Studies in non-teleost vertebrates have found microRNAs (miRNAs) to be essential for proper gonadal development. However, comparatively little is known about their role during gonadal development in teleost fishes. So far in zebrafish, a model teleost, transcript profiling throughout gonadal development has not been established because of a tiny size of an organ in juvenile stages and its poor distinguishability from surrounding tissues. We performed small RNA sequencing on isolated gonads of See-Thru-Gonad line, from the undifferentiated state at 3 weeks post fertilization (wpf) to fully mature adults at 24 wpf. We identified 520 gonadal mature miRNAs; 111 of them had significant changes in abundance over time, while 50 miRNAs were either testis- or ovary-enriched significantly in at least one developmental stage. We characterized patterns of miRNA abundance over time including isomiR variants. We identified putative germline versus gonadal somatic miRNAs through differential small RNA sequencing of isolated gametes versus the whole gonads. This report is the most comprehensive analysis of the miRNA repertoire in zebrafish gonads during the sexual development to date and provides an important database from which functional studies can be performed.

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Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography–tandem mass spectrometry (LC–MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3–4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

The role of sex steroid regulation in gonadal maturation is a very complex process that is far from being fully understood. Hence, we have investigated seasonal changes in gonadal expression of estrogen receptors (ERs) in Atlantic cod (Gadus morhua L.), a batch spawner, throughout the annual reproductive cycle. Three nuclear ER partial cDNA sequences (esr1, esr2a, and esr2b) were cloned and all esr transcripts were detected mainly in liver and gonads of fish of both sexes. In situ hybridization of esrs along with germ cell (vasa) and gonadal somatic cell markers (gonadal soma-derived factor (gsdf), 3b-hydroxysteroid dehydrogenase (3bhsd), and anti-Mü llerian hormone (amh) for testicular, or gsdf for ovarian somatic cells) showed that all three esrs were preferentially localized within interstitial fibroblasts composed of immature and mature Leydig cells in testis, whereas they were differentially expressed in both follicular cells and oocytes in ovary. Quantitative real-time PCR analysis revealed a sexually dimorphic expression pattern of the three esr paralogs in testis and ovary. A significant increase in esr2a expression was identified in testis and of esr2b in ovary, whereas esr1 transcripts were elevated in both testis and ovary in February and March before the spawning period. The localization and sexually dimorphic expression of esr genes in gonads indicate a direct function of estrogen via ERs in gonadal somatic cell growth and differentiation for Leydig cell in testis and follicular cells in ovary throughout the annual reproductive cycle in Atlantic cod. Key Words" androgen receptor (ar) " Atlantic cod " cytochrome P450 aromatase (cyp19a1a)" estrogen receptor (esr)" sexual maturation

Zebrafish are an important model species in developmental biology. However, their potential in reproductive biology research has yet to be realized. In this study, we established See-Thru-Gonad zebrafish, a transparent line with fluorescently labeled germ cells visible throughout the life cycle, validated its gonadal development features, and demonstrated its applicability by performing a targeted gene knockdown experiment using vivo-morpholinos (VMOs). To establish the line, we crossed the zf45Tg and mitfa(w2/w2); mpv17(b18/b18) zebrafish lines. We documented the in vivo visibility of the germline-specific fluorescent signal throughout development, from gametes through embryonic and juvenile stages up to sexual maturity, and validated gonadal development with histology. We performed targeted gene knockdown of the microRNA (miRNA) miR-92a-3p through injection of VMOs directly to maturing ovaries. After the treatment, zebrafish were bred naturally. Embryos from miR-92a-3p knockdown ovaries had a significant reduction in relative miR-92a-3p expression and a higher percentage of developmental arrest at the 1-cell stage as compared with 5-base mismatch-treated controls. The experiment demonstrates that See-Thru-Gonad line can be successfully used for vertical transmission of the effects of targeted gene knockdown in ovaries into their offspring.