OLD EGGS FOR NEW – DOES THE ADULT MAMMALIAN OVARY EVER MAKE DE NOVO OOCYTES?

Aims/Objectives: Reproductive biology is a vast and ever expanding field and within it, the possibility of female germline stem cells in the adult mammalian ovary has become the subject de jour. These cells – defined as “a unique cell population committed to producing gametes for the propagation of the species” – could hold the key to allowing women who have had fertility problems to become pregnant.

This project aims to look more specifically at the oogonial stem cells (OSCs) – the original scientific dogma disputing their existence and the new cutting edge research isolating these proposed cells and thus tackling the long held idea that there is a fixed number of female eggs. We will look at debate surrounding the OSCs actions in vivo – do they produce oocytes? And if they do, are they viable? In addition we will examine the potential uses of them in the future and what obstacles need to be overcome to make these a reality. This is a dynamic area of research and one that holds great promise for the future.

This site was made by a group of University of Edinburgh Biomedical Students, with the help of Maria Camacho, who studied this subject over 10 weeks as a part of the Reproductive Systems course. This website has not been peer reviewed. We certify that this website is our own work and that we have authorisation to use all the content (e.g. figures / images) used in this website.

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INTRODUCTION

Female germ cell development and folliculogenesis

Germline stem cells (GSCs) are required in both sexes to produce mature gametes.

In males, sperm are produced continuously from the onset of puberty by

spermiogenic stem cells in the testes. In contrast, it has been postulated that

females are born with all of the oocytes they will ever have, and that there are no

oogonial stem cells (OSCs) which produce gametes in vivo in adult females.

Germ Cell Specification and Sex-Specific Changes

Germ cell specification is a process that is currently best understood in mice (1).

Cells in the proximal epiblast of the developing blastocyst respond to signals from

extraembryonic tissues which induces PGC (Primordial Germ Cell)

specification. Only cells directly in contact with the extraembryonic ectoderm begin

to express Blimp1/Prdm1 and it is these cells which become the PGCs. PGC

specification requires the repression of somatic gene expression e.g. Hox genes,

and promotion of germ-cell specific gene expression e.g. Stella, Sox2. After

gastrulation, the germ cells must then proliferate and migrate through the hindgut

and dorsal mesentery to the gonadal ridge (2) (See figure 1: step 1). This migration is

driven by chemoattractant signals such as SDF-1.When the germ cells reach the

developing gonads, massive epigenetic reprogramming occurs, involving the

removal of histone modifications and DNA methylation and allowing sex-specific

imprinting to occur for either spermatogenesis or oogenesis. These PGCs being to

undergo mitosis and increase in number (Figure 1, step 2).

XX germ cells lack genes on the Y chromosome which would direct gametogenesis

towards the formation of sperm. Signals produced by neighbouring somatic cells

(such as granulosa cells in females and Sertoli cells in the male) influence the sexual

differentiation of the germ cells. Female germ cells (oogonia) then initiate meiosis

which continues until the arrest that occurs shortly after birth in mice (Figure 1, step

3).

Figure 1: Formation of the Follicular Reserve (3)

Follicle Development

Figure 2: Folliculogenesis (3)

Meiosis I begins in early embryonic development, but arrests in the diplotene stage

of prophase I. This results in the creation of primordial follicles. During meiotic arrest,

these primordial follicles become primary/preantral follicles as they grow and

increase in diameter from around 20µm to 200-400µm (see Figure 2). The growing

oocyte secretes glycoproteins which condense around it to form the zona pellucida

(ZP). The ZP separates the oocyte from the granulosa cells, which nourish the

growing gamete via cytoplasmic processes. The granulosa is, in turn, surrounded by

theca cells. This stage of development occurs continuously until the menopause, and

is not dependent on gonadotropins, and can therefore occur even before the onset of

puberty.

Preantral follicles that do not undergo atresia will transition into antral follicles. The

granulosa cells proliferate and follicular fluid begins to fill the gaps between these

cells. This results in the formation of a follicular antrum. The oocyte then begins to

synthesise RNA and proteins. Within the follicle, the oocyte becomes surrounded by

fluid, which is then encapsulated by a collection of granulosa cells known as the

cumulus oophorus. The oocyte remains connected to the outer granulosa cells by a

thin stalk of cells.

The follicles rely on hormones to develop. Larger, more mature follicles need less

follicle stimulating hormone (FSH) to survive. As FSH levels fall, this leads to the

apoptosis of smaller follicles, leaving usually one dominant follicle known as the pre-

ovulatory follicle. This follicle acquires luteinising hormone (LH) receptors, and at the

LH surge (around day 14 in the cycle), will rupture, releasing the oocyte. During the

late preovulatory stage, meiosis I resumes, before arresting in metaphase of meiosis

II. Only at fertilisation does the oocyte complete meiosis II. (4)

Atresia is the process by which follicles which do not proceed to ovulation

degenerate and are re-absorbed. It is a hormonally-controlled, apoptotic process

which is implicated in the decline of the ovarian reserve from birth until the

menopause. Investigation into the rate of atresia has been implicated in the debate

over the existence of OSCs (5), as the central dogma would suggest that once

follicles undergo atresia, no more can be produced to replace them.

Germ Cell Markers

As primordial germ cells mature and differentiate, different cells will express different

proteins and can therefore be determined using specific cell markers. Germ cell

markers have played an important role in determining whether OSCs exist as the

method to isolate these proposed cells relies on these markers, specifically DDX4

(5). DDX4, also known as VASA, is an evolutionary conserved gene known to exist

in mice and humans (6). Other important markers include those that are meiotic

specific because if all germ cells undergo meiotic arrest during foetal development,

there should be none of these markers present in adult cells.

Using combinations of these markers allows for specific cell types to be determined.

Table 1: Most Common Germ Cell Markers Used (7,8)

HISTORICAL PERSPECTIVE

Before publication of the Zuckerman (1951) paper, the prevailing belief was that

oocytes were produced throughout adult life. However since then, there has been a

change in belief towards one of a fixed pool of oocytes that did not self-renew. This

timeline gives an outline of changing views throughout the past few centuries

regarding the presence of OSCs.

Click on the timeline below for a larger image.

EVIDENCE FOR THE PRESENCE OF OSCS

Evidence for neo-oogenesis across species

Belief in Zuckerman’s dogma (9) is thought to be unjustified due to a lack of detailed

studies in ovaries, (10) hence, neo-oogenesis is currently being studied across many

species. Oogenesis occurs continually throughout the reproductive life of all

amphibians, most reptiles, but few mammals. It is suggested that some oocytes in

adulthood are produced de novo from OSCs found in the germinal epithelium of the

ovary (10), and are constantly destroyed by atresia. (11) In adult mammalian

ovaries, 70-95% of oocytes are in various stages of atresia, implying that

supplementation by new follicles is required for continued fertility.

One of the main arguments which shows the potential for the presence of OSCs and

adult oogenesis, surrounds the discrepancy between the rate of atresia and the

number of remaining non-atretic follicles in the mouse ovary (5). If the follicles

underwent the rate of atresia originally demonstrated, (12) ovarian reserve would be

depleted by young adulthood. Approximately 89 follicles were believed to be lost

each day, leading to a total of 2136 follicles lost in 24 days. However, the Tilly group

found that only 294 follicles were lost in a 24 day assessment period (5) and has

since been supported by other collaborators (13).

Furthermore, cattle follicles remain constant in number during the ‘prime

reproductive period’ (PRP), suggesting that newly formed follicles replace those in

atresia. (11) It has been proposed that follicular renewal occurs throughout the PRP

across the animal kingdom (10), reinforcing the Tilly group’s position.

Large ovoid cells resembling germ cells were found on the ovarian surface

epithelium (OSE), believed to be OSCs. These cells express MVH, a germ cell

specific marker, which confirms that they are of germ cell lineage and thus could

have the potential to cause adult oogenesis. Further work by the Tilly group suggests

these cells originate from the bone marrow and peripheral blood (14) although this

was later disproved by Eggan et al. (25)

It has also been shown that these cells are actively proliferating, as they incorporate

5-bromodeoxyuridine (BrdU) at a level higher than that expected for simply

mitochondrial replication or DNA repair. Thus these cells are actively proliferating

and are of germline origins, supporting oogenesis (5). Zou (15) further proved that

cells undergo mitosis by analysis of BrdU incorporation. The same result was also

generated in human OSCs, confirming division occurs (12).

Various researchers were also able to isolate these cells from both mice and

humans, and found they express numerous germ-cell specific markers. (15) (Figure

1.)

Figure 1. Table of germ cell specific markers found in mice OSCs, and their

characterisation.

The expression of these markers confirms that these isolated cells are of germ cell

lineage. They also show high telomerase activity, only found in proliferating germ

cells. (8)

Perhaps one of the most important findings supporting the existence of OSCs is that

they can form oocytes not only in vitro, but also in vivo (15). As shown in Figure 2,

upon isolation, OSCs were transfected with green fluorescent protein (GFP) and

injected back into the ovarian tissue. A few days later, GFP positive oocytes at

varying stages of development were found, which must have originated from the

isolated OSCs. These chimera mice were also able to produce healthy, fertile

offspring through natural mating, revealing that OSCs have the potential to form

fertilisable oocytes in mice, something that had not been believed to be possible.

Figure 2. Method detailing the transplantation of GFP OSCs into sterilised mice

ovaries. (Reprinted by permission from Macmillan Publishers Ltd: Nature Medicine,

advance online publication, 06 March 2012 (doi: 10.1038/sj.nm.2699))

Pigs

Similar results can also be seen in pigs, where cells characteristic of PGCs are

present in the OSE -porcine PGC-like Putative Stem Cells (PSCs). These

cells maintain germ cell identity in vitro, as shown by a number of cell markers

(Figure 4), and differentiate into oogonial-like cells which form clusters, supporting

the potential for neo-oogenesis. (18)

Figure 4. Cell marker expression in the ovaries of pigs

Oct4 is expressed throughout the PSC colonies, whereas Stella expression occurs

only in surrounding cells similar to what can be seen in early follicular development

(18), suggesting folliculogenesis has taken place. The PSCs may also undergo a

cytoplasmic to nuclear translocation of Oct4, as has been previously seen with PGCs

in mice and humans. Therefore porcine PSCs may be equivalent to PGCs, which are

able to form oogonial cells and form follicles, supporting the argument for neo-

oogenesis.

The Marmoset Monkey

The marmoset monkey is very primitive and has a differential ovary structure to most

mammals with the introduction of the Indifferent Cortical Zone, found beneath the

OSE. This contains germ cell nests containing both oocytes (post-meiotic) and

oogonia (pre-meiotic), as shown by specific germ cell marker expression. (Figure 3.)

Figure 3. Germ cell markers expressed in the ovary of marmoset monkeys.

Thus, the neonatal marmoset ovaries contain a significant population of proliferating

pre-meiotic germ cells which form oogonia. Monkeys generally have an absence of

pre-meiotic germ cells in the adult ovary, (16) so evidence in the marmoset may

therefore suggest a possibility for neo-oogenesis to occur in others. For example,

SCP3-a meiotic marker-is expressed in the OSE of both monkey and human PRPs.

Humans

Human OSCs are also able to produce oocytes, in vitro (10). These oocytes are

able to maintain germ cell identity and homogeneity throughout culture (17), as

shown by specific gene expression. (Figure 5.)

Figure 5. OSC specific marker expression in the human ovary.

This expression is consistent with PGCs, (19) and suggests oocytes are formed from

germline stem cells.

Numerous OSCs are found within the OSE layer of the human foetal ovary, and new

follicles are seen to form by associating with granulosa cells in the ovarian cortex.

(11) These OSCs are able to fertilise and produce embryos, as seen in vitro by the

production of 4 cell embryos, and development to polarised blastocysts. In

summary, human OSCs produce oocytes which are able to undergo

folliculogenesis and further fertilised in culture.

While in vitro studies are essential, in vivo testing will confirm if these cells play a

role within the body. In vivo studies have revealed the presence of OSCs in humans,

where they can be propagated (19) to form germ cells. (11) OSCs exist in women of

reproductive age (17), and during the PRP, these can be consistently obtained from

the OSE derived crypts (11) (similar to intestinal crypt stem cells) (20) of

cryopreserved ovarian tissue. Repetition of the GFP experiment (15) using human

OSCs produced oocytes which became surrounded by somatic cells, as seen in

vitro. This is again seen with OSCs derived from PRP surgical patients, which could

be returned to form follicles. LHX8 and YBX2 – oocyte specific markers- expression

confirms that these form oocytes. This proves that human OSCs are able to produce

oocytes in vivo, which can stimulate folliculogenesis, and supports human neo-

oogenesis (5). Tissue samples from older women produced the same result (17),

showing OSCs are present beyond the PRP. However, follicular renewal is believed

to be terminated after the PRP by immune changes in the OSC niche. This would

explain why the presence of OSCs does not allow continuing fertility in women

beyond the natural age of menopause, with any remaining follicles after the PRP

being stored until exhaustion with menopause. (10)

Both mice and human OSCs show matched gene expression profiles, along

with similarities in size and morphology, most notably the expression of Ybx2/YBX2

– a diplotene stage oocyte specific marker – essential for meiotic progression and

gametogenesis. (19) This confirms that oocytes able to reach meiosis 2 are

produced from the OSCs, suggesting neo-oogenesis has occurred, and that the

oocytes produced are viable and can undergo ovulation. Thus neo-oogenesis can be

seen in humans as it is in mice, and therefore may be just as likely in other

mammalian species.

EVIDENCE AGAINST THE PRESENCE OF OSCS

Even though there has been increasing evidence for the presence of OSCs,

evidence disputing their existence and discrediting the original findings against the

central dogma have also been on the rise. The fact that the preliminary findings have

not been consistently reproducible, with many alternative interpretations available,

puts the existence of these putative OSCs into suspicion. Regardless, if these OSCs

do exist, many studies have found they play no active role in natural folliculogenesis

in adults.

Reproducibility problems

Many researchers (24,26) were unable to reproduce the results of Johnson et

al. (14), claiming that upon doxorubicin treatment (depletes primordial follicles),

follicles are lost within 24 hours, but start to regenerate rapidly 36 hours post

treatment with a complete restoration by 2 months. Using the same mouse strain

and methods, only a depletion of the primordial follicles but no restoration of their

reserves was observed (24,26) and thus the conclusion of de novofolliculogenesis

from stem cells (14) could not be supported nor replicated.

It was also reported that in mice, approximately 77 new follicles are formed every

day (5), results that could not be reproduced by other researchers (24,27) and have

been further disproved by a mathematical model looking at two different scenarios –

the existence of a stem cell pool with 77 new follicles produced a day, and the

generally accepted central dogma of a finite oocyte pool (27). Only the latter model

was able to successfully reproduce the decreasing numbers of oocyte observed in

normal life of mice from postnatal day 6 through to 12 months (27). Using SSEA-1 as

a stem cell marker (as described by (5)) this group did not see any stem cell activity

in the mice, further supporting the idea that no oocytes are formed de novo and a

finite oocyte pool is enough to support female’s fertility throughout her reproductive

life (27).

Another proposal that was disproved was the idea of OSCs originating from the bone

marrow (14). Parabiosis of a wild-type mouse and a mouse ubiquitously expressing

green fluorescent protein (GFP) showed that no GFP-expressing oocytes were

formed in the wild-type mouse, even though these mice with joined circulation

exhibited chimerism of the blood cells and ovulated at the same time (25). If OSCs

were present in the bone marrow and contributed to the production of new oocytes

and primordial follicles, one would expect to see GFP-expressing oocytes in the wild-

type mouse (25).

Issue with Methods/Alternative interpretations

The inability to reproduce many of the results in favour of OSCs existence could be

due to the lack of proper markers for these cells, different methods used and even

incorrect/alternative interpretation of the results obtained. Many papers suggest that

using an antibody against DDX4 is not appropriate to isolate OSCs from an ovarian

cells population, as DDX4 is a cytoplasmic protein and thus is not expressed on the

cell surface (29). That makes it unable to bind cells and isolate them from the

ovarian tissue, suggesting that we do not even know what cells have been isolated

and used in many experiments using the DDX4 antibody.

If the above is true, it could suggest that the cells isolated might be any other ovarian

cells which inexplicably express DDX4 on their surface and are able to generate

cells morphologically similar to oocytes upon appropriate stimulation in vitro. Cells

from other organs have been found to be able to produce oocyte-like cells in

vitro (31,32); if the cells that have been isolated are residues of embryonic stem cells

that are found within the ovary (28), it would not be surprising if they could be

stimulated to produce oocyte-like cells under the appropriate conditions, which may

never occur in vivo. However, these cells might not be able to support the production

of viable offspring upon fertilization.

Additionally, it had been proposed that the mice strain used to show fast follicular

depletion in the absence of OSCs is not representative of normal follicle dynamics,

as this strain exhibits abnormally high levels of follicular depletions (30). The

increased number of atretic follicles observed was also considered by some to be

due to the harsh fixation technique used, damaging more follicles and making more

of them look atretic than would be observed normally (30).

Neo-oogenesis: Not a Natural Physiological Process

The conversion of ovarian surface epithelial (OSE) cells and reprogramming cells to

germ-like cells in vitro does not confirm the existence of OSCs and the process of

neo-oogenesis in vivo (3).Several recent studies have performed in vivo procedures

with the aim of proving whether or not these cells exist, with many results

discrediting the existence of OSCs.

One method to investigate these proposed OSCs involved live-cell imaging.

Zhang et al. performed a lineage trace of DDX4-expressing genes using an in vivo,

endogenous genetic approach and found that there were no mitotically active female

OSCs in the postnatal mouse ovary (21). Transplanting reporter mouse ovarian cells

into a 2 month old wild-type mouse resulted in follicle development but none of the

follicles observed were chimeric therefore suggesting that de novo folliculogenesis,

while supported by the ovary is not an active physiological process. The lineage

tracing experiment also revealed that DDX4-expressing cells from postnatal mouse

ovaries did not proliferate or form colonies thus indicating these cells did not enter

mitosis. A similar approach performed by a 2013 study used an inducible lineage-

labelling method and found, in contrast to the Johnson et al. (5) report describing fast

follicular turnover, that follicle turnover of individual follicles was relatively slow and

experienced similar stability with the total count of primordial follicles (23). This slow

measured turnover also indicated that at 4-wks old, mice had more than a sufficient

amount of follicles to sustain oogenesis for their reproductive lifespan, without the aid

of OSCs.

John et al. took a novel, indirect approach to the subject by examining the effects of

FOXO3 on follicle assembly (22). Foxo3 is a transcription factor known to inhibit

primordial follicle activation and the study reported that a FOXO3-/- mutant resulted in

the early onset of ovarian failure. Discussion of the work deduced that if de

novo folliculogenesis did occurred in the ovary, the researchers would have

expected to see the mutant ovaries undergoing activation in later life and follicles

developing at different stages. However this was not observed and the mass

primordial follicle activation in early life caused mutant sterility, suggesting that there

was no evidence of de novo follicle regeneration.

Yuan et al. used rhesus monkeys as a model to test for neo-oogenesis within

primates (3). This study monitored the expression of mitotic and germline cell

markers (homologous to those used to detect SSCs in the testes) in monkey ovaries

at different ages. Their work found no existence of proliferating germline stem cells in

the postnatal ovaries at any age. While trying to identify OSCs in the ovary, the

authors found cells in the OSE that exhibited proliferative properties (shown by

expression of proliferative markers), suggesting that these cells are stem cells.

However, they did not express laminA (a differentiation marker) nor DAZL (a germ-

cell marker), proposing that these cells are somatic stem cells rather than OSCS.

The lack of proliferating OSCs suggested there is no evidence for neo-oogenesis in

the adult monkey.

All of the evidence above puts into dispute the idea that these proliferating germ cells

exist. The techniques used to isolate these cells need to be investigated further as

the foundations on which they are based are unstable. Even if they do exist, so far

none of the evidence we have found suggests they play an active part in oogenesis.

POTENTIAL BENEFITS OF OSCS

Causes of infertility

Decreased fertility is defined as the inability to achieve a successful pregnancy within

at least twelve unprotected cycles (33). According to a UK-based population study,

one in six women confirmed that they had difficulties in conceiving and 2.4% of

women had never conceived a child (34). More and more women are currently

seeking treatment for infertility and in 40% of women with fertility problems, the

cause is unknown (35). However, there are some common causes of infertility, some

of which can be treated. 33% of women with sub fertility have ovulatory disorders,

primarily polycystic ovarian syndrome (PCOS), which due to an imbalance of

gonadotrophins results in an inability to ovulate. Chormosomal abnormalities, for

example Turner syndrome, are another cause of infertility. In Turner syndrome,

ovaries are usually non-functional which means that women are universally infertile

(36). Maternal age is also a factor which reduces fertility as female fecundity

decreases by 50% between the ages of 25 and 35. Deterioration of oocyte quality is

suggested to be the cause of infertility as this can lead to aneuploidy and in turn

destruction of the embryo (37). Other causes of infertility include tubal defects and

endometriosis (35).

Current therapies, including assisted reproductive technology (ART)

Fortunately, in vitro fertilisation (IVF) has meant that women with fertility problems

now have the chance to become pregnant and bear children. Since 1978, success

rates in IVF have improved dramatically, mostly due to the shift from multiple embryo

transfer to elective single embryo transfer (38). Multiple embryo transfer often leads

to multiple pregnancies and as a result, low birthweight and preterm birth. By

implanting a single embryo instead, these risks are effectively eliminated and lead to

comparably increased live birth rates. However, success rates in women over the

age of 40 still remain reasonably low, yielding live birth rates of 6% in women aged

43 (39). We can increase these birth rates via oocyte donation, from 53% using own

oocytes to 59% with oocyte donation (40). According to a recent study, outcomes are

still significantly worse in women over 45, with live birth rates of 55.8% in women

aged 40-44 and 52.7% in women aged 45-49 (41). However, not all women with

fertility problems require in vitro fertilisation. Some anovulatory disorders can be

treated with hormone therapies, for example women with hypogonadotrophic

hypogonadism (35). Women with endometriosis can improve their fertility by having

surgery. However despite improvements in management of infertility and IVF

technology, pregnancy outcomes for women of increased maternal age are bleak.

Oogonial stem cells (OSCs) and fertility

Despite the ongoing success and development of fertility treatments significant

hurdles still persist, most notably difficulties that arise with advancing maternal

age.The proposal that OSCs are a potential solution to infertility is a new and

exciting prospect that has accelerated investigation within this area despite the

physiology and mechanisms of human OSCs still to be confirmed. Nonetheless,

OSCs have sparked interest within reproductive medicine and may provide a novel

approach to treating infertility. OSCs may support reproductive health in women with

either premature ovarian insufficiency (POI) or age-related infertility (42,43.) When

considering how exactly OSCS could be used in treatment, the primary cause of

infertility should be considered.

New eggs

In cases where the ovarian reserve is depleted, OSCs could be matured to produce

viable oocytes. A promising in vitro technique shown by Telfer 2012 (44) has

developed primordial follicles into mature oocytes suitable for IVF. Combining

primordial formation from OSCs with in vitro folliculogenesis and maturation would

permit production of mature oocytes that could be used in IVF (45). This may allow

infertile women to conceive their own child without need for egg donation (17). In

vitro folliculogenesis of OSCs to mature oocytes may offer a solution to fertility

preservation in women or children undergoing chemotherapy as it eliminates the risk

of returning malignant cells post cancer treatment (42). Culturing OSCs into viable

oocytes for IVF is also a relevant treatment for both women with POI, due to

unknown early depletion of the ovarian reserve, and women post-menopause, who

may still maintain a population of OSCs. Older women using their own eggs for IVF

may have less risk of age-related aneuploidy with OSC folliculogenesis.

Menopause: replenish stores

Reproductive ageing is accompanied with diminished oocyte stores however studies

have revealed the presence of OSCs in postmenopausal women.(46). OSCs of

these women could undergoin vitro folliculogenesis as mentioned earlier, although

the discovery of a possible oocyte source has sparked questions concerning the

mechanisms involved with menopause. If OSCs exist, why do women enter the

menopause? Are there unknown contributing factors that trigger OSC maturation?

What prevents OSC development in menopausal women?. Understanding of the

local factors involved in OSC activation and folliculogenesis could be used to

replenish oocyte numbers in depleted ovaries. Investigation into OSC maturation in

vivo could help us understand the underlying cause of menopause and manipulate

this knowledge for use in clinical treatments (42 ,17).

Improving egg quality

Oocytes of older women are associated with reduced mitochondrial numbers and

ATP levels which is thought to contribute to defects in oocyte physiology e.g.

irregular chromosomal separation. AUGMENT (autologous germline mitochondrial

energy transfer) is a procedure that could solve this issue by isolating mitochondria

from OSC’s and transplanting into an oocyte from the same women during

intracytoplasmic sperm injection (ICSI).This could supply an older oocyte with

sufficient energy required for successful fertilisation and embryo development

leading to an increase in pregnancy rates for women of increasing age (47).

Conclusion

The potential of OSCs use within fertility treatments is undoubtedly exciting with the

possibility of replenishing and sustaining ovarian stores, improving egg quality and

strengthening our understanding of ovarian physiology. However there is

considerable controversy surrounding OSCs; their existence is unresolved, OSC

physiology and mechanisms have yet to be confirmed and possible treatments are

theoretical, experimental and will not solve all types of infertility (48).Before OSCs

are used clinically, extensive research is needed into the conditions required for

OSC development & folliculogenesis as well as confirming production of viable

oocytes that can be fertilised and develop into healthy embryos (49). OSCs hold the

potential for novel treatments acting to prolong fertility and postpone menopause to

be developed in the future.

DISCUSSION & CONCLUSION

In recent years, there has been a large volume of research into the existence of

OSCs, suggesting that these cells do exist in mammals (such as mice) and

contribute to neo-oogenesis. However, the origins of these cells are not currently

known, and whether they exist or perform neo-oogensis in humans remains to be

elucidated.

Assuming these cells do exist, they would only present in very small numbers

(0.05% cells of the ovarian tissue population) (53) and it is still unclear whether they

are active or can only produce oocytes upon appropriate stimulation in vitro. Some

research groups have shown that upon culturing the putative OSCs in vitro they can

be re-transplanted into animals and viable offspring can be produced (15).This, in

humans, would be very helpful in the treatment of infertility, however it does not

suggest that there are active OSCs under normal in vivo conditions which contribute

to neo-oogenesis.

Current research shows cells exist within the ovary that exhibit stem-cell-like

properties. This is a controversial area of research and there have been problems

with reproducing the results of key experiments, such as those of the Tilly group (5).

It has also been argued that there is a lack of appropriate markers for OSCs. For

example, there has been debate as to whether Ddx4 (the human equivalent of MVH)

is suitable OSC marker, as it exists within the cytoplasm, and so it’s use as a marker

on the surface of the ovary is questionable. This issue has been an integral part of

the debate (21,54).

With regards to mammalian animal models, mice have been the main subject of

research within this field of study. The OSCs generated from mice have been shown

to express multiple markers of transcription and germ cell markers to show that they

are stem cells. Although caution is required in drawing conclusions from experiments

in other species, some of these markers e.g. MVH have human germ cell

equivalents meaning that they can still be considered as valuable models.

Porcine models fulfil the same criteria. They can be used as exemplars for

generation of OSCs and do express markers however the comparison of these to

humans must be considered. Primates are the closest model in terms of human-like

oocyte expression and manipulation. Their markers are equivalent; however, female

marmosets have an Indifferent Cortical Zone, which humans do not. This implies that

primate models, whilst they are closely related to humans, cannot be entirely

representative of human ovarian function. The obvious gold standard would be

human in vivo experiments; however numerous ethical difficulties arise when

considering this.

As discussed in previous sections, one of the potential uses for OSCs would be to

replenish the oocyte population in women who have already depleted their ovarian

reserve, notably women with premature ovarian failure (POF) and post-menopausal

women. One of the key ethical issues that has been debated in recent years is

whether or not older women should have access to assisted reproductive

technologies (ART). Once again, this area of research brings to light a host of ethical

issues surrounding human reproduction.

It is conceivable that older parents would be more financially secure and have more

time to spend with potential children after retirement (50). However, older women are

at a much greater risk during pregnancy of certain conditions including hypertension

and cardiovascular diseases that can lead to maternal mortality (51). It is also

important to consider the ethical implications that these decisions have on any

potential child. There is a risk that older parents would die earlier, leaving the child

without parental support at an earlier age (52). The Human Fertilisation and

Embryology Authority (HFEA) have an important role in providing guidance to help

the scientific community to work through these ethical issues.

In order to clarify the mechanisms of OSC development into oocytes it must be

determined whether OSCs are producing viable oocytes continually (until ovarian

insufficiency) or if oocyte development can only be induced experimentally.

Understanding the niche and environmental conditions essential for OSC

development in vivo is potentially a key step in reproductive health. If OSCs are

continually producing new oocytes, it would raise the question as to why women go

through the menopause at all. It is suggested that the deterioration of immune

system with age could be associated with a loss of OSC development, on the

condition that the cells exist and are active during reproductive life (54). Further

research may also present a link between an altered niche and premature ovarian

insufficiency thus providing a possible answer to the unknown aetiology of POF.

Discovering the niche and environmental conditions of OSC development is

fundamental to understanding the requirements, mechanisms and interactions of

OSCs and hence the future of reproductive medicine.

The research in this area is continually evolving and the debate is fierce. Most

recently, in August this year, Tilly’s group continued the debate with a paper

concerning the use of Ddx4 as a marker (54), in response to criticism by Liu’s group

(21). For now, further examination is required to prove whether or not the OSCs

exist and function in the mammalian ovary.

Conclusion

Increasing evidence suggests that a pool of germline stem cells exists within the

ovaries of many mammalian species, but further work is required before the issues

surrounding this controversial topic are resolved. Investigation into the markers used

to isolate the cells is required and, if these cells play an active role in vivo, work must

be carried out to understand their mechanisms of development, microenviromental

requirements and cell signalling. The answer will eventually come from integration of

all of the unique viewpoints of different researchers and from further advancements

in technology. Perhaps the most important aspect of this research will be concluding

whether or not these cells play an active role in oogenesis and if they can be used

clinically to aid in the treatment of infertility.

LAY SUMMARY

Since the 1950s, it has been the central dogma of female reproductive biology that

women are born with a finite pool of eggs that will determine their reproductive

potential. In 2004, this idea was challenged with the discovery of potential germ stem

cells, oogonial stem cells (OSCs) in mice. Since this breakthrough, the existence of

these cells has been greatly debated, with supporting evidence for each side of the

argument. Many animal models have been used to test the presence of OSCs in

mammals, including in vitro human studies. Our task was to further investigate this

controversial topic, exploring both sides of the argument and examining current

research. We also looked into the future contributions these cells could play in aiding

fertility. These include extending a woman’s ability to have children past the

menopause as well as improving fertility in women with premature ovarian

insufficiency, where menopause occurs at a much earlier age. While it is widely

believed at this point that these cells do exist, their importance in human

reproduction and development is yet to be determined.

CONTRIBUTIONS

Contributions:

Fraser Barratt – Germ cell development and Folliculogenesis, General Editing

Lewis Finlayson – Aims/Objectives, Historical Perspective, References, General

Editing

Amanda Morris – Evidence for OSC Existence

Aoibheann Bradley – Evidence for OSC Existence, Website maintenance

Christina Mackie – Benefits of OSCs

Elizabeth Undrell – Benefits of OSCs, Website maintenance, General Editing

Magda Mareckova – Evidence against OSC Existence, Germ Cell Markers, General

Editing

Sophie Nash – Evidence against OSC Existence, Germ Cell Markers, General

Editing

All members worked together for Discussion, Conclusions and Lay Summary

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