Epigenetics and Beyond
ISIS Report 23/03/09
Epigenetic Inheritance through Sperm Cells, the Lamarckian Dimension in Evolution
New findings on the molecular mechanisms whereby epigenetic changes acquired
during development can be transmitted to the next generation via sperm cells
are vindicating Lamarck’s theory of evolution that had been completely eclipsed
by Darwin’s followers for over a century Dr.
Mae-Wan Ho
A fully
referenced version of this article is posted on ISIS members’ website. Details
here
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Lamarck versus Darwin
For nearly a century, the overwhelming majority of biologists held firmly
to the ‘neo-Darwinian’ dictum that organisms are strictly determined by their
genetic make-up, which is essentially isolated from the environment, so characteristics
acquired during one’s lifetime can never be transmitted to the next generation.
Thus, if one of your parents trained very hard to become an Olympic
tennis champion, you would not expect be born with particularly strong arms
or be a child prodigy in tennis unless you happened to have inherited the
right genes from that parent, and it would not have mattered if he/she trained
for the championship or not. That was because genes were supposed to remain
constant except for rare random mutations; random in the sense that
the mutations bear no relationship to the environment, so your parents’ experience
can never influence your life.
Neo-Darwinism is derived from a combination of Darwin’s theory
of natural selection and Mendel’s theory of genes determining the characteristics
of organisms. Neo-Darwinism is opposed to ‘neo-Lamarckism’, derived from Lamarck,
who proposed that non-random epigenetic variations arising from the
organism’s experience during development are the
stuff of evolution, as they can be transmitted to the next generation
Notable critics of neo-Darwinism such as British evolutionary and developmental
biologist Conrad Waddington had realised that the Lamarckian dimension in evolution
cannot be ignored, which was why he put ‘epigenesis’, i.e., development at centre
stage. He proposed that the intrinsic dynamics of development - the “epigenetic
landscape” - was the real source of novel non-random variations for evolution
[1] (see Beyond
neo-Darwinism: the Epigenetic Approach to Evolution [2], ISIS scientific
publication, for further details.).
Since then, findings in molecular genetics had made neo-Darwinism increasingly
untenable in both development and evolution [3, 4] (see Evolution,
ISIS scientific publications, and Living
with the Fluid Genome, ISIS publication). While the dynamics of development
remains one of the hardest problems still in search of a solution, Lamarckian
mechanisms abound at the molecular level [4, 5] (Life
Beyond the Central Dogma series, SiS 24).
New research reveals that you could be getting, not so much the
right genes, but ‘retro-genes’ from your Olympic tennis champion father.
That is a very significant finding, because mother’s influence through the
egg cell and the womb, has long been recognized as an important part of the
environmental (non-genetic) input for development, while the father was supposed
to contribute nothing before birth except his genes. Now, it appears that
the father’s experience, too, could result in ‘retro-genes’ that might be
passed on via the sperm.
Retro-genes delivered via sperm cells
‘Retro-genes’ are generated by the process of reverse transcription, in which
transcribed RNA, modified, amplified and tested by the individual’s experience,
are ‘back copied’ into complementary DNA (cDNA), constituting new genetic
messages that may be delivered in sperm cells to the egg at fertilization
[6].
This process has been shown to occur during in vitro fertilization,
regardless of whether intact sperm cells are incubated with exogenous DNA
or RNA molecules. The reverse-transcribed sequences transferred to embryos
at fertilization are propagated in mosaic fashion in the tissues of founder
animals, and further transmitted to their offspring, where they are maintained
as low-copy number structures (episomes) outside the chromosomes.
It is now widely accepted that sperm cells of virtually all animal
species can take up DNA molecules and deliver them to the egg at fertilization;
this has been exploited to make genetically modified animals with variable
efficiency. But the fate of sperm-bound DNA after delivery to the egg is still
unclear, in particular, whether foreign nucleic acids become integrated into
the host genome, or remain outside the chromosome.
Lab experiments indicate that non-integrated episomal structures
are frequently generated when foreign DNA molecules are directly incubated
with intact sperm cells that are then used to fertilize eggs. Integration
in the host genome is rare under those conditions, and so far, claimed by
a single research group experimenting on swine. The same group also later
reported the transmission of non-integrated sequences.
In contrast, integration seems to be favoured with protocols
that avoid direct interaction between the exogenous nucleic acid molecules
and the sperm membrane, such as wrapping the foreign nucleic acids in membrane
lipids, or incubating foreign DNA with sperm cells without membranes followed
by microinjection into the eggs.
Sperm-mediated gene transfer depends on reverse transcription
Corrado Spadafora at the University of Rome in Italy has identified most
of the factors involved in this sperm-mediated gene transfer (SMGT) [6]. The
foreign nucleic acids are taken in and reach the nuclear scaffold of sperm
cells, where they are rearranged by nucleases and undergo recombination that
eventually leads to integration in the sperm genome. Analysis revealed that
integration occurs in one, or only very few, preferred sites in the mouse
genome, and is therefore very infrequent. External DNA molecules activate
one or more nucleases in the sperm, which heavily degrade the foreign DNA,
and eventually also cleave a minor chromatin component. These findings suggest
that discrete sites of nuclease sensitivity exist within the otherwise tightly
packed chromatin of mature sperm cells that are preferential targets for integrating
foreign DNA.
The fraction of mouse sperm chromatin that closely resemble active
chromatin of somatic cells in being nuclease-sensitive and has very low level
of methylation are also unexpectedly enriched in retrotransposon DNA, among
which, the most abundant are reverse transcriptase (RT)-encoding LINE1 sequences.
This intriguing finding prompted Spadafora’s research team to incubate mouse
sperm cells with foreign RNA molecules, and then search for evidence of reverse
transcription into cDNA.
In one of these experiments, they incubated sperm cells with
RNA transcribed from a construct expressing a b-galactosidase (b-gal) reporter gene. They then used this to fertilize eggs in
vitro, and produced a F0 founder generation, followed by a F1 progeny
by normal breeding. Direct PCR analysis of DNA samples from both F0 and F1
animal populations confirmed that b-gal
containing cDNAs were generated in sperm, delivered to oocytes, and propagated
in mosaic fashion through embryonic development in various tissues of the
adult animals and transmitted to the next generation.
Remarkably, these sequences are maintained stably as low-copy
number episomes (<1 copy per genome), and inherited in non-Mendelian, mosaic
fashion. Most significantly, the expression of the b-gal protein was detected in a variety of tissues
in both F0 and F1 animals.
Subsequently, the researchers found that an RT-dependent process
is triggered not only when sperms are incubated with RNA, but also when they
are exposed to DNA. They incubated sperm cells with a retrotransposing cassette
containing the DNA construct with enhanced green fluorescence protein (EGFP)
as reporter gene interrupted by a g-globin
intron placed in the opposite orientation to that of the EGFP. In order to
become expressed, the reporter gene must go through reverse transcription.
First the DNA construct interacts with the sperm and is taken up into the
nucleus where it is transcribed; the primary RNA is then spliced (to remove
the interruption g-globin intron) and finally reverse-transcribed
to cDNA containing intact EGFP sequence. Interestingly, only a small proportion
of the newly synthesized cDNAs is retained within the sperm, while most of
it is released into the incubation medium, and available for further interaction
with sperm cells. Eventually, a steady state is reached in which the vast
majority of the sperm cells are associated with foreign cDNA as extrachromosomal,
low-copy number episomes. These are transcribed and the EGFP reporter gene
expressed in various tissues of the adult animals.
Rare integration events may occur in the matrix-bound chromosomal
DNA which are nuclease sensitive and integration-prone, as opposed to non-matrix
bound chromatin.
These findings are impressive, but they only involve artificial
in vitro conditions in which sperms are incubated with foreign DNA
or RNA. There is so far no evidence that the same processes would take place
in vivo. Or is there?
Epigenetic inheritance in vivo
Another team of researchers led by Minoo Rassoulzadegan at the French National
Institute for Health and Medical Research (INSERM) in Nice recently reported
on a case of non-Mendelian RNA-mediated inheritance of extra-genomic information
in mice [7]. The mutant engineered by a LacZ insertion in the Kit gene
coding for tyrosine kinase receptor results in mice that die shortly after
birth in the homozygous state, while heterozygotes survive with white patches
on the tail and feet. The surprising finding is that some of the offspring
of such mice, which inherited two wild-type copies of Kit, still exhibited
the white patches characteristic of the mutant animals. Similar results are
obtained from mating heterozygous mutants to wild-type mice, regardless of
the sex of the parent carrying the mutant gene. The mutant phenotype appearing
in homozygous wild-type offspring of heterozygous parents is associated with
reduced expression of wild-type Kit mRNA, concomitant with an accumulation
of Kit RNA transcripts without poly-A tail (pre-mRNA) of abnormal size
in tissues, most easily detected by staining for RNA in the sperm. Microinjection
of that RNA into fertilized eggs induced a heritable white tail phenotype.
Thus, phenotypes are not exclusively due to chromosomal genes
but depend on information apparently stored in a stable class of RNA molecules,
which may depend on RNA-dependent RNA polymerase enzyme.
Commenting on these findings, Spadafora thinks it is possible
that the propagation and expansion of RNA goes through a DNA intermediate
via reverse transcriptase [6]. Such RT-mediated replication and expansion
not only takes place in sperm cells and eggs, but perhaps also in embryos
and in differentiated somatic cells, given that RT is now known to operate
throughout embryogenesis.
Somatic hyper-mutation hypothesis revisited
The proposal that RNA mediates epigenetic inheritance is not new. Immunologist
Ted Steele, currently at Australian National University Canberra, first put
forward such a theory in his book, Somatic Selection and Adaptive Evolution:
On the Inheritance of Acquired Characters, published in 1981 [8], based
on the then highly controversial experimental results demonstrating the inheritance
of immunological tolerance through the male line. While some of us cheered
from the side lines [4], Steele and his fellow researchers were vehemently
dismissed and attacked by the establishment throughout the 1980s and 1990s
when they continued pushing back the frontiers [8], and are now in danger
of being fully vindicated.
Essentially, Steele and colleagues proposed that immune responses
to foreign antigens provoke high rates of mutation (hyper-mutation) in white
blood cells through cycles of transcription and error-prone reverse-translation
until high affinity antigen-binding antibodies are formed. Cells forming such
antibodies are selected by the foreign antigen resulting in the enormous clonal
expansion of those cells.
Steele thinks that as the Kit-specific effects in the
work of Rassoulzadegan and colleagues are transmitted at least to the second
generation, the phenomenon must depend on more than just RNA stability. He
is convinced that a reverse transcription and genomic integration step must
intervene at some stage to fix the DNA in the germline [9]. In the case of
Spadafora’s important work, Steele thinks “The [rare] centromeric integration
site Spadafora describes may be peculiar to his type of in vitro uptake
system [and may be the organism’s way] to quarantine potential genetic effects
of exogenous foreign DNA in seminal fluid.”
But what about the ‘natural’ route, involving somatic RNA/DNA delivered to
developing sperm or spermatogonia?” Steele asks. That is the crucial question
as far as adaptive evolution is concerned.
For more on the problem of development and evolution in the light of the new
findins in epigenetics see [11] Development and Evolution Revisited (ISIS scientific
preprint).For more on the problem of development and evolution in the light
of the new findins in epigenetics see [11] Development
and Evolution Revisited (ISIS scientific preprint).
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