The role of mitochondrial DNA copy number in mammalian fertility

Abstract

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000-50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

FIG. 1.

Distribution of mitochondrial DNA copy number in wild-type, heteroplasmic, cleavage-stage embryos. Embryos contain an average of 161 000 (SD, 73 000) copies of mtDNA (range, 11 000–428 000 copies; n = 219).

FIG. 2.

Distribution of mitochondrial DNA copy number in germline knockout embryos: comparison of mtDNA copy number in wild-type (WT), heteroplasmic, cleavage-stage embryos from Tfam^+/− heteroplasmic females (A), Tfam^fl/+;Zp3-Cre⁺ heteroplasmic females (B), and Cox10^+/− females (C). Embryos from Tfam^+/− heteroplasmic females (A; black) contained an average of 69 000 copies of mtDNA (n = 74), whereas those from Tfam^fl/+;Zp3-Cre⁺ heteroplasmic females (B; black) contained an average of 18 000 copies (n = 96). Embryos from heteroplasmic Cox10^+/− females (C; black) contained an average of 424 000 copies of mtDNA (n = 54).

FIG. 3.

Defining the critical developmental threshold of mtDNA copy number. Mitochondrial DNA quantification of biopsied blastomeres from cleavage-stage (8-cell) embryos was performed to estimate mtDNA content in cleavage-stage embryos before transfer to surrogate recipients. Embryos were grouped into low-copy or high-copy groups and then transferred to separate uterine horns of the surrogate. In some cases, it was possible to assign copy number in individual embryos by examining the heteroplasmy measurements in the developed postimplantation embryo. Normal, stage-appropriate morphological development was scored as positive; aborted and developmentally delayed embryos were scored as negative. All images were acquired under the same original magnification (×25).

FIG. 4.

Embryo transfer and developmental outcome. Bar graphs represent the mean frequency of implantation and survival, scored by normal morphological and developmental progression following embryo transfer of nonbiopsied controls, Low-copy group embryos (n = 19; mtDNA copy number range, 3558–55 000), and high-copy group embryos (n = 15; mtDNA copy number range, 56 000–160 000). Low-copy and high-copy groups were determined to be statistically different (P > 0.001), but the developmental outcome between the nonbiopsied control embryos and the high-copy group were not (P = 0.52).

FIG. 5.

Measurement of sperm function and allelic bias in Tfam heterozygous males. Standard measures of sperm motility (average path velocity [VAP], progressive velocity [VSL], track speed [VCL], amplitude of lateral head displacement [ALH], beta cross frequency [BCF], measure of the departure of the cell path from a straight line [STR], measure of the departure of the cell track from a straight line [LIN], and percentage motility [Percent Motile]) and sperm count were assessed in caudal epididymal samples from Tfam heterozygous males (A). Bar graphs represent the results as a percentage of wild-type littermate controls of VAP. No statistically significant differences (Student t-test) were observed between groups for any of the measurements. A paternal transmission bias for the deleted Tfam allele from Tfam^+/− males was observed in these sperm samples as well as in the offspring of recessive testcrosses (B). Floxed Tfam, wild-type Tfam, and maternally transmitted deleted Tfam alleles segregated in mendelian fashion (data not shown).

FIG. 6.

Critical threshold of mtDNA copy number during embryogenesis. The model for the evolution of mtDNA copy number in the female germline indicates a critical threshold for mtDNA set at approximately 50 000 copies of mtDNA in the mature oocyte. Subthreshold embryos can progress normally through cleavage but spontaneously abort after implantation. At E6.0, the stage at which mtDNA replication resumes, the cells of the embryo proper would be expected to contain an average of 215 copies, assuming equal distribution of mtDNA in the cells of the developing embryos. Tfam^fl/+;Zp3-Cre⁺ oocytes (∼18 000 copies of mtDNA; red) are successfully fertilized and progress through cleavage but fail to develop normally following implantation because of mitochondrial insufficiency.