Preantral Folliculogenesis Flashcards
(39 cards)
How can we study reproduction?
What are some challenges with each method
1.Using animal models
These can be :
Mono-ovulatory
Poly-ovulatory
(We need to always check how many offspring these animals will produce to make the model similar to humans
2.Genotype/phenotype associations in naturally occurring mutations or from knock-out mice.
(for e.g for early follicle growth)
3.Culturing tissue -whole ovaries,slices,biopsies,large follicles ,small follicles,cells
These can be difficult in human because of limited supply of tissue
Primary Cells are also difficult to obtain
granulosa cell lines are available but not theca cell lines available
Where do primordial germ cells originate from?
Early on during 3-4 weeks human embryo the epiblast (primitive ectoderm) is one of two distinct layers arising from the inner cell mass) cell in the yolk sac at the base of all allantois (an extension of the posterior wall of the yolk sac) differentiate into PGC
What will happen to the PGCS which in the 5-6 weeks human embryo?
The mitotically dividing (increase in numbers)PGCS will migrate along the dorsal mesentery of the hind gut and colonise the genital ridge.
This will become the reproductive gonads depending on sex differentiation
Chemotactic substance is secreted by the ridge to attract the PGCS.This maybe the Kit ligand because the receptor cKit has been found on the surface of PGCS
What will happen to the PGCS to form oocytes?
Mitotically dividing PGCS will reach the genital ridge :
If female=oocytes
This will let them form syncytia (nests).
They have cytoplasmic bridges which connect them together to form syncytia and this is thought to allow the exchange of organelles e.g. ER and mitochondria
What is the role of retinoic acid ?
This is the biological active variant of Vitamin A and has been found to be a key extrinsic regulator of germ cell entry into meiosis.
These cells have been dividing through mitosis in order to increase in number and stop to enter meiosis.
What gene has been identified to be important in the process of oocyte formation from PGCS ?
DAZL expression has been found to increase before meiosis at 9-14 weeks gestation.
We know this because in Knock out mice germ cells do not develop past primordial germ cell stage
and mutations in DAZL have been associated with sub fertility
What happens following meiosis of cells whjich are developing following 16 week gestation?
At this stage there is cyst breakdown /primordial follicle formation
This stage will happen before birth in humans whereas in mice it was found to happen following birth
Describe the nest breakdown to form primordial follicles
Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF) – these somatic cells go on to become the granulosa cells. This occurs postnatally in mice, in humans this always occurs before birth.
Numerous transcription factors identified in mice & human follicle formation e.g FIGLA, Nobox & Activin βA…
FIGLA k/o female mice sterile.
Activin βA expression ↓ just before nest breakdown.
Important signalling pathways: KIT signalling, Notch and TGFβ.
Hormones : FSH promotes and E2 /P oppose primordial follicle formation
Describe embryo menopause and some of the reasons this is caused
In utero prior to birth, there is a mass reduction in the oocyte reserve by apoptosis to form the primordial follicles which represent the entire pool of germ cells available during reproductive life of the female.
This is called the ovarian reserve
Predicted range: 35,000-2,500,000 primordial follicles.
Loss of PF prior to birth…
Aneuploidies
Errors in follicle formation – naked oocytes.
Chromosome damage – unrepaired DNA damage.
Dysfunctional spindle.
Germ cell selection – selecting good quality oocytes.
Where are primordial follicles initially located and how do the migrate as they grow towards ovulation?
These are located in the avascular ovarian cortex
Blood vessels are found in the central hilum of the ovary
As the follicles grow they will move towards the blood supply towards the vascular section of medulla.
Following selection of dominant follicule and ready to ovulate, it will move out towards periphery and outer cortex
What are the stages of follicle growth ?
1..Resting
This is when they are in meiotic arrest
A cohort of these will initiate growth
2.Pre-antral
Following initiation , the follicles will grow in slow and controlled manner to create pre antral follicles. This will last around 65 days and is gonadotrophin independent.
3.Recrutiment
At the early antral stage the follicles will no begin to require FSH so a cohort will be recruited.
-Antral
4.Selection
The selection of the dominant follicle
(ovulatory)
Describe each of the preantral follicle stages
Primordial Follicles 🡪 oocyte with layer of flattened granulosa cells.
The growth of the follicle will occour by expansion of the oocyte and proliferation of granulosa cells.
These begin to change in shape and number
At this stage the oocyte is still in meiotic arrest but becomes metabolically active and begins to make RNA /protein for future stages
Transitional 🡪 oocyte surrounded by growing granulosa cells, visible mixture of flattened and cuboidal granulosa cells.
Primary 🡪 oocyte surrounded by a single layer of cuboidal granulosa cells.
Secondary 🡪oocyte develops second layer of granulosa cells and zona pellucida forms between the oocyte and granulosa cells (thick glycoprotein coat). Surrounding precursor theca cells condense around the follicle. A basement membrane forms, separating the granulosa and theca and remaining stromal cells.
Why can we not see theca cells in the real images of the preantral follicles?
This is because when digesting the tissue , the theca cells also get digested
Why are the follicles called pre-antral?
They do not contain an antrum which is the portion of an ovarian follicle filled with follicular fluid.
What are the classifications of the pre antral follicle stages?
Class 1 Primordial follicle Transitional follicle Primary follicle Secondary follicle Multi-laminar follicle
(the last two are sometimes both referred to as secondary follicles)
How can we isolate and classify pre-antral follicles?
Elective caesarean section patients consented for ovarian cortical biopsy
( outer portion of the ovary)
This is then digested in collagenase +DNAse for 1 hour
Collagenases are enzymes that break the peptide bonds in collagen
Follicles are the digested out of the stroma and placed individually in drops of media
What are the morphological changes which take place from primordial to primary transition changes ?
- There is a change in the granulosa cells
- Massive increase in oocyte growth and activity
- controlled and very slow process
Describe the process of Zona pellucida formation
Define the zona pellucida
This is a thick extra-cellular coat separating the egg from surrounding gc.
Zona pellucida formation is a marker of follicle/oocyte growth.
Human follicles made up of four zona pellucida proteins:
ZP1, ZP2,Zp3,ZP4
They will form a thick glycoprotein coat but it is still permeable to large macromolecules
Follicle extensions continue through it.
Describe what the cross section of a preantral follicle structure would be
Outside in: Capillaries Theca layer Basal lamina Granulosa cell layer Zona pellucida Oocyte (in meiotic arrest) Nucleus
What are the communicatiosn which exist between oocytes and granulosa cells ?
There is intracellular communication between oocyte and GC via gap junctions that penetrate ZP as well as communication via connexions i.e. Cx43 between GC and Cx37 between GC & oocyte. The gap junctions are withdrawn when the follicle restarts meiosis
Where can primordial follicles be found ?
These are found along the cortical region very close to the surface.
Avascular - no blood supply
What stage are primordial follicles arrested in ?
They are arrested in the dictytate stage of meiosis
the resting phase in the oogenesis in certain mammals
What are the consequences of primordial follicles not having a blood supply?
This means the follicles are not subject to blood-borne influences such as hormones
What does the basal lamina do to the primordial follicle ?
Basal lamina around the follicle will create a microenvironment for the granulosa cell and oocyte.
Not in contact with other cells in the ovary
therefore the important factors required for initiation are thought to come from the follicle itself
