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Oocytes spend the majority of their lifetime in a primordial, dormant state. Unlike many somatic cell types and mature oocytes, the cellular and molecular biology of primordial oocytes is largely unexplored. Yet, studying their cellular biology is necessary to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age.A hallmark of primordial oocytes in many species is the Balbiani body, a non-membrane bound compartment that contains the majority of mitochondria in the oocyte cytoplasm. The Balbiani body has been proposed to be essential for maintaining mitochondria in a healthy state during long-lasting dormancy, however, the architecture and function of the mammalian Balbiani body remains unknown.Here, we develop enabling methods for live-imaging based comparative characterization of Xenopus, mouse and human primordial oocytes. We show that primordial oocytes in all three vertebrate species contain active mitochondria, Golgi apparatus and lysosomes. We further demonstrate that human and Xenopus oocytes have a Balbiani body characterized by a dense accumulation of mitochondria in their cytoplasm. However, despite previous reports, we did not find a Balbiani body in mouse oocytes. Instead, we demonstrate what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi apparatus that is not functionally associated with oocyte dormancy. Our work provides the first insights into the organisation of the cytoplasm in mammalian primordial oocytes, and clarifies relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.

Selection against deleterious mitochondrial mutations is facilitated by germline processes, lowering the risk of genetic diseases. How selection works is disputed: experimental data are conflicting and previous modelling work has not clarified the issues. Here we develop computational and evolutionary models that compare the outcome of selection at the level of individuals, cells and mitochondria. Using realisticde novomutation rates and germline development parameters, the evolutionary model predicts the observed prevalence of mitochondrial mutations and diseases in human populations. We show the importance of organelle-level selection, seen in the selective pooling of mitochondria into the Balbiani body, in achieving high-quality mitochondria at extreme ploidy in mature oocytes. Alternative mechanisms debated in the literature, bottlenecks and follicular atresia, are unlikely to account for the clinical data, because neither process effectively eliminates mitochondrial mutations under realistic conditions. Our findings explain the major features of female germline architecture, notably the longstanding paradox of over-proliferation of primordial germ cells followed by massive loss. The near-universality of these processes across animal taxa makes sense in light of the need to maintain mitochondrial quality at extreme ploidy in mature oocytes, in the absence of sex and recombination.

Fragmentation of the Balbiani body inXenopusoocytes engenders a region extending from the germinal vesicle (GV) towards the vegetal pole that is enriched in mitochondria. This area is later transversed by RNA that is being localized to the vegetal cortex. Inhibition of mitochondrial ATP synthesis prevents the perinuclear formation of these RNA transport granules that can be reversed by the nonhydrolyzable ATP analog, adenosine 5’-(βγ-imido) triphosphate. The protein composition and sensitivity of the transport granules to hexanediol indicate that they are liquid phase condensates. Mitochondria in the remnants of the Balbiani body produce a region of elevated ATP that appears to act as a hydrotrope to support the perinuclear phase transition leading to granule formation.SummaryMitochondria in the remnants of the Balbiani body produce elevated levels of ATP required for the formation of liquid phase condensates containing RNA transport granules.

In mammalian females, the transition between quiescent primordial follicles and follicular development is critical for maintaining ovarian function and reproductive longevity. In primary oocytes of mouse quiescent primordial follicles, Golgi complexes are organized into a spherical structure, the Balbiani body. Here, we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1A. Proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological disassembly of the B-body triggers translation of stored mRNAs and activates primordial follicles in culture and in vivo mouse model. Thus, primordial follicle quiescence is maintained by the B-body, and translationally inactive B-body-stored mRNAs may be regulated by 5’-capping.

SummaryIn recent years, it has become clear that phase separation represents an important class of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures range from a single large aggregate (Bb), to a dispersed state and back to relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts directly with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents a new mechanism for how prion-like protein-aggregations can be regulated and highlights the biological relevance of such regulatory events.

Xenopus oocytes provide an excellent model system for understanding the cis-elements and protein factors that carry out mRNA localization in vertebrate cells. More than 20 mRNAs have been identified that localize to the vegetal cortex during stages II–IV of oogenesis. The earliest localizing RNAs are presorted to a subcellular structure, the Balbiani body (also called the mitochondrial cloud in Xenopus), of stage I oocytes prior to entering the vegetal cortex. While some evidence has suggested that diffusion drives RNA localization to the Balbiani body, a role for temperature and metabolic energy in this process has not been explored. To address this issue, we developed a quantitative assay to monitor RNA localization in stage I oocytes. Here we show that the rate of RNA accumulation to the Balbiani body is highly dependent on temperature and the intracellular concentration of ATP. In fact, while ATP depletion severely impairs RNA localization, increasing the intracellular concentration of ATP by a factor of two doubles the localization rate, indicating that ATP is limiting under normal conditions. We also show that RNA localization in stage I oocytes is reduced by inhibition of kinesin II, and that the Xcat-2 RNA localization element recruits kinesin II to the Balbiani body. We conclude from these studies that the energy state of the cell regulates the rate of RNA localization to the Balbiani body and that this process, at least to some extent, involves kinesin II.