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LESSON III PART II
DIA 1
Before the imprinting genes were codified, Surani was able to demonstrate the
complementarity of the paternal and maternal genome related to their existence by studying
the development behaviour of androgenota and gynogenota embryos.
The experiment is quite simple.
First of all they had made androgenota and gynogenota embryos.
To obtain an embryo with a diploid chromosome makeup totally paternal or maternal, Surani
started from the zygote cell.
At this stage the two genomes are distinct and in mice they are also easily visible.
So, using a micromanipulator, Surani was able to remove one pronucleus to each zygote and
replace it with a new PN of the same gender.
The reconstituted zygotes with a diploid paternal (androgenota) or maternal genome
(gynogenota) were transferred then into a syncronized female.
DIA 2
Differently from normal embryos, andro and gynogenote fetuses interrupt early their
development.
Approximately after 2 weeks from embryo transfer both fetuses died.
An interesting thing was observed: the to fetuses died for different reasons.
The ginogenota fetus displayed a normal and regular growth but an incomplete development
of the placenta anexa that were unable to sustain further development.
On the contrary, death was caused in the androgenota embryos by exactly opposite reasons:
the androgenota embryo died suffocated by an huge overgrowth of the placenta annexa and
displayed a clear delay in growth.
Although Surani did not have any molecular evidence of the existence of the imprinted genes,
he hypothesized that the normal development of AN embryo in mammals can be obtained
exclusively from the combination of paternal and maternal genomes.
This experiment demonstrated to him that in mammals the two genomes are clearly
functionally complementary. In particular, the maternal genes seem to be essential for
stimulating a normal development fetal development while the paternal ones are
essential to drive fplacenta anexa growth.
DIA 3
The reason of this reproductive failure was now clear at the beginning such as the molecular
genome mechanimsl.
Now it is clear that embryo development is related to the correct transcription of imprinted
genes that control synthesis of proteins strictly related to fetal and fetal annexa development
such as growth or tissue differentiation factors.
One of these genes, for example, is the Insulin-like growth factor (IGF) 2 that is responsible for
the development of placenta annexa belonges to this class of gene.
The IGF 2 gene during fetal development is transcribed exclusively from the paternal allele
since it is imprinted during the oogenesis.
DIA 4
LESSON WORK
DIA 5
The monoallelic transcription of imprinted genes is crucial in mammals for a harmonic
embryo and fetal development. Some processes is regulated by a precise concentration of
proteins that can be obtained exclusively when a gene is trascribed in a monallelic way as it
happens for imprinted genes.
The role of this class of gene must not be considered to be limited, however, to fetal life
but it persist after birth and during the adult lifetime.
In particular, these genes are involved in a correct differentiation of tissues like CNS, bone,
muscle and liver that differentiated during the growing pre-pubertal phase of the organism.
In addition, a their role has been demonstrated later in female adult lifetime when some of
imprinted genes control the relational behavior between mother and child during the
lactation.
So imprinted genes are key crucial genes that were selected during the evolution of
mammals class in order to increase the variability in animal that use a sexual
reproduction by imposing the simultaneously presence of two individuals
DIA 6
Another biochemical modification that involve the chromatin in this case the
eterochromatin during the growth phase is the elongation of the end portion of each
chromosomes.
This end portion, named telomeres, have a great biological relevance since they regulate cell
viability and cell senescence
by protecting DNA deterioration that could occur as a consequence of repeated processes of
cell divisions.
DIA 7
As a consequence of repetitive division the DNA is shortened and this potentially could
determine the lost of infromation. This does not occur since telomeres exist.
Telomeres are in fact no coding sequences inserted at the end of DNA in order to
preseve unaltered the information enclosed into the genome during the DNA
replication.
This end portion are named telomeres and are constituted by highly repeated and no
codifing DNA sequences that in vetertebrated is synthetized on a template of 6
nucleotide bases of Tymidine Tymidine Adenine Guanine Guanine.
This template can be repeated several times and linked at 3‘i strand ends of DNA .
The telomeres in most of the cells is shortened at each DNA synthesis as the
consequence of the incomplete action of the polimerase enzyme.
But this doen not represent a problem since they doesn not contian trascribable genes
DIA 8
The presence of teleomeres is essential to avoid that genomic information can be lost
during the DNA replication.
Indeed, during this process the DNA replication, the polimerase is not able to duplicate
the 5’ strand end portion of chromosome and, as a consequence, during the DNA
synthesis the chromosome are shortened.
This phenomenon does not involve, on the contrary, the portion of DNA containing
genome information that are preserved from telomeres.
The biologocical function of telomeres is indeed to avoid the lost of genome information
(proteins) during cell proliferation.
DIA 9
In most of the cells, the replication is followed by telomores shorthened until they
reach a critical size and the cell becomes senescence. Indeed, when the length of the
telomeres are under the threshold values the check point machinery switch on the apotosis
parhway and the cell undergoes a programmed died process.
In few cells the telomeres is actively elongated after each duplication as the
consenquence of the presence of a dedicated enzyme the telomerase.
These few cells that have the telomerase are immortal cells.
The telomerases is a specific ribonucleoprotein enzyme that syntheised these no
codifing sequences,.
This enzyme works as an inverse trascriptase able to add the sequence of TTTAGGG to the 3’
strand end of DNA thus eleongating the chromosome with terminal no codifing sequences .
DIA 10
Telomerases activity is present in very few cells of the adult organims.
All the cells that have active telomerases are charachterized by self-renewal properties since
they can divede without incur in senescence.
These cells are
embryonic stem cells
Fetal and adult stem cells
Cancer stem cells
And gametes
DIA 11
Now the question is what happen to oocyte telomeres during the growth phase?
In detail, the female gamete actively elongates telomeres during the growth phase in order to
prepare its-self to sustain the early embryonic divisions which will occur during the early
stage of embryo development without the activation of the embyo telomerases.
As you can see in this slide the telomerescan be visualized with the FISH (fluorescence in situ
hybridization) technique using a specific human genome probe that recognized the TTTAGGG
sequences.
The fluorenscence is then used to have idea of the dimension of the telomeres.
As you can see in the slide, Tlemomeres are short structures in of sheep oocytes collected fom
preantral follicles whereas it reach their maximum dimensione in oocyte with a SN chromatin
configuration.
This results indicates that the telomeres are actively elongated untill the oocyte reached its
final dimension and the chromatin assume a mature large configuration represented by the
SN one
DIA 12
And in fact, This process of telomeres elongation is strctly related to the presence and
localization of the telomerase.
The ribonlucease enzyme is always present into the oocyte even if itremains active only until
it is localized into the nucleus, the subcellular district where it works by elongating the
telomeres.
An exclusive nucleus telomerase localization is shown in growing oocytes collected from
preantral and early antral follicles. Then, the telomeras translocates into the ooplasm into SN
oocytes collected by early antral follicles to become exclusively cytoplasmatic from later stage
of oogenesis until the embryo reaches the blastocyst stage. The telomerase approximately in
blastocyst stage transocates again into the nucleus starting again to actively elongate the
embryo telomeres.
DIA 13
The fully grown oocyte, indeed, has actively elongated its telomeres that are significantly
longer than those recorded in other typology of cells.
The longer telomeres are required in fertilized oocyte to sustain the fast embryo division that
at the beginning of embryo development occurs without the activation/traslocation of
telomerase into the nucleus.
DIA14
A reduced LENGTH of telemores has been correlated signficntly with human embryos that
have problem of fragmentation, early block of embryo development
DIA 15
It is now clear that the succes of embryogenesis is strongly related with the cytoplasmic and
nuclear modifications which occur during the oogenesis.
The zygote inherits from the matured oocyte a storage of:
1. Macromolecules
2. Organelles
3. Remodelled chromatin
4. Epigentic arranged DNA
That are required to transform the zygote in a totipotent cells after fertilization.
The totipotency, which is the ability to differentiate in a alive organism, can be express if the
oocyte at the end of the oogenesis is able to responde after fertilization at all the:
• Nutritional
• Energetic
• Synthetic
• Regulatory
requires that the early stage embryo should sustain before embryonic genome
activation occurring at divisions. 3-4