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Science of Reproduction > Preantral Folliculogenesis > Flashcards

Preantral Folliculogenesis Flashcards

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1
Q

What methods are used to investigate folliculogenesis?

A
  • Animal models
  • K/O / natural mutations
  • Cultures, biopsies, slices etc.
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2
Q

What are the drawbacks of using animal models to investigate folliculogenesis?

A

may not accurately reflect human fertility

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3
Q

Why is culturing whole ovaries / taking biopsies / slices not always feasible?

A

Very difficult in human due to limited tissue supply

Primary cells difficult to obtain; granulosa cell line but no suitable theca cell line

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4
Q

Outline how to form gene k/o mice

A
  1. Inject defined mutations into blastocysts (embryonic stem cell)
  2. Create chimeric mice
  3. Breed them as homozygous for mutations
  4. Examine phenotype
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5
Q

When do follicles first develop?

A

3-4 week human embryo

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6
Q

How does follicle development begin?

A

Epiblast cell in yolk sac at base of allantois differentiate into PGC (primordial germ cells)

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7
Q

Explain how a 5-6 wk embryo PGCs develop

A

PGCs divide mitotically

Migrate along dorsal mesentery of hind gut to colonise genital ridge

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8
Q

How are PGCs drawn towards the genital ridge?

A

chemotactic substance secreted by ridge to attract PGCs

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9
Q

What is the proposed mechanism of action of PGC migration to the genital ridge?

A

maybe Kit ligand (KL) as the receptor cKit is present on surface of PGCs

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10
Q

When does primordial follicle formation occur in mice?

A

Primordial follicle formation in mice occurs AFTER birth

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11
Q

When does primordial follicle formation occur in humans?

A

In Humans primordial follicle formation occurs BEFORE birth`

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12
Q

Outline the 3 steps of primordial follicle formation

A
  1. germ cell cyst formation
  2. meiotic arrest
  3. cyst breakdown - primordial follicle formation
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13
Q

Describe how the germ cell cyst is formed

A

cytoplasmic bridges between mitotically-dividing oocytes form syncitia or “nests”

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14
Q

What is the role of the germ cell cyst?

A

Maybe exchange organelles eg. ER and mitochondria

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15
Q

What regulates the entry of germ cells into meiosis?

A

Retinoic Acid (biologically active variant of Vit.A) has been identified as key extrinsic regulator of germ cell entry into meiosis

DAZL gene also seen to have effect on meiosis entry

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16
Q

What is the evidence of DAZL genes regulatory effects on meiosis?

A

DAZL expression ↑before meiosis at 9-14 weeks gestation.

DAZL k/o mice germ cells don’t develop past PGC
DAZL mutations in human assoc. subfertility

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17
Q

How does the germ cell cyst breakdown?

A

Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF)

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18
Q

What transcription factors are thought to regulate primordial follicle formation?

A

Numerous transcription factors identified in mice & humans

e. g.
- FIGLA
- Nobox
- Activin βA (TRKβ receptor)

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19
Q

What is the evidence of FIGLA TF activity in PF formation?

A

FIGLA k/o female mice sterile with no Priomordial Follicles

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20
Q

What evidence supports activin βA role in PF formation?

A

Activin βA expression ↓ just before nest breakdown

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21
Q

What evidence shows TRKβ receptor activity in PF formation?

A

if k/o → loss of oocytes → “streak” ovaries

contrast with male as can have testes with no sperm

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22
Q

What signalling pathways regulate PF formation?

A

Coordination of KIT, Notch and TGFβ signalling pathways

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23
Q

Which hormones play a role in PF formation?

A

FSH promotes PGC formation and E2 and P oppose

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24
Q

What is the ovarian reserve?

A

Formed primordial follicles represent entire pool of germ cells available during females reproductive life

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25
What is the estimated range of follicles a woman has?
Predicted range: 35,000-2,500,000 primordial follicles | mathematical modelling & histological counting
26
When does follicle growth resume?
Once puberty established, follicle growth occurs continuously until all follicles are depleted ⇒ menopause
27
Why may women lose follicles before birth?
Loss of follicles before birth is thought to be related to germ cell selection
28
What is the purpose of germ cell selection?
to select highest quality oocytes for ovarian reserve
29
Where are primordial follicles located?
Primordial follicle located in avascular ovarian cortex
30
Where is the ovaries blood supply found?
Blood vessels located in the central hilum of the ovary
31
What is the significance of the ovarian blood supply in PF growth?
s follicles grow they migrate towards blood supply and vascular central medulla
32
Describe the movement of follicles once DF is selected
Once DF selected, moves out to periphery in the outer cortex ready for ovulation
33
Outline the stages of follicle growth
Gonadotrophin independent: - resting - Initiation : pre-antral Gonadotrophin dependent: - Recruitment : antral - Selection: ovulatory
34
What are the resting follicles?
Resting follicles are in meiotic arrest
35
How is follicle growth initiated?
Cohort of follicles initiate growth everyday once puberty is established - cause unknown
36
What does the initiation of follicle growth cause?
Growth initiation leads to controlled slow maturation into preantral follicles
37
How long does follicle maturation into pre-antral follicles take?
~lasts 65 days | Gonadotrophin independent
38
Why is antral stage gonadotropin dependent?
Early antral stage requires LH/FSH for antrum formation
39
How is DF selected?
Some follicles recruited into menstrual cycle and grow - from this cohort DF is selected
40
Describe the structure of the preantral follicle
Oocyte surrounded by single layer of flattened granulosa cells no antrum
41
Describe the ways a follicle can grow
- expansion of oocyte | - proliferation of granulosa cells
42
Describe the structure of a primary follicle (translational)
Granulosa cells undergo structural changes from flattened → cuboidal
43
How does oocyte grow despite still in meiotic arrest?
Oocyte also grows due to metabolic activity
44
How does a primary follicle become a secondary follicle?
Secondary follicle once another layer of granulosa cells have been acquired
45
How does the theca layer form?
Theca formation begins from precursor cells condensing around the follicle Zona pellucida also forms
46
Outline the technique to isolate pre-antral follicles
1. Elective caesarean section, patients consented for ovarian cortical biopsy 2. Biopsy taken to lab and dissected into many smaller pieces 3. Pieces digested in collagenase +DNAse for <1hr - to digest thick and tough ovarian stroma 4. Tease apart digested tissue with v fine acupuncture needles 5. Isolate follicles and transfer to drops of media in a dish 6. Vies under high magnification microscopes
47
What are the alternative follicle classifications used?
When no antrum present = preantral Antral formation = antral follicles / tertiary follicles When follicles are ready to be ovulated they are called preovulatory
48
What morphological changes occur when follicles transition form primary to secondary?
1) Change in granulosa cells (~15 cuboidal GC) 2) Massive increase in oocyte growth & activity 3) Controlled & very slow process
49
Where do follicles move in the ovary as they grow?
As they grow they move away from collagen-rich ovarian cortex towards perimedullary zone of the ovary, where ECM is of lower density.
50
What is the zona pellucida?
ZP is a thick extra-cellular coat separating the egg from surrounding gc
51
What does Zona pellucida formation tell us?
ZP formation is a marker of follicle/oocyte growth
52
What 4 proteins make up human zona pellucida?
Human follicles made up of four ZP proteins: ZP1, ZP2, ZP3, ZP4
53
Describe the structure of the zona pellucida
Permeable to large macromolecules (gap junctions) Follicle extensions continue through it
54
How does ZP allow connections to oocyte still
ZP doesn't completely separate the egg from the surrounding GC It has projections that make connections between GC and Oocyte
55
Describe the preantral follicle structure
Oocyte still in meiotic arrest Thick glycoprotein zona pellucida coating Several layers of granulosa cells Basal lamina separating granulosa cells from theca
56
When does theca formation occur?
Theca formation initiated at secondary follicle stage | Theca well vascularised unlike granulosa cells
57
Describe structure of theca
Theca well vascularised unlike granulosa cells
58
How does intracellular communication occur in oocyte?
Via connexin proteins ie. - Cx43 between GC - Cx37 between GC and oocyte Via gap junctions that penetrate ZP
59
Where are primordial follicles located?
Primordial follicles located in the cortical region very close to the surface and is avascular
60
Why are PF located in the ovarian cortex away from blood supply?
not exposed to bloodborne influences
61
What is the role of the basal lamina around the follicle?
Basal lamina around the follicle creates microenvironment for gc & oocyte i.e not in contact with other cells in the ovary
62
What are the 3 possible fates of the PF?
- Remain quiescent, die out directly at dormant stage - Begin development but arrest and later undergo atresia - Develop, mature & ovulate

Science of Reproduction (32 decks)

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