Zebrafish sex: a complicated affair

Abstract

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.

Keywords: Danio rerio; fish; gonad differentiation; polygenic sex determination; sex chromosome; teleost.

Figure 1:

Inferring the type of sex chromosomal system by progeny sex ratio as a result of genome manipulation or artificial sex reversal. (A) Performing gynogenesis on male heterogametic species will produce an all-female (XX) F₁ offspring. (B) For the female heterogametic species (assuming full viability of all genotypes), gynogenesis will produce F₁ progeny with about 1:1 sex ratio and all F₁ females will have WW sex chromosomes. [In case of WW lethality, an all-male (ZZ) F₁ offspring will be produced, whereas partial survival of WWs will yield intermediate results]. When F₁ females (WW) are crossed with normal males (ZZ), their F₂ offspring will be all female (ZW). (C) When hormone- or temperature-based masculinization is performed in a male heterogametic species, crossing of sex-reversed neo-males (srXX) with normal females will produce all-female F₁ offspring. (D) For female heterogametic species (assuming full viability of all genotypes), the expected phenotypic sex ratio in the F₁ progeny, from crossing the sex-reversed neo-male (srZW) with normal female (ZW), will be 25% males and 75% females. Among the F₁ females, one-third of them will have WW sex chromosomal pair that does not normally occur in nature and yield an all-female offspring when crossed with a normal male (ZZ). (If WWs showed lethality, then 33% ZZ males and 67% ZW females will be expected, whereas their partial survival will yield intermediate results).

Figure 2:

A simplified mechanistic model for a PSD system based on the involvement of four autosomal genes. In this theoretical PSD system, protein products of four genomic loci determine sex. Two of the protein products perform a function that pushes the gonad toward femaleness D and E loci, while the remaining two are proteins with pro-male function A and B loci. For simplicity, it is assumed that (i) for every locus there is a strong (larger shape and upper case letter) and weak (smaller shape and lower case letter) effect allele; (ii) the effect of the four strong alleles are equal and the same is true for the four weak alleles at a lower level and (iii) the four products do not exert any direct effect on the functions of each other. If we assign a binary code to the alleles (strong—1 and weak—0), then the outcome could be predicted by simply comparing the sum of male and female alleles (assuming that in case of a tie the individual would continue to develop into a female). In this system, a weak male that has two strong male alleles and one strong female allele (A) could produce sperm cells of different sexual genotypes [see (B) and (C) for examples], among them some that would have excess of pro-female alleles [see (C)]. Similarly, a weak female with one strong male and two strong female alleles (D) could also produce oocytes with excess strong female alleles (E) or more strong male alleles (F). Symbols with different shapes label genes located on four different autosomes. Ratios of strong male to strong female alleles are indicated in brackets.

Figure 3:

The combination of parental ‘SD allele sets’ have a profound effect on the sex ratio of the offspring. Using bigenic genotyping data from the offspring groups analyzed by Bradley et al. (Supplementary Table 4 of [24]), we have created imaginary crosses using one male genotype with three different female genotypes. The parental genotypes were chosen in such a way that they could only produce a single genotype per cross that was among those detected by the authors. Wide range of sex ratios obtained and two opposite sexes with the same SD genotype both indicate a higher level of complexity than what could be explained by participation of just two loci in the SD process. On the pie charts, checkered background indicates males, and solid background indicates females.

Figure 4:

Offspring sex ratios show much higher level of variability for zebrafish (PSD) than Nile tilapia (CSD). (A) Crossing a single zebrafish male with four different female partners yielded offspring groups with very different sex ratios (13.1–81.8% males; data are from [26]). (B) Crossing a Nile tilapia male (XY) with four different female partners (XX) yielded offspring groups with very similar sex ratios (data are from [116]). Checkered bars indicate males, and solid bars indicate females.

Figure 5:

Shift in the balance of pro-male and pro-female pathways will determine direction of gonad differentiation in zebrafish. Three major developmental pathways (Tp53-apoptosis, NF-кB and canonical Wnt) have been shown to participate in the process. The number of germ cells also has a profound effect on the final outcome (data not shown). In the males (left), numbers of primary oocytes originating from the ‘juvenile ovary’ are low, tp53 is upregulated, apoptotic processes are fully active and the NF-кB as well as canonical Wnt signaling pathways are both downregulated. The hormonal balance is shifted toward maleness. In the females (right), the number of primary oocytes originating from the ‘juvenile ovary’ is high, the NF-кB pathway and canonical Wnt signaling pathways are both upregulated, whereas tp53 is downregulated. Apoptosis is inhibited and the hormonal balance is shifted toward femaleness. The timing and causative effects of these processes are not fully understood.