosomal dynamics.

Molecular condensates provide new paradigms in biology, but their cellular regulation is unclear. Condensates undergo phase separation, decreasing their solubility and compartmentalizing their content. In vertebrate oocytes, RNA-protein (RNP) granules form condensates by phase separation, but the underlying mechanisms are unknown. RNP granules localize to the Balbiani body (Bb), a conserved membraneless organelle, that establishes oocyte polarity. Bb loss results in symmetrical eggs and embryonic lethality. Bb granules aggregate around the centrosome in a nuclear cleft, prior to assembling the mature structure. The Bucky ball (Buc) protein nucleates Bb granules and is essential for Bb formation. Howe ver, the dynamics, mechanisms, and regulation of Bb granule nucleation are unclear. While the mature Bb structure was shown to be a rigid, amyloid-like, phase-separated structure in Xenopus, the early phase separation dynamics prior to maturation are completely unknown. Here, by live, genetic, super-resolution microscopy, and FRAP analyses in zebrafish ovaries, we establish that Buc phase-separates Bb granules and that microtubules play multiple stepwise roles in controlling Buc phase separation and Bb formation specifically at the early nuclear cleft stages. We show that Buc first phase-separates into dynamic liquid droplet-like granules that fuse to form the main Bb aggregate. We demonstrate that early aggregated Buc exhibited dynamic turnover and that this turnover requires dynein-mediated trafficking of Buc on a transient lattice of microtubules that we identified. At later stages, microtubules encapsulated the Bb, indicating a structural role. Thus, microtubules organize multiple steps in Bb condensation. Moreover, in the mature Bb, Buc was stable and required for Bb amyloid formation, finalizing Bb condensation. We found for the first time by live imaging, ThT-positive presumptive amyloid b-sheets in the mature zebrafish Bb, that we re absent in buc-/- oocytes. Thus, providing the first genetic evidence for Buc dependent formation of presumptive amyloid fibrils in the Bb. Molecular condensation is often viewe d as a self-assembly process. Here, we propose a novel paradigm for the cellular control over condensation mechanisms by microtubules in development. The regulation of Bb assembly and disassembly in the context of phase separation allows studying these mechanisms in physiological conditions, advancing their understanding in neurodegenerative disease and female reproduction.

Meiosis is a cellular program essential for the production of haploid gametes. A hallmark of meiosis is chromosomal pairing via synaptonemal complexes, and a major focus traditionally has been to understand synaptonemal complex formation. However, chromosomal pairing also depends on cytoplasmic counterparts that tether and rotate telomeres on the nuclear envelope, shuffling chromosomes and mechanically driving their homology searches. Rotating telomeres slide on perinuclear microtubules and are ultimately pulled towards the centrosome, forming a configuration called the zygotene chromosomal bouquet. The bouquet is universally conserved and is essential for pairing and fertility. However, despite its discovery in 1900, how the cytoplasmic counterparts of bouquet formation are mechanically regulated has remained enigmatic. Here, by studying zebrafish oogenesis, we report and comprehensively characterize a previously unrecognized cilium in oocytes, which we term the zygotene cilium. We show that the zygotene cilium specifically connects to the bouquet centrosome and constitutes a cable system of the cytoplasmic bouquet machinery. Farther, zygotene cilia extend throughout the germline cyst, a conserved cellular organization of germ cells. By analyzing multiple ciliary mutants, we demonstrate that the zygotene cilium is essential for chromosomal pairing, germ cell morphogenesis, ovarian development and fertility. We further show that the zygotene cilium is conserved in both male meiosis in zebrafish, as well as in mammalian oogenesis. Our work uncovers the novel concept of a cilium as a critical player in meiosis and sheds new light on reproduction phenotypes in ciliopathies. Furthermore, most cells in metazoans are ciliated and exhibit specific nuclear dynamics. We propose a cellular paradigm that cilia can control chromosomal dynamics.