Studies On Histology Of Early Gonadal Development, And Molecular Cloning And Expression Of Foxl2 And Cyp19a1b Genes In Northern Snakehead, Channa Argus

Careful histological observations of the gonadal morphogenetic process are of primary importance for a precise understanding of the mechanism of gonadal sex differentiation. Forkhead box L2 (Foxl2) is a member of the winged helix/forkhead transcription factors superfamily, involved in ovarian development, granulosa cell differentiation and the maintenance of ovarian function. Foxl2 gene mutations cause eyelid malformations and premature ovarian failure. Cytochrome P450 aromatase (Cypl9) is a steroidogenic enzyme and can catalyze the conversion from androgens to estrogens. And it is extremely important to gonadal development and differentiation of vertebrates. Most teleosts have two forms of the aromatase gene. One cyp19 isoform is designated as cyp19a1a and the other as cypl9a1b. The former encodes the gonadal aromatase P450aromA and the latter encodes the brain aromatase P450aromB. Cyp19a1a plays an important role in the synthesis of estrogen and ovarian differentiation, Cypl9a1b is closely related to the biosynthesis of estradiol-17β (E2) in the brain, and may be involved in the development of central nervous system development and regulation of sexual behavior. Channa argus is one of the most commercially important fish species in China. Male individuals of C. argus grow faster than female ones at the same age. In order to explore sex differentiation mechanism of this species and lay the foundation for manual manipulation of the sex ratio, we investigated the early gonadal development and sex differentiation by histological analysis and also identified Cypl9alb and Foxl2 cDNA. We analysed Foxl2 expression patterns by qPCR during gonadal sex differentiation and the localisation of the Foxl2 protein by immunohistochemistry in mature gonads of C. argus. The main results are as follows:1. Histology of early gonad development in C. argusTwo primordial germ cells (PGCs) were present under the mesonephric duct at 6 dph (day post hatching), forming the primitive gonads. From 15 dph to 21 dph, the gonads projected into the abdominal cavity. Gonad size and number of germ cells increased dramatically by active mitosis. The gonads did not exhibit any morphological characteristics indicative of a differentiating ovary or testis at the moment. An apparent change in larval gonad histology occurred at 27 dph. The presumptive initial ovarian cavity formation was indicated by the presence of two elongated aggregations of germ cells in the gonads at 27 dph. In contrast to ovarian development, the presumptive testis had germinated to be two aggregations of stromal cells at 27 dph. At 30 dph, the ovarian cavity was completely formed and oogonia were undergoing active mitosis to become oocytes. At 34 dph, ovarian gonads were observed to contain numerous oocytes at chromatin nucleolus phase. Some slit-like spaces in the central stromal tissue of testes formed the efferent duct anlages at 34 dph, and blood vessels were observed in the dorsal region of testes. At 48 dph, numerous peri-nucleolus oocytes were found in the ovarian gonad and some spermatogonia were undergoing mitotic proliferation in the testicular gonad.2. Cloning and sequence analysis of Cypl9a1b and Foxl2 cDNA in C. argusA 1432 bp partial length Cypl9a1b cDNA was obtained, including a 120 bp 3’-UTR and a 1311 bp coding region encoding a putative 436 amino acids (GenBank accession no. KF746073). C. argus Cyp19a1b amino acid sequence showed a high level of homology with Trichogaster trichopterus (89%), Rhabdosargus sarba (88%) and Scatophagus argus (88%). The similarity was 69%～85% to other teleosts. The similarities were low (55%～56%) to rat and human. Alignment of known Cyp19a amino acid sequences from vertebrates showed that C. argus Cyp19a1b amino acid sequence contained three functionally conservative regions. A 1966 bp full length Foxl2 cDNA was obtained, including a 213 bp 5’-untranslated region (5’UTR), an 832 bp 3’UTR and a 921 bp opening reading frame (ORF) encoding a putative 306 amino acids (GenBank accession no. KF746072). Channa argus Foxl2 amino acid sequence showed the highest similarity to Epinephelus merra (97%). The similarity were 81%～95% to other teleosts. The similarities were 61%～79% to rat and human. The forkhead domain of Foxl2 was highly conserved among these vertebrate species.3. Tissue distribution of Cypl9alb and Foxl2 mRNA and expression analysis of Foxl2 gene during the early development stages in C. argusSemi-quantitative RT-PCR and qPCR revealed that Cypl9a1b mRNA had high levels of expression in the pituitary and low levels of expression in the intestine of both female and male. There was a high expression level in the brain of male, but no expression in the brain of female and in other detected tissues of sexes. Expression of Foxl2 mRNA in females had the highest tissue-specific level in ovary, followed by a high level in pituitary and gill, and a very low level in other tested tissues. The expression level of Foxl2 mRNA in males was the highest in gill, followed by a high level in pituitary, a moderate level in brain, whereas the expression levels were very low in testis and other tested tissues. The expression of Foxl2 mRNA increased significantly from a moderate level at 1 dph to the highest level at 4 dph, and then decreased significantly to a moderate level at which it was maintained until 11 dph. Afterwards, the expression level decreased significantly to a low level at 17 dph and was maintained until 34 dph with a minor increase at 23 dph, and further decreased significantly to a very low level at 41 dph until 48 dph.4. Localisation of Foxl2 protein in the mature gonads of C. argusIn the ovary of the adults, Foxl2 immunoreactivity was detected in the granulosa cells surrounding the oocytes but not in the oocytes. In contrast, no specific signals were detectable in the testis.