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

Abstract

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, 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 underlying 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 exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of 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. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

Fate Decisions of Chicken Primordial Germ Cells (PGCs): Development, Integrity, Sex Determination, and Self-Renewal Mechanisms

“…Recent studies have shown characteristic avian features in the sex determination of PGCs. Ichikawa et al conducted RNA-seq analysis using male and female PGCs harvested from embryonic chicken blood at HH stage 17 (after 2.5 d of incubation) and gonads at HH stage 25–26 (after 4.5 d of incubation) and HH stage 30 (after 6.5 d of incubation) [ 5 ]. The gene expression profiles obtained in that study suggest that several W-chromosome-linked genes are expressed in a cell-autonomous manner in female PGCs before they settle into the gonads ( Figure 2 b) [ 5 ].…”

“…Ichikawa et al conducted RNA-seq analysis using male and female PGCs harvested from embryonic chicken blood at HH stage 17 (after 2.5 d of incubation) and gonads at HH stage 25–26 (after 4.5 d of incubation) and HH stage 30 (after 6.5 d of incubation) [ 5 ]. The gene expression profiles obtained in that study suggest that several W-chromosome-linked genes are expressed in a cell-autonomous manner in female PGCs before they settle into the gonads ( Figure 2 b) [ 5 ]. Furthermore, alterations in gene expression profiles were observed during gonadal sex determination in female PGCs prior to that in male PGCs, and female-biased genes were enriched in several metabolic processes ( Figure 2 b) [ 5 ].…”

“…The gene expression profiles obtained in that study suggest that several W-chromosome-linked genes are expressed in a cell-autonomous manner in female PGCs before they settle into the gonads ( Figure 2 b) [ 5 ]. Furthermore, alterations in gene expression profiles were observed during gonadal sex determination in female PGCs prior to that in male PGCs, and female-biased genes were enriched in several metabolic processes ( Figure 2 b) [ 5 ]. In contrast, Rengaraj et al showed male-biased gene expression in chicken PGCs collected from early embryos around HH stage 17–34 (after 2.5–8 d of incubation) ( Figure 2 b) [ 14 ].…”

“…Avian PGCs have characteristic developmental features, such as migration into the gonads using the vascular system [ 2 ]. Chicken PGCs have also shown a unique sex determination mechanism in which PGC-intrinsic factors may occur in a cell-autonomous manner [ 3 , 4 , 5 ]. In addition, several studies have revealed the self-renewal mechanism of chicken PGCs, resulting in the establishment of stable culture protocols for chicken PGCs [ 6 , 7 ].…”

The Transcriptome of Chicken Migratory Primordial Germ Cells Reveals Intrinsic Sex Differences and Expression of Hallmark Germ Cell Genes

“…Recently, the transcriptomes of male and female PGCs isolated from blood at different embryonic stages showed transcriptome divergence [ 16 ]. To date, the germ cell-specific transcriptome of in vitro propagated chicken PGCs in a serum-free medium and the sexual differences in the gene regulatory networks at this stage for male and female PGCs have not been described.…”