Global survey of protein expression during gonadal sex determination in mice

Abstract

The development of an embryo as male or female depends on differentiation of the gonads as either testes or ovaries. A number of genes are known to be important for gonadal differentiation, but our understanding of the regulatory networks underpinning sex determination remains fragmentary. To advance our understanding of sexual development beyond the transcriptome level, we performed the first global survey of the mouse gonad proteome at the time of sex determination by using two-dimensional nanoflow LC-MS/MS. The resulting data set contains a total of 1037 gene products (154 non-redundant and 883 redundant proteins) identified from 620 peptides. Functional classification and biological network construction suggested that the identified proteins primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. The data set contains potential novel regulators of gonad development and sex determination not revealed previously by transcriptomics and proteomics studies and more than 60 proteins with potential links to human disorders of sexual development.

Fig. 1.

Distribution of peptide counts used for protein identifications. Proteins identified by a single peptide were manually validated.

Fig. 2.

Subcellular localization of embryonic gonad proteins. Intracellular locations of 656 identified proteins were determined using IPA software.

Fig. 3.

Functional classification of embryonic gonad proteins. Generic functions were assigned to 656 mapped gonadal proteins using Ingenuity software.

Fig. 4.

Molecular roles of embryonic gonad proteins. For specific molecular functional analysis, 310 genes were matched to 49 significant functions (p < 0.05) using IPA. The 15 most significant functions are shown with the number of associated genes identified for each functional group.

Fig. 5.

Biological network constructed from embryonic gonad proteins. Using IPA software to predict coordinated networks of gene products, 342 genes were found to be components of 24 significant biological networks. Depicted is the highest scoring network containing 33 genes identified in the gonad proteome (shown in gray). The functions of this network include RNA post-transcriptional modification, gene expression, and RNA trafficking. Solid lines represent established direct interactions; dotted lines represent indirect interactions.

Fig. 6.

Chromosomal distribution of ESTs from embryonic gonad protein data set. Chromosomal positions for 867 genes encoding the identified proteins were ascertained from the mouse EST library.

Fig. 7.

Tissue expression of ESTs from embryonic gonad protein data set. Expression profiles in adult reproductive and related tissues for 696 genes collated from the mouse EST library are shown. All sites of expression were included for any gene found in multiple tissues.

Fig. 8.

Comparison of present screen and two other proteomics screens of embryonic gonads/PGCs. A Venn diagram comparing the present data set (Ewen) with those of Wilhelm et al. (35) and Han et al. (34) is shown.

Fig. 9.

Comparison of present screen and transcriptomics screen of embryonic gonads. A Venn diagram comparing the present data set (for proteins with available microarray identifiers; Ewen) with a transcriptomics data set from Small et al. (26) is shown.