Zebrafish sex: a complicated affair

B RIEFINGS IN FUNC TIONAL GENOMICS . VOL 13. NO 2. 172^187 doi:10.1093/bfgp/elt041 Zebrafish sex: a complicated affair Woei Chang Liew and La¤szlo¤ Orba¤n Advance Access publication date 21 October 2013 Abstract Keywords: polygenic sex determination; sex chromosome; gonad differentiation; teleost; fish; Danio rerio ZEBRAFISH SEX: LOTS OF QUESTIONS AND ONLY A FEW ANSWERS Teleosts (ray-finned fishes) form the largest group of extant vertebrates with more species than the rest of the other vertebrates combined [1]. The rich diversity of teleosts is observed not only in their phenotypes and behavior (review: [2]), but also in the varieties of their reproductive processes that seem to utilize all known sex determination (SD) mechanisms described for other vertebrates (see reviews [3, 4]). It is believed that the common ancestor of teleosts diverged from that of land vertebrates about 410 million years ago (Mya), well before the split of placental mammals from the latter lineage (ca. 180 Mya) [5]. Therefore, it is not surprising that the evolution of sex determination systems between teleosts and the well-studied mammals show substantial differences. The diversity of reproductive systems in teleosts allows them to contribute to comparative studies on the evolution of SD mechanisms. One of the most popular model organisms among teleosts is the zebrafish (Danio rerio). Natural habitats of the zebrafish stretch from South Asia (e.g. Pakistan, Nepal and India) to Southeast Asia (e.g. Myanmar) [6]. This small-bodied freshwater species is most commonly found in slow or stagnant waters, such as rivers, ponds and paddy fields [6, 7]. The ambient water temperature at these natural habitats typically ranges from 26–32 C [7]. Among the reasons that make zebrafish a popular laboratory model is its short generation time. In the laboratory, zebrafish reach reproductive maturity at around 3–4 months. Despite its relatively small size, the species is quite fecund: mature females kept under ideal conditions often produce 200–300 eggs regularly on a weekly basis. However, offspring sex Corresponding author. László Orbán, Reproductive Genomics Group, Temasek Life Sciences Laboratory, Singapore. Tel: þ65-68727413; Fax: þ65-6872-7007; E-mail: Woei Chang Liew is a PhD candidate at the Orbán group at TLL. His research interest is in sexual development of teleosts and other vertebrates with special focus on the area of sex determination. La¤szlo¤ Orba¤n has been educated in Hungary and in the USA. He has been studying (zebra)fish reproduction for over 20 years. Currently, he is a group leader at Temasek Life Sciences Laboratory (TLL, Singapore). ß The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. Zebrafish sex is complicated ZEBRAFISH SEX IS DETERMINED PRIMARILY BY GENETIC FACTORS AND NOT ENVIRONMENTAL ONES In vertebrates, sex is determined either by genetic mechanisms (genetic sex determination or GSD; reviews: [3, 18, 29]) or by the environment (environmental sex determination or ESD; reviews: [3, 4, 8, 12, 15, 30]). Although SD has only been analyzed in a small subset of the 32 000 fish species (reviews: [3, 15]), there are plenty of examples for species with GSD (e.g. Japanese medaka [31], threespine stickleback [32] and Patagonian pejerrey [33]) and ESD (e.g. American eel [34] and bluehead wrasse [35]). In some teleosts, SD (or gonad differentiation) can be overridden by environmental effects, most often temperature (thermal effect (TE) on GSD; review: [36]). In GSD, the sex of an individual is determined primarily by genes/chromosomes inherited from the parents. There are two major forms of GSD: (i) SD by a single sex chromosomal pair or chromosomal sex determination (CSD) and (ii) SD by several (i.e. more than one) genetic factors or polygenic (multigenic) sex determination (PSD). In our opinion, the latter includes those with multiple sex chromosome types (e.g. several Lake Malawi cichlid species [37]), weak sex chromosomes easily and often overridden by autosomal modifiers (e.g. X/YþA or ZWþA according to Devlin and Nagahama [3]) and those regulated by several autosomal loci without any sign of sex chromosomes (e.g. European seabass [38]). To find out whether zebrafish uses GSD or not, we conducted several experiments [26]. Repeated mating of the same breeding pairs yielded offspring groups with very similar sex ratios, even when they were reared in uncontrolled environmental conditions (i.e. variations were expected in rearing density, amount of feed and ambient water temperature [26]). We also performed a selective breeding experiment, whereby factorial crosses were made with four to six siblings in every generation and brooders for the new generation were chosen from the family that produced the highest bias toward the required direction. With this approach, we generated families with severely biased sex ratios within a short period of time (three to four generations), especially toward male excess [26]. Strong influence of parental genotype on the sex of zebrafish offspring was also described by Abozaid et al. [39, 40]. They observed that offspring from gynogenetic males showed stronger effect of heatinduced masculinization than progenies sired (...truncated)