Abstract
In females of many species, over half of the germ-cell (oocyte) population
dies by apoptosis before birth1. For example, germ-cell numbers
peak at 5–7
106 at week 20 of gestation in humans,
but drop to less than 1106 in the early neonatal period2, 3. Apparent germ-cell wastage occurs on a similar scale in female
rodents, falling from 6.4104 at day 17.5 of pregnancy
to 1.9104 shortly after birth4. Krakauer
and Mira5 have interpreted this death of germ cells as a developmental
solution to the accumulation of mutations in mitochondria, proposing that
prenatal oocyte apoptosis effectively removes oocytes carrying mutant mitochondria.
Here we test whether mitochondria can actually influence oocyte fate by microinjecting
small numbers of mitochondria into mouse oocytes and find that this prevents
these cells from undergoing apoptosis. We also show that a common mitochondrial
DNA deletion occurs more frequently in unfertilized, as compared with fertilized,
human oocytes.
We used oocytes from FVB female mice, because these oocytes undergo inherently
high rates of apoptosis in vitro6. After being denuded
of somatic (granulosa) cells, each oocyte was microinjected individually,
either with buffer or with about 5103 mitochondria purified
from non-apoptotic follicular granulosa cells of female mice that had been
primed 46 hours beforehand with a single injection of equine chorionic gonadotropin
to promote granulosa-cell viability7. After culturing for 24
hours, 70% of the oocytes that had either not been microinjected or had been
microinjected with buffer underwent apoptosis8, 9, whereas only
36% of the oocytes microinjected with mitochondria initiated apoptosis (Fig. 1). As a single mouse oocyte contains about 110
5 mitochondria10, these findings are striking, considering
that the total mitochondrial pool per microinjected oocyte was only increased
by some 5%.
Figure 1: Effect of microinjecting mitochondria on the occurrence of spontaneous apoptosis in murine oocytes incubated in vitro for 24 hours.
The magnitude of apoptosis was equivalent in oocytes not microinjected
(control, CON) and those microinjected with buffer alone. However, microinjection
of approximately 5103 purified mitochondria (MITO) suppressed
the activation of programmed cell death in cultured oocytes by 50%. The total
number of oocytes used in each group is indicated over the respective bar
(mean
s.e.m., n=3 independent experiments; *P<0.05
).
We then used the polymerase chain reaction to assay for a common deletion of 4,977 base pairs in mitochondrial DNA in 72 human primordial follicles isolated from ovarian biopsies of women aged 20–49. Deletions were detected in 14 follicles (R.G.G. et al., unpublished results), although without the correlation with age reported in long-lived somatic cells11. In parallel studies, the frequency of deletions in human embryos was lower than in oocytes, suggesting that superior oocytes had been selected for fertilization (R.G.G. et al., unpublished results).
Although these two pieces of evidence support the hypothesis of Krakauer and Mira5, several issues remain unresolved. We agree that one function of prenatal germ-cell loss could be to remove oocytes with defective mitochondrial genomes; however, these oocytes probably represent a very small percentage of the total oocyte pool lost before birth, and thus additional functions for prenatal oocyte death need to be considered1.
The proposed mitochondrial mutations must also be defects carried forward from the previous generation(s), as it is implausible that so many oocytes could be lost prenatally as a result of mitochondrial DNA mutations accumulating between oogenesis and parturition. This may create a paradox, however, because Krakauer and Mira5 predict that the organism will arise from an oocyte free of mitochondrial DNA defects. In many animal species, the mitochondria present in all cells of the organism are derived from replication of the original maternal (oocyte-derived) mitochondria, implying that all cells in the newly developing embryo, including the germ line, would possess mitochondria free of DNA mutations.
Krakauer and Mira5 also do not account for the continued
postnatal loss of oocytes in many species. The oocyte population in human
females declines mainly as a result of the degeneration (atresia) of follicles
housing each oocyte to about 3105 at puberty, 2.510
4 at age 37, and 1103 at menopause12.
A similar situation occurs in postnatal rodent ovaries, although the number
of follicles is proportionately smaller13.
The events responsible for the initiation of postnatal follicular atresia do not universally depend on the oocyte as the prime factor. Atresia is driven by oocyte apoptosis in the early (primordial, primary, preantral) stages of development6, 13, 14, but somatic granulosa cells of the follicle are the first to undergo apoptosis during atresia of follicles at later stages of development, including the point of preovulatory selection14. Krakauer and Mira5 do not explain how mutations in the mitochondrial DNA of an oocyte might trigger apoptosis in the surrounding granulosa cells as a means to remove that follicle from the ovulatory pathway, particularly in the light of the fact that gonadotropins prevent atresia of maturing follicles by direct action on the follicular somatic cells14.