Download LESSON IV first part File - Progetto e

DIA 1
Several relevant events occur during fetal oogenesis:
first of all, the female or male gamete lineage is defined during the fetal life,
secondly the number of gametes increases passing from a few cells to approximately 12 million of gametes immediately before or after birth depending of mammalian
species
, finally, what is really important, the reproductive cells undergo a dramatic change in
genome makeup either in term of genome variability and epigenetic asset.
Several relevant events occur during fetal oogenesis:
first of all, the female or male gamete lineage is defined during the fetal life,
secondly the number of gametes increases passing from a few cells to approximately 1-2
million of gametes immediately before or after birth depending of mammalian species
, finally, what is really important, the reproductive cells undergo a dramatic change in
genome makeup either in term of genome variability and epigenetic asset.
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Now we are going to begin by focusing the attention on the origin of gametes.
The primordial germ cells are differentiated very early during fetal life.
The presence of primordial gametes is documented in mice approximately 7 days after
fertilization when PGC are visualized for the first time in extra-embryonic district.
The PGC are pluripotent stem cells, which are able to differentiate into all the tissues of
the organism but they are unable, differently from early stage embryos, to differentiate
into a live organism.
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Since oogenesis is strictly related to body size does not surprise to find that the PGC
appear during the fetal life of mice, pig and humans after 7, 13 and 21 days
respectively.
They appear as an aggregate of pluripotent cells located in the mesodermal layer.
More in detail, they originate in the mesodermal layer of the yolk sac wall, in response
to growth factors released from cells of the ectodermal layer.
Immediately after differentiation, the PGC start to proliferate and in some day they
move towards the embryo in response to local chemiotactic stimuli
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The PGC start to migrate leaving the yolk sac to reach the primitive genital ridge at day 12 in
mice.
They undertaken this long journey travelling along the wall of the hindgut.
During the journey the PGC proliferate by increasing further in number.
The PGC that reach the primitive gonads at day 12 are approximately 1-50 thousand cells, so
100 times more than the original ones (about 50 cells).
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The PGC during this long journey, in addition, undergoes a profound epigenetic
remodeling.
The chromatin of these cells undertake a dramatic de-methylation that involve all gene
sequences including the imprinted ones.
Indeed, when the cells reach the genital tract at day 12 all the methylation markers
have been completely erased (iresd) and the PGC displaying a whole de-methylated
DNA
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The question is Why are the PGC de-methylated and What does this process functionally
mean?
The PGC immediately after their differentiation display, such as all the cells of embryo and
extraembryonic tissues, a somatic genome.
This mean that the PGC trascribe the imprinted genes in a monoallelic manner since they
present a complementary methylation.
It is clear that this somatic epigenetic organization can not last for long time, since differntly
from PGC the gametes must organize a specific parental imprint.
The gametes, indeed, must develop a complementary parental genome for what concern
imprinted gene sequences in order to promote complementary between gamete genomes
essential to sustain reproduction.
To this aim, the PGC undergo a process of de-methylation that interest any genes. Thanks to
this process all pre-existing epigentic markers are completely erased.
This event occurs in PGC during the phase of migration.
Indeed, the PGC that reached the primitive genital tract (day 12) have lost all the methyl
groups on the DNA.
The methyl group will be inserted into the DNA later during the post natal period and some of
them will be used to impose the permanent parental markes on the imprinted genes.
In female gametes, the process will keep long time.
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In summary, the epigenetic remodeling and in particular the DNA methylation is a very
dynamic process during the oogenesis .
Indeed, if we look at the degree of DNA methylation of imprinted genes of gametes like in this
slide,
We can observe that early during the fetal life the status of methylation of imprinting genes in
PGC are similar to those of all other somatic cells.: it involves either male e female imprinted
genes (2x).
The major epigenetic transformation occurs during the fetal oogenesis when all epigenetic
markers are completely erased also the gene sequences of imprinted genes and the DNA is
transformed into un-methylated structure (0x).
This status may persist for long time until, the primary imprinting is defined during the postnatal oogenesis when the oocyte are recruited.
At different stage of growth and maturation phases the maternal specific epigenetic markers
are inserted by following a gene specific kinetic.
At the end of oogenesis reach into the matured oocytes, all the imprinted genes are
methylated (1x), whereas the same genes remain complementary un methylated in the male
gamete.
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Lesson case
- The maternal and paternal primary imprinting are laid down during germ cell development
that you can recognize in the pink and blue shaded area, respectively)
When oocyte and sperm are fully mature (before fertilization) the correct/final pattern of
DNA methylation on imprinted genes is reached (the final degree of methylation must be
indicated with four pink and blue shared ovals).
- After fertilisation (yellow-shaded area) both parental genomes during the early stage of
embryo development, undergo global de-methylation even if the epigenetic process does not
involve the imprinted sequences. At this stage the imprinted genes are protected from the
process of de-methylation.
- Analogously, this is not the case for the imprinted genes of PGC during fetal development.
By considering this premises:
1. Complete the figure introducing the correct degree of methylation in the pink
shared area (post natal oogenesis) by inserting the pink colour inside the ovals
that indicated the degree of methylation related to maternal imprinted genes
(female imprints, pink ovals) of:
2. Complete the figure introducing the correct degree of methylation in the yellow
shared area (mebryo/fetal development) in order to describe the status of
methylation of male and female imprinting sequences
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The maternal (pink shaded region) and paternal (blue shaded region) imprints are laid down
during germ cell development so that by the time the oocyte and sperm are fully mature the
correct pattern of DNA methylation is present on the genome (female imprints, pink ovals;
male imprints, blue ovals). After fertilisation (yellow-shaded area), both parental genomes
undergo global demethylation of non-imprinted sequences: imprinted genes are protected
from this process. During early embryo development the imprinted genes of both the somatic
and PGC retain the parental imprints. From E11.5 the primordial germ cells begin to undergo
demethylation to erase the inherited parental imprints, but the somatic cells of the embryo
maintain the parental imprints through embryo development and into adulthood. The process
of PGC demethylation is complete by E13. Subsequent reprogramming of the germ cells
occurs when the gender-specific imprinting patterns are once more laid down.
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The sex gamete differentiation is another important aim of fetal oogenesis.
When PGC appear during the early stage of fetal development they are sexual
undifferentiated cells.
The chromosome makeup of somatic cells of the primitive genital ridge are responsible of
this sex specific lineage gamete differentiation.
The somatic cells, indeed, are responsible of PGC differentiation towards spermatogonia or
oogonia lineages
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Gamete differentiation is promoted by the transcription of a gene localized in the
sexual Y chromosome.
This gene is expressed by somatic cells showing a Y chromosome. These cells become
Sertoli cells, the main supporting cells of spermtogenesis, and they will be
repsponsable of the sex specific transformation that involve gonad cells and
reproductive apparatus.
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Through their secretion activity, Sertoli cells are responsable of the in situ
differentiation of the PGC toward the spermatogonia.
In addition, Sertoli factors promote another improtant sex related cell lineage, the
Leyding cells.
The leyding cell have an active role in gonad since they are mianly involve in the
steroidogenesis by releasing high level testorene
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The Sertoli cells are also responsable for the differentiation of the internal male genital
organs.
First of all, the Sertoli cells actively secrete the anti-Mullerian hormone that inhibites the
development of Mullerian ducts from which oviduct and uterus could differentiate.
The Sertoli paracrine influence is also involved in the differentiation in male gonades of
Leyding cells a steroigenetic active cells.
Immediately after the development Leydig cells the level of testorone increases and this is a
key event to stimulate the differentiation of glands supporting the male genital system and in
parallel inhibit the degenration of seminufourus tubules
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In the absence of Y chromosome, the PGC move spontaneously towards the female
gamete differentiation and the relative genital ridge is transformed into the female
reproductive apparatus
This component transform the synaptonemal as a ladder like complex displaying a central
element (blue one), transverse filaments (thin black lines) connecting the lateral elements
(red) that have previously anchored the chromatids loops.
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Without the transcription of the Y related gene, the genital ridge spontaneously differentiated
into the female reproductive system.
First of all, in the absence of high level of anti Mullerian hormone secreted by Sertoli cells, the
Mullerian ducts evolved.
The failure of the paracrine role of Lending cells, in addition, it is responsible of the
regression of seminiferous tubules.
As a consequence aft that, the simplified female genital tract is developed composed of two
gonads and two serial ducts represented by the oviduct and uterus.
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The Y sexual chromosome is responsable of the gender defintion of the embryo.
More in detail, a specific gene named SRY = Sex-determining Region of Y is localized on
this sexual chromosome that is responsable of the processes male lineage
differentiation.
The localization in the genital ridges of some cells containing the Y chromosome is
enough to trigger the sexual male differentiation process even if in the presence of
more than one X chromosome.
In the absence of any Y chromosome, the genital ridges differentiate spontaneously
towards the female reproductive system even if only one X chromosome is present
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This concept is clarify from this experiment where the SRY gene is inserted into the nucleus of
a female zygote
The transgenic embryo express the SRY genes even if in the presence of two sexual XX
chromosomes.
As a consequence of that the PGC undergo differentiation towards male gamete lineage and
the primitive gonads develop a complete male reproductive apparatus.
This male transformation is clearly appreciable by observing the external genitalia of the
transgenic mouse that are completely similar to those of a XY male mouse.