Preantral Folliculogenesis

Question 1

What techniques can be used to investigate folliculogenesis?

Why is it important to understand the techniques used to investigate folliculogenesis?

Answer 1

1) Animal models = mono-ovulatory/poly-ovulatory. A common way to study reproduction is by using animal models. Although animal models are very useful (and crucial for disease processes), we have to be careful, particularly in regards to female reproduction, when considering animal models because many animals are poly-ovulatory (produce litters). These models may not very accurately reflect what is going on in a human. There are mono-ovulatory species which will predominantly produce one or twin offspring, like sheep or cow, but those are big species that are not very easy to use in a laboratory. Monkeys are ideal but there are a lot of ethical issues with using and maintaining them. There are pros and cons to everything.
2) Genotype/phenotype associations in naturally occurring mutations or from knock-out mice. Can look at naturally occurring mutations of particular genes in humans and then follow the phenotypic outcome or association of that mutation. Can also create KO mice. This has been of great utility in reproductive biology. There are still limitations with using mice. In terms of especially early follicle growth, knockout mice have proved very useful.
3) Culture of whole ovaries/slices/biopsies/large follicles/small follicles/follicular cells, e.g. theca = Very difficult in human because of limited supply of tissue, Primary cells difficult to obtain; There is a granulosa cell line, but to date, no one has been able to create a suitable theca cell line. There was one, but then it seemed to stop propagating and working. Culturing tissues is a very useful technique. Cryopreservation has given access to a lot more human tissue than before. In the past, women would also often have an oophorectomy (their ovaries removed) because of various gynaecological issues, but we don’t want to induce early menopause so moving away from this.

• These are the challenges and issues we face to investigate folliculogenesis in particular, but especially in female reproduction.
• We’re going to have to read and critically evaluate research papers and studies related to reproduction throughout the course of this module. Understanding these various techniques is quite important.

Question 2

How are gene k/o mice made?

Answer 2

• Introduce defined mutations into the embryonic stem cell by injecting into the blastocysts, create chimeric mice, breed them to be homozygous for the mutations and then examine the phenotype.

Question 3

Where do eggs come from?

Answer 3

• In humans, primordial germ cells are recognised and identified very early on (about three to four weeks) in a human embryo. These epiblast cells in the yolk sac at the base of the allantois will differentiate into primordial germ cells.
• They will continue dividing mitotically to increase in numbers. In a 5 to 6 week human embryo, the mitotically dividing PGCs will migrate along the dorsal mesentery of the hindgut to colonise the genital ridge, which will become the presumptive gonads depending on the sex differentiation.
• The movement is kind of amoeboid in nature and it is thought that there is a chemotactic substance secreted by the ridge to attract the primordial germ cells towards it. Current thinking is that this substance may be Kit ligand. It may be Kit ligand (KL) as the receptor cKit is present on surface of PGCs.

Question 4

How are primordial follicles formed?

Answer 4

• Those primordial germ cells that have migrated and divided mitotically increase in numbers. They colonise the genital ridge. In the case of a female embryo, this becomes an ovary. Those germ cells become oogonia.
• The syncytia (cytoplasmic bridges) between nests break down and surrounding somatic cells invade to surround the oogonia and form the primordial follicle.
• KO mice studies show that primordial follicle formation is regulated by a few genes listed.
• PF formation regulated through the following:
  1) Numerous transcription factors identified in mice & human eg FIGLA, Nobox & Activin βA
  2) FIGLA k/o female mice sterile with no PF; FIGLA is obviously responsible in the process of forming primordial follicles.
  3) Activin beta A expression is downregulated before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if k/o → loss of oocytes → “streak” ovaries, contrast with male as can have testes with no sperm. There is a big difference between the female and male knockouts of this receptor. In males, they can have testes with no sperm. In females, if there are no eggs or follicles, the ovaries do not fully develop (just form streak structures).
  4) Co-ordination of signalling pathways: KIT, Notch and TGFβ; it is thought that several signalling pathways are required to coordinate and are important for the formation of the primordial follicles.
  5) Hormones – FSH promotes, while E2 and P oppose; FSH is also thought to promote formation of primordial follicles, while estradiol and progesterone are thought to oppose it.
• Some of these studies are coming from larger animals, like monkeys and baboons, so some may be a bit more relevant to humans while others may not.

Question 5

What is the predicted range of primordial follicles within the ovarian reserve?

Answer 5

• There is germ cell migration, colonisation of the ovary and formation of the follicles occurring (huge numbers; estimated to go up to 7 million). Just before birth, there is massive loss (apoptosis) of the oocytes and follicles so that at birth, a woman is born with her entire stock of primordial follicles that she will have for the rest of her reproductive life. Once puberty is established, there is growth of follicles occurring as a continuum until all the follicles are depleted and the women is then stated as entering menopause. Once formed, those primordial follicle represent the entire pool of germ cells available to a woman during her reproductive life. This is known as true ovarian reserve (ovarian capability that you were born with).
• Using mathematical modelling and histological counting, e.g. from post-mortem, miscarriages, foetal tissue etc., the range of follicles is estimated to be between 35,000 to 2.5 million. There is a huge range; probably reflects the various methods of estimation and is not necessarily an accurate reflection of what is happening. However, it is known that it varies from woman to woman, and therefore it can be quite tricky to give an estimate of somebody’s ovarian reserve (don’t know what they’re actually born with).

Question 6

What is the germ cell selection theory?

Answer 6

• “Germ Cell Selection” = to select oocytes of highest quality
• The reason that there is loss of follicles and oocytes before birth is thought to be related to germ cell selection theory. The ovaries are able to select the oocytes of highest quality to establish true ovarian reserve.

Question 7

What are four possible reasons for the massive reduction in follicle numbers prior to birth in the human ovary?

Answer 7

1) Failure of mitosis/meiosis involving defective chromosome spindle function
2) Unrepaired DNA damage during egg/follicle formation
3) Insufficient pre-granulosa cells resulting in naked oocytes which degenerate
4) Degeneration of oocytes during nest breakdown and follicle formation.

Question 8

Why would a woman have low ovarian reserve?

Answer 8

• In utero:
  1) Insufficient mitosis to make the initial pool large?
  2) Failure of mitosis/meiosis involving defective chromosome spindle function
  3) Unrepaired DNA damage
  4) Naked oocytes/insufficient pre-granulosa cells
  5) Degeneration of oocytes during nest breakdown and follicle formation
  6) Mutations in genes involved in follicle formation
• Postnatal
  1) Chromosomal defects (laid down in utero)
  2) Altered hormonal signalling
  3) Autoimmune diseases
  4) Environmental genotoxins

Question 9

How is ovarian reserve assessed in clinical practice?

Answer 9

• Use ultrasound to count number of 2-8mm follicles at start of cycle (early follicular phase)- AFC and correlate with serum markers FSH, AMH, E2 and Inhibin B used to determine “functional ovarian reserve”, but does not indicate true PF reserve, i.e. what you are born with; low numbers of antral follicles are a sign of ovarian ageing, observable earlier than a rise in FSH serum level. Can count antral follicles using ultrasound since they have fluid-filled spaces.
• True ovarian reserve is the number of primordial follicles but no way of knowing this; this is the clinical solution.

Question 10

How does the anatomy of the ovary support the primordial follicles as they develop?

Answer 10

• All of the resting primordial follicles, which are in meiotic arrest, are located in this avascular ovarian cortex. The blood vessels are in the central hilum of the ovary, as shown in this H&E stained section of the ovary. This then has implications for thinking about follicle growth. As follicles grow, they will move in towards the blood supply and towards the vascular central medulla. Can see some of the large antral follicles moving closer in (zoomed out image). Once there is selection of the dominant follicle and it is ready to ovulate, it starts to move out again towards the periphery (outer cortex). Moves to the surface; ready for ovulation.

Question 11

What are the stages of follicle growth?

Answer 11

• There are resting follicles which are in meiotic arrest. A cohort of these follicle initiate growth every day once puberty is established. What causes this initiation is unknown but it is something that will be tackled in subsequent slides.
• Once the follicles have initiated growth, they grow in a very slow and controlled manner to form pre antral follicles (lasts over 65 days) and do not need gonadotrophins for this growth (will discuss what controls this growth since it is not LH and FSH).
• Once they reach the early antral stage and have started to form an antrum, they need FSH to continue growing. A cohort of these follicles are then recruited into the menstrual cycle. They will grow up and, from that cohort, the dominant follicle is selected. This stage is gonadotrophin-dependent, both for recruitment and further growth of antral follicles, dominant follicle selection and ovulation.
• The follicle that was ovulated this month would have initiated and started its growth nearly three cycles beforehand.

Question 12

What are the stages of the preantral follicle?

Answer 12

• Beginning with 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 these granulosa cells. Once the follicle is set to initiate growth, the granulosa cells start to change in appearance and number.
• The initial change is that they go from being flattened to more cuboidal. Growth of the oocyte also occurs. The oocyte is in meiotic arrest but it becomes metabolically active and starts to lay down RNA and proteins for future growth and stages. Initiation of growth and start of formation of one or two cuboidal granulosa cells means that this follicle is now called a transitional follicle.
• It continues to grow; single layer of granulosa cells but they are all now cuboidal = a primary follicle.
• Another layer of granulosa cells forms along with theca formation from precursor cells that are condensing around the follicle = secondary follicle. The zona pellucida also starts to form. A full secondary follicle has two layers of granulosa cells. The theca forms and there is a definitive basement membrane which separates the theca from the granulosa cells.
• Different classification systems to be aware when reading papers. Some call all follicles Primary follicles if they have a primary oocyte i.e. whilst still in meiotic arrest and once completed meiosis 1 and ejected 1st polar body, known as Secondary follicles because they have a secondary oocyte. Important to clearly state which classification system is being used in assessments. These can all be classified as preantral follicles because there is no antrum and they are all class 1 follicles according to some categorisation stages. However, in some books or research studies, follicles with more than one layer of granulosa cells are known as secondary follicles.

Question 13

What technique can be used to Isolate Pre-antral Follicles?

Answer 13

• This bit of ovarian cortical biopsy (Elective Caesarean section patients consented for ovarian cortical biopsy) is brought to the laboratory and dissected out into many smaller pieces.
• Those pieces are digested in collagenase and DNAse for under an hour. It has to be digested because the ovarian stroma is very thick and tough, so it needs to be digested away before primordial follicles can be collected. The digested tissue is then teased apart using fine acupuncture needles. The follicles are isolated out and transferred into drops of media in a dish so they can be observed under high power magnification.
• These are actual pictures of human preantral follicles that have been isolated out from ovarian cortex biopsies. The scale bar is around 10 microns. Can see the oocytes in the primordial follicle with a single layer of flattened granulosa cells. The transitional follicle still has flattened granulosa cells around one edge but they have started to become cuboid on the other side. The primary follicle has a slightly more expanded oocyte with a single layer of expanded cuboidal granulosa cells. The secondary follicle has two layers of granulosa cells. The zona pellucida can be seen clearly along with the basement membrane. No theca cells can be seen in these pictures because the thecas are digested away when retrieving the follicles. Several layers of granulosa cells form in order to produce a multilaminar follicle. These are all preantral follicles because they have no antrum.

Question 14

What technique can be used to Isolate Pre-antral Follicles?

Answer 14

• This bit of ovarian cortical biopsy (Elective Caesarean section patients consented for ovarian cortical biopsy) is brought to the laboratory and dissected out into many smaller pieces.
• Those pieces are digested in collagenase and DNAse for under an hour. It has to be digested because the ovarian stroma is very thick and tough, so it needs to be digested away before primordial follicles can be collected. The digested tissue is then teased apart using fine acupuncture needles. The follicles are isolated out and transferred into drops of media in a dish so they can be observed under high power magnification.
• Follicles dissected out of stroma & placed individually in drops of media
• These are actual pictures of human preantral follicles that have been isolated out from ovarian cortex biopsies. The scale bar is around 10 microns. Can see the oocytes in the primordial follicle with a single layer of flattened granulosa cells. The transitional follicle still has flattened granulosa cells around one edge but they have started to become cuboid on the other side. The primary follicle has a slightly more expanded oocyte with a single layer of expanded cuboidal granulosa cells. The secondary follicle has two layers of granulosa cells. The zona pellucida can be seen clearly along with the basement membrane. No theca cells can be seen in these pictures because the thecas are digested away when retrieving the follicles. Several layers of granulosa cells form in order to produce a multilaminar follicle. These are all preantral follicles because they have no antrum.

Question 15

What are the alternative follicle classifications?

Answer 15

1) Preantral/ Class I
2) Antral/ tertiary
3) Preovulatory/ Graafian

Question 16

What are the morphological changes that occur in the transition from a primordial follicle to a primary follicle?

Answer 16

• There is a very distinct morphological change as the follicles initiate growth from becoming primordial to becoming a primary follicle.

1) Change in granulosa cells (~15 cuboidal granulosa cells)
2) Massive increase in oocyte growth & activity. 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.
3) Controlled & very slow process (thought to be a stop/start mechanism)

Question 17

How does the zona pellucida form?

Answer 17

• As the follicle and oocyte inside starts to grow, there is formation of the zona pellucida.
• ZP formation is a marker of follicle/oocyte growth
• ZP is a thick extra-cellular coat separating the egg from surrounding gc
• Human follicles are made up of four ZP proteins = ZP1, ZP2, ZP3, ZP4
• Even though they form this thick glycoprotein coat, they are permeable to large macromolecules because they have gap junctions and follicle extensions continue through. This is not only important to allow macromolecules and nutrients to get through, but also to allow the oocyte to remain in meiotic arrest. As seen in previous lectures, cyclic GMP needs to come through from the granulosa cells into the oocyte to keep it in meiotic arrest.
• Permeable to large macromolecules
• Follicle extensions continue through it
• The ZP does not completely separate egg from surrounding granulosa cells as it has projections that make connections between GC and oocyte.

Question 18

Describe the structure of a preantral follicle.

Answer 18

• A cross section can be seen in a multilaminar or fully formed preantral follicle. This is the oocyte with the nucleus of the oocyte still in meiotic arrest (in meiosis I arrest). There is a thick glycoprotein zona pellucida coat, several layers of granulosa cells and a basal lamina (basement membrane) which separates the theca cells from the granulosa cells. The theca starts to form after the secondary follicle stage (defined by two layers of granulosa cells); this classification was determined by a French scientist = the Gougeon method classification. Unlike the granulosa cells, the theca is very well vascularised.
• Even though there is this thick ZP coat, there is a lot of intracellular communication between the oocyte and those granulosa cells. This communication occurs between oocyte and GC via gap junctions that penetrate the zona pellucida.
• There is also communication between granulosa cells. This communication occurs via connexin proteins – the predominant ones are Cx43 (allows for communication and messaging between GC) and Cx37 (between GC and oocyte).

Question 19

What processes and factors are involved in activation of the primordial follicle? What causes initiation of follicle growth?

Answer 19

• Many ideas have come about from various research studies and experiments. Two main ideas =
  1) initiation is regulated by loss of an inhibitor. One ideas is that follicles are under a constant inhibitory influence. Resting follicles under constant inhibitory influence (local paracrine/autocrine factors) to remain dormant. This can be local paracrine factors coming from adjacent follicles or the surrounding ovarian cortical stroma or autocrine factors coming from within the follicles themselves. These factors exert constant inhibition and prevent the primordial follicles from growing so that they remain dormant.

2) initiation is regulated by stimulatory factor/s. The other theory is that something needs to stimulate the follicles to grow. These stimulatory factors can again be coming from the micro environment or from the blood. As the primordial follicles are in the cortex and away from the blood supply, there can be a gradient of diffusion from the centre to the periphery. Some bloodborne influence is coming in, diffuses along and activates the primordial follicles that are resting in the cortex. From the microenvironment (other follicles, stromal cells) and/or blood, there is a gradient of diffusion from centre to periphery.

• It is now believed to be a combination of both of these = inhibition and stimulation. As there is a decrease in whatever inhibitor exerts its influence on the follicle pool, it allows the stimulatory follicles to take over and activate. In other words, as inhibitory factors decline, there is an increase in the effects of stimulatory ones. It is also dependent on the size of the PF pool (ovarian reserve) and the ratio at which it enters the growing pool.
• “Production-line” hypothesis = this theory suggests those that have entered meiotic arrest first in foetal ovary, will initiate growth first.
• It is known that the initiation of follicle growth is also dependant on the size of the primordial follicle pool and the ratio at which it enters the growing pool. When women enter the perimenopausal period in their late 30s and early 40s, as the primordial pool has decreased, more and more follicles activate and enter the growing pool at a faster rate, so depletion occurs at a faster rate.
• Also have to take into account the extracellular matrix or surrounding stromal cells.

Question 20

Where is the primordial follicle located?

Answer 20

• Primordial follicle arrested in dictyate stage of meiosis
• Primordial follicles located in ovarian cortex & have no blood supply. As a consequence, they are not subject to blood borne influences.
• The primordial follicles are all to be found bunched together along the outer cortical region, very close to the surface, which is quite avascular.
• As they are located in the ovarian cortex, where there is no blood supply, the follicles are not subject to bloodborne influences. In addition, the basal lamina around the follicles creates a microenvironment for the granulosa cells and the oocytes, limiting its contact with other cells in the ovary. This means that any factors involved in initiation of growth of these follicles must be coming from within the ovary itself.
• Basal lamina around the follicle creates microenvironment for gc & oocyte i.e not in contact with other cells in the ovary.

Question 21

What are the three possible fates of the primordial follicle?

Answer 21

• Primordial Follicle in the ovary has 3 possible fates:
  1) To remain quiescent and die out directly at dormant stage
  2) To begin development but arrest and later undergo atresia (this could be at any stage, e.g. primary, multilaminar etc.
  3) Could be the one to develop, mature & ovulate
• There is massive loss of follicles at all the stages.

Question 22

What is the importance of the extracellular matrix?

Answer 22

• ECM consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides
• ECM turns over and remodelled during folliculogenesis to allow for growing follicle. The primordial follicles are in the cortex. As they grow, they moved into the medulla. When the dominant follicle is selected and it starts to become a preovulatory follicle, it moves back towards the surface of the ovary to allow it to be ovulated. The extracellular matrix is very dynamic in its nature, allows for this remodelling and turnover to allow growing follicles through; not a static, rigid structure.
• May regulate follicle growth especially interactions between gc & oocyte
• Mechanical stimuli are communicated rapidly throughout follicle as various cell types are physically connected eg via connexins. This could be a combination of mechanical signals compressing it but also chemical diffusion of substances from the ECM into the cells.
• Also have to take into account the extracellular matrix of surrounding stromal cells.
• This is because the structural architecture around the follicle plays a crucial role in regulating follicle growth. In other words, it is this three dimensional matrix. Key to this is the extracellular matrix which surrounds the follicles, consisting of a mixture of proteins. It exerts force onto the follicle to maintain its integrity and also potentially to keep it quiescent.
• In vitro experiments show that when follicles are taken out of the ovary and kept in a dish, the oocyte can pop out; culture much better with some kind of supporting matrix around them.

Question 23

Which genes have been implicated in primordial follicle activation?

Answer 23

1) Endocrine disruptors BPA (Bisphenol A), Genistein, DES (diethylstilbestrol) – inhibits nest breakdown
2) Nest breakdown & Primordial Follicle Assembly = FIGLA (human & mice), Zona Pellucida 1-4 (human & mice), Activin βA & BDNF (human), AMH? (mouse), Oestrogen? (baboons, human)
3) Primordial Follicle Maintenance/Repression = PTEN, FOXO3 (forkhead family), AMH (Anti-Mullerian Hormone), SDF-1 (stromal derived factor)
4) Primordial Follicle Activation = KIT ligand & KIT receptor (cKIT), FOXL2, NOBOX (newborn ovary homeobox), SOHLH 1&2 (transcription factors)

• Primordial follicle formation occurs when the nest breaks down, allowing for surrounding cells to invade and surround the oogonia. They formed the flattened granulosa cells and so the primordial follicle is formed. Several genes have been implicated in nest breakdown and primordial follicle assembly and some have been mentioned previously.
• Primordial follicles are formed postnatally in mice and AMH is very important for this primordial formation in mice (not thought to act in the same way in humans at this stage with regard to primordial follicle formation). - Another interesting factor is oestrogen. Nest breakdown and primordial follicle formation occurs postnatally in mice when oestrogen levels drop.
• In larger mammals and humans, this occurs in utero (before birth, much earlier on). In humans, oestrogen levels increase and reach their peak in the third trimester, but this nest breakdown occurs much earlier than the third trimester. Majority of oestrogen from the mother will not reach the foetus, because it is aromatised by the placenta (don’t want a male foetus becoming oestrogenised). How important is oestrogen in humans with regards to primordial follicle formation? There was an interesting study done in baboons whereby they were given aromatase inhibitors. These inhibitors prevented the conversion of androgens to oestrogens, so there were not very high levels of oestrogens. Clearly saw a decrease in primordial follicle formation and more nests were visible; somehow oestrogen must be getting across/made available and involved in nest breakdown at this stage.
• Likewise, endocrine disruptors which mimic oestrogen (occupy the oestrogen receptor and prevent oestrogen from acting) also inhibit nest breakdown.
• There are several genes which are involved in primordial follicle maintenance or repression. It is inhibitory factors which keep the primordial follicle as a primordial follicle. On the contrary side, there are genes involved in activating primordial follicles and their progression to primary
• Careful in using murine models since primordial follicle assembly occurs after birth, whereas in human primordial follicle formation occurs in utero.
• In mice germ-cell, nest breakdown occurs postnatally and is due to loss of mammalian E2.
• In larger mammals occurs before birth (in utero) so not entirely due to E2, but may have a role as E2-deprived baboon (via administration of aromatase inhibitor in late gestation) have less PFs but increased numbers of germ cell clusters. Also thought that this is how EDC work.

Question 24

What factors are important in the formation of a primary follicle from a primordial follicle?

Answer 24

• PTEN and FOXO3 are oocyte derived factors as is cKIT (the receptor for KIT ligand). KL is granulosa derived.
  FOXO3 (forkhead transcription factor) is found in nucleus bound to Cyclin D2, which keeps the follicle in arrest and prevents it entering cell cycle. How is this brake overcome? KL produced from gc, binds to it’s receptor (cKIT) and activates PI3 kinase. This then results in conversion of PIP2 to PIP3, which activates AKT which then phosphorylates FOXO3. Phosphrylated FOXO3 moves out of the nucleus releasing Cyclin D2 and allowing the cell cycle to progress. Upstream of FOXO3 is PTEN which facilitates conversion of PIP3 to PIP2, which the opposes actions of Akt to keep follicle in arrest. Aided by other factors like AMH and SDF-1 produced from other follicles and stroma.
• This pathway is really important in primordial follicle activation. There is the resting primordial follicle. Within the nucleus of the oocyte, there is a transcription factor called FOXO3. FOXO3 binds to cyclin D2 and keeps the follicle in arrest. It prevents it from entering into the cell cycle when it is bound to cyclin D2. As the follicle activates, KIT ligand is produced in the granulosa cells and this binds to its receptor, CKIT (which is present in the oocyte), and activates PI3 kinase. This then mediates the conversion of PIP2 to PIP3. This then phosphorylates AKT and leads to the phosphorylation of FOXO3. When FOXO3 is phosphorylated, it comes out of the nucleus, releasing cyclin D2 and allowing for activation of the cell cycle. Interestingly, there is another factor in the oocyte called PTEN. PTEN prevents that conversion of PIP2 to PIP3 (maintains PIP2 within the oocyte). Hence, it prevents the activation of AKT and that whole signalling pathway; keeps the primordial follicles in that repressed state. Adding to this is also AMH coming from surrounding follicles and SDF-1 coming from the surrounding stroma.

Question 25

What are the inhibitory factors that repress primordial follicles?

Answer 25

1) Oocyte-derived factors (inhibitory):
- PTEN (tumour suppressor gene) → inhibits signalling by Akt/PI3K signalling pathway; loss of PTEN (KO mice) → global activation of primordials
- FOXO3a (transcription factor) → also part of PI3K and restrains follicle activation. FOXO3 k/o have global activation of primordials.
- SDF-1 (stromal-derived factor) chemokine → inhibits follicle activation in autocrine/paracrine fashion

2) Granulosa-derived factors (inhibitory):
- AMH (anti-Müllerian hormone) → acts in paracrine fashion to inhibit primordial follicle initiation. Comes from growing follicles; k/o have less stock of primordial follicles & more growing follicles. Therefore, knocking out AMH = loss of brake on primordial follicle activation so most of them will grow.

Question 26

What are the stimulatory factors that activate primordial follicles?

Answer 26

1) Granulosa-derived factors (stimulatory):
- KL (KIT ligand aka stem cell factor SCF) secreted from granulosa cells → evidence that KL may inactivate Foxo3a and allow cell cycle progression

2) Oocyte-derived factors (stimulatory):
- cKIT (KIT ligand tyrosine kinase receptor) in oocytes → necessary for follicle activation. Newborn mice injected with antibody to cKIT that blocks interaction with KL = do not progress beyond primordial follicle stage

Question 27

What factor is involved in granulosa cell proliferation and is important in the development of preantral follicles from primary follicles?

Answer 27

• FOXL2 is another transcription factor that belongs to the forkhead family of transcription factors. It is involved in granulosa cell proliferation. - Women who have mutations in the gene have a condition known as type 1 BPES and also POF; they get primary follicles but these follicles do not progress to further preantral stages. Type 1 BPES is a condition that affects the development of the eyelids, giving them characteristic phenotypic features to do with their eye opening, droopy eyelids and the way that the skin folds up. The gene that causes these particular eye defects is also linked with progression of primary follicles through the preantral stages.
• FOXL2 (forkhead transcription factor) ⇒ granulosa cell proliferation
• FOXL2 -/- have Type 1 BPES and POF (premature ovarian failure) = no progression of follicles to secondary stage
• In mice, FOXL2-/- form PF but gc proliferation is interrupted and there are NO secondary follicles.
• BPES: affects development of eyelids
• Blepharophimosis = narrowing of eye opening
• Ptosis = droopy eyelids
• Epicanthus Inversus Syndrome = upward fold of the skin of the lower eyelid near the inner corner of the eye

Question 28

Summarise preantral folliculogenesis.

Answer 28

• There are primordial follicles which are resting (oocytes in meiotic arrest with flattened granulosa cells). It is thought that this arrest is maintained by certain inhibitory factors.
• Once these factors are overcome, it can activate various stimulatory pathways/factors. They start to grow; oocyte grows in size, flattened granulosa cells become cuboidal.
• Progression is then determined by various genes and when they enter into the menstrual cycle, there also activins, inhibin, gonadotrophins and AMH also plays a role.

Question 29

What genes and factors have been implicated in preantral follicle progression?

Answer 29

• Many other genes are involved in preantral follicle progression, for example GDF9 and BMP-15. Both of these genes belong to the TGF beta superfamily which includes AMH, inhibins and activins. These genes and the family are interesting because there is real species difference. For example, knockout of GDF9 in mice shows that follicles will not progress beyond the primary stage. Similarly, knocking out BMP-15 in mice causes them to be sub-fertile (not a complete reduction in follicles/loss of fertility), but the same mutation in sheep results in a very profound infertility. There are species differences! Similarly, some women who have POF have shown mutations in BMP-15 and GDF-9 when they have done gene sequencing, but this is very rare. In other words, women with POF where follicles are not progressing or deplete early are not necessarily linked to these mutations (seem to have much bigger effects in in mice and other animal species).
  Have mentioned the connexin genes. Cx37 is the gene that is responsible for encoding the connexin proteins that connect the granulosa to the oocyte. Cx43 connects granulosa cells to each other. Mutations in genes encoding Cx37 result in infertility because they disrupt communication between the granulosa cells and the oocyte.
  Similarly, insulin, androgens and insulin-like growth factors also have profound effects on follicles; can really stimulate granulosa cell proliferation and follicle growth, but too much can be detrimental (particularly when looking at PCOS).
  There are other nerve growth factors and neurotrophins that have been implicated in progression of follicles. There are neurotrophin receptors on the oocyte.

Question 30

Can the ovary form new oocytes?

Answer 30

• This is a contentious topic that has been reverberated within the world of reproductive biology over the last few years.
• Tend to follow the dogma that women are born with their complete stock of follicles (ovarian reserve) and once this stock is depleted, they undergo menopause (can’t create new ones). However, in 2004, a series of papers came out that seemed to indicate that under the ovarian surface epithelium, there was a germline stem cell niche which was capable of regenerating the ovary (filling it up with follicles if depleted). This created a lot of controversy because similar structures have been found in humans, cows and other species, but there were no consistent markers. While it could be shown in the mice, it was thought that they did not contribute to their fertility. Some people thought they were just left over oocytes trapped from ovary formation or when they didn’t form follicles. The debate still rages because people are still keen on looking for these structures, trying to prove that they exist and are capable of forming eggs and follicles. The issue is even if they did exist, they do not seem to contribute in any way to fertility of mice. While in women the age of puberty has got younger and younger, very much linked with nutrition and obesity, the age of menopause has not really changed (remained fixed over centuries).

Question 31

What is the importance of gonadotrophins during basal follicular growth?

Answer 31

• The preantral stage is gonadotrophin independent. This can be seen when follicular growth still occurs in physiological and pathological states where circulation gonadotrophin levels are still low.
  1) e.g. physiological= infancy (pre-pubertally), pregnancy. In these physiological states, FSH levels are very low, yet preantral follicles can be seen at all stages in the ovary.
  2) e.g. pathological= Kallman’s syndrome, anovulatory PCOS. The same applies to these pathological conditions.
  3) FSHß & FSHR k/o mice have normal preantral growth
  4) inactivating mutations of the FSH receptor = follicle growth to antral stage, but less follicles. The gene that encodes for the FSH receptor is mutated; the receptor that is formed will not be able to bind FSH/may not be activated in the same way when FSH binds. Still see follicle growth to antral stages but there are much less follicles. (Aittomaki K, 1996; Tapanainen, 1998; Touraine, 1999)
• FSHR have been found on primary stage follicles using the same technique describes before = may not be coupled to 2nd messenger system (Otkay et al, 1997; Rice et al, 2007). FSHR found on primary stage follicles but may not be coupled to the second messenger system; could be starting to be expressed but the protein may not be fully functional and formed. It does appear that FSH is not essential for preantral follicle growth, but low tonic levels of FSH keep the follicles much healthier and in a better state to respond, i.e. healthy pool of selectable follicles.

Question 32

Summarise the factors involved in the progression of primary follicle progression (to preantral stage).

Answer 32

1) Oocyte-derived factors:
- GDF-9 (growth differentiation factor-9). K/o = no progression beyond primary
- BMP-15 (bone morphogenetic protein-15). K/o mice sub-fertile ≡ equivalent mutation in sheep (Inverdale sheep FecXi) profound infertility; think about species difference
- Cx37 (connexin 37) gap junction protein (between oocyte & gc). K/o failed folliculogenesis

2) Granulosa-derived factor:
- Cx43 (connexin 43) gap junction protein (between gc & gc). K/o deficient in germ cells and no progression beyond 1°/2° stage

3) Extra-follicular factors:
- Insulin & IGF-1 & IGF-II →
increase primary stage follicles in cultured human ovarian cortex
- NGF (Nerve Growth Factor) → K/o have ↓no. growing follicles → no correlation in domestic animals/humans

• Culturing human ovarian cortex in insulin or IGFs, there will be an increase in the number of primary stage follicles. In other words, it pushes through that activation and movement towards primary stage. Similarly, with nerve growth factor, KO mice have a decrease in growing follicles, but there is no correlation in domestic animals or humans (approach some of this with a critical eye, especially when looking at different literature for ICAs).

Question 33

Do androgens have a role in early follicle growth?

Answer 33

• In mice, culturing ovaries and follicles in testosterone rapidly upregulates the intra-oocyte PI3K/Akt/FOXO3 pathway → increases the ratio of primary to primordial follicles more than two fold. In other words, it increases activation and initiation of follicle growth.
• monkeys treated with androgens have more primary follicles and increased FSH receptors on the follicles
• Can do the opposite experiment by blocking androgen receptors. Inhibiting AR in bovine ovaries prevents primary to secondary follicle transition (Yang & Fortune, 2006, BoR). Androgens will only bind to their receptor and blocking the androgen receptor in cow ovaries prevents primary to secondary follicle transition. This shows that androgens can be quite important in pushing the follicles through.
• Is there a human equivalent?
• This is seen in PCOS. Androgen receptors are present on preantral follicles from the very early stages. Can carry out quantitative PCR on isolated human follicles to measure first beta actin (to ensure the mRNA was all intact) and then androgen receptor. Collected follicles at different stages; can see the androgen receptors were not found on the primordial follicles but as they started to grow, the amount of androgen receptors was increasing. This showed that once human preantral follicles activate growth, they are capable of producing androgen receptor which could bind androgens. It is known that women with PCOS, they have high levels of androgen (hyperandrogenaemia) and an increased number of primary and growing follicles.

Question 34

How are oocytes and follicles formed?

Answer 34

• As those mitotically dividing primordial germ cells colonise the genital ridge, they will differentiate to form oocytes and they start to build these nests if the embryo is destined to become female. They have cytoplasmic bridges around them which connect them together to form syncitia or “nests”. It is thought that the role of the syncitia or “nests” is to exchange organelles.
• Now known that retinoic acid, a biologically active variant of vitamin A, is a key extrinsic regulator of germ cell entry into meiosis. These cells have been dividing by mitosis to increase in number, then they stop and enter into meiosis. Retinoic acid regulates this entry into meiosis. KO mice have been used to show that there is another gene which seems to be quite crucial in this whole process = DAZL.
• DAZL expression increases before meiosis at about 9 to 14 weeks gestation. Knocking out DAZL in mice, then the germ cells don’t develop past the primordial germ cell stage; they won’t form these oogonia. In humans, DAZL mutations are associated with sub-fertility. This is a good example of seeing genotype/phenotype associations in humans and then confirm it by creating knockout mice of that particular gene to see what stage and processes it is affecting.
• When writing an ICA, include genotype/phenotype association in humans and investigate to see whether it has been confirmed with KO mice for details of the actual process.
• There is then cyst breakdown and primordial follicle formation. Those syncytia break down and surrounding cells will infiltrate. They come around the oogonia to start forming primordial follicles. In humans, this occurs before birth. However, in mice, this occurs after birth. This is an important consideration when using models - how relevant/important is the model. Mice are very useful, but this critical difference has to be recognised; primordial follicle formation occurs before birth in humans (human gestation line) and in mice, primordial follicle formation occurs after birth (mouse gestation line). Can see embryonic day 10.5, embryonic day 13.5 and post-natal day 1. There is then nest breakdown and primordial follicle formation.