Gabriele Zaffagnini

Mammalian oocytes undergo an extended growth phase, during which transcription gradually declines. To sustain meiotic divisions and early embryonic development, growing oocytes must accumulate maternal stores and tightly regulate translation. Using immunofluorescence, mass spectrometry, electron microscopy, RNA sequencing, and live ex vivo ovarian tissue–slice imaging, we identified a transient ribonucleoprotein-organelle compartment in growing mouse oocytes, which we named the Zollo body. Although it resembles the Balbiani body observed in nongrowing oocytes of many vertebrate species, the Zollo body arises during growth and shows nascent translation and phospho-mTOR enrichment. Pharmacological dissolution of the Zollo body arrests follicle and oocyte growth in cultured ovarian tissue slices. Thus, the Zollo body is a transient, translation-associated compartment required for normal follicle and oocyte growth.

Oocytes, female germ cells that develop into eggs, are among the longest-lived cells in the animal body. Recent studies on mouse oocytes highlight unique adaptations in protein homeostasis (proteostasis) within these cells. However, the mechanisms of proteostasis in human oocytes remain virtually unstudied. We present the first large-scale study of proteostatic activity in human oocytes using over 100 freshly donated oocytes from 21 healthy women aged 19–34 years. We analysed the activity and distribution of lysosomes, proteasomes, and mitochondria in both immature and mature oocytes. Notably, human oocytes exhibit nearly twofold lower proteolytic activity than surrounding somatic cells, with further decreases as oocytes mature. Oocyte maturation is also coupled with lysosomal exocytosis and a decrease in mitochondrial membrane potential. We propose that reduced organelle activity preserves key cellular components critical for early embryonic development during the prolonged maturation of human oocytes. Our findings highlight the distinctive biology of human oocytes and the need to investigate human-specific reproductive biology to address challenges in female fertility.

Mammalian oocytes undergo a long growth phase in the ovary, during which transcriptional levels gradually decrease. Growing oocytes must therefore accumulate maternal stores and regulate their translation to achieve successful divisions and early embryo development. Using immunofluorescence, mass spectrometry and electron microscopy, we identified a novel and transient compartment, the Zollo Body, in late growing mouse oocytes, constituted of RNPs and organelles. Morphologically, this structure resembles the Balbiani body found in most vertebrate species but it stains positively for nascent translation and active phospho-mTOR. RNAseq analysis and dry mass measurements of growing oocytes with or without this compartment further support its key role in boosting translation, allowing growing oocytes to avoid cytoplasmic dilution despite their rapid size increase, ultimately ensuring their developmental potential.

Oocytes spend the majority of their lifetime in a primordial state. The cellular and molecular biology of primordial oocytes is largely unexplored; yet, it is necessary to study them to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age. 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 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 that what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi that is not functionally associated with oocyte dormancy. This study provides the first insights into the organization of the cytoplasm in mammalian primordial oocytes, and clarifies the relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.

Macroautophagy/autophagy mediates the degradation of ubiquitinated aggregated proteins within lysosomes in a process known as aggrephagy. The cargo receptor SQSTM1/p62 condenses aggregated proteins into larger structures and links them to the nascent autophagosomal membrane (phagophore). How the condensation reaction and autophagosome formation are coupled is unclear. We recently discovered that a region of SQSTM1 containing its LIR motif directly interacts with RB1CC1/FIP200, a protein acting at early stages of autophagosome formation. Determination of the structure of the C-terminal region of RB1CC1 revealed a claw-shaped domain. Using a structure-function approach, we show that the interaction of SQSTM1 with the RB1CC1 claw domain is crucial for the productive recruitment of the autophagy machinery to ubiquitin-positive condensates and their subsequent degradation by autophagy. We also found that concentrated Atg8-family proteins on the phagophore displace RB1CC1 from SQSTM1, suggesting an intrinsic directionality in the process of autophagosome formation. Ultimately, our study reveals how the interplay of SQSTM1 and RB1CC1 couples cargo condensation to autophagosome formation.

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.

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Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.