In Vitro Storage of Functional Sperm at Room Temperature in Zebrafish and Medaka.

The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

The many faces of the bouquet centrosome MTOC in meiosis and germ cell development.

Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.

Stage Specific Transcriptomic Analysis and Database for Zebrafish Oogenesis.

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.

Control of meiotic chromosomal bouquet and germ cell morphogenesis by the zygotene cilium.

A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the "zygotene chromosomal bouquet." Here, we identified the "zygotene cilium" in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics.

A transcriptional time-course analysis of oral vs. aboral whole-body regeneration in the Sea anemone Nematostella vectensis.

BACKGROUND: The ability of regeneration is essential for the homeostasis of all animals as it allows the repair and renewal of tissues and body parts upon normal turnover or injury. The extent of this ability varies greatly in different animals with the sea anemone Nematostella vectensis, a basal cnidarian model animal, displaying remarkable whole-body regeneration competence. RESULTS: In order to study this process in Nematostella we performed an RNA-Seq screen wherein we analyzed and compared the transcriptional response to bisection in the wound-proximal body parts undergoing oral (head) or aboral (tail) regeneration at several time points up to the initial restoration of the basic body shape. The transcriptional profiles of regeneration responsive genes were analyzed so as to define the temporal pattern of differential gene expression associated with the tissue-specific oral and aboral regeneration. The identified genes were characterized according to their GO (gene ontology) assignations revealing groups that were enriched in the regeneration process with particular attention to their affiliation to the major developmental signaling pathways. While some of the genes and gene groups thus analyzed were previously known to be active in regeneration, we have also revealed novel and surprising candidate genes such as cilia-associated genes that likely participate in this important developmental program. CONCLUSIONS: This work highlighted the main groups of genes which showed polarization upon regeneration, notably the proteinases, multiple transcription factors and the Wnt pathway genes that were highly represented, all displaying an intricate temporal balance between the two sides. In addition, the evolutionary comparison performed between regeneration in different animal model systems may reveal the basic mechanisms playing a role in this fascinating process.