Effects of hypoxia on sex determination and differentiation in the Japanese medaka (Oryzias latipes)

Abstract

The gender of a fish is not only determined by its genotype, but may also be affected by environmental variables which can alter sex differentiation, including both testicular and ovarian development. Recent laboratory work for this current research has, for the first time, demonstrated that hypoxia can affect the expression of certain genes regulating steroidogenesis during embryonic development, thereby disrupting the hormonal balance and sex differentiation, subsequently leading to a male–biased F1 generation in zebrafish. Using the freshwater medaka (Oryzia latipes) as a study model, a hypothesis was tested that hypoxia can affect the expressions of the sex–determining gene, DMY and the gene which regulates germ–cell development, vasa, during early embryonic development, thereby affecting sex differentiation and sex determination, and contributes to the observed male–biased F1 generation. In the first experiment, medaka embryos were exposed to hypoxia (1.5±0.1 mg O2 L–1) and normoxia (≥5.8 mgO2 L–1) for nine days. 94% of the embryos survived hypoxia for the entire experimental period. Significant inductions of three hypoxia–responsive genes (HIF–1α, EPO and VEGF) were found at stage 21 (HIF–1α: fold–change = +1.3; EPO: fold–change = +2.1; VEGF: fold–change = +1.3; p ≤ 0.001) as compared with the normoxic embryos. A significant reduction in HIF–1α expression was also found at Stage 39 (fold–change = –2.6; p < 0.001), as were sustained inductions of EPO and VEGF (EPO: fold–change = +1.4, p = 0.006; VEGF: fold–change = +1.4; p < 0.001). The above results show that molecular responses could be induced and yet embryos can survive and develop at this hypoxic level. In the second experiment, medaka embryos were allowed to develop under the same hypoxic (1.5±0.1 mg O2 L–1) normoxic level (≥5.8 mgO2 L–1) levels for 90 days throughout their embryonic development (from Stage 10 to Stage 44), and expression of DMY and vasa at developmental Stages 9, 21, 39, 40 and 44 were determined using Q–PCR. In the hypoxic treatment, DMY was significantly up– regulated at Stage 39 (fold–change = +4.2, p < 0.001) and Stage 40 (fold–change = +1.5, p = 0.023), whereas expression of vasa was significantly down–regulated at Stage 39 (fold–change = –2.3, p < 0.001), Stage 40 (fold–change = –4.3, p = 0.012) and Stage 44 (fold–change = –1.2, p = 0.003), but no change was observable at Stage 9 and Stage 21. After 90 days of hypoxic exposure, 54% of genotypic females (with XX chromosomes) in the chronic hypoxia treatment showed testicular development and 77% of genotypic females exhibited male phenotypic characteristics, as shown by the sexual dimorphic characteristics of dorsal and anal fins, spines on male anal fin rays and were further confirmed by histological examination of the gonads. The results implied that hypoxia can delay or inhibit the proliferation of primordial germ cells (PGCs) and also up– regulate the expression of the DMY gene from embryonic Stage 39 onward. These alternations in the gene expression pattern may, in turn, favor the genotypic females’ (with XX chromosomes) development into phenotypic males. For the first time, this research has demonstrated that hypoxia can alter the expression of genes controlling sex determination and germ cell production during early embryonic development in vertebrates, and change genotypic females with XX chromosomes into phenotypic males.

Date of Award	2 Oct 2008
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Shiu Sun Rudolf WU (Supervisor)