Regulation of early gonocyte differentiation in zebrafish

Abstract

Zebrafish have been and continue to be an important model organism for studies of fundamental biology and biomedicine, including reproductive development and the cell intrinsic and extrinsic mechanisms regulating early gonocyte differentiation. Wild zebrafish strains determine sex using a ZW genetic system wherein the maternally inherited sex chromosome determines the embryo's sex. Like other species, including humans, regulation of conserved autosomal genes is crucial for gonocyte and sexual differentiation. How these conserved factors are regulated by the diverse mechanisms found throughout the animal kingdom is an active area of investigation. Domesticated zebrafish strains lack the ZW sex determination system found in wild strains and undergo gonocyte and sexual differentiation through a process exclusively governed by autosomal genes and nongenetic influences like environmental factors. Through mutational analysis, molecular genetics, and RNA sequencing, our understanding of the complexity of oocyte and spermatocyte differentiation has become clearer. In this review, we explore the most recent studies of the conserved and divergent mechanisms of gonocyte differentiation between wild and domesticated zebrafish as well as possible adaptations related to their domestication. Further, the contributions of individual genes and their molecular genetic hierarchy in regulating gonocyte differentiation are discussed and related to other species where relevant. We also address the recent characterization of a novel oocyte-progenitor and its potential implications in gonad differentiation. Finally, the role of gonocyte-extrinsic mechanisms, specifically communication between differentiating gonocytes and surrounding somatic gonad cells and the influence of resident and infiltrating immune cells, is discussed.

Keywords: bipotential gonad; gonadogenesis; indeterminant gonad; ovary; sexual differentiation; testis.

Figure 1. Gonadogenesis in wild and domesticated zebrafish.

(A) Wild ZZ, ZW, WW, and domesticated laboratory zebrafish strains develop indeterminate gonads composed of gonocytes and germline stem cells. ZZ indeterminate gonads directly develop into testes, whereas wild ZW, WW, and lab strains establish a bipotential gonad containing oocyte-like cells [1]. The number of germ cells in the bipotential gonad has been linked to its developmental potential into either an ovary or testis, with fewer cells biasing to the latter [2]. Given the correct cues, the ZW, WW, and lab strain bipotential gonads can differentiate into either an ovary or testis. (B) Chromosome 4 has been partitioned into four transcriptionally distinct regions [3]. Overall, these regions are more highly expressed in testis, with region 2 being almost entirely transcriptionally silent in ovaries. Within region 4 of chromosome 4, the sar4 locus contains an rDNA region termed M-rDNA that is highly expressed in ovaries [4]. Percentages represent the number of protein-coding genes expressed in that given region and the exact number of genes is indicated in parentheses. Gene expression data were compiled from [3].

Figure 2. Meiotic progression defects in zebrafish mutants.

Prophase I in zebrafish begins with leptotene, where sister chromatids condense and pair via cohesin proteins. The cells then progress from zygotene and initiate synaptonemal complex (dark blue) formation, which is fully intact and supports chromosome cross-overs in pachytene. In diplotene, chromosome cross-over is completed and the cells enter meiotic arrest [29]. The gonocyte differentiation trajectories of zebrafish mutant for the meiotic machinery and RNAbp genes discussed in this review are shown for oogenesis (striped, pink arrows) and spermatogenesis (light blue arrows). The arrows indicate the overall meiotic progression of mutant cells during prophase I, and the red dotted line indicates the most advanced stage of differentiation detected. The * indicates the inferred latest stage of meiosis I that the indicated mutant germ cells reach based on the available published data. RNAbp, RNA-binding protein.

Figure 3. Estrogen synthesis and follicle maturation in oogenesis.

In the indeterminate gonad, the precursor granulosa cells (orange) express Cyp17a1 and Cyp19a1a to initiate the first wave of estrogen synthesis in oogenesis. As the indeterminate gonad transitions into a bipotential gonad, Gdf9 in the early oocyte, likely positively regulated by Nobox, prompts expression of Inhbaa [76] by a currently unknown mechanism and inhibits anti-Müllerian hormone (AMH) production [77]. Inhbaa forms a homodimer, Activin A, which promotes follicle-stimulating hormone (FSH) expression by the pituitary gland to promote the second wave of estrogen synthesis. Upon ovary differentiation, the established granulosa cells (dark pink) express Cyp17a1 and Cyp19a1a to sustain estrogen synthesis while oocyte Gdf9 inhibits granulosa AMH production. FSH promotes estrogen synthesis and sufficient estrogen levels feedback to suppress further FSH production in mammals (reviewed in [78]). Based on the available genetic evidence, we hypothesize that this feedback loop is conserved in zebrafish.