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Mitochondria Aging

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#1 JonesGuy

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Posted 28 February 2004 - 06:54 AM

Alright, almost 100% of your mitochondria comes from your mother. However, the DNA of the mitochondria is entirely separated from the nuclear DNA.

In our germ cells (the ones producing sperm and eggs) the nuclear DNA is protected from aging.

But what protects the mitochondria DNA? Why doesn't the egg have the same 'aged' DNA as the mother?

Any papers I could read? Any professors who've done this type of research?

Sorry for being a bit lazy here. I suddenly realised that I didn't know the answer (while I was driving). I can't do the research myself for awhile. I'm hoping for a quick answer before I dive in-depth in to the research (in a couple days ... sadly, I don't have the computer/papers access for a bit).

#2 Cyto

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Posted 28 February 2004 - 08:19 AM

My personal slapstick answer.

* no population doublings and minimal housekeeping proceses (low activity)
* a lot is condensed so if a double strand break occurs then closer localization will allow bonds to reassociate (like in Deinococcus radiodurans)
* multitude of apoptic signaling cascades inside the mitochondria itself (Apaf1, Smac/Diablo, cyt c., AIF, endo G)

Course, eggs still do get old and can have defects later on as the mother gets older.

#3 kevin

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Posted 28 February 2004 - 08:33 AM

Here's an abstract that you might find interesting.

Sci Aging Knowledge Environ. 2003 Feb 26;2003(8):PE4. Related Articles, Links

Germline genomes--a biological fountain of youth?

Walter CA, Walter RB, McCarrey JR.

The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA. walter@uthscsa.edu

The fusion of male- and female-derived gametes initiates the phenomenal process of producing a highly complex mammalian organism. Successful reproduction is so important that mammals invoke a battery of protective mechanisms for the germ cell lineages that function to maximize genetic integrity while still allowing genetic diversity and adaptation. Protective mechanisms likely include, but are not limited to, robust DNA repair to safeguard genetic integrity and apoptosis to remove cells with intolerable levels of DNA damage. Analyses of spontaneous mutant frequencies are generally consistent with germline DNA being stringently maintained relative to somatic tissues. Despite the rigorous protection afforded germ cells, genetic integrity is observed to decline with increased maternal and paternal age. It is not yet clear whether cells in the germ line truly age or whether other processes decline or become dysfunctional with age. For example, in a younger animal, the differentiation and/or utilization of germ cells with lower genetic integrity might be disallowed, whereas in an older animal, such cells might slip past these quality-control mechanisms.

Publication Types:
Review, Tutorial

PMID: 12844546 [PubMed - indexed for MEDLINE]

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#4 Cyto

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Posted 28 February 2004 - 08:43 AM

Didn't find anything I really wanted to find...

Some neat things though:

Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway (2004)
DNA Repair

Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.

The effect of aging and caloric restriction on mitochondrial protein density and oxygen consumption (2004)
Experimental Gerontology

It has been proposed that part of the anti-aging mechanism of caloric restriction (CR) involves changes in mitochondrial function. To investigate this hypothesis, mitochondria from various tissues of male Brown Norway rats (fully fed and CR) were isolated and respiration rates determined. In mitochondria from liver, heart, brain and kidney, there were no significant effects of CR on state 4 mitochondrial respiration rate. Further experiments using liver mitochondria under a variety of incubation conditions confirmed that CR does not alter mitochondrial respiration rate in this tissue. However, the respiration rate of mitochondria from brown adipose tissue (BAT) of CR animals was approximately three-fold higher compared to mitochondria from fully fed controls. Mitochondrial protein density was significantly higher in liver tissue of CR animals; it was significantly lower in heart and unchanged in BAT. It is concluded that whilst CR results in tissue-specific changes in mitochondrial respiration rate, these effects do not explain the CR-induced changes in free radical production reported previously for these organelles.

#5 Mind

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Posted 27 May 2004 - 02:22 PM

Here is an article related to mitochondria and aging. Reading this, I was surprised how much I have already learned from being a member here at Imminst. Much of this "news" review" of the experiments I already knew (ie. current theories of aging), just from reading the forums here. So this article may be "old news" for some of you, but it is confirmation that genetic mutations in mitochondria cause aging.

Researchers zero in on a cause of aging
Experiment points to defect in cells
By Alice Dembner, Globe Staff  |  May 27, 2004

Taking a major step toward identifying one cause of aging, researchers have shortened the life of mice and created signs of old age by injecting a small genetic defect in the mice's mitochondria, the tiny power plants within each cell.

The experiment in Sweden offers the first hard evidence of a decades-old theory that mutations in the mitochondria are one of the causes of age-related illnesses. Earlier research had shown that such defects build up in people as they grow old, but scientists were not sure whether that was a cause or a symptom of aging.

''What we have now is this clear-cut cause-and-effect relationship," said Dr. Nils-Goran Larsson, a genetics professor at the Karolinska Institute in Stockholm and the senior author of the study. ''It will provide a completely new angle to treat aging-related problems."

Other researchers, however, said the theory won't be fully proven until experiments extend the life span of mice by eliminating defects in the mitochondria.

Larsson said the new results could lead to the development of drugs or procedures to ward off the changes to the mitochondria. A number of companies are already working on other compounds, potentially related to mitochondria, to tackle the diseases of old age.

The study, published in today's issue of the journal Nature, comes as researchers are unlocking many of the mysteries of aging. Scientists have identified a number of genes that affect aging in animals. They have linked the natural shortening of telomeres, the tail ends of chromosomes, to shorter life spans. In addition, researchers have added years to the lives of mice by increasing their ability to disarm byproducts of metabolism called free radicals, which can damage cells.

Since mitochondria also generate free radicals, the latest work may also propel work on the free-radical theory, researchers said.

A separate path of research is focusing on factors that regulate life span, rather than on the causes of aging. Altering the genes that control insulin receptors can double the life span of worms, researchers have shown.

David Sinclair, a molecular geneticist at Harvard Medical School who works on the regulators of aging, said the Swedish experiment ''suggests -- but doesn't prove -- that mitochondria might really play an integral part in the aging process" and he suggests that the regulators will prove more important in combating aging than the causes. ''We believe that manipulating the regulators of aging will have a much larger effect on longevity because these regulators could potentially slow down all the causes of aging, not just one," Sinclair said.

However, Dr. George M. Martin, a geneticist at the University of Washington in Seattle who wrote an article accompanying the Swedish study, said the paper makes ''an important contribution because . . . changes in the mitochondrial DNA may be among the more important processes of aging."

Mitochondria are the structures in each cell of the body that convert fat and sugar into usable energy. If they severely malfunction, humans eventually die. The theory that damage to the mitochondria caused aging was first proposed in 1972, according to Sinclair. Evidence piled up that these changes were common in older people and animals, but no one had attempted to prove the connection.

In the Swedish experiment, researchers genetically engineered the mice to have a defect in their mitochondria. They replaced a single amino acid, which disabled the ability of the mice to find and correct errors in their mitochondrial DNA. As the mitochondria replicated themselves as part of normal growth and development, errors built up.

Starting at young adulthood (24 weeks), the mice began to show symptoms of premature aging, including weight loss, hair loss, curvature of the spine, osteoporosis, and enlargement of the heart. They lived an average of 48 weeks, and all had died by the age of 61 weeks. Normal lab mice can live up to two years. Designing the experiment and carrying it out took a painstaking four years, Larsson said. ''I now know why no one had done it [before]," he said.

Larsson said next steps would include researching the underlying mechanisms and figuring out how they relate to other possible causes of aging. In addition, he said, the mice would be used to design ways to counteract the problem using drugs, diet, or genetic alterations.

Original at boston globe

#6 manofsan

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Posted 27 May 2004 - 07:47 PM

I gotta wonder, though -- the results seem to show that mitochondrial deterioration are a big part of aging, but are they the totality of it? I don't think so.

Anyhow, if you can even lick 60% of the aging problem, then that's a big deal. So where do we go from here?

Why are mitochondrial DNA more fragile or prone to breakdown than regular DNA? Or is it instead the case that mitochondrial DNA & function are more critical to health than the rest of the genome?

Any speculations?

#7 ocsrazor

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Posted 27 May 2004 - 09:48 PM

The article Mind posted is HUGELY important to aging research. This is a true breakthrough and a vindication for Aubrey de Grey who has been telling people for years that mitochondrial malfunction was a major cause of aging. I will try to comment more after I read the scientific publication.


PS Yes manofsan, Mitochondrial DNA is much more susceptible to damage than nuclear DNA.

#8 reason

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Posted 27 May 2004 - 10:22 PM

I was pretty impressed too. This work took four years, so I give them another four years or so to demonstrate the results of fixing up mitochodrial damage in mice.

Isn't one of the ExI list people (Joao maybe?) working on a mitochondrial gene-alteration technology that's been all hush-hush up until recently?

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#9 JonesGuy

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Posted 27 May 2004 - 10:54 PM

See, the problem is though, that we directly inherit mitochondria from our mothers. So why aren't my mitochondria (from my mom) 30years old when I get them? And why aren't they 60years old now?

Aging in our germ cells is protected aggressively until puberty, and then less so as we age. There are really active mechanisms protecting against our DNA deterioration. But what stops the mitochondria from aging? I'm just not sure.

#10 Lazarus Long

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Posted 28 May 2004 - 02:54 AM

Peter it is now almost a year since we sat and had that "confidential discussion about "Mitofection" and I have been waiting for the results patiently. Did you meet this year with Rafal?

#11 kevin

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Posted 28 May 2004 - 03:46 AM

Here's a little more info on the study posted by mind from Sage Knowledge Environment. If anyone wants to borrow my copy of the paper, please do..

Sci. Aging Knowl. Environ., Vol. 2004, Issue 21, pp. nf55, 26 May 2004
[DOI: 10.1126/sageke.2004.21.nf55]

Symphony of Errors
Sloppy proofreading of mitochondrial DNA spurs a premature demise

Esther Landhuis

Document URL: http://sageke.scienc...ll/2004/21/nf55

An orchestra's sound quality suffers whether each member of the cello section flubs a particular pitch or scattered violinists play random notes offkey. Similarly, cells can atrophy when they amass many identical mitochondrial DNA defects or, according to new work, multiple haphazard flaws. The findings support the much-debated theory that accumulation of assorted blunders in the mitochondrial genome underlies aging. Because this type of slip-up commonly occurs in mammals, the results could hold significance for humans.

Cells routinely sustain assaults from reactive oxygen species (ROS), which mitochondria produce as they convert food to fuel (see "The Two Faces of Oxygen"). Because of their location, mitochondrial genomes, separate from the bulk of DNA in the cell's nucleus, bear the brunt of the ROS attack. Furthermore, unlike the nucleus, which boasts an arsenal of DNA-proofreading proteins, mitochondria direct scant resources toward error-checking; the burden falls on a single protein, DNA polymerase-. For these reasons, presumably, glitches accrue in mitochondrial DNA faster than in nuclear DNA. Some researchers had proposed that they hasten an organism's decline, but they might instead just build up over time. Because each cell contains hundreds to thousands of mitochondria, many scientists had assumed that mutations in individual mitochondria wouldn't cause trouble because other mitochondria in the same cell could pick up the slack; a defect would hurt only if mitochondria containing it greatly outnumbered those that didn't. Such events occur: In single cells from older people, the same mutation sometimes appears in most mitochondria, suggesting that one flawed mitochondrial genome can eventually swamp others (see "Go Forth and Multiply"). But some researchers wondered whether the accumulation of multiple random mutations would induce age-related defects.

Trifunovic and colleagues addressed the issue by genetically engineering a mouse that stockpiles mitochondrial DNA mutations indiscriminately. To accelerate the buildup of these mistakes, the team replaced the DNA polymerase- gene with one that encodes a variant that proofreads poorly. Genetically altered mice copied mitochondrial DNA as efficiently as normal animals did but accumulated more deletions and three- to fivefold more single DNA-base changes, evenly distributed across a sample gene. The mutant mice died sooner, on average, and prematurely developed a host of aging-related conditions such as weight loss, osteoporosis, anemia, reduced fertility, and compromised mitochondrial function in the heart.

"They're really pinning down a cause of aging," says Matthew Longley, a biochemist at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina. This experiment provides the best evidence to date that the accumulation of random mitochondrial DNA mutations induces aging-related dysfunction, he says. Jeff Stuart, a mitochondrial bioenergeticist at Brock University in Ontario, Canada, praises the study's design because it enabled the researchers to document mitochondrial failure and aging-associated defects in young mice; most previous experiments had examined the mitochondria of older animals, in which other aging-related conditions might have confounded the observations, Stuart says. A finer understanding of the mechanisms that link mitochondrial DNA damage to aging-related defects could lead to therapeutics that keep us playing a youthful tune.

May 26, 2004


1. A. Trifunovic et al., Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417-423 (2004). [Abstract] [Full Text]

Citation: E. Landhuis, Symphony of Errors. Sci. Aging Knowl. Environ. 2004 (21), nf55 (2004).

#12 reason

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Posted 28 May 2004 - 06:47 AM

Isn't one of the ExI list people (Joao maybe?) working on a mitochondrial gene-alteration technology that's been all hush-hush up until recently?

It was Rafal in a post on May 06; I just dug it up:

I have some news for afficionados of mitochondria:

Recently at the Mitochondria and Neurodegeneration Meeting in Fort
Lauderdale our research group at Gencia Corporation unveiled a method
for manipulating mitochondrial genomes, protofection. I have been
obliquely hinting at it for some time on Extropy but now I can say more:
we can place mitochondrial genomes in living cells, in vitro and in
vivo, including reporter genes and other genetic cargo, express proteins
in the mitochondria, and even export them to other subcellular
locations. We can actually stably make a huge amount of protein without
ever entering the nucleus, or using a viral and immunogenic vector, or a
synthetic and cytotoxic cationic lipid. This capability will be most
salient to anybody familiar with the keywords "X-SCID", and "insertional
mutagenesis". The research has been submitted for publication and I hope
to be able to post the paper here as soon as it's published.

Additionally, we found that some possibilities for mitochondrial
dysfunction treatment envisioned previously on theoretical grounds, can
actually be made to work, at least in cell culture, and should work in
adult mammals as well. But I will leave you guessing....

Suffice it to say that some of our wildest hopes seem to be coming true.


Randall Parker is commenting on it too, while I think of it, giving good press for Aubrey de Grey as usual:


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#13 manofsan

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Posted 28 May 2004 - 07:10 PM

Cool stuff. Forgive my ignorance, but does anyone know a little more about what Mitochondrial DNA looks like? Is it organized into chromosomes, plasmids, or what?
Does it interact with ribosomes via mRNA, does it use transcriptase enzymes and all that stuff?

Does it have telomeres? (presumably, those are only a chromosomal feature)

Someone on Usenet sci.bio.technology commented to me that one reason mitochrondria may be more vulnerable to breakdown is due to the fact that they are the site of oxidative activity in the cell.

I responded back that if this is the reason, then the specifics of this oxidative activity should be more correlated to specific types of free-radicals being produced.
In which case, shouldn't there be specific types of anti-oxidants more suited to combatting these particular free-radicals?

Also, do you remember that recent post about the rats who lost weight because of a genemod that increased the number of mitochondria per cell? That's almost analogous to a regenerative/cloning type of effect.

I'm thinking that increasing the number of mito's per cell would create more redundancy against damage to mitochondria. Even if some get damaged, at least others will be around to survive and pass on their functional DNA.

What do you think?

#14 manofsan

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Posted 28 May 2004 - 07:38 PM

Oh yeah, you know how excessive damage to nuclear DNA can trigger apoptosis? Could a similar mechanism be engineered for mitochondria, to specifically keep them pure? (ie. mitochondrial suicide as opposed to killing the whole cell)

And that also reminds me -- if mitochondrial DNA is separate from nuclear DNA, then can I assume that mito DNA damage cannot similarly trigger apoptosis for the entire cell?

If so, no wonder mito DNA deteriorates faster, since there's no apoptosis for quality control, like there is for nuclear DNA.

#15 olaf.larsson

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Posted 28 May 2004 - 07:46 PM

The mitochondria has circular genome with ~16000bp. But the repair mechanisms are not as good as for nuclear genome. The mitochondria has its own bacterialike ribosomes and own t-rna. It also has a alternative genetic code diffrent from the nucleus. Most proteins in the mitochodria are coded in nuclear DNA.

#16 kevin

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Posted 28 May 2004 - 08:27 PM

Link: http://dir.niehs.nih...B_Copeland.html
Here's a superb primer on mitochondrial replication. The obvious thing that comes to my mind regarding the information in Reason's post is what affect inserting better DNA repair genes into the mitochondrial DNA might have.

Mitochondrial Replication

William C. Copeland, Principal Investigator

Mitochondria are the oxidative energy factories of the mammalian cell, and they contain small chromosomes derived from early bacteria. Mammalian mitochondrial DNA is 16 times more prone to oxidative damage and evolves 10-20 times faster than nuclear DNA. Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. More than 40 million people are infected by the human immunodeficiency virus worldwide. Although antiviral therapy effectively extends the life of individuals, the death toll continues to rise: 3 million people, the highest number since the epidemic began, died from AIDS in 2000. Current antiviral nucleoside-analog therapy against HIV results in compromised mitochondrial function due to inhibition of the mtDNA polymerase. The two primary goals of this project are to understand the contribution of the replication apparatus towards the production and prevention of mutations in mtDNA and to understand the inhibition of mitochondrial function by anti-AIDS drug therapies.

Click the image for larger picture
Posted ImageHuman mitochondrial DNA is replicated by the two-subunit DNA polymerase g (g). Human pol g is composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3´-5´ exonuclease proofreading activity, and 5´dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, the pol g participates in DNA replication and DNA repair. We have cloned, overexpressed, purified, and characterized the human pol g catalytic subunit, p140, and the p55 accessory subunit. The accessory subunit functions as a processivity factor and enhances DNA binding of the holoenzyme. Since the genetic stability of mitochondrial DNA depends on the accuracy of pol g, we investigated the fidelity of DNA replication by pol g with and without exonucleolytic proofreading and its p55 accessory subunit. Pol g has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides.

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mitochondrial DNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the catalytic subunit of pol g. Since Y955 is a highly conserved residue critical for nucleotide recognition among Family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (kcat) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (KM). The Y955C derivative is twofold less accurate for base-pair substitutions than wild-type pol g despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C pol g is consistent with the accumulation of mtDNA mutations in patients with PEO.

To see a complete listing of mutations in the gene for the human DNA polymerase gamma catalytic subunit, please visit the Human DNA Polymerase Gamma Mutation Database.

The mitochondrial respiratory chain is a source of endogenous reactive oxygen species (ROS), and oxidative modification of biomolecules, including proteins, can alter their normal functions. Since pol g is associated with DNA within the mitochondrial matrix, this enzyme is subject to oxidation in vivo by hydrogen peroxide and iron ions associated with mtDNA. The effect of H2O2 on the enzymatic activities and DNA binding efficiency of pol gamma has been examined. Hydrogen peroxide inhibits the DNA polymerase activity of the p140 subunit and lowers its DNA-binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H2O2 treatment offers protection from oxidative inactivation. Pol g can be detected as one of the major oxidized proteins in the mitochondrial matrix, and the degree of oxidation correlates with a decline in polymerase activity. These results suggest that pol gamma is a target for oxidative damage by ROS which may impair mitochondrial DNA replication and repair.

Human DNA Polymerase q: Exogenous DNA damaging agents such as cisplatinin, nitrogen mustard, and psoralen create interstrand DNA crosslinks. Such non-coding lesions must be repaired to ensure accurate replication of the genome and viability of the organism. The Drosophila mus308 mutation, which confers hypersensitivity to nitrogen mustard but not the monofunctional agent methyl-methane sulfonate, identified a DNA polymerase likely involved in processing DNA crosslinks. Based on homology to the Drosophila mus308 gene and another Family A DNA polymerase, DNA polymerase g, we previously cloned and expressed the cDNA for human DNA polymerase q. Recent analysis of the gene for human DNA polymerase q has identified a 9 kb coding region, encoding the full length DNA polymerase q with a molecular weight of 300 kDa. Amino acid sequence alignments predict DNA polymerase, ATP binding and/or hydrolysis, helicase activity, and 3´ to 5´ exonuclease functions for this enzyme. We previously have cloned and expressed cDNA constructs of 6.5 and 3 kb, which have produced functional DNA polymerase polypeptides of 200 and 100 kDa. These have been produced in baculovirus and in E. coli. The substrate specificity and accuracy of DNA synthesis in vitro were determined. Unlike many of the other newly discovered DNA polymerases, we determined that polymerase q synthesizes DNA with high fidelity. Site directed alteration of conserved amino acids have identified the polymerase activity in these polypeptides

#17 manofsan

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Posted 28 May 2004 - 08:37 PM

Why is it necessary for mitochrondia to keep their own separate DNA at all? Is this just a holdover from the days when mito's were independent organisms? Or is there a useful purpose for mitochondria having their own separate DNA?

What would happen if all the mitochondrial DNA was instead incorporated into nuclear DNA? Wouldn't the advanced characteristics of nuclear DNA improve things, if the mito genes were brought under its umbrella?

Are there any examples of this elsewhere in the biological world? Or do all creatures have mito's which have their own DNA?

Perhaps such a shift would impair the mito functions, since they might benefit from their own onsite DNA & ribosomal synthesis.

#18 kevin

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Posted 28 May 2004 - 09:09 PM

It is becoming clear that reactive oxidative species are responsible for damaging the mitochondrial DNA and other components within the matrix at an accelerated rate in comparison to the damage sustained by other components within the cytoplasm. With this in mind, the search has been increasing for antioxidants which target the mitochondria more than the rest of the cell, with some success.

Posted ImageA mitochondrial based inherited disorder called Friedrich's Ataxia which has loss of sensory neurons and hypercardiomyopathy as its worst consequences is being treated with a molecule related to idebenone, an analogue of conenzyme Q-10 that is sold as an "anti-aging" chemical.

The molecule dubbed MitoQ and is under investigation by Dr. Michael Murphy at Otago University, New Zealand. They have used the basic structure of idebenone and modified it with in order to have it target the mitochondria thus concentrating the anti-oxidant capabilities of the molecule where it is most effective in prevent ROS damage.

Here's a good paper with plenty of diagrams and good explanations of the disorder and how the chemical works freely available in PDF format.

Mitochondria-targeted antioxidants protect Friedrech Ataxia fibroblasts from endogenous oxidative stress more effective than untargeted antioxidants. - Aug, 2003

where it says

The mitochondria-targeted antioxidant MitoQ was several hundredfold more potent than the untargeted analog idebenone. The mitochondria-targeted antioxidant MitoVit E was 350-fold more potent than the water soluble analog Trolox. This is the first demonstration that mitochondria-targeted antioxidants prevent cell death that arises in response to endogenous oxidative damage. Targeted antioxidants may have therapeutic potential in FRDA and in other disorders involving mitochondrial oxidative damage.

Transfection of genes into germline cells may make the mitochondria of future generations much more efficient and less damaging, but as Aubrey has said.. "I've already got aging" and it's nice to know that there may be some easily managed interventions available in the near future. The drug MitoQ may be made available to patients with Friedrichs Ataxia by the end of 2004. A support forum for the disorder can be found here.

#19 kevin

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Posted 28 May 2004 - 09:50 PM

Link: http://www.funkdafie...ochondrial.html

Here's an excellent article describing the process of "Reductive evolution of the mitochondrial genome: how and why?"

Reductive evolution of the mitochondrial genome: how and why?

Though mitochondria have their own genomes, most of the genes involved in the structure and function of these organelles are located in the nucleus. These genes have come to occupy the nucleus through evolutionary processes, having initially all been contained within the mitochondria. Each has undergone a five-step process: escape from the mitochondrion; journey through the cytosol to the nucleus; integration in the nuclear genome; routing of the gene’s product; and loss of the gene’s mitochondrial copy. Several broad forces have worked to favor this relocation of the genome, including the instability of mitochondrial DNA, the danger posed by redox functions of the mitochondria, and the disadvantage of asexual reproduction. However, this evolution has not gone to completion, as several common genes remain in all mitochondria. These genes remain due to their products’ inability to be safely and efficiently routed back to the mitochondria, their products’ need for special mitochondrial regulation, and the ability of plant mitochondria to counteract the drawbacks of asexual reproduction. These various opposing forces and complex physical processes have conjoined to evolve the ancestral endosymbiont to the present-day mitochondrion.

#20 bacopa

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Posted 13 June 2004 - 07:00 AM

Kevin thank you for all the mitochondirial information, as I'm trying to become more versed in aging theories your contributions are invaluable and I appreciate them very much. Perhaps my own brain will reach a singularity whereby the more wordy science papers will suddenly make total sense! [lol]

#21 kevin

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Posted 07 November 2004 - 01:48 AM

Link: http://www.sciencema...tract/1102521v1

Interesting evidence that single mutations in mitochondrial tRNA is linked to a variety of 'age' related disorders, possibly explaining their linkage.

A Cluster of Metabolic Defects Caused by Mutation in a Mitochondrial tRNA
Hypertension and dyslipidemia are risk factors for atherosclerosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercholesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicative of mitochondrial inheritance. Analysis of the mitochondrial genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5' to the mitochondrial tRNAIle anticodon. Uridine at this position is nearly invariate among tRNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders.

#22 olaf.larsson

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Posted 08 November 2004 - 06:52 AM

We miss some very important information, why???
Becouse if it would be the way we understand it the ofspring would inherit old defective mitochondria from the mother. This clearly is not the case. So the question is: How do old mitochondria from mother become new mitochodria in the fetus??

#23 Lazarus Long

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Posted 12 December 2004 - 06:31 AM

Wolfram I addressed why the defective mtDNA of the aged mother is not an issue because all the oocytes are created when the mother is still a fetus but I was curious about why it still doesn't decay over multiple generations and I went looking for articles and found this.

Full Text with many useful links

Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence
Jonathan Van Blerkom
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA and Colorado Reproductive Endocrinology, Rose Medcial Center, Denver, Colorado 80220, USA

Correspondence should be addressed to J Van Blerkom, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA; Email: vanblerk@spot.colorado.edu


Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo. While their role in ATP production has long been known, only recently has their contribution to oocyte and embryo competence been investigated in the human. This review considers whether such factors as mitochondrial complement size, mitochondrial DNA copy numbers and defects, levels of respiration, and stage-specific spatial distribution, influence the developmental normality and viability of human oocytes and preimplantation-stage embryos. The finding that mitochondrial polarity can differ within and between oocytes and embryos and that these organelles may participate in the regulation of intracellular Ca2+homeostasis are discussed in the context of how focal domains of differential respiration and intracellular-free Ca2+regulation may arise in early development and what functional implications this may have for preimplantation embryogenesis and developmental competence after implantation.


In clinical in vitro fertilization (IVF), current investigational efforts are directed to understanding why a high proportion of oocytes result in developmentally incompetent embryos. Studies of embryo performance during the pre-implantation stages show high frequencies of abnormal development and early demise, with further losses seen after uterine transfer as measured by outcome per embryo.

There is a growing consensus of opinion that much of this embryonic wastage originates from oocyte chromosomal and subtle cytoplasmic defects whose adverse developmental consequences are not expressed until well after fertilization. The notion that mitochondrial dysfunctions or abnormalities in the oocyte may be a critical determinant of human embryo developmental competence has gained currency from recent studies in which defects at the structural and mitochondrial DNA (mtDNA) levels have been identified. Likewise, the number of mtDNA copies has been shown to differ between human oocytes (in the same cohort) by over an order of magnitude, and for the early embryonic stages developmentally significant differences in mitochondrial numbers between blastomeres can result from disproportionate inheritance during the cleavage stages.

Structural, spatial and genetic dysfunctions that affect the capacity of mitochondria to produce ATP by oxidative phosphorylation could have pleiotropic affects on early human development that, as described below, may include the normality of spindle organization and chromosomal segregation, timing of the cell cycle, and morpho-dynamic processes such as compaction, cavitation and blastocyst hatching. Mitochondrial dysfunctions that may initiate or contribute to the activation of apoptosis have also been suggested to be a proximal cause of human oocyte wastage and early embryo demise.

Because the normalcy of critical nuclear and cytoplasmic activities may be determined by mitochondria, it is not surprising that their role in early human development as related to outcome in IVF treatments has become a subject of clinical and basic research interest (Christodoulou 2000, Howell et al. 2000, Jansen 2000a,b, Cummins 2002, 2004, Brenner 2004, Chinnery 2004, Eichenlaub-Ritter et al. 2004). From a basic science viewpoint, the extent to which mitochondria contribute to or actually determine oocyte and embryo competence must be better understood if proactive clinical therapies such as oocyte mitochondrial donation/replacement (Cohen et al. 1997) are to be considered acceptable treatments for certain types of infertility in which a mitochondrial association has been clearly identified (Brenner 2004, St John et al. 2004).

    Mitochondria as genetic forces in early human development 

Mitochondrial transmission across generations is uniparental
It has long been known that the human mitochondrial genome is 16 560 kb of double stranded DNA that encodes 13 proteins in the respiratory chain, and 22 unique transfer RNAs and 2 ribosomal RNAs (Clayton 2000, Trounce 2000). Although not without some controversy (Cummins 2004), mitochondria are inherited through the maternal lineage with paternal mitochondria arriving at fertilization targeted for destruction primarily by ubiquitin-dependent proteolysis (Schwartz & Vissing 2002, 2003, Johns 2003, Sutovsky 2004).

The maternal transmission of mitochondria between generations is the genetic basis for the inheritance of certain debilitating or ultimately lethal metabolic disorders in the human (Chinnery & Turnbull 1999, Christodoulou 2000, Leonard & Schapira 2000, Chinnery 2004), and heterogeneity in mtDNA is used in forensic medicine to assist in the identification of individuals and in anthropology to trace the origins and geographical dispersal of populations.

All of the mitochondria in the mature oocyte (metaphase II, MII stage) arise from the clonal expansion of an extremely small number of organelles present in each primordial germ cell that after colonization of the forming ovary, expands by mitosis to form numerous progeny that can be identified as ‘nests’ of primordial oocytes (Makabe & Van Blerkom 2004). By some estimates (Jansen 2000b), <10 mitochondria may be the progenitors of the tens to hundreds of thousands of organelles present in the human oocyte at fertilization. That mitochondrial transmission across generations is uniparental and that procreation requires their significant numerical expansion presents some potentially unique biological challenges. For example, it has been argued that this uniparental replication might be expected to follow the ultimately lethal consequences of Muller’s ratchet hypothesis (Muller 1964, and concisely discussed by Jansen 2000a), whereby species extinction is an inevitable consequence of asexual reproduction owing to the accumulation of deleterious mutations by random genetic drift. Indeed, because of maternal inheritance and high mutation frequency, the human mtDNA should be prone to Muller’s ratchet. Counteracting this natural entropic tendency to mutational degradation and extinction is the severe reduction in maternal germline mtDNA copy number in the primordial germ cell, a phenomenon generally known as the ‘mitochondrial bottleneck’ (Bergstrom & Pritchard 1998). As a result of the reduction in progenitor organelles, the accumulation of mtDNA mutations is diminished with certain mutations, such as those that could adversely affect replication or metabolic capacity being eliminated by natural selection or ‘dying out’ through oogenesis (Hoekstra 2000).

However, others have argued that there is not a single mitochondrial bottleneck at the outset of oogenesis, but rather an active selection process that occurs throughout oogenesis and early embryogenesis and involves multiple stage-specific bottlenecks and differential patterns of mitochondrial segregation (Howell et al. 2000). The occurrence of individuals with maternally inherited metabolic diseases (OXPHOS diseases) resulting from known mtDNA mutations demonstrates that (a) the bottleneck is not an effective natural means of eliminating oocytes carrying potentially lethal mitochondrial genetic defects and (b) that developmental competence does not require that the mitochondrial complement be genetically normal or even capable of normal levels of respiration (oxidative phosphorylation).

    Mitochondrial transmission between generations is not necessarily monogenomic: homoplasmy and heteroplasmy 

As maternally inherited organelles, the mtDNA genotype(s) in the embryo is largely determined by what existed in the few mitochondria contained within the primordial germ cell and resulting primary oocyte. If the expansion of the mitochondrial population during oogenesis involves an identical genome, the MII oocyte and resulting embryo would be expected to be homoplasmic. Heteroplasmy occurs when two or more different mitochondrial genotypes occur in the same cell, whether a primordial germ cell, oogonia, oocyte or blastomere. Heteroplasmy per se does not imply an adverse condition if the mtDNA mutations are benign with respect to function, but can become problematic and have cytopathological consequences if the mutant form(s) has reduced respiratory capacity and occurs at toxic levels (mutant load). While heteroplasmy as a factor in human infertility or early embryo demise is a current issue in reproductive medicine (Brenner 2004, Cummins 2002, 2004), it is necessary to note that adverse developmental consequences of mtDNA mutations become relevant only when they affect mitochondrial activities (e.g. replication and respiration) at levels that are inconsistent with cell survival or normal function (Christodoulou 2000). Indeed, the threshold levels at which OXPHOS diseases become clinically significant are usually quite high (Chinnery & Turnbull 1999, Leonard & Schapira 2000, Trounce 2000).

Heteroplasmy, detected by highly sensitive analysis of mtDNA in the oocytes of certain women, has been related to infertility by virtue of the occurrence of certain mtDNA genotypes such as the ‘common deletion’ mtDNA 4977, which in one report was proposed to increase with maternal age and negatively affect competence in women >40 years old (Keefe et al. 1995). However, in a recent review of the relationship between competence and the various types of mtDNA mutations detected in human oocytes obtained by ovarian hyperstimulation for IVF, Brenner (2004) found no compelling evidence to suggest that any occurred at loads which could compromise outcome. This is not to say that mtDNA is unimportant in the establishment of competence or as an etiology of infertility but rather, that additional investigation is needed to validate such interpretations, especially if proactive therapies (e.g. cytoplasmic transfer) are contemplated in an IVF treatment cycle. In this respect, Brenner (2004) described some promising leads related to point mutations in the control region of the mitochondrial genome responsible for replication. These mutations seemed to increase in frequency in the oocytes of certain women, especially those of advanced reproductive age and, if confirmed, could be an important and unrecognized factor in outcome because mitochondrial replication does not begin until after implantation. Therefore, replication defects would not be expected to compromise preimplantation embryogenesis, but depending upon mutant load, could manifest as post-implantation demises described as chemical (transient elevation of human chorionic gonadotropin levels) or anembryonic pregnancies (no fetal pole detected by ultra-sonography).

Experimental approaches to the question of whether specific mtDNA defects in human oocytes cause postim-plantation demise require some formidable challenges to be overcome. For example, are unused blastocysts from IVF programs, even if available, suitable material to screen for mtDNA defects and should the trophoblast and inner cell mass (ICM) be analyzed separately? For patients with a history of repeated chemical or anembryonic pregnancies, is it ethical to use IVF protocols to generate multiple blastocysts such that some could be transferred or cryopreserved while others are used for mtDNA analysis? If specific mtDNA mutations that affect competence during the pre- and postimplantation stages are clearly identified, screening and ethical issues become moot as it would be expected that the same protocols used for embryo biopsy and preimplantation genetic diagnosis as applied to chromosomes and nuclear DNA (Verlinsky & Kuliev 2000) would be applicable to mtDNA.

However, an assessment of whether a particular mtDNA mutation could influence competence and outcome requires the ability to accurately quantify the mutant load. This is especially evident when it is considered that some mtDNA-related OXPHOS diseases clinically manifest only when a genetic defect occurs at high load (Christodoulou 2000), while for others the severity of the clinical symptoms is proportional to the mutant load (Dahl et al. 2000). Whether the finding that mtDNA copy numbers that can vary by over an order of magnitude between MII oocytes in the same cohort (see below) presents another challenge for the application of mitochondrial analysis in clinical IVF, remains to be determined.

   Mitochondria as metabolic forces in early development 

Mitochondrial fine structure and metabolic activity
It has long been known from transmission electron microscopy (TEM; Sotelo & Porter 1959, Baca & Zamboni 1967) that mitochondria in mammalian oocytes and early embryos have a unique fine structure in which a spherical profile, dense matrix and relatively few cristae are indicative of an undeveloped state (for reviews see Van Blerkom & Motta 1979, Makabe & Van Blerkom 2004). What makes recent TEM studies of human oocytes and embryos clinically relevant is the possibility that structural abnormalities detected in certain infertile women could be associated with mitochondrial dysfunctions that reduce their metabolic activity and may, therefore, be an important etiology of oocyte or embryo incompetence (Motta et al. 2000, for review). This may be especially relevant in women of advanced reproductive age as reported by Muller-Hocker et al.(1996).

Similar to other mammals (Van Blerkom & Motta 1979), mitochondria in fully-grown human oocytes are the most abundant organelles detected by electron microscopy (Fig. 1A) and occur as spherical/ovoid elements <0.5 µm in diameter (Dvorak et al. 1987). Typically, these mitochondria contain only a few short cristae that rarely penetrate an electron-dense matrix (Fig. 1B and C). This phenotype persists through the cleavage and late morulae stages of human embryogenesis in vitro before a gradual transition to an elongated form with a matrix of low-to-moderate electron density is observed (arrows, Fig. 1D and E). An increased number of lamellar cristae that completely traverse the inner mitochondrial matrix is generally characteristic of mitochondria actively engaged in ATP production by oxidative metabolism, and this profile represents the predominant form seen at the blastocyst stage in most mammals. For the human preimplantation embryo developing in vitro, serial section TEM analysis has shown that at the blastocyst stage virtually all cells contain (albeit in different proportions) both undeveloped and well-developed mitochondria (Fig. 1D).

Unlike the situation that prevails in other mammals such as the mouse and rabbit (Van Blerkom & Motta 1979), for the human blasto-cyst the fully developed mitochondrial phenotype shown in Fig. 1E may predominate in some cells and be comparatively scarce in others (Sathananthan et al. 1993, Van Blerkom 1993). It is not known whether the apparent cell-specific differences in the state of mitochondrial differentiation observed in human blastocysts are related to the conditions of culture and therefore not representative of the in vivo situation. Alternatively, they could be a normal aspect of early human development and represent developmentally significant differences in mitochondrial activity within the embryo, perhaps related to differential cell function, as discussed below for the mouse blastocyst.

While most of this study was related to difficulties arising from invitro fertilization it overlapped a discussion we have been separately having about the process of Mitochondrial aging and I thought it relevant to this thread and meriting raising the again the questio of do we yet fully understand what is happening to the mtDNA during oocyte formation?

What I find interesting is that something that apparantly happens during the formation of the oocyte also appears to reset the mtDNA clock.

So how is this done?

Why is obvous.

#24 Lazarus Long

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Posted 12 December 2004 - 08:22 AM

Here is another article alluding to something that should be of interest to us with respect to Mitochondrial aging that is not clearly understood but that we need to.


Molecular aspects of oogenesis

Mitochondrial DNA rearrangements in human oocytes and embryos

Jason A. Barritt1,3, Carol A. Brenner1, Jacques Cohen1 and Dennis W. Matt2

1 The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, Gamete and Embryo Research Laboratory, Livingston, New Jersey and 2 Medical College of Virginia, Virginia Commonwealth University, Department of Obstetrics and Gynecology, Richmond, VA, USA


Human mitochondrial DNA (mtDNA) rearrangements, including more than 150 deletions and insertions, accumulate with age and are responsible for certain neuromuscular diseases. Human oocytes, arrested for up to 50 years, may express certain mtDNA rearrangements possibly affecting function.

Investigations have previously shown a single mtDNA rearrangement (dmtDNA4977) in human oocytes. Sequencing of other rearrangements and their correlation with maternal age have not been performed in human oocytes or embryos. Here we use a nested PCR strategy of long followed by short polymerase chain reaction (PCR) that amplifies two-thirds of the mitochondrial genome. mtDNA rearrangements were detected in 50.5% of the oocytes (n = 295) and 32.5% of the embryos (n = 197). This represents a significant difference in the percentage of mtDNA rearrangements between oocytes and embryos (P < 0.0001). Twenty-three novel mtDNA rearrangements with deletions, insertions and duplications were found.

There was no significant age-related increase in the percentage of human oocytes or embryos that contained mtDNA rearrangements. Significant reductions in the number of oocytes containing mtDNA rearrangements occurred as oocyte development progressed from germinal vesicle to the mature metaphase II oocyte (P < 0.05). These findings are discussed as they relate to mitochondria, mtDNA, and ATP production in human oocytes and embryos.


mtDNA rearrangements and age

Population age variation was analysed by MWRST to verify that there was no statistically significant age difference in the sampled populations of oocytes and embryos. The mean ± SEM of the maternal ages of 235 oocytes analysed was 33.0 ± 0.3 years, and showed no significant difference from the 196 embryos analyzed (33.2 ± 0.3 years).

The donor age of MII oocytes (n = 184) with mtDNA rearrangements (30.5 ± 0.6 years; mean ± SEM) was significantly different (MWRST; P < 0.0001) from the donor age of MII oocytes without mtDNA rearrangements (33.4 ± 0.3 years). The presence of mtDNA rearrangements in oocytes from donors aged <38 years was 84/192 (43.8%). The presence of mtDNA rearrangements in oocytes from donors aged 38 years was 14/40 (35.0%). 2-Analysis revealed no age-related change in the presence of mtDNA rearrangements in human oocytes. The donor age of MII oocytes (n = 182) with dmtDNA4977 (31.1 ± 0.8 years) was not significantly different from the donor age of MII oocytes without dmtDNA4977 (32.8 ± 0.3 years).

The donor age of embryos (n = 196) with mtDNA rearrangements (33.2 ± 0.5 years) was not significantly different from the donor age of embryos without mtDNA rearrangements (33.2 ± 0.4 years). The presence of mtDNA rearrangements in embryos from donors aged <38 years was 48/155 (31.0%). The presence of mtDNA rearrangements in embryos from donors aged 38 years was 16/42 (38.1%). 2-analysis revealed no age-related difference. The donor age of embryos (n = 196) with dmtDNA4977 (32.5 ± 0.6 years) was not significantly different from the donor age of embryos without dmtDNA4977 (33.4 ± 0.4 years).

Mitochondrial rearrangements during oocyte development

When the oocytes were grouped by meiotic stage [ovarian, germinal vesicle (GV), and MI arrested at prophase of meiosis I, compared with MII oocytes arrested at metaphase of meiosis II] there was a significant difference by Fisher exact test (P = 0.048), with 32/53 and 107/230 respectively containing mtDNA rearrangements. The presence of mtDNA rearrangements in the ovarian and GV oocytes grouped together was 65.0% (26/40), and this was significantly different (Fisher exact test; P = 0.022) from the presence of mtDNA rearrangements in the MI and MII oocytes grouped together, 46.5% (113/243).

The presence of mtDNA rearrangements in oocytes isolated from ovarian tissues was 85.7% (6/7); in GV oocytes collected from IVF was 60.6% (20/33); in MI oocytes collected from IVF was 46.2% (6/13); and in MII oocytes collected from IVF was 46.5% (107/230).

Mitochondrial rearrangements and embryo quality

Statistical analyses found no significant differences based on mtDNA rearrangements for percentage of fragmentation, number of cells, or the presence of multinucleated cells. There was a significant difference based on mtDNA rearrangements between embryos considered sub-optimal without distinct morphological characteristics and embryos considered slow-developing (P < 0.05), with the slow embryos having fewer mtDNA rearrangements (13/53, 24.5%) than the normally dividing embryos (16/31, 51.6%).


Approximately half of human oocytes and one-third of embryos contain single mtDNA rearrangements. Multiple mtDNA rearrangements occurred in fewer samples, but again, at least twice as often in oocytes than in embryos. Over 20 new rearrangements were detected in this study. Although the differences between oocytes and embryos confound the idea of a fertilization `bottleneck', one has to be careful in drawing conclusions, since the material selected here may contain an inherent bias common in human embryo research: apparently normal MII oocytes and normal embryos were not included due to their obvious need in the clinical process. Moreover, a study of bottleneck mechanisms and timing is dependent on the determination of quantifiable mtDNA mutations in single cells, rather than the presence of any mutation. The latter is important since the human oocyte contains an abundance of mitochondrial genomes and the presence of just one mutation may be insignificant. Nevertheless, trends observed in this work confirm the likelihood of a selection mechanism aimed at removing cells containing any mutated mtDNA during or shortly after fertilization.

DNA rearrangements/embryos/mitochondrial deletions/mitochondrial DNA/oocytes

Bingo ;))


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Posted 12 December 2004 - 09:32 AM

So your theory, LL, is that certain mitochondrial DNA rearrangements act to reset the oocyte and that imperfect rearrangements are eliminated? Explain please.

Whilst we are on topic also check this out:

Mech Ageing Dev. 2004 Oct;125(10-11):755-65.
Age-dependent modulation of DNA repair enzymes by covalent modification and subcellular distribution.

Szczesny B, Bhakat KK, Mitra S, Boldogh I.

Sealy Center for Molecular Science, University of Texas Medical Branch, 6.136 Medical Research Building, Route 1079, Galveston, TX 77555, USA.

Chronic oxidative stress is generally believed to be a major etiologic factor in the aging process. In addition to modulation of signaling processes and oxidation of cellular proteins and lipids, reactive oxygen species (ROS) induce multiple damages in both nuclear and mitochondrial genomes, most of which are repaired via the DNA base excision repair pathway. 8-Oxoguanine (8-oxoG), a major ROS product in the genome, is excised by 8-oxoG-DNA glycosylase (OGG1) and the resulting abasic (AP) site is cleaved by AP-endonuclease (APE1) in the initial steps of repair. Here, we provide data showing that differences between young and aged cells' efficiency in import of OGG1 and APE1 may be responsible for age-associated increase in DNA damage in both nuclear and mitochondrial compartments. It is also evident that age-dependent changes in covalent modifications of APE1 by acetylation regulate its action as a transcriptional repressor of many Ca(2+)-responsive genes by binding to nCaRE, in addition to its endonuclease activity. Thus, ROS-induced altered signaling is responsible for age-dependent changes in post-translational modifications and import of DNA repair enzymes into nuclei and mitochondria (mt), which in turn affect repair of their genomes.

Basically saying that as we get older we can't get enough DNA repair enzymes into where they are needed (sites of nuclear and mitochondrial DNA).

#26 Lazarus Long

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Posted 12 December 2004 - 10:11 AM

I am suggesting that we may have two qualitatively different models of cumulative mutagenic damage. One is associated with developmental cellular function and replication. A part of it is from oxidative stress and another part from errors associated with DNA damage but also we may have another evolutionary model for mtDNA damage and repair, one that influences oocyte formation and viability.

It is this second possible repair process that corrects during the formation of the oocyte that interests me because it is correcting the possible mtDNA mutations that are cumulative over generations and must be correcting to a kind of template for a base norm that resists mutation. It is a potentially different repair process than the one described above and generally understood. If it exists it is probably mostly a selection and elimination process but also does appear to involve the concentration of cytoplasm and mitochondria for the formation of the oocyte when the cytoplasm is concentrated there and removed for the most part from the remaining polar bodies.

Even if this is the result of a *selection process* only (and not a repair) then there does appear to be a kind of matching process against a standard norm at work. However if my suspicion is correct then we may be looking at a secondary repair process designed to ensure viability.

I do not suggest the process described in the article you offer is mutually exclusive. I simply wonder if we are looking at two distinctly different possible repair mechanisms with possibly very different modalities. Oocytes do not age for the same reasons as most other cells and they are formed during fetal growth. They do not age in the woman because of replication (though environmental stress can be a factor) they should age over generations; but they don't.

The bottleneck issue is suggested as causing possible extinction in asexual reproduction but this is not the case either so it simply raises a flag to my attention as an area deserving further investigation.

If there is a distinctly different process of mtDNA rejuvenation/repair occurring during oogenesis then might that not be identifiable and replicated?

And if that process was one that lead to an alternative model for mtDNA repair than what the cell normally does could this offer a treatment we haven't been looking at?

You see oogenesis is the result of meiotic division but normal cellular growth and replacement is the result of mitotic division so there is good reason to suspect two different mechanisms may be at work. I am simply a curious fellow and love a good mystery and this one appears to be an area that is still vague in terms of our overall understanding. It may simply be another of the many dead ends but what if it isn't?

Edited by Lazarus Long, 12 December 2004 - 06:32 PM.

#27 Lazarus Long

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Posted 12 December 2004 - 05:19 PM

OK I have slept on this observation and I can now at least offer one hypothesis for *why* there might be two different models of mtDNA aging and repair at work. I think the answer is in part found in an evolutionary correction mechanism and second in the very differences between meiotic and mitotic cellular division necessary for the production of gametes.

You see male and female gametes are produced for very different adaptive environments and while they need one another to complete their life cycle they exist by two very different methods in two very different environments so to speak.

Second, before I get too deep into the issue of male gametic production and whether some of the male mtDNA might get involved in zygotic development lets focus in on one simple, identifiable, and testable hypothesis: mtDNA does not ever reproduce meiotically, it is always reproduced through mitosis even during meiosis.

So why is this relevant and important?

During oogenesis when the cells are splitting nuclear DNA meiotically forming haploid gametes only one of these resulting divisions actually forms the oocyte and the remaining ones form polar bodies that are not viable and essentially are the left over nuclear DNA without the cytoplasm because most of the cytoplasm concentrates into just the one oocyte (egg) and as we do know the mtDNA is all located in the cytoplasm, not the nucleus.

Second clue, mtDNA is passed down from mother to child through what is essentially a clonal process and is not recombined (there may be examples of deviations from this generality) with the fathers' mtDNA in the sperm so it is always reproducing mitotically.

Third, mtDNA is essentially a form of bacterial gene and may demonstrate some types of bacterial sexuality that we might induce or discover but the normal reproductive form for this type of DNA is mitotic asexual reproduction. While plasmids and episomal budding and assimilation may be theoretically possible it does not appear to be a part of the normal reproductive life cycle of mtDNA. (It could for example be the result of paternal mtDNA introduced at fertilization that goes on to contribute some mtDNA toward zygotic development under some probably pathological instances by making episomal bodies available to the maternal mtDNA genome but this would be the exception rather than the norm).

Fourth, over many generations because mtDNA is essentially reproducing from generation to generation asexually through mitotic division there should without some correction method in place be an accumulation of errors that results in extinction due to the *Bottleneck Hypothesis*. This does not appear to be the case and I should add that we do observe healthy mutagenesis of the mitochondrial genome that can be assimilated and passed on to subsequent generations so this raises the additional question of how is this being done?

The suggestion is that mtDNA concentrates in the eventual oocyte for two basic reasons.

The first is to increase the viability of the oocyte by making available to it a larger than normal concentration of mitochondria necessary for the intense high energy demand period that occurs initially after fertilization and before there is the ability to tap the mother's metabolism through placental development.

The second is to reduce the waste (mtDNA genetic survival) if the development of polar bodies are essentially a dead end but this raises the question of how does the cell select which gamete to make the oocyte?

In other words there may be an evolutionary survival mechanism at work for mtDNA to select the host most likely to go on during meiosis.

The interesting thing is that after the female gamete forms an oocyte, cell reproduction (and all apparent metabolism) stops and all the mtDNA created during this process goes dormant till the next life cycle, which *normally* only occurs *after* fertilization of the next generation during the offspring's fetal development.

The dormant mtDNA can and does *live* (remain viable) for decades but does not induce even the first mitotic division until it is activated by the introduction of the male gamete. This raises another question of how does the mtDNA of the oocyte understand (chemically) to both turn on and off?

This mitotic division during meiosis is then repeated just once during the fetal growth cycle (only in the female) utilizing stem cells to form the next generation of oocytes and is not a part of the general development and physiology of the mother.

In other words this background mtDNA mitotic life-cycle of the oocyte has a very long periodicity only occurring in females and only once in each womans' life actually prior to her birth. This is why we do not observe the process as a part of *normal* senescence. However this strategy can be understood to reduce the impact of mitotic senescence that would still accumulate over generations but by itself it should not be able to eliminate it.

It should be noted that meiotic division also does occur in males for the purpose of sperm production but this is a very different life-cycle for combined nuclear meiosis and cytoplasmic mitosis. The sperm is not a long term survivor but reproduces by a sort of *mayfly method* of massive populations compared to female oocytes. Spermatogenesis does not produce the equivalent of polar bodies during meiosis so it doesn't concentrate cytoplasm but only stores enough energy to make one journey in its life cycle that is its one chance at glory in a race against time, environment, and its brothers. It is also a different strategy of quantity versus quality of gamete. Female gametes are significantly smaller in number but built to last and male gametes are manufactured by the billions but are generally disposable soma.

However while in oogenesis we see the concentration of cytoplasm and in spermatogenesis we do not, in both cases we are actually observing nuclear DNA meiosis simultaneous with mitochondrial mitosis. When this is occurring for the male there is less of a need to correct mtDNA since the male gametic mtDNA will not be a significant part of zygotic development but there is a real need for a correction method to be in play for female gametic (re)production and I wonder that it is no small coincidence this is occurring during fetal development utilizing natal stem cells for the purpose.

It also should be noted that results of recent experiments in parthenogenesis suggest that stem cells can be used for the creation of gametes and I will try and find those studies if no one can place them here first.

So in conclusion for mtDNA to evolve with the host and remain viable without significant accumulated damage over thousands of generations even with the hyper-long mitotic periodicity noted (one mitotic division per generation) implies the need for a secondary correction modality that is still unrecognized.

It is also the case that male and female gametes are demonstrating very different life-cycle strategies that are polar opposites yet compliment one another by perhaps ensuring the efficiency of sexual reproduction as a method to enhance reproductive selection to incorporate successful mutation into the genome. But it also does also beg the question of why the life-cycles of male and female gametes are so very different yet complimentary?

Add to this another question: How is the host's nuclear DNA contributing to regulating mitochondrial asexual behavior through a dominant sexually determinant model?

In other words it is logical to presume something in the X/Y chromosomal characteristics of the nuclear DNA is contributing to mtDNA life-cycle regulation altering it from long period to an extremely short period reproductive cycling but the trigger and regulating mechanism is most likely to be found in paternal/maternal DNA not in the mtDNA specifically.

These very different reproductive strategies of male and female gametes reflect a combined evolutionary methodology of selection for quality of oocyte versus quantity of sperm. It also reflects two entirely different models for selection with respect to *fitness* in consideration of evolutionary models as sperm must compete against themselves and their environment to survive a very short existence that is literally a race against time, environment, and one another while oocytes do not experience this competitive race aspect but are selected for viability and genetic quality control, slowing down and hoarding reserves of energy against their single tenuous monthly opportunity to survive and reproduce that is dependent on mating with the winner of the male fitness race. For this reason while there might be an mtDNA correction method at work during meiosis for both genders it is logical to suggest that it is more likely present and to be found in oogenesis.

#28 olaf.larsson

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Posted 12 December 2004 - 10:55 PM

An alternative to correction would be that mito function is measured somehow and oocytes with non-vital mitos would die. Some of the mitos in wannebe oocytes could hypotheticaly be in some kind of resting state and not used as normal mitos for energy production, thus lowering the mutationrate. Thank you all for the long and very interesting articles and texts.

#29 manofsan

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Posted 13 December 2004 - 01:48 PM

Hmm, so this corrective mechanism in oogenesis might maintain quality control from one generation to the next. But aging is about defect-accumulation within the lifespan of the individual organism. And we're presuming that the organism got all the proper DNA at conception, so we're just trying to help it retain what it already has from birth.

I don't care about helping my germ-line and distant descendants hang onto what I have. They can sweat it out for themselves. I want to know about how I can hang onto what I already got at birth. ;)

It's good that we're doing more to decipher mito quality control, though.

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#30 Cyto

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Posted 14 December 2004 - 10:57 PM

Well, since the amino acid code for codons is degenerate this can provide some buffer (multiple codes for X amino acid). Saying that mutations is what would lead to a lower localization of repair prots like OGG1 just doesn't seem right, I like the area of investigation proposed in the "age-Dependant modulation of DNA repair...": The possible change in post translational modifications throughout the aging process sounds like a great avenue to pursue. Heat shock proteins like cytosolic Hsp90, which has the evidence to support it in this case, also can provide another buffer to cover ectopic amino acids. Hsp90 is usually involved in the end-phase "tight packing" of a (ie: glucocorticoid receptor) and this has been shown that the tight packing ability being present allots for higher cryptic genetic variability in the gene itself. surrounding charges proximally can cover others. Anything have homology to the Hsp90 that associates with the mito? emmmm, but I don't know the answer to this. I can only throw out some other things. The selection of female gametes sounds good and with the number of mitochondria that can be present in a cell sounds like there is another buffer there. Also I would think that something like female gametes would have more mature mitochondria in the long run, less dependence on mass amounts due to the larger surface area. I would like to know how frequent the formation of secondary lysosomes are with mitochondria in oocytes, when a lysosome merges with another organelle to degrade.

I dont read up on mitochondria too much so I can't procure as great a theory as I would like, but this already looks like a lot of factors going from the [starting binary fission] to [degradation] of the mitos.

I'll be able to think about it more when finals are done with. [tung]

Edited by Bates, 14 December 2004 - 11:32 PM.

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