Stage Specific Transcriptomic Analysis and Database for Zebrafish Oogenesis

Abstract

Oogenesis produces functional eggs and is essential for fertility, embryonic development, and reproduction. The zebrafish ovary is an excellent model to study oogenesis in vertebrates, and recent studies have identified multiple regulators in oocyte development through forward genetic screens, as well as reverse genetics by CRISPR mutagenesis. However, many developmental steps in oogenesis, in zebrafish and other species, remain poorly understood, and their underlying mechanisms are unknown. Here, we take a genomic approach to systematically uncover biological activities throughout oogenesis. We performed transcriptomic analysis on five stages of oogenesis, from the onset of oocyte differentiation through Stage III, which precedes oocyte maturation. These transcriptomes revealed thousands of differentially expressed genes across stages of oogenesis. We analyzed trends of gene expression dynamics along oogenesis, as well as their expression in pair-wise comparisons between stages. We determined their functionally enriched terms, identifying uniquely characteristic biological activities in each stage. These data identified two prominent developmental phases in oocyte differentiation and traced the accumulation of maternally deposited embryonic regulator transcripts in the developing oocyte. Our analysis provides the first molecular description for oogenesis in zebrafish, which we deposit online as a resource for the community. Further, the presence of multiple gene paralogs in zebrafish, and the exclusive curation by many bioinformatic tools of the single paralogs present in humans, challenge zebrafish genomic analyses. We offer an approach for converting zebrafish gene name nomenclature to the human nomenclature for supporting genomic analyses generally in zebrafish. Altogether, our work provides a valuable resource as a first step to uncover oogenesis mechanisms and candidate regulators and track accumulating transcripts of maternal regulators of embryonic development.

Direct Male Development in Chromosomally ZZ Zebrafish

“…S2Y-DD). In situ hybridization had previously detected gja11 expression in follicle cells surrounding Stage II oocytes (cortical alveolus stage, 140–340 μm (Bogoch et al, 2022), which were not present in our samples), but not in Stage IB oocytes (Liu et al, 2022); our data detected gja11 expression at earlier stages presumably due to increased sensitivity. The oocyte partner of Gja11 could be encoded by gjc4a.1 ( cx44.2 ) or gjc4b ( cx43.4 ), which were expressed strongly and rather specifically in 30dpf oocytes (Santos et al, 2007) (Supplementary Fig.…”

“…Oocyte stages recovered are limited by the scRNA-seq protocol, which passes cells through a 40µm filter. According to recent staging criteria (Bogoch et al, 2022), we would recover only oocytes from the earliest two stages, the symmetry-breaking stage (8–20 μm Selman Stage IA, oogonia and meiotic oocytes from leptotene to pachytene), and early Selman Stage IB, nuclear cleft, 15–50μm, oocytes from pachytene to mid-diplotene (Selman et al, 1993, Bogoch et al, 2022)). Comparison of gene expression patterns with other transcriptomic analyses of zebrafish oocytes (Wong et al, 2018, Zhu et al, 2018, Can et al, 2020, Bogoch et al, 2022) are difficult because at least some of the oocyte sizes we examined were not analyzed or were mixed with larger oocytes, and in other studies, oocyte sizes were listed only in relative terms, so comparison wasn’t possible (Cabrera-Quio et al, 2021).…”

The Balbiani body is formed by microtubule-controlled molecular condensation of Buc in early oogenesis

“…To culture oocytes, we isolated them form adult ovaries, where they are abundant (Fig. 2A), using our isolation protocol 8,50 , as also described above (Fig. 2A).…”

“…Finally, we wanted to biochemically determine the solubility state of Buc granules and test whether they constitutes a separate non-soluble phase in physiological conditions. We previously developed a method for a size-based isolation of stage specific oocytes from ovaries 8 , which is successfully used for biochemical and omics analyses 44,50 . We first used this method to isolate oocytes at sizes of 50-100 μm in diameter, which represent mature Bb stages, from Buc-GFP transgenic ovaries and confirmed that all oocytes showed a clear signal of Buc-GFP in the mature Bb (Fig.…”

“…Ovaries were collected from adult females at 6-8 months post-fertilization. Stage specific oocyte isolation from ovaries was performed as described in 8,50 Briefly, oocytes are first isolated from ovaries by short enzymatic digestion (3 mg/ml Collagenase I, Sigma-Aldrich, #C0130;…”