Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro

Abstract

Our understanding of human germ cell development is limited in large part due to inaccessibility of early human development to molecular genetic analysis. Pluripotent human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been shown to differentiate to cells of all three embryonic germ layers, as well as germ cells in vitro, and thus may provide a model for the study of the genetics and epigenetics of human germline. Here, we examined whether intrinsic germ cell translational, rather than transcriptional, factors might drive germline formation and/or differentiation from human pluripotent stem cells in vitro. We observed that, with overexpression of VASA (DDX4) and/or DAZL (Deleted in Azoospermia Like), both hESCs and iPSCs differentiated to primordial germ cells, and maturation and progression through meiosis was enhanced. These results demonstrate that evolutionarily unrelated and divergent RNA-binding proteins can promote meiotic progression of human-derived germ cells in vitro. These studies describe an in vitro model for exploring specifics of human meiosis, a process that is remarkably susceptible to errors that lead to different infertility-related diseases.

Figure 1

Fluorescence-activated cell sorting (FACS) of VASA-GFP positive and negative populations. (A): FACS plots and representation of the percentage of VASA-GFP positive cells we found for each cell line. Data are represented as mean ± SEM. (B): Representative VASA immunostaining of GFP-positive and -negative sorted populations. The scale bar represents a distance of 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; PE, phycoerythrin.

Figure 2

Gene expression in VASA-GFP positive and negative sorted cell populations. (A): Comparison of early germ cell markers expression profile among GFP-positive and -negative sorted populations. (B): Comparison of late and meiotic germ cell markers expression profile among GFP-positive and their respective negative sorted populations. Asterisks represent significant differences (*, p, < .05) with controls. Data are represented as normalized fold change mean ± SEM. Abbreviations: DAZL, Deleted in Azoospermia Like; DMC1, dosage suppressor of MCk 1; GFP, green fluorescent protein; MLH1, MutL homolog 1; SCP3, synaptonemal complex protein 3.

Figure 3

Expression of early and late germ cell markers in cells with and without ectopic expression of VASA (iVASA) 7 days postdifferen-tiation. (A): Comparison of expression of early germ cell markers. (B): Comparison of expression of late and meiotic germ cell markers. Asterisks represent significant differences (*, p < .05) with controls. Data are presented as normalized fold change mean ± SEM. Abbreviations: DAZL, Deleted in AZoospermia Like; DMC1, dosage suppressor of MCk 1; MLH1, MutL Homolog 1; SCP3, synaptonemal complex protein 3.

Figure 4

Analysis of the putative 1N cell populations from cell lines with ectopic expression of VASA (iVASA), 14 days post-transduction. (A): Representative DNA-fluorescence-activated cell sorting plot for the isolation of the putative 1N population and percentage of putative 1N cells we were able to isolate from each cell line. Sperm control was used to set the sorting parameters. (B): Representative double VASA and ACROSIN immunostaining of 1N sorted cells and percentage of cells positive for each marker. (C): Representative fluorescence in situ hybridization results for probes against chromosomes 16 (green) and 18 (red) over putative 1N sorted cells and percentage of haploid cells found in each cell line based on the analysis of these two probes. Scale bar = 10 μm. Asterisks represent significant differences (*, p < .05) with controls. Data are presented as mean ± SEM. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Synaptonemal complex (SCP) formation analysis over cell lines with ectopic expression of DAZL (iDAZL), VASA (iVASA), and DAZL+VASA (iDAZL+iVASA) and differentiated for 14 days. (A): Representative SCP3 staining of meiotic spreads. Scale bar = 10 μm. (B): Percentage of cells with punctuated (leptotene) and elongated (diplotene, pachytene, or zygotene) SCP3 staining patterns. Abbreviations: CENPA, centromeric protein A; DAPI, 4′,6-diamidino-2-phenylindole; DAZL, Deleted in Azoospermia Like; SCP3, synaptonemal complex protein 3.

Figure 6

Analysis of the DNA-FACS-sorted putative 1N cell populations with ectopic expression of DAZL (iDAZL), VASA (iVASA), and DAZL+VASA (iDAZL+iVASA), 14 days postdifferentiation. (A): Percentage of putative 1N cells isolated from each cell line. (B): Percentage of haploid cells confirmed by FISH in 1N sorted populations. (C): Percentage of 1N sorted cells positive for VASA and ACROSIN staining. Asterisks represent significant differences (*, p < .05) with controls. Data are presented as mean ± SEM. Abbreviations: DAZL, Deleted in AZoospermia Like.

Figure 7

CpG methylation analysis at the DMR of H19. (A): Diagrams represent methylation status of each CpG dinucleotides on individual DNA clones. Lines represent different clones and columns are different CpG dinucleotides. Methylated CpGs are represented as filled circles and unmethylated CpGs are represented as open circles. Empty CpG sites represent the CpGs that could not be determined. (B): Percentage of methylated CpGs found in each condition at the DMR of H19. Asterisks represent significant differences (*, p < .05) with controls. Abbreviation: DMR, differentially methylated region.