Control of epigenomic landscape and development of fetal male germ cells through L-serine metabolism

Abstract

Sex-specific metabolic characteristics emerge in the mouse germ line after reaching the genital ridges around embryonic day 10.5, coinciding with sexual differentiation. However, the impact of such metabolic characteristics on germ cell development remains unclear. In this study, we observed the specific upregulation in male fetal germ cells of D-3-phosphoglycerate dehydrogenase (PHGDH), the primary enzyme in the serine-glycine-one-carbon metabolism, along with an increase in a downstream metabolite, S-adenosylmethionine (SAM), crucial for protein and nucleic acid methylation. Inhibiting PHGDH in fetal testes resulted in reduced SAM levels in germ cells, accompanied by increases in the number of mouse vasa homolog (MVH/VASA)-positive germ cells and the promyelocytic leukemia zinc finger (PLZF)-positive undifferentiated spermatogonia ratio. Furthermore, PHGDH inhibition led to a decrease in the methylation of histone H3 and DNA, resulting in aberrations in gene expression profiles. In summary, our findings underscore the significant role of certain metabolic mechanisms in the development of male germ cells.

Keywords: Cellular physiology; Developmental biology; Epigenetics.

Graphical abstract

Figure 1

Changes in SGOC metabolism during germline differentiation (A) Schematic representation of metabolic pathways around PHGDH. Cyan denotes components more abundant in male germ cells than female germ cells. (B) Immunostaining analysis depicting changes in PHGDH expression during fetal germline differentiation. Testes and ovaries were examined at E12.5 and E16.5. (C) Quantification of relative fluorescence signal intensity of PHGDH in VASA-positive germ cells and surrounding somatic cells in fetal gonads (total of 29–30 cells from three biological replicates). Graphs present relative fluorescence intensity, with the fluorescence intensity of VASA-negative somatic cells at each fetal age set as 1. (D) Schematic illustration of metabolic pathways around SAM. Cyan indicates components more abundant in male germ cells than female germ cells. (E) Immunostaining analysis revealing changes in SAM abundance during fetal germline differentiation. Testes and ovaries were examined at E12.5 and E16.5. (F) Quantification of relative fluorescence signal intensity of SAM in VASA-positive germ cells and surrounding somatic cells in fetal gonads (total of 30 cells from three biological replicates). Graphs present relative fluorescence intensity, with the fluorescence intensity of VASA-negative somatic cells at each fetal age set as 1. Data information: values represent mean ± SE of three independent experiments. Statistical significance indicated as N.S. (not significant), ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test). Scale bar: 50 μm. Refer also to Figures S1 and S2.

Figure 2

Effects of PHGDH inhibition in fetal testicular organ culture (A) Schematic illustration of the fetal testes organ culture experiment. (B) Phase-contrast microscopic image of the testes on day 14 of organ culture. (C) Immunostaining of VASA and PLZF in testes on day 14 of organ culture. Rectangular areas correspond to enlarged images in the insets. (D–F) Changes in the number of VASA-positive cells (D), number of PLZF-positive cells (E), and PLZF-positive rate calculated by dividing the number of PLZF-positive cells by the number of VASA-positive cells (F) following PHGDH inhibition in testes on day 14 of organ culture. Graphs present relative number of positive cells and PLZF-positive rate with the number in the culture with CBR set as 1 (two tiling sections for each replicate). (G) Immunostaining analysis showing changes in SAM abundance following PHGDH inhibition in cultured testes at day 7. (H) Quantification of relative fluorescence signal intensity of SAM in VASA-positive germ cells normalized to the surrounding somatic cells in cultured testes at day 7 (total of 30–33 cells from three biological replicates). (I–K) Changes in the number of VASA-positive cells (I), number of PLZF-positive cells (J), and PLZF-positive rate calculated by dividing the number of PLZF-positive cells by the number of VASA-positive cells (K) following the addition of SAM in testes on day 14 of organ culture. Graphs present relative number of positive cells and PLZF-positive rate with the number in the culture with CBR set as 1 (two tiling sections for each replicate). Data information: values represent mean ± SE of three to six biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test for I, J, and K, and unpaired Student’s t test for D, E, F, and H). Scale bar: 500 μm (B), 100 μm (C), and 50 μm (G). Refer also to Figure S2.

Figure 3

Impact of PHGDH inhibition on gene expression in male germ cells (A) Schematic representation of the gene expression analysis in germ cells derived from cultured testes. (B) RT-qPCR analysis of gene expression in germ cells isolated from cultured testes. Relative gene expression levels in germ cells treated by the PHGDH inhibitor CBR-5884 with or without SAM supplementation compared to those treated with DMSO are presented. (C) Illustration outlining the induction of germline stem (GS) cells from cultured testes. (D) GS cell colonies derived from VASA-RFP-positive germ cells in cultured testes at day 14. (E) Visualization of GS cell colonies through VASA-RFP fluorescence. Colonies were observed across the wells using tiling analysis, with rectangular areas corresponding to enlarged images in the insets. (F) Quantification of the relative number of GS colonies per well. The number of colonies from germ cells treated with DMSO was normalized to 1. Data information: values represent the mean ± SE of three to four (B) and five (E) biological replicates. Statistical significance is denoted by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (unpaired Student’s t test). Scale bar: 200 μm (D) and 1 mm (E). Refer also to Figure S3, Tables S1, and S2 for additional details.

Figure 4

Effects of PHGDH inhibition on epigenomic regulation (A, C, and E) Immunostaining analysis illustrating changes in H3K4me3 (A), H3K9me2 (C), and H3K27me3 (E) due to PHGDH inhibition with or without SAM supplementation in cultured testes at day 14. (B, D, and F) Quantification of relative fluorescence signal intensities of H3K4me3 (B), H3K9me2 (D), and H3K27me3 (F) in PLZF-positive spermatogonial cells, normalized with surrounding somatic cells in cultured testes at day 14 (total of 30 cells from three biological replicates). Data information: values represent the mean ± SE of three biological replicates. Statistical significance is denoted by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test). Scale bar: 50 μm. Refer also to Figure S4.

Figure 5

Male perinatal germ cell properties in Phgdh-cKO mice (A) Schematic depiction of the generation of Phgdh-cKO mice. (B, D, and F) Immunostaining analysis illustrating changes in PHGDH (B), SAM (D), and H3K4me3 (F) due to Phgdh KO in testes at E19.5. (C, E, and G) Quantification of relative fluorescence signal intensities of PHGDH (B), SAM (D), and H3K4me3 (F) in VASA-positive germ cells normalized with surrounding somatic cells at E19.5 (total of 30–33 cells from three biological replicates). (H) Detection of VASA-positive germ cells throughout the testicular section by tiling analysis at E19.5. (I) Quantification of the number of VASA-positive germ cells per unit area in Phgdh wild-type and homo-cKO testes at E19.5 (five sections for each replicate). Data information: values represent the mean ± SE of three (C, E, G) and five (I) biological replicates. Statistical significance is indicated by N.S. (not significant), ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test for C, E, G, and unpaired Student’s t test for I). Scale bar: 500 μm (I), 50 μm (C, E, and G). Refer also to Figure S5.

Figure 6

Spermatogonial transition in Phgdh-cKO mice (A, C, and F) Immunostaining analysis illustrating changes in apoptosis (A), undifferentiated spermatogonia (C), and spermatogonial differentiation (F) due to Phgdh KO in testes at P7. (B, D, and G) Quantification of the ratio of germ cells exhibiting a positive signal for active caspase-3 in VASA-positive germ cells (B), for PLZF within VASA-positive germ cells (D), or for c-KIT within SALL4-positive spermatogonia (G) in testes at P7 (two sections, four areas for each replicate). (E and H) Quantification of germ cells exhibiting a positive signal for PLZF and/or VASA (E) or for SALL4 and/or c-KIT (H) per unit area in wild-type or Phgdh-KO testes at P7 (two sections, four areas for each replicate). Data information: values represent the mean ± SE of three biological replicates. Statistical significance is indicated by N.S. (not significant), ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test for E and H, and unpaired Student’s t test for B, D, and G). Scale bar: 50 μm. Refer also to Figure S6.