| 【Objective】To investigate the molecular mechanism of sex reversal in female Western mosquitofish
(Gambusia affinis) under the effect of gestodene (GES) by studying the development of gonadal cell differentiation and
gene expression changes involved in the process of sex reversal in female mosquitofish at the single-cell transcriptome
level.【Method】Sexually mature female mosquitofish were exposed to 500 ng/L GES after culture and domestication for
2 months , and samples were taken at the 4th and 8th week to observe morphological changes, and single-cell suspensions
were made from the gonads of normal female controls, male controls, and samples exposed for 4 and 8 weeks, respectively, for single-cell transcriptome sequencing and analysis.【Result】The morphology of GES-treated female mosquitofish showed
gonopodium, and single-cell transcriptome sequencing revealed completely different cellular composition of gonads between
male and female mosquitofish. The gonads of male mosquitofish consist mainly of fibroblasts, male germ cells, epithelial cells
and primordial germ cells, whereas the gonads of female mosquitofish consist mainly of endothelial cells, fibroblasts, oocytes
and primordial germ cells. After 4 and 8 weeks of exposure, a small amount of male germ cells appeared in the gonads of
female mosquitofish, and the number of primordial germ cells increased significantly. Further analysis revealed that several
crucial genes related to the sex-differentiation were differentially expressed in primordial germ cells. Cell trajectory analysis
showed that some primordial germ cells showed characteristics of differentiation towards males, in which 2 477 genes showed
different expression patterns in two different different directions of differentiation. The results of KEGG pathway enrichment
analysis revealed that signaling pathways, such as Toll-like receptor, TGF-beta, Notch, etc., were involved in the process of
reversal of primordial germ cells towards maleness. Glycine, serine, threonine, cysteine, methionine metabolism, amino sugar
and nucleotide sugar metabolism and other pathways were also related to this process. 【Conclusion】After exposure to GES
solution, female mosquitofish showed morphologically masculinized anal fins, and some primordial germ cells in the gonads were
transformed to male germ cells. Differential expression of amh, fabp3, dmrt1, wt1a, wt1b, cyp19a1a and fgf16, which are key
genes for sex determination, was observed in the primordial germ cells. Toll-like receptor, TGF-beta, Notch, and some amino
acid metabolism signaling pathways were involved in the process of sex reversal. |
