The evolution of phenotypic differences between males and females, which are often spectacular, has been a subject of intense scrutiny since Darwin [1
]. Several well-studied, often-integrated forms of sexual dimorphism include morphological [1
], behavioral [2
], and physiological [3
] differences. Clearly, the evolutionary mechanisms ultimately responsible for sexual dimorphism (i.e., sexual selection [4
], sex-specific ecological selection [4
], and sexual conflict [5
]) are of great interest. However, a complete understanding of these processes is impossible without knowledge of the proximate genetic and genomic underpinnings of sex-limited phenotypes.
Several proximate mechanisms can account for the phenotypic differences between males and females. For instance, fixed genetic differences between males and females via heteromorphic sex chromosomes [6
] or a sex-determination locus provide one basis for sexual dimorphism. In this case, the two sexes possess partially distinct genomes. However, phenotypic sexual dimorphism may also be mediated by sex differences in gene expression
when a key transcript differs in abundance between males and females. These two mechanisms are by no means mutually exclusive, as sex-specific aspects of the genome result in downstream sex differences in gene expression at sex-shared loci, especially when the original sex-unique genes are highly pleiotropic (e.g. they affect multiple developmental pathways). Sexes need not have distinct genomes for sexual dimorphism to exist, however, because species characterized by environmental sex determination nevertheless maintain a considerable degree of sex-based phenotypic differentiation with respect to primary and often secondary sexual traits [7
]. In these cases of non-genetic sex determination, sex differences in gene expression are obviously important sources of sexual differentiation and dimorphism.
Some interesting gene expression patterns with regard to sex have been reported over the past several years, initially in Drosophila melanogaster
and later in other taxa (see a recent review of sex-biased gene expression by Ellegren and Parsch [10
]). One observation is that of those genes that demonstrate sex-biases in expression level, more tend to be male-enriched than female-enriched [11
] (but see [12
]). This high level of observed sexual dimorphism in gene expression is mostly attributable to differences between testis and ovary [11
]. Furthermore, male-enriched genes are more divergent in their expression levels among species than are female-enriched or sex-unbiased genes [17
]. These patterns, in addition to the discovery that male-enriched genes also demonstrate faster rates of DNA sequence evolution relative to female-enriched and sex-unbiased genes [18
], have been interpreted as a general signature of stronger sexual selection on males. This "male sex drive" hypothesis, formally proposed by Singh and Kulathinal [19
], is consistent with findings across several animal taxa. However, additional independent tests of this hypothesis should be carried out before it is accepted as a general pattern of evolution.
In this study we take advantage of the zebrafish as a model of vertebrate reproduction to test predictions under the male sex drive hypothesis. Environment, hormones, and genetic components likely influence sex differentiation in Danio rerio
, but the precise roles and interactions of these factors with respect to reproductive development remain unclear [20
]. Takahashi [22
] originally described zebrafish gonad differentiation as a transition from a two-weeks-post-fertilization ovary-like precursor to either the mature ovary or the highly differentiated testis. This transition from ovary-like precursor to testis in males is mediated by oocyte apoptosis, which is generally complete by 29 days post-hatching [23
]. More recently it has been shown that some male zebrafish exhibit few ovary-like features and lack ovary-typical gene expression during gonadal development [24
]. In fact, males vary dramatically in the developmental timing and abundance of ovarian features (genetic and morphological) leading up to testis formation, and there is even substantial variation within sibling groups [21
]. Sexual maturity in zebrafish is attained well after gonad differentiation, and usually is complete when individuals reach 23-25 mm standard length (approximately 75 days post-hatching for domesticated strains) [25
One advantage to zebrafish is that Affymetrix GeneChip® technology is readily available, permitting the assessment of large-scale patterns of expression in adults and their gonads. The Zebrafish Genome Array design is based on sequence information from RefSeq (July 2003), GenBank (release 136.0, June 2003), dbEST (July 2003), and UniGene (Build 54, June 2003). With approximately 14,900 transcripts represented on the array, this technology can provide a representative sample of sex differences in gene expression patterns. Our goal was to compare gene expression patterns between testes and ovaries as well as between male and female somatic tissue. A collateral benefit to these comparisons was that we were also able to identify genes within each sex that were up- or downregulated in the gonads. Under the male sex drive hypothesis, we expected more genes upregulated in males relative to females. We predicted many of these genes to be gonad specific, but also expected to find some genes expressed at different levels in the somatic tissues of males compared to females.
While our study is the first to explicitly address the male sex drive hypothesis in Danio rerio
, several recently published microarray studies of gene expression in zebrafish have addressed aspects of sexually dimorphic gene expression and gonad specific expression patterns. In general these studies have revealed that the quantities of particular transcripts often differ significantly in adult males and females, at the level of the whole body [26
], the gonads [27
], the brain [28
], the liver [30
], and other tissues [28
]. However, these studies do not necessarily agree with ours on all points related to patterns relevant to the evolution of sex-biased gene expression in zebrafish, so we will return to this topic in the discussion.