1. Research Background:

How can a baby be born from a decades-old cell? Oocytes are the female germ cells, which develop into fertilizable eggs. In most mammals, oocytes are formed only before birth and are used for the entire fertile life of the individual. In other words, each of us derives from an oocyte that was as old as our mom at the time of our conception. This means several decades!

However, oocytes are not eternal. Starting on average in the fourth decade of their life, women experience a decay in their fertility, mostly due to decreased quality of their oocytes. This phenomenon is called “reproductive aging”. Reproductive aging is becoming a growingly urgent societal problem, as in many European Countries women are increasingly delaying maternity. However, the causes of reduced oocyte quality with advanced maternal age are still largely unknown.

We are interested in the mechanisms that allow mammalian oocytes to remain healthy for decades, and in the causes of their eventual demise during reproductive aging. For this, we primarily focus on protein homeostasis.

Protein homeostasis (proteostasis) involves the degradation of misfolded, damaged, and aggregated proteins, and it is essential in all cell types. Proteostasis defects are associated with a number of pathological conditions, including aging-related diseases such as neurodegeneration. Long-lived cells such as neurons are particularly sensitive to proteostasis decay during aging. How proteostasis is maintained in long-lived mammalian oocytes is mostly unknown. Our main goal is to understand how proteostasis preserves cytoplasmic integrity in mammalian oocytes and how its demise influences reproductive aging.

2. Research questions addressed by the group:

At fertilization, the newly formed embryo inherits all its cytoplasm from the oocyte, upon which the initial steps of embryonic development rely. Thus, oocytes need to maintain their cytoplasm damage-free to allow reproduction.

We are interested in the proteostasis mechanisms that allow oocytes to maintain their cytoplasm intact during their prolonged life.

We have recently discovered that mouse oocytes store protein aggregates in large non-membrane-bound compartments, that we have named EndoLysosomal Vesicular Assemblies (ELVAs) (Zaffagnini et al., 2024). ELVAs harbor all the major degradative systems in the cell, including endosomes, autophagosomes, lysosomes and proteasomes, embedded in a liquid-like matrix. ELVAs show emergent properties respect to their individual components, such as an autonomous regulation of their degradative activity. Largely inactive in immature oocytes, ELVAs activate protein degradation shortly before fertilization, thereby disposing of the stored aggregates. Forced inheritance of aggregates in the embryo impairs embryonic development, indicating that ELVAs play a fundamental role in maintaining oocyte quality to ensure reproduction. Our results defined a new paradigm to contrast protein aggregation in long-lived oocytes and unveiled a novel “super-organelle” with a fascinating, yet largely unknown, biology.

Our current research focuses on how ELVAs are assembled, regulated, and eventually dissolved during early embryonic development. For this, we use mouse oocytes and embryos, in vitro reconstituted systems, and cultured cells as complementary experimental models. We then combine biochemistry and cell biology, employing live-cell and super-resolution imaging, oocyte microinjections, single-cell proteomics, and lipidomics, to understand how ELVAs function.

3. Possible projects:

We offer a range of interconnectedand interdisciplinaryprojects to study the assembly, regulation, and dissolution of ELVAs, a newly discovered super-organelle that maintains proteostasis in mouse oocytes. We also welcomeprojects proposed by the candidates, that fit within the scope and the expertise of the lab.

Possible projects:

• How are ELVA activation and relocation regulated during oocyte maturation and aging?ELVAs relocate to the oocyte cortex and activate protein degradation upon oocyte maturation. Unlike somatic cells, in which endolysosomal positioning is achieved through microtubule-based transport, ELVAs relocate depending entirely on the actin cytoskeleton. Blockade of oocyte maturation delays ELVA relocation and prevents their activation, indicating that these processes are tightly coupled. This project will study the signaling events and the mechanisms responsible of ELVA relocation and activation, and their dysregulation during reproductive aging. For this, the candidate will perform live oocyte microinjections, imaging, single particle tracking, proteomics and lipidomics. This research will provide fundamental insight into how ELVAs couple the degradation of stored aggregates to their metabolic state and cell cycle stage of the oocyte.
• How is vesicle trafficking regulated inside ELVAs?
  Efficient vesicle trafficking is essential during endocytosis and autophagy to achieve cargo degradation. For this, the transport and fusion of endosomes, autophagosomes, and lysosomes are tightly regulated processes. In ELVAs, endolysosomal vesicles are embedded in a liquid-like matrix formed by RUFY1, inside which they must move and fuse with each other to achieve degradation of the stored aggregates. This project aims to understand how endolysosomal trafficking is regulated inside the liquid-like matrix of ELVAs. The project will entail the in vitro reconstitution of ELVAs using purified components, combined with validations in cultured cells and live mouse oocytes. This research will uncover the biochemical principles that regulate the endolysosomal trafficking inside a liquid-like matrix.
• How is ELVA dissolution coupled to lysosomal exocytosis?
  ELVAs disappear during the first embryonic division, in concomitance with a wave of lysosomal exocytosis (i.e., fusion of lysosomal vesicles with the plasma membrane). Blockade of lysosomal exocytosis in mouse embryos impairs embryonic development, indicating that it is necessary for proper development. This project aims to dissect the mechanisms and functions of lysosomal exocytosis in the early mouse embryo, using a combination of high-resolution live imaging, in vitro fertilizations, artificial egg activations, and pharmacologic manipulations. This research will elucidate the role of lysosomal exocytosis during early mammalian embryonic development.

4. Applied Methods and model organisms:

We use mouse oocytes, in vitro reconstituted systems, and cultured cells as model systems for our research. We employ an array of interdisciplinary techniques, including live and super-resolution imaging, quantitative image analysis, oocyte microinjections, recombinant protein expression and purification, single-cell proteomics, and lipidomics.

5. Desirable skills and qualifications:

We are looking for highly motivated, curious, and enthusiastic PhD students with strong analytical skills and logical thinking. A background in biochemistry, molecular, cell, or developmental biology is desirable. Previous experience with oocyte/embryo manipulation, imaging, quantitative image analysis, or protein purification would constitute a bonus. Students should be willing to perform procedures on live mice, including manipulation, intraperitoneal injections, and humanely sacrificing. All the specific training required to perform research in the lab will be provided.

6. References and key publications:

• Zaffagnini et al., 2024. Mouse oocytes sequester aggregated proteins in degradative super-organelles. Cell. 2024 Feb 29;187(5):1109-1126.e21. doi: 10.1016/j.cell.2024.01.031.