Sex determination in the Squalius alburnoides complex: an initial characterization of sex cascade elements in the context of a hybrid polyploid genome

Abstract

Background: Sex determination processes vary widely among different vertebrate taxa, but no group offers as much diversity for the study of the evolution of sex determination as teleost fish. However, the knowledge about sex determination gene cascades is scarce in this species-rich group and further difficulties arise when considering hybrid fish taxa, in which mechanisms exhibited by parental species are often disrupted. Even though hybridisation is frequent among teleosts, gene based approaches on sex determination have seldom been conducted in hybrid fish. The hybrid polyploid complex of Squalius alburnoides was used as a model to address this question.

Methodology/principal findings: We have initiated the isolation and characterization of regulatory elements (dmrt1, wt1, dax1 and figla) potentially involved in sex determination in S. alburnoides and in the parental species S. pyrenaicus and analysed their expression patterns by in situ hybridisation. In adults, an overall conservation in the cellular localization of the gene transcripts was observed between the hybrids and parental species. Some novel features emerged, such as dmrt1 expression in adult ovaries, and the non-dimorphic expression of figla, an ovarian marker in other species, in gonads of both sexes in S. alburnoides and S. pyrenaicus. The potential contribution of each gene to the sex determination process was assessed based on the timing and location of expression. Dmrt1 and wt1 transcripts were found at early stages of male development in S. alburnoides and are most likely implicated in the process of gonad development.

Conclusions/significance: For the first time in the study of this hybrid complex, it was possible to directly compare the gene expression patterns between the bisexual parental species and the various hybrid forms, for an extended set of genes. The contribution of these genes to gonad integrity maintenance and functionality is apparently unaltered in the hybrids, suggesting that no abrupt shifts in gene expression occurred as a result of hybridisation.

Figure 1. Unrooted Neighbour-Joining tree obtained among the DMRT1 family and other proteins that share the conserved DM domain.

Dre-DMRT1- Danio rerio; Tru-DMRT1- Takifugu rubripes; Omy-DMRT1- Oncorhynchus mykiss; Ola-DMRT1- Oryzias latipes; Hsa-DMRT1- Homo sapiens; Gga-DMRT1- Gallus gallus; DMRT2-Hsa- H. sapiens; DMRT2-Ola- O. latipes; Hsa-DMRT3- H. sapiens; DMRT3-Tru- T. rubripes; DMRT3-Dre- D. rerio. Bootstrap values are shown above the branches. ▴ S. pyrenaicus (PP); • S. alburnoides (AA).

Figure 2. Unrooted Neighbour-Joining tree obtained among the teleost Wt1 proteins and other WT1 orthologs.

Dre-a-WT1- Danio rerio, Omy-a-WT1; Omy-b-WT1- Oncorhynchus mykiss, Aja-WT1- Anguilla japonica; Ola-WT1- Oryzias latipes; Hsa-WT1- Homo sapiens, Gga-WT1- Gallus gallus, Mmu-WT1- Mus musculus, and Rno-WT1- Rattus norvegicus. Bootstrap values are shown above the branches. Bootstrap values are shown above the branches. ▴ S. pyrenaicus (PP).

Figure 3. Unrooted Neighbour-Joining tree obtained among the DAX1 family.

Dre-DAX1- Danio rerio, Dla-DAX1- Dicentrarchus labrax, Ola-DAX1- Oryzias latipes, Ony-DAX1- Oreochromis niloticus, Hsa-DAX1- Homo sapiens, Mmu-DAX1- Mus musculus and Gga-DAX1- Gallus gallus. Dax2 protein of Oreochromis niloticus (Oni-DAX2). Bootstrap values are shown above the branches. ▴ S. pyrenaicus (PP) ; • S. alburnoides (AA).

Figure 4. Unrooted Neighbour-Joining tree obtained among the FIGLA family of proteins.

Dre-FIGLA- Danio rerio, Tni-FIGLA- Tetraodon nigroviridis, Kma-FIGLA- Kryptolebias marmoratus, Hsa- FIGLA- Homo sapiens, Rno-FIGLA- Rattus norvegicus, and Xtr FIGLA- Xenopus (Silurana) tropicalis. Bootstrap values are shown above the branches. ▴ S. pyrenaicus males (PP-M); ▵ females (PP-F) and • S. alburnoides (AA).

Figure 5. Expression patterns of candidate genes in the adult gonads S. pyrenaicus and S. alburnoides.

Dmrt1: (a) S. pyrenaicus (PP genotype); (b) S. alburnoides (AA genotype) and (c) S. alburnoides (PA genotype) testis; (d) S. alburnoides (PA genotype) and S. alburnoides (PAA genotype) ovary with (e) antisense and (f) sense probes. Wt1: (g) S. pyrenaicus (PP genotype); (h) S. alburnoides (AA genotype) and (i) S. alburnoides (PA genotype) testis; (j) S. alburnoides (PA genotype) and (k) S. alburnoides (PAA genotype) ovary. Dax1: (l) S. alburnoides (AA genotype), (m) S. pyrenaicus (PP genotype) and (n) S. alburnoides (PAA genotype) ovary. Examples of areas with positive signal are indicated by black arrows; examples of negative cells are highlighted with grey arrowheads. Germ cells (GS), early perinuclear (P), cortical alveolar (CA) and vitellogenic (V) oocytes. Scale bar = 100 µm (a, b, e, f, g, h, k, l, m); scale bar = 200 µm (c, d, i, j, n).

Figure 6. Expression patterns of figla in the adult gonads S. pyrenaicus and S. alburnoides.

(a) S. alburnoides (AA genotype) testis; (b) S. alburnoides (PAA genotype) ovary and S. pyrenaicus (PP genotype) ovary with (c) antisense and (d) sense probes (positive signals in primary oocytes exemplified with black arrows; lower expression in early stage oocytes exemplified with grey arrows; cells in different maturation stages, not expressing the transcript are indicated by grey arrowheads). Germ cells (GS), early perinuclear (P), cortical alveolar (CA) and vitellogenic (V) oocytes. Scale bar = 100 µm (a, c, d); scale bar = 200 µm (b).