Bogoch, Y. | Search bioRxiv

bioRxiv2025

A Novel mTOR-Stat3-Stathmin Pathway Establishes Oocyte Polarization Competence by Orchestrating Centrosome Regulation and Microtubule Organization

In animals and plants, egg production is essential for fertility, reproduction, and embryonic development. The production of functional eggs in sufficient numbers for a female lifespan requires dynamic and precisely coordinated cellular and developmental programs in early oogenesis. However, our understanding of their underlying regulatory mechanisms is critically lacking. Here, we aimed to identify overlooked essential regulators of early oogenesis in zebrafish. First, we present the establishment of a long-term ovary culture system which enables physiological oocyte development from oogonia to primordial follicles in cultured ovaries, providing an invaluable ex-vivo platform for rapid investigation. Next, we utilized this system for robust functional screening of candidates from stage-specific oocyte transcriptomic data. We identified mTOR, Stat3, and Stathmin as novel regulators of oocyte polarity. By using a combination of genetics, pharmacological manipulations ex-vivo, and rescue experiments, we established an essential mTOR-Stat3-Stathmin pathway that orchestrates centrosome regulation and microtubule organization to facilitate oocyte polarity. Microtubules control the localization and condensation of the essential polarity regulator Bucky ball (Buc). Loss of mTOR or Stat3 functions, as well as overactivation of Stathmin (a microtubule destabilizing protein) resulted in aberrant centrosome regulation and destabilization of microtubules, leading to dispersed mis-localized Buc condensates and loss of polarity. We show that mTOR acts upstream of Stat3 in oocytes, and that inhibition of Stathmin in stat3-/-or mTOR deficient ovaries rescued both cytoskeletal and polarity defects. We propose a novel mTOR-Stat3-Stathmin pathway which through cytoskeletal regulation, provides oocytes with polarization competence, a step likely widely conserved in biology. mTOR, Stat3, and Stathmin are known for their roles in cellular growth and cancer. Our work reveals their novel unpredicted functions in cell polarity during animal post-embryonic development.

developmental biology↗

bioRxiv2023

The piRNA protein Asz1 is essential for germ cell and gonad development in in zebrafish and exhibits differential necessities in distinct types of RNP granules

Germ cells are essential for fertility, embryogenesis, and reproduction. Germline development requires distinct types of RNA-protein (RNP) granules, including germ plasm in embryos, piRNA granules in gonadal germ cells, and the Balbiani body (Bb) in oocytes. However, the regulation of RNP assemblies in zebrafish germline development are still poorly understood. Asz1 is a piRNA protein in Drosophila and mice. Zebrafish Asz1 localizes to both piRNA and Bb granules, with yet unknown functions. Here, we hypothesized that Asz1 functions in RNP granule assemblies and germline development in zebrafish. We generated asz1 mutant fish to determine the roles of Asz1 in germ cell development. We show that Asz1 is dispensable for somatic development, but essential for germ cell and gonad development. asz1-/- fish developed exclusively as sterile males with severely underdeveloped testes that lacked germ cells, demonstrating that Asz1 is essential for spermatogenesis. Mechanistically, we provide evidence to conclude that zygotic Asz1 is not required for primordial germ cell specification or migration to the gonad, but is essential for germ cell survival during post-embryonic gonad development, likely by suppressing the expression of germline transposons. Increased transposon expression and morphologically mis-organized piRNA granules in asz1 mutants, argues that zebrafish Asz1 functions in the piRNA pathway. We generated asz1;tp53 fish to partially rescue ovarian development, revealing underdeveloped mutant ovaries with defective oocytes, and that Asz1 is also essential for oogenesis. We further showed that in contrast with piRNA granules, Asz1 is dispensable for Bb granule formation, as shown by normal Bb localization of Buc and dazl. By uncovering Asz1 as an essential regulator of germ cell survival and gonadogenesis in zebrafish, and determining its differential necessity in distinct RNP granule types, our work advances our understanding of the developmental genetics of reproduction and fertility, as well as of RNP granule biology. Author SummaryGerm cells undergo a highly dynamic developmental program that begins in the early embryo and continues through juvenile and adult life. Identifying functional regulators and deciphering the developmental mechanisms of germ cells are critical for advancing our understanding of fertility and reproduction, as well as their associated diseases. Here, we identified Asz1 as an essential regulator of germ cell and gonad development in zebrafish. We demonstrate that zygotic Asz1 is dispensable for the specification and migration of primordial germ cells in the embryo, but is necessary for germ cell survival in the developing gonad, likely by protecting them from transposable elements. Upon loss of asz1, expression of germline transposons was induced, and piRNA granules were mis-organized, suggesting a conserved role for zebrafish Asz1 in the piRNA pathway. We show that Asz1 is required for both spermatogenesis and oogenesis. However, unlike RNA-protein (RNP) granules of the piRNA pathway, Asz1 was not required for RNP granules of the Balbiani body in differentiating oocytes, revealing its differential necessity in distinct types of germline RNP granules. In mice, Asz1 was shown to be essential for spermatogenesis but not oogenesis, but its functions in human gonads are unclear. Our work reports the functional requirements of Asz1 in both sexes in zebrafish, contributes to our knowledge of developmental reproduction biology, and sheds new light on the complexity of RNP granule assemblies.

developmental biology↗