Preantral Folliculogenesis Flashcards
Folliculogenesis- brief overview
Initiation of growth of primordial follicles
Will progress through the stages
Recruitment of antral follicles into the menstrual cycle
Selection of the dominant follicle from the cohort of smaller antral follicles
Maturation and ovulation of the dominant follicle
Conversion into the CL / degeneration
Investigating folliculogenesis (techniques)
Animal models – mono-ovulatory/poly-ovulatory
Need to be careful as many animals are poly-ovulatory (produce litters) so the models may not reflect humans
Genotype/phenotype associations in naturally occurring mutations or from knock-out mice
You can have natural occurring mutations of genes in humans, you can then follow the phenotypic association of the mutation.
Or can create KO mice
Culture tissue of whole ovaries/slices/biopsies/large follicles/small follicles/cells
Very difficult in human because of limited supply of tissue
Primary cells are difficult to obtain; granulosa cell line but no suitable theca cell line
Where do eggs come from?
Primordial germ cells are seen very early on: about 3-4 weeks in the human embryo.
Epiblast cells in yolk sac at base of allantois differentiate into PGC (primordial germ cells)
Then at 5-6 old human embryo
The PCGs will divide Mitotically to increase in number.
The PGCs then migrate along dorsal mesentery of hind gut to colonise genital ridge (which will become the gonads) → it is believed that a chemotactic substance secreted by ridge to attract PGCs towards it.
It maybe Kit ligand (KL) as the receptor cKit is present on surface of PGCs
Formation of oocytes and follicles
As the mitotically dividing primordial germ cells colonise the genital ridge. If the embryo is destined to become female they will differentiate to form oocytes, and they will start to build nests.
They have cytoplasmic bridges around them which connects them together to form syncitia (or nests).
The role of the nests is thought to be to exchange organelles (like ER or mitochondria
Role of retinoic acid in germ cell entry into meiosis
Retinoic Acid (the biologically active variant of Vitamin A) has been identified as key extrinsic regulator of germ cell entry into meiosis
So the cells have been dividing by mitosis to increase in number, they then stop and enter into meiosis. (which is regulated by retinoic acid)
DAZL expression
Using KO mice it has been shown that there is another gene crucial in the process called DAZL.
DAZL expression ↑before meiosis at 9-14 weeks gestation. DAZL k/o mice germ cells do not develop past primordial germ cell stage, DAZL mutations in human assoc. sub-fertility. So see genotype/phenotype associations in humans and then confirm it using KO mice.
Cyst breakdown and primordial follicle formation
The syncitia nests breakdown and the surrounding cells will infiltrate around the oogonia to start forming primordial follicles.
In humans primordial follicle formation occurs before birth. However in MICE this occurs after birth.
Formation of primordial follicle
The primordial germ cells that have migrated and are mitotically dividing to increase in number, colonise the genital ridge which will then become an ovary. The germ cells will become oogonia, then the cytoplasmic bridges between the nests breakdown and the surrounding somatic cells will invade to surround the oogonia to form the primordial follicle.
Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF)
PF formation regulated through the following:
Numerous transcription factors identified in mice & human eg FIGLA, Nobox & Activin βA
FIGLA gene k/o female mice are sterile with no PF.
Activin βA expression ↓ just before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if k/o → loss of oocytes → “streak” ovaries, contrasts with male as can have testes with no sperm
You also need Co-ordination of several signalling pathways: KIT, Notch and TGFβ
Hormones : FSH promotes formation of primordial follicles and E2 and P oppose it
Embryo-Menopause
There is germ cell migration, colonisation of the ovary and formation of the follicles occurring.
There are large numbers going up to 7m.
Just before birth there is mass apoptosis of the oocytes and follicles
- At birth a woman is born with her entire stock of primordial follicles that she will have for the rest of her reproductive life.
- This loss is due to:
Failure of mitosis/meiosis involving defective chromosome spindle function
Unrepaired DNA damage during egg/follicle formation
Insufficient pre-granulosa cells resulting in naked oocytes which degenerate
Degeneration of oocytes during nest breakdown and follicle formation.
Once puberty is established, you get growth of
follicles occurring continuously until all the follicles are depleted and the woman enters into menopause.
Once formed primordial follicles represent the entire pool of germ cells available during reproductive life of the female – known as “ovarian reserve”. Predicted range: 35,000-2,500,000 primordial follicles (mathematical modelling & histological counting)
Loss of primordial follicles is related to…
The loss it thought to be related to the “Germ Cell Selection theory” – to select oocytes of highest quality
Allows the ovary to select the oocytes of the highest quality to establish the true ovarian reserve
Briefly describe the anatomy of the ovary
All of the resting primordial follicles which are in meiotic arrest are located in the avascular ovarian cortex.
Blood vessels are in the central hilum of the ovary
As follicles grow, they will move inwards towards the blood supply and towards the vascular central medulla
Once there is selection of the dominant follicle and it is ready to ovulate, it then moves out again toward the outer cortex and surface ready for ovulation.
Stages of follicle growth
Have resting follicles that are in meiotic arrest.
A cohort of these follicles will initiate growth every day once puberty is established. What causes this initiation is still unknown.
Once the follicles have initiated growth, they grow in a very slow and controlled manor to form preantral follicles.
This process lasts over 65 days
This process is gonadotrophin independent
Once the follicles have reached the early antral stage, and have stated to form an antrum, they need FSH to continue growth.
So a cohort of these follicles are recruited into the menstrual cycle.
These will grown, then from this cohort, the dominant follicle is selected.
This is gonadotrophin dependant
The follicle that is ovulated would have initiated its growth nearly 3 cycles beforehand.
Preantral Follicle (Stage 1)
In the primordial follicle there is the oocyte, surrounded by a single layer of flattened granulosa cells.
Growth of the follicle will occur by expansion of the oocyte and proliferation of the granulosa cells.
Once the follicle initiates growth, the granulosa cells start to change in appearance and number. This is called a transitional cell – initiation of growth and formation of 1 or 2 cuboidal granulosa cells.
They go from being flattened to more cuboidal.
There is also growth of the oocyte.
As it continues to grow you will get a single layer of cuboidal granulosa cells, it is now known as a primary follicle.
It then acquires a second layer of granulosa cells.
You start to get theca formation too, which comes from precursor cells which are condensing around the follicle.
You will also see formation of the zona pellucida.
SO… When you have a full secondary follicle (with two layers of granulosa cells) you then have theca formation, you have a definitive basement membrane which separates the theca from the granulosa cells.
Preantral Follicle (Stage 2)
Primordial follicle
Can see the oocyte with a single later of flattened granulosa cell
Transitional follicle
Can still see flattened granulosa cells, but some are starting to become cuboidal
Primary follicle
More expanded oocyte. Have a single later of expanded granulosa cells which are all cuboidal.
Secondary follicle
Has two layers of granulosa cells
Can see the zona pellucida and the basement membrane.
Multilaminar follicle
Has several layers of granulosa cells forming
Different classification systems – need to be aware when reading papers. Some call all follicles Primary follicles if they have a primary oocyte i.e. whilst still in mieotic arrest and once completed mieosis 1 and ejected 1st polar body, known as Secondary follicles because they have a secondary oocyte. (some call them secondary follicles when you get more than 1 layer of granulosa cells forming)
Technique to isolate pre-antral follicles
Elective Caesarean section patients consented for ovarian cortical biopsy
The piece is dissected out into many smaller pieces in the lab
It is then Digested in Collagenase +DNAse for under 1hr
As the ovarian stroma is very thick
The follicles are then dissected out of the stroma and placed individually in drops of media in a dish
They are then looked at under high power magnification.
Alternative Follicle Classification
Some papers will class them all as pre-antral or class 1 follicles
At antral formation they are called antral follicles or tertiary follicles
When they are ready to be ovulated they are fall graafian or preovulatory follicle
Primordial -> Primary Transition Morphological changes
There are distinct morphological changes as follicles initiate grown from primordial follicles to becoming primary follicles:
1) Change in granulosa cells (~15 cuboidal granulosa cells)
2) Massive increase in oocyte growth & activity
3) Controlled & very slow process
Also as they start to grow they move away from the collagen-rich ovarian cortex towards the perimedullar zone of ovary, where the ECM is of lower density.
Zona Pellucida formation
As the follicle and oocytes start to grow you get formation of the Zone Pellucida:
ZP is a thick extra-cellular coat separating the egg from surrounding granulosa cells
ZP formation is a marker of follicle/oocyte growth
Human follicles made up of four ZP proteins:
ZP1, ZP2, ZP3, ZP4
Even though they form a thick glycoprotein coat, they are permeable to large macromolecules. (gap junctions)
Follicle extensions continue through it
Preantral Follicle Structure
What you would expect to see in a multilaminar or pre antral follicle:
Oocyte with nucleus still in meiotic arrest
Thick glycoprotein ZP
Several layers of granulosa cells
Basal laminar – which separates the granulosa cells from the theca
The theca has started to form around the secondary follicle stage, secondary follicle = 2 layers of granulosa cell.
The theca is very well vascularised unlike the granulosa
Even though there is a thick ZP coat, there is a lot of Intracellular communication between oocyte and granulosa cells via gap junctions that penetrate the zona pellucida.
But there is also communication between granulosa cells. This communication is formed by connexin proteins:
Also communication via connexins i.e. Cx43 between GC and Cx37 between GC & oocyte
Location of the Primordial Follicle
The primordial follicles are found along the cortical region, close to the surface which is quire avascular. They group together in this region.
Primordial follicles are arrested in dictyate stage of meiosis
Primordial follicles located in ovarian cortex & have no blood supply. Consequences?
This means thee follicles are not subject to blood borne influences
In addition the basal lamina around the follicle creates microenvironment for granulosa cells & oocyte i.e not in contact with other cells in the ovary
This shows that any factors that are involved in initiation of growth of the follicles must be coming from within the ovary itself.
Fate of the Primordial Follicle
Primordial Follicle has 3 possible fates:
To remain quiescent and die out directly at dormant stage
To begin development but arrest and later undergo atresia
To develop, mature & ovulate
What causes initiation of follicle growth?
Initiation is regulated by loss of an inhibitor
Resting follicles under constant inhibitory influence (local paracrine(adjacent)/autocrine factors(within follicle) to remain dormant. Where from?
Initiation is regulated by stimulatory factor/s
Need something to stimulate the follicles to grow
Can come From the microenvironment (other follicles, stromal cells) and/or blood
Could be Gradient of diffusion from centre to periphery
Changes to what may initiate follicle growth?
However we now think that it is a combination of both inhibition and stimulation:
So as you get a decrease in the inhibitor, it will allow the stimulating factors to dominate and activate the follicles.
Is also a “Production-line” hypothesis: those that enter meiotic arrest first in foetal ovary, will initiate growth first
Initiation of follicle growth is Also dependent on size of primordial follicle pool and ratio at which enters the growing pool.
As women come into the perimenopausal period, as the PF pool has decreased, you get more follicles activating and entering the growing pool at a Faster rate.
