Eggs in the Balance

Nature Genetics 28, 300–301 (1 August 2001) | doi:10.1038/91039
Martin M. Matzuk

Smoking destroys oocytes (egg quality)

Conclusions: The study suggest that smoking and environmental pollution destroys oocytes, turning them into mutants! The research shows how this can lead to early menopause, as the primordial follicles disappear.

Martin M. Matzuk Departments of Pathology, Molecular and Cellular Biology, and Molecular and Human
Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.

Polycyclic aromatic hydrocarbons (PAH), found in cigarette smoke and air pollution, interact with the aryl hydrocarbon receptor (Ahr) to cause reproductive defects. Mice lacking either Ahr or the pro-apoptotic protein Bax have an increased number of primordial follicles, and these mutant oocytes are resistant to PAH toxicity. A new study shows that the Bax promoter contains two core Ahr response elements, which are required for PAH stimulation of Bax promoter activity in oocytes. Thus, the toxic effects of PAH in oocytes are mediated directly by Ahr induction of the Bax pathway.

Twenty years ago, Mattison1 showed that PAH xenobiotics, including 2, 3, 7, 8-tetrachlorodibenzo-p-dioxins (TCDD) and benzo(a)pyrene (a potent carcinogen and mutagen in cigarettes), cause oocyte loss. PAH exert their effects through the aryl hydrocarbon receptor (Ahr), a ligand-activated member of the Per-ARNT-Sim (PAS) transcription factor family that also contains a basic helix-loop-helix DNA-binding domain2. Upon ligand binding to its PAS domain, cytoplasmic Ahr translocates to the nucleus, where it dimerizes with the Ahr nuclear translocator and directly activates gene expression. Mice lacking Ahr are viable and can produce offspring, but have smaller livers and many hepatic pathologies3. In addition, they do not respond to TCDD by induction of the cytochrome P450, 1a1, and show only a 25% induction of the cytochrome P450, 1a2, compared with wildtype controls. Thus, induction of these cytochromes by TCDD requires Ahr.

Life and death in the balance. Anti-apoptotic factors (for example, Bclx) support oocyte survival, whereas pro-apoptotic factors (for example, Bax) mediate oocyte destruction. The mouse and human Bax promoters contain consensus DNA binding elements for the aryl hydrocarbon receptor (Ahr). Polycyclic aromatic hydrocarbons (PAH), which are environmental toxins, would shift the life/death oocyte balance by binding to the Ahr, induce Bax expression in oocytes, and lead to oocyte death.

Bob Crimi
Members of the Bcl2 family regulate apoptosis and are found in species as divergent as nematodes (for example, Caenorhabditis elegans) and humans. The Bcl2 and Bclx subgroup is anti-apoptotic, whereas the Bax subgroup is pro-apoptotic. Bcl2-/- mice have kidney, lymphocyte, and hair abnormalities, and half die by six weeks of age4. Bax-/- mice have B- and T-cell hyperplasia, and a block in spermatogenesis leading to male infertility5. On page 355 of this issue, Tiina Matikainen and colleagues6 show a direct link between Ahr and Bax in the regulation of oocyte survival.

Bax and Bclx in ovarian physiology
Between embryonic day 8.5 (E8.5) and E11.5 in the mouse, primordial germ cells migrate over a tortuous route from the allantois along the hindgut and to the genital ridge7. Their number rapidly increases8 from 100 (E8.5) to 3,000 (E11.5) to 22,000 (E13.5). At E13.5, many of the germ cells of the female embryo enter prophase I of meiosis, at which time a significant number undergo apoptosis9. The second major period of germ cell apoptosis in the ovary occurs between postnatal day 7 (P7) and P14 (ref. 9). A similarly dramatic attrition of oocytes occurs between birth and puberty in humans. Only recently have we begun to understand the genetic pathways and molecular mechanisms regulating germ cell apoptosis and survival.

Bax is a pro-apoptotic protein expressed in granulosa cells and oocytes of several mammalian species10. In mice lacking Bax11, there are similar numbers of non-atretic primordial (dormant) or primary (early growing) follicles in the ovaries compared with wildtype controls at P4. In contrast, the numbers of non-atretic primordial and primary follicles are increased significantly at six weeks of age. As a result, Bax-/-mice have a prolonged ovarian lifespan such that 1.5−1.75-year-old females continue to have primordial, primary, and more mature follicles, some of which can be fertilized in vitro and develop into embryos.

Bclx is an anti-apoptotic protein expressed ubiquitously during development. Hennighausen and colleagues12 showed that E12.5−E15.5 Bclx-/- embryos have a decreased number of germ cells due to increased apoptosis—indicating that decreased expression of a critical anti-apoptotic factor (Bclx) shifts the balance in favor of pro-apoptotic factors. Germ cells at E12.5 and E13.5 express Bax, suggesting that it is the putative pro-apoptotic protein. The relationship between Bclx and Bax was proven when Bax-/-Bclx-/- mice were found to have a significantly increased number of follicles at P1, compared with Bclx hypomorphic or wildtype females. Bax-/-Bclx-/- females are also fertile, in contrast with Bclx hypomorphs. Thus, during embryogenesis or postnatal life, anything that alters the balance towards pro-apoptotic Bax—including 'reproductive toxins'—would cause increased oocyte loss.

A link between Ahr and Bax
Ahr protein is present in granulosa cells and oocytes at all follicle stages13, suggesting that signaling through the receptor may have a role in ovarian follicle development. Ovaries of Ahr-/- mice13, 14 have a twofold increase in the number of primordial follicles at P2−P4. Interestingly, the number of antral follicles at eight weeks is decreased. These findings indicate that, like Bax ablation, Ahr deficiency helps to maintain oocytes.
Matikainen and colleagues6 demonstrate why the Bax-/- and Ahr-/- ovarian phenotypes are similar. Analysis of the 5' region of the mouse Bax promoter reveals two consensus Ahr response elements2 (GCGTC) in close proximity to one another. Four core response elements are also present in the human BAX promoter. When the nucleotide three bases downstream of the response element is an adenine residue, dioxin (an environmental toxin) is able to activate the gene.

In the mouse, the nucleotides three bases downstream of the two response elements are guanine and cytosine, suggesting that dioxin cannot activate Bax. Indeed, mouse oocytes are normally insensitive to dioxin, and oocyte microinjection experiments with a construct containing the wildtype Bax promoter confirm that dioxin is ineffective at induction. Furthermore, mutation of these critical downstream nucleotides to adenine renders the promoter responsive to dioxin. Thus, two nucleotides in the Bax promoter would seem to be the sole 'protection' against damage mediated by dioxin.Matikainen et al.6 further show the effects of the PAH 9,10-dimethylbenz(a)antracene (DMBA) on oocytes and on Bax levels (see figure). Intraperitoneal delivery of DMBA causes a 72% increase in Bax mRNA levels after 24 hours, and a concomitant increase in Bax protein in oocytes. Treatment with DMBA or a DMBA metabolite also leads to a decrease in the number of non-apoptotic oocytes in cultured wildtype mouse ovaries. However, treatment of AhR- or Bax-deficient ovaries with the potent DMBA metabolite has no effect on the number of non-apoptotic oocytes of primordial follicles, confirming that the Ahr-Bax pathway is required for PAH-induced cell death. Finally, DMBA has a similar effect on human ovarian cortical tissue that is grafted subcutaneously into immunodeficient mice.

As the world's population increases, the consumption of fossil fuels and the production of air pollution will also rise. Aside from the risk of global warming, the generation of PAH and other environmental toxins will continue to cause more health problems. Likewise, there is an increased risk of PAH-related reproductive and developmental defects due to the rising incidence of smoking in young women15. We now know that these toxic PAH compounds signal through a Ahr/Bax-regulated pathway to cause their destructive effects on oocytes. It will be important to identify the endogenous ligand that binds to the Ahr. This would provide a potential means to prevent oocyte loss and also to prolong the reproductive lifespan of human females.

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