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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
Q

What is the estimated range of follicles a woman has?

A

Predicted range: 35,000-2,500,000 primordial follicles

mathematical modelling & histological counting

26
Q

When does follicle growth resume?

A

Once puberty established, follicle growth occurs continuously until all follicles are depleted ⇒ menopause

27
Q

Why may women lose follicles before birth?

A

Loss of follicles before birth is thought to be related to germ cell selection

28
Q

What is the purpose of germ cell selection?

A

to select highest quality oocytes for ovarian reserve

29
Q

Where are primordial follicles located?

A

Primordial follicle located in avascular ovarian cortex

30
Q

Where is the ovaries blood supply found?

A

Blood vessels located in the central hilum of the ovary

31
Q

What is the significance of the ovarian blood supply in PF growth?

A

s follicles grow they migrate towards blood supply and vascular central medulla

32
Q

Describe the movement of follicles once DF is selected

A

Once DF selected, moves out to periphery in the outer cortex ready for ovulation

33
Q

Outline the stages of follicle growth

A

Gonadotrophin independent:

  • resting
  • Initiation : pre-antral

Gonadotrophin dependent:

  • Recruitment : antral
  • Selection: ovulatory
34
Q

What are the resting follicles?

A

Resting follicles are in meiotic arrest

35
Q

How is follicle growth initiated?

A

Cohort of follicles initiate growth everyday once puberty is established - cause unknown

36
Q

What does the initiation of follicle growth cause?

A

Growth initiation leads to controlled slow maturation into preantral follicles

37
Q

How long does follicle maturation into pre-antral follicles take?

A

~lasts 65 days

Gonadotrophin independent

38
Q

Why is antral stage gonadotropin dependent?

A

Early antral stage requires LH/FSH for antrum formation

39
Q

How is DF selected?

A

Some follicles recruited into menstrual cycle and grow - from this cohort DF is selected

40
Q

Describe the structure of the preantral follicle

A

Oocyte surrounded by single layer of flattened granulosa cells
no antrum

41
Q

Describe the ways a follicle can grow

A
  • expansion of oocyte

- proliferation of granulosa cells

42
Q

Describe the structure of a primary follicle (translational)

A

Granulosa cells undergo structural changes from flattened → cuboidal

43
Q

How does oocyte grow despite still in meiotic arrest?

A

Oocyte also grows due to metabolic activity

44
Q

How does a primary follicle become a secondary follicle?

A

Secondary follicle once another layer of granulosa cells have been acquired

45
Q

How does the theca layer form?

A

Theca formation begins from precursor cells condensing around the follicle
Zona pellucida also forms

46
Q

Outline the technique to isolate pre-antral follicles

A
  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
Q

What are the alternative follicle classifications used?

A

When no antrum present = preantral
Antral formation = antral follicles / tertiary follicles

When follicles are ready to be ovulated they are called preovulatory

48
Q

What morphological changes occur when follicles transition form primary to secondary?

A

1) Change in granulosa cells (~15 cuboidal GC)
2) Massive increase in oocyte growth & activity
3) Controlled & very slow process

49
Q

Where do follicles move in the ovary as they grow?

A

As they grow they move away from collagen-rich ovarian cortex towards perimedullary zone of the ovary, where ECM is of lower density.

50
Q

What is the zona pellucida?

A

ZP is a thick extra-cellular coat separating the egg from surrounding gc

51
Q

What does Zona pellucida formation tell us?

A

ZP formation is a marker of follicle/oocyte growth

52
Q

What 4 proteins make up human zona pellucida?

A

Human follicles made up of four ZP proteins:

ZP1, ZP2, ZP3, ZP4

53
Q

Describe the structure of the zona pellucida

A

Permeable to large macromolecules (gap junctions)

Follicle extensions continue through it

54
Q

How does ZP allow connections to oocyte still

A

ZP doesn’t completely separate the egg from the surrounding GC
It has projections that make connections between GC and Oocyte

55
Q

Describe the preantral follicle structure

A

Oocyte still in meiotic arrest

Thick glycoprotein zona pellucida coating

Several layers of granulosa cells
Basal lamina separating granulosa cells from theca

56
Q

When does theca formation occur?

A

Theca formation initiated at secondary follicle stage

Theca well vascularised unlike granulosa cells

57
Q

Describe structure of theca

A

Theca well vascularised unlike granulosa cells

58
Q

How does intracellular communication occur in oocyte?

A

Via connexin proteins ie.

  • Cx43 between GC
  • Cx37 between GC and oocyte

Via gap junctions that penetrate ZP

59
Q

Where are primordial follicles located?

A

Primordial follicles located in the cortical region very close to the surface and is avascular

60
Q

Why are PF located in the ovarian cortex away from blood supply?

A

not exposed to bloodborne influences

61
Q

What is the role of the basal lamina around the follicle?

A

Basal lamina around the follicle creates microenvironment for gc & oocyte i.e not in contact with other cells in the ovary

62
Q

What are the 3 possible fates of the PF?

A
  • 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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