Preantral folliculogenesis Flashcards
what has to be achieved to reproduce?
· Differentiation into male or female.
· Sexual maturation from an infant to a sexually mature person.
· Production, storage of eggs and release of sufficient supply of eggs and sperm.
· Correct number of chromosomes in eggs and sperm - both must be haploids to produce a diploid when they come together.
· Eggs and sperm have to meet - need the right environment to aid transport.
· Creation of new individual with genes from both parents - genetic variability during meiosis (crossing over and random alignment).
what are primordial germ cells (PGCs)?
· Cells that will become eggs or sperm are called PGCs.
· PGCs are first identifiable in the yolk sac of the developing foetus at around 3 weeks.
· PGCs undergo many cycles of mitosis:
o They migrate to the genital ridge in the foetus.
o The genital ridge becomes the gonads, therefore, PGCs are bipotential - they can become testes and ovaries.
o Further differentiation of the PGC depends on the development of the gonads e.g. ovaries or testes.
what are oocytes?
· If PGCs migrate to the area which will become the ovaries, they become oocytes - these increase in number via mitosis. When it migrates to the ovaries and the area has received signals to produce ovaries, the PGCs become oogonia.
o Oogonia are egg-precursors, diploid and multiply by mitosis.
what happens when oogonia enter the first stage of meiosis?
no more divisions occur and they become primary oocytes. The primary oocytes remain in the first phase of meiosis until it is ovulated or dies.
give an overview on mitosis
REFER TO NOTES
- Chromosomes are replicated during S-phase of the cell cycle–> 2 chromatids attached by a centromere.
- Chromosomes align one behind the other–> spindle fibres form, attach to the chromatids and contract = pull apart the chromatids, breaking the centromere = 1 chromatid to each centriole–> forms a new cell with original number of chromosomes.
give an overview on meiosis
REFER TO NOTES
- Chromosomes replicate similar to mitosis, but instead they align up next to each other.
- During meiosis 1) chromosomes pulled apart to the centrioles.
- During meiosis 2) chromatids pulled apart.
The primary oocytes remain arrested in meiosis 1 until you have a surge in LH and ovulation occurs. Older eggs are more problematic as their chromosomes have been aligned for much longer on the spindles.
where are primary oocytes localised?
Primary oocytes are packed into the outer layer of the ovary, in the cortex
what are primary oocytes surrounded by?
· protective cells and protective layers, never on their own.
· In the foetal ovary, the surrounding cells condense around the oocyte and differentiate into the granulosa cells—> secrete an acellular layer called the basal lamina. The whole structure is known as the primordial follicle.
what is folliculogenesis?
· Folliculogenesis is the growth and development of follicles from the earliest resting stages as laid in the foetus through to ovulation.
· Most of the follicles in the ovary are not growing - after puberty, only a few grow each day out of the resting phase, as a continuum.
· As the follicles start to grow, the granulosa cells multiply and the oocytes secretes another protective acellular layer called the zona pellucida, which stays attached after ovulation. Therefore, sperm have to get through the zona pellucida to fertilise the oocyte.
· Once growth of the follicles has started, a second layer of cells then differentiate around the granulosa cells, the theca cells.
Describe follicle growth
· The factors controlling initiation of growth and the early stages are unknown - it’s not hormonal, mainly autocrine and paracrine. The follicles and surrounding follicles release factors - these can be inhibitory or stimulatory in nature.
EARLY GROWTH = FSH-independent, driven by these local factors.
LATE GROWTH = FSH-dependent.
· Therefore, in FSH-deficient patients (pregnant or on contraceptives) or those with mutations of FSH receptors will still get continual early growth of the follicles, but then they die - this is why contraceptives don’t preserve your fertility, you still get continual early growth.
· As follicles start to grow, they rapidly increase in diameter and granulosa cell divisions increase BUT gaps begin to form in these granulosa cell layers —>known as antrums - these fill with follicular fluid.
what is one way we can we characterise follicle growth?
We can characterise follicle growth: 1) absence of antrum 2) presence of antrum.
Follicles with an antrum are known as antral/secondary follicles.
Describe antral development
Stage 1) antrums form and get filled with follicular fluid.
Stage 2) fluid spaces fuse together and expand.
Stage 3) fluid separates the granulosa cells, into the mural and cumulus GCs.
Stage 4) can grow up to 20mm.
As the follicle grows, the oocyte is displaced to one side.
how can follicles be visualised?
Follicles can be visualised using ultrasound, but it’s only the follicles which are larger than 1-2mm which can be visualised. People think contraceptives preserves fertility but actually what happens is, the follicles are arrested in the early growth phase which is FSH-independent, therefore they’re too small to be picked up by ultrasound but they’re still there.
describe follicle initiation and follicle recruitment
· A cohort of early follicles leaving the resting pool and grow continuously–> follicle initiation.
· They will NOT continue to grow unless they reach the size at which they respond to changes in FSH that occur in the menstrual cycle–> follicle recruitment.
· The human pelvis is only designed to carry a single foetus, therefore, from the group that is recruited, only one will be selected for ovulation - this is the dominant follicle.
what do we mean when we say “many follicles to one”?
· Majority of the eggs will die during reproductive life, even before puberty, but majority will die during puberty.
· There are thousands of primordial follicles most die through atresia, and very few make it into the menstrual cycle.
· But only one follicle is ovulated (dominant follicle) which has more FSHr so it grows even during low levels of FSH and the FSHr has a more efficient downstream IC cascade.
describe the antral follicle
· The follicle is avascular until when the theca is formed–> forms vasculature which allows blood borne influences to act on the follicles e.g. hormones, cortisol, insulin, etc. This suggests a lot of stuff comes into play during the later stages of antrum growth, in the presence of theca cells.
follicles produce steroids. Describe where the receptors are and which cells produce which steroid
· Theca cells have LHr–> produces progesterone and androgens which goes into circulation or into granulosa cells.
· Granulosa cells have FSHr–>stimulates aromatase which converts the androgens into oestrogen (allows granulosa cells to continue proliferating).
Known as the 2-cell, 2-gonadotrophin theory.
· The dominant follicle develops LHr on the granulosa cells so it starts to produce progesterone.
· Oestrogen and progesterone feedbacks to the hypothalamus and anterior pituitary–> decreases levels of FSH and LH.
give an overview on preantral follicle stages
A single layer of flattened granulosa cell surrounds the oocyte. Growth is determined by changing shape of granulosa cell and increased proliferation of granulosa and oocyte.
Flattened granulosa form cuboidal (transitional) so a single layer of cuboidal granulosa cells around the oocyte, at this point it is considered primary.
Multiplication of layers, ZP forms around oocyte and other cells in the ovarian cortex, which condenses around the follicle and form the theca (secondary), a defined basement membrane separating it from granulosa. It is metabolically active even though it is in meiotic arrest, stops growing at the end of the preantral stage.
how can we investigate folliculogenesis?
- Animal models - mono-ovulatory (e.g. sheep and monkeys) and poly-ovulatory (e.g. rabbits and mice, however produce litter which is not an accurate representation of reproduction).
- Introduce defined mutations into embryonic stem cell by injecting into blastocysts, breed them to be homozygous for the mutations and then examine the phenotype.
- Genotype/phenotype associations in naturally occurring mutations or from knock-out mice.
- Culture of whole ovaries/slices/biopsies/large follicles/small follicles/cells.
- Very difficult in human because of limited supply of tissue
- Primary cells difficult to obtain; granulosa cell line but no suitable theca cell line (inherent or induced mutations to keep growing them)
describe the formation of oocytes
- As the PGCs divide, they form nests (cytoplasmic bridges between dividing oocytes). The purpose of this is to exchange organelles between the oocytes e.g. mitochondria and ER.
- Eventually, you get breakdown of the nests to allow the individual oogonia to form primordial follicles.
- DAZL + Retinoic acid are thought to be involved in nest breakdown.
- Retinoic acid is thought to regulate entry into meiosis (from mitosis).
- DAZL expression increases before meiosis at 9 - 14 weeks of gestation. DAZL K/O mice germ cells don’t develop past the PGC, they don’t go onto form primordial follicles. In humans, DAZL mutations associated with sub-fertility.
Therefore, this highlights the importance of both of these genes for entry into meiosis.
what is the difference between humans and mices (regarding the formation of cytoplasmic bridges)?
In humans: PGCs divide along the ridge and colonise the ridge. Cytoplasmic bridges form between the dividing oocytes to form nests of germ cells. They then enter into meiosis + nests are broken down = primordial follicles are formed. Therefore, we are born with all the primordial follicles.
In mice: This process doesn’t occur until after birth - made it problematic to research this area.
Describe the formation of primordial follicles
The germ cell nests are broken down and the surrounding somatic cells (pre-granulosa cells) invade to surround oogonia to form primordial follicles. This process is highly regulated through transcription factors e.g. FIGLA, Nobox and Activin βA.
o FIGLA is important for primordial follicle formation as female FIGLA K/O mice develop no primordial follicles
o Activin βA expression decreases just before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if K/O → loss of oocytes = “streak” ovaries.
· Co-ordination of signalling pathways: KIT, Notch and TGFβ, and hormones is also required (FSH promotes, while oestrogen and progesterone oppose formation of primordial follicles).
NOTE: As these primordial follicles have entered meiosis, they are now under arrest.
what is the follicle reserve?
· Once all the primordial follicles are formed, the entire pool of germ cells available during reproductive life of the female = ovarian reserve (varies from 35,000 - 2.5million follicles).
Just before birth, there is a massive loss of follicles. Why does this occur?
- Failure of mitosis/meiosis (forming defective chromosome spindles).
- Unrepaired DNA damage.
- Insufficient pre-granulosa cells (naked oocytes).
- Degeneration of oocytes during nest breakdown + follicle formation.
= “Germ cell selection”, allows us to select oocytes of the highest quality.
