Emergence of breeding tubercles and puberty onset in male zebrafish: evidence for a dependence on body growth.

The presence of breeding tubercles (BTs) on the pectoral fins has been investigated as a typical male secondary sexual characteristic (SSC) that distinguish males from females in adult zebrafish. Nonetheless, the earliest occurrence of these tubercles and its association with puberty onset and body growth remain unclear. In this study, using morphological, histological and statistical analyses, the authors examined the first appearance of BTs and puberty onset in male zebrafish, with particular emphasis on the potential impact of body growth on them. The results of this study revealed that BTs distributed along the first five branched pectoral fin rays were the earliest manifestation of male SSCs, which is significantly strongly correlated with body weight (R2  = 0.9609, P < 0.001), and could be used as a "gold standard" for the earliest sex distinction (<0.1 g in weight). Using the first appearance of BTs (<0.20 mm2 ) as a metric, the authors established that male puberty commenced at a body weight of c. 0.056 ± 0.015 g or a standard length of 10.99 ± 1.051 mm (mean ± S.D.). In this study, the authors thus established a simple method that can be used to sex live zebrafish at the pubertal stage and provides the first evidence for the relationship of BTs and male puberty initiation with body growth. These findings will accordingly lay a foundation for exploring mechanisms of the SSCs and male puberty onset in zebrafish and other teleost fish.

In vivo assessment of gonad status, secondary sex characteristics and spawning in transparent Casper zebrafish.

Important aspects of vertebrate reproduction, such as gametogenesis, involve changes in organs found deep internally and thus not easily studied directly in most living vertebrates due to obscuring pigment and overlying tissues. Transparent lines of zebrafish, especially the Casper double mutant, allow direct observation and analysis of reproductive events in the gonads in vivo. The natural production of fertilized eggs in zebrafish is a complex process involving oogenesis, spermatogenesis, mating behavior, endocrine and neurological processes with inputs from the environment including light, temperature and nutrition. While these factors play important roles, the hypothalamic-pituitary-gonadal axis (HPGA) is central in the regulation of embryo output. Endocrine disrupting compounds (EDCs) include a variety of pollutants often present in the environment. EDCs may have direct effects on the HPGA or indirect effects through toxic action on supporting organs such as the liver or kidney. Estrogenic compounds such as diethylstilbestrol (DES) have been reported to affect reproduction in a variety of species including man. In this study, the effects of DES on reproduction were determined in a novel way by using transparent Casper zebrafish that allow direct visualization of gonad status over time. Changes in gonad status with DES treatment were correlated with effects on secondary sex characteristics (i.e., genital vent size and breeding tubercles) spawning and embryo production. The results suggest that the Casper zebrafish is a useful model for studying dynamics of reproductive events in vertebrate gonads in vivo and for determining effects of EDCs on zebrafish reproduction.

Regeneration of breeding tubercles on zebrafish pectoral fins requires androgens and two waves of revascularization.

Sexually dimorphic breeding tubercles (BTs) are keratinized epidermal structures that form clusters on the dorsal surface of the anterior rays of zebrafish male pectoral fins. BTs appear during sexual maturation and are maintained through regular shedding and renewal of the keratinized surface. Following pectoral fin amputation, BT clusters regenerate after the initiation of revascularization, but concomitantly with a second wave of angiogenesis. This second wave of regeneration forms a web-like blood vessel network that penetrates the supportive epidermis of BTs. Upon analyzing the effects of sex steroids and their inhibitors, we show that androgens induce and estrogens inhibit BT cluster formation in intact and regenerating pectoral fins. Androgen-induced BT formation in females is accompanied by the formation of a male-like blood vessel network. Treatment of females with both androgens and an angiogenesis inhibitor results in the formation of undersized BT clusters when compared with females treated with androgens alone. Overall, the growth and regeneration of large BTs requires a hormonal stimulus and the presence of an additional blood vessel network that is naturally found in males.