Prediction of sex-determination mechanisms in avian primordial germ cells using RNA-seq analysis

Abstract

Avian sex is determined by various factors, such as the dosage of DMRT1 and cell-autonomous mechanisms. While the sex-determination mechanism in gonads is well analyzed, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism of sex-determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also displayed sex-biased expression, and the number of female-biased genes detected was higher than male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Stimulation with retinoic acid against cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination of avian PGCs possess aspects of both cell-autonomous and somatic cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.