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Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes - PubMed

  • ️Fri Jan 01 2021

Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes

B Soygur et al. Sci Adv. 2021.

Abstract

Meiosis is critical to generating oocytes and ensuring female fertility; however, the mechanisms regulating the switch from mitotic primordial germ cells to meiotic germ cells are poorly understood. Here, we implicate intercellular bridges (ICBs) in this state transition. We used three-dimensional in toto imaging to map meiotic initiation in the mouse fetal ovary and revealed a radial geometry of this transition that precedes the established anterior-posterior wave. Our studies reveal that appropriate timing of meiotic entry across the ovary and coordination of mitotic-meiotic transition within a cyst depend on the ICB component Tex14, which we show is required for functional cytoplasmic sharing. We find that Tex14 mutants more rapidly attenuate the pluripotency transcript Dppa3 upon meiotic initiation, and Dppa3 mutants undergo premature meiosis similar to Tex14 Together, these results lead to a model that ICBs coordinate and buffer the transition from pluripotency to meiosis through dilution of regulatory factors.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Figures

Fig. 1
Fig. 1. 3D mapping of meiosis in mouse fetal ovaries.

(A) Schematic summarizing 3D analysis method for ovaries. Total germ cell population is illustrated in green, while meiotic germ cells are depicted in red and magenta. Following whole-mount immunofluorescence and 3D visualization of embryonic ovaries, individual germ cells were identified. The ovary was divided into seven segments based on distance and angle from origin, respectively. In M-L distribution graph, bins 1 and 7 are most lateral, and 3 to 5 are medial (toward the middle) regions of the ovary. In A-P distribution graph, bin 1 is anterior, and 7 is posterior. (B) Oct4-GFP revealed homogeneous distribution of germ cells at E14.5, whereas (C) OCT4 protein showed mild posterior skewing (P = 0.0274). STRA8-expressing (D) and SYCP3-expressing (F) cells were homogeneously distributed, indicating that most germ cells completed premeiotic DNA replication and started synaptonemal complex assembly. Dashed rectangle in (C) shows borders of zoom in (Bi). (Cii), (Diii) to (iv), and dashed yellow line defines borders of the OCT4-expressing cell cluster. (G) SYCP1 expression was detected in very few cells at E14.5. Dashed rectangle in (G) shows borders of zoom in (i) and (ii). (E) Quantification of OCT4+, STRA8+, SYCP3+, and SYCP1+ germ cells in whole-mount at E14.5. Adjacent to each immunostaining is the graph of M-L (top) and A-P (bottom) distribution with identical color scheme. Primary y axes (on the left) indicate germ cell numbers in each bin, dashed lines represent individual ovaries, and color-coded thick lines show mean value for each antibody. Secondary y axes (on the right and colored in gray) present the percentage of OCT4, STRA8, and SYCP1 cells (normalized to total germ cells) in each bin. At least n = 4 ovaries (from two litters and two embryos) were analyzed for each time point.

Fig. 2
Fig. 2. Earlier meiotic initiation and a new radial meiotic wave in embryonic mouse ovaries.

Detection of TRA98 (A), OCT4 (B), STRA8 (C), and SYCP3 (D) proteins in E12.5 ovaries by wholemount immunofluorescence. Sparse STRA8+ and SYCP3+ cells were identified as early as E12.5. White dashed rectangle in (D) indicates zoom at right. (Di) SYCP3+ meiotic germ cells at E12.5 are delineated by yellow dash with TRA98 stain in (Dii). Graphs show anterior skew of SYCP3+ (P = 0.0404) but not STRA8+ cells [(C); P = 0.8070] at E12.5. More SYCP3+ cells were detected in the medial part (segments 3 to 5) than lateral parts (1, 2, 6, and 7) (P = 0.0002) at E12.5. (E) Quantification of germ cells with each marker at E12.5 and E13.5. Wholemount immunolocalization of TRA98 (F), OCT4 (G), STRA8 (H), and SYCP3 (I) at E13.5. Uptick of STRA8+ and SYCP3+ germ cells was confirmed at E13.5, with STRA8+ cells enriched in the anterior (P = 0.0546). (J) SYCP3+ cells congregated in clusters in the anterior-medial part of the ovary [shown in transverse optical section view in (Ii) to (Iiii)] with significant A-P bias (P = 0.0001), although clusters were also noted near the posterior tip. (J) and (K) show SYCP3+ spots in the ventral and dorsal views of the E13.5 ovary, respectively, with graph showing the highest numbers in the middle segment and increased numbers in the dorsal compared to ventral region (P = 0.0003). Primary y axes (on the left of each graph) indicate germ cell numbers in each bin, dashed lines represent individual ovaries, and color-coded thick lines show mean value for each antibody. Secondary y axes (on the right and colored in gray) present percentage of STRA8 and SYCP3 cells (normalized to total germ cells) in each bin. At least n = 3 ovaries (from two litters and three to four embryos) were analyzed for each time point. ***P < 0.0001.

Fig. 3
Fig. 3. ICBs orchestrate meiotic initiation and the radial meiotic wave in differentiating female germ cells.

(A) Increased numbers and scattering of SYCP3+ cells were observed in Tex14 mutant (Tex14−/−) at E13.0 compared to SYCP3+ clusters in the core of WT ovaries. (B) At E13.5, SYCP3+ cells were increased in number in Tex14−/− ovaries and more advanced toward the posterior, compared to WT. (C) Similar germ cell numbers were observed in WT and Tex14 mutant in E12.5, E13.0, and E13.5 ovaries using TRA98 or Oct4-GFP. n.s., not significant. (D) Percentage of SYCP3+ cells in each ovary segment at E13.0 and E13.5. WT indicated in blue and Tex14−/− in black. (E) An increased number of SYCP3+ cells was detected in lateral segments (1 to 3 and 5 to 7) of Tex14−/− ovaries compared to WT at E13.0 (P = 0.0003) and E13.5 (P = 0.0335) supporting the absence of the radial wave in Tex14−/−. (F) Similar SYCP3 expression pattern was seen E16.5 WT and Tex14−/− ovaries, suggesting that meiosis progressed normally in later stages. Graphs corresponding to each immunostaining are adjacent, with WT indicated in blue and Tex14−/− in black. Dashed lines represent individual ovaries, and thick lines show the mean value for each genotype. (G) Analysis of different stages in MPI between genotypes showed that meiotic progression is comparable at E16.5. Insets (i) to (iv) in (A) and (i) and (ii) in (F) are forelimbs collected from WT and Tex14−/− mutants for accurate staging and age-matched comparison of embryos. WT includes genetically WT or heterozygous animals. MPI, Meiotic prophase I. At least n = 4 ovaries (from two litters and three embryos) were analyzed for each genotype and time point. *P < 0.05, ***P < 0.0001. ns, not significant.

Fig. 4
Fig. 4. Cytoplasmic sharing through ICBs is absent in Tex14−/− fetal ovaries.

(A) Experimental approach to detect germ cell cytoplasmic sharing using recombination-induced multicolor clonal labeling. (B) Schematized formation of bicolored germ cell clones with R26R-Rainbow;Pou5f1Cre-ER in the presence and absence of ICBs through cytoplasmic sharing of FPs in WT and Tex14−/− genetic backgrounds, respectively. (C) Endogenous expression of FPs in E13.5 R26R-Rainbow;Pou5f1Cre-ER;Tex14+/+ and R26R-Rainbow;Pou5f1Cre-ER;Tex14−/− ovaries reveals divergent clone organization. Magnified image below shows a bicolor clone positive for mCerulean and mOrange in a R26R-Rainbow;Pou5f1Cre-ER;Tex14+/+ E13.5 ovary. Although germ cells expressing different FPs were closely juxtaposed in some cases, bicolored clones were not observed in E13.5 R26R-Rainbow;Pou5f1Cre-ER;Tex14−/− ovaries. (D) The mean clone size in 214 clones from E13.5 R26R-Rainbow;Pou5f1Cre-ER;Tex14+/+ ovaries was 8 (mCerulean, 64 clones; mOrange, 33 clones; mCherry, 177 clones). (E) Table of total labeled cells, estimated total labeled clones (based on mean clone size of 8), total bicolored clones, and estimated percentage of bicolored clones in R26R-Rainbow;Pou5f1Cre-ER;Tex14+/+ (n = 3) and R26R-Rainbow;Pou5f1Cre-ER;Tex14−/− (n = 4) ovaries at E13.5 (see fig. S4D for the breakdown of total number of cells per genotype).

Fig. 5
Fig. 5. Dissecting the transcriptional signature of Tex14+/− and Tex14−/− ovaries during the time of meiotic initiation.

(A) Experimental design schematic. (B) Visualization of single-cell clustering by tSNE shows 10 germ cell and 12 somatic cell clusters in Tex14+/− and Tex14−/− ovaries represented by different colors. FACS, fluorescence-activated cell sorting. (C) Expression of germ cell marker Dazl identifies germ cell clusters. Pluripotent germ cells are defined by expression of Pou5f1 transcript; meiotic germ cells are defined by Sycp3 transcript. Dashed lines indicate cells with higher levels of Pou5f1 or Sycp3. (D) Integration of previously published E13.5 and E14.5 C57BL/6 datasets (24) with E13.5 Tex14+/− and Tex14−/− datasets. (E) Dazl identifies germ cell clusters, pluripotent germ cells are defined by expression of Pou5f1, and meiotic germ cells are defined by Sycp3 transcript. (F) Scatter plot of differentially expressed genes between pluripotent and meiotic clusters in Tex14+/−. (G) Dot plot graph of highly expressed genes (Rhox5, Actb, Dppa3, Phlda2, and Rec8) in the pluripotent clusters compared to meiotic clusters in E13.5 C57BL/6, E14.5 C57BL/6, E13.5 Tex14+/−, and Tex14−/− integrated datasets. Dot size indicates percentage of cells expressing the transcript, while colors denote expression level in cells. Expression of genes associated with the pluripotent cluster was the lowest in Tex14 mutant germ cells, whereas expression of meiotic markers Stra8 and Sycp3 was similar in E14.5 C57BL/6, E13.5 Tex14+/−, and Tex14−/−.

Fig. 6
Fig. 6. Reciprocal expression of SYCP3 and DPPA3, precocious meiosis in Dppa3 mutants, and methylation changes in Tex14 mutants link ICBs, the epigenetic reprogramming during meiotic initiation.

(A) WT germ cell cluster and (B) Tex14 mutant germ cell cluster (yellow outlines) expressing SYCP3 (magenta) at E13.5 lack DPPA3 (red), whereas SYCP3− cells express DPPA3 (arrows). (C) SYCP3 expression in Dppa3+/− and Dppa3−/− ovaries at E14.5, with quantification in (D) (n = 6 for WT C57BL/6, n = 2 for Dppa3+/−, and n = 4 for Dppa3−/− ovaries). (E) mOrange+ clone with uniform SYCP3 (magenta) in R26R-Rainbow;Pou5f1 Cre-ER;Tex14+/+ ovary. R26R-Rainbow;Pou5f1 Cre-ER;Tex14−/− exemplary mCherry and mCerulean clones (yellow outlines) do not homogeneously express SYCP3 (white arrows indicate SYCP3−cells). (F) Model for coordination of meiotic initiation through ICBs in E13.0 fetal germ cells depicts dilution of regulatory factors in mitotic-meiotic transition. Cytoplasmic sharing may synchronize the pluripotent state of germline cysts in WT by increasing cytoplasmic volume, as meiotic transcripts must be expressed by a quorum to initiate meiosis. Without ICBs, germ cells expressing meiotic transcripts enter meiosis individually, leading to earlier meiotic onset in Tex14−/−. (G) LINE-1 ORF1p expression, as a proxy of DNA demethylation, shows increased scattering (white arrows) and frequency (H) in Tex14−/− compared to Tex14+/+ ovaries at E13.5. (I) Ripley’s K function analysis shows the clustered distribution of LINE-1 ORF1p+ germ cells in WT compared to Tex14 mutant (†P = 0.002). Embryo forelimbs in (G) compare ages. n = 5 ovaries were analyzed in both cases. (J) Methylation of GRR genes is similar between Tex14−/− female germ cells and Tex14+/− or Tex14+/+ controls at E13.0 (n = 1 knockout, n = 2 control) but reduced at E13.5 (n = 2 knockout, n = 2 control) by WGBS. WT includes genetically WT and heterozygous animals in (J).

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