New evidence confirms that the mitochondrial bottleneck is generated without reduction of mitochondrial DNA content in early primordial germ cells of mice

Abstract

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells.

Figure 1. Isolation of single PGCs from 7.5 dpc early bud (A–H) and late bud (I–N) stage Blimp1-mRFP embryos.

(A,C,E,G,I,K,M) Light microscopy images. (B,D,F,H,J,L,N) Fluorescent microscopy images. At both EB and LB stages mRFP was detected as a cluster in the posterior part of the embryo (dashed rectangle) and in the visceral endoderm (arrowheads). For isolating PGCs, the posterior regions bearing PGCs were cut off carefully to remove the visceral endoderm (E,F,K,L). The resulting tissue fragments were disaggregated through trypsinization, and single PGCs with fluorescence were collected unambiguously using micromanipulators under a fluorescent microscope (G,H,M,N).

Figure 2. Stella expression in the posterior region of Blimp1-mRFP transgenic mouse embryos.

Blimp1-mRFP (red, left column), Stella (green, middle column), and overlapped images (right column) at the EB (top row) and LB (bottom row) stages.

Figure 3. Alkaline phosphatase staining pattern of cells from the posterior region of Blimp1-mRFP embryos.

(A,B) Cells from EB stage. (C,D) Cells from LB stage. (A,C) Blimp1-mRFP negative cells. (B,D) Blimp1-mRFP positive cells. Scale bar, 20 µm.

Figure 4. mtDNA copy number in single cells of 7.5 and 13.5 dpc mouse embryos (logarithmic scale).

Open black circles, primordial germ cells (PGCs); Open grey circles, somatic cells. Each circle represents a single cell. Horizontal lines indicate mean values. EB, early bud stage. LB, late bud stage. F, M, and Soma denote samples from female PGCs, male PGCs, and gonadal somatic cells, respectively.