Current Research on The Ovarian Stem Cells: a Brief Review

Adult mammalian ovary has been under watch for over 10 years now since it was proposed to home stem cells that experience postnatal oogenesis amid reproductive period like spermatogenesis in testis. Ovarian stem cells are situated in the surface epithelium of adult and menopausal ovary and in addition in ovary with early failure. Ovarian stem cells contain two particular populaces including round, very small-like embryonic cells (VSELs which express nuclear OCT-4 and other pluripotent and primordial germ cells particular markers) and somewhat bigger ovarian germ stem cells (OGSCs with cytoplasmic OCT-4 which are identical to spermatogonial stem cells in the testicles). These stem cells can unexpectedly differentiate into oocyte like structures in vitro. Stem cells can also be derived from bone marrow and thus can act as an alternative source. The ovarian stem cells express FSHR and react to FSH by experiencing self-renewal, clonal extension, and starting neo-oogenesis and primordial follicle assembly. VSELs are largely quiet and were reported to survive chemotherapy and start oogenesis in mice when exposed to FSH. This rising understanding and further research in the field will help advancing novel methodologies to oversee ovarian pathologies and furthermore towards oncofertility.

Introduction

The idea that a female is born with a fixed pool of follicles was confronted by Professor Tilly in 2004 and his coworkers who revived the essentiality of the theme of postnatal oogenesis and proved to us that the rate of loss of oocytes in mice ovary because of atresia and ovulation were infact replenished by a continuous supply of immature oocytes which keeps up a steady state [2]. These perceptions supported the idea of ovarian immature stem cells and postnatal oogenesis and a few minds were drawn into this zone of research.

First significant advance was to demonstrate the presence of stem cells in the ovary trailed by how they work under ordinary conditions prompting postnatal oogenesis, and how they result in different pathologies like ovarian disappointment, menopause, and cancer. Likewise, it ended up relevant to examine whether undeveloped cells present in the adult mammalian ovary could be controlled to recapture ovarian capacity under certain particular conditions, for instance, after oncotherapy in tumor survivors. Postnatal follicular regeneration in mouse ovary [3] and ovary surface epithelium (OSE) as a source of germ cells amid fetal stage ovary was accounted for in the past [4, 5]. It was therefore suggested that OSE is the dynamic site of starting point for neoplasms and right around 90% of ovarian tumors emerge from the OSE [6]. Different techniques like label retaining cells, Hoechst-dye barring side population affirmed the stem cells [7– 9] and a novel population of stem-like cells coexpressing Lin28 and Oct-4 in epithelial ovarian growths have been stated [10]. Flesken-Nikitin et al. [11] verified the presence of stem cells in the OSE in the hilum locale as the niche for ovarian cancer cells. Present paper gives a short review of our present knowledge on ovarian stem cells, their source and characteristics, and how they are involved in postnatal oogenesis alongside an remarkable development that they express follicle stimulating hormone receptors (FSHR) and are modulated by FSH to exhibit self-renewal, clonal extension to form germ cell nests, proliferation, differentiation, and primordial follicle (PF) assembly in adult ovary. Adult Mammalian Ovary harbours Stem Cells, Progenitors, and Germ Cell Nests The adult mammalian ovary is responsible for providing mature and competent oocytes for reproduction. In addition, it is responsible for the secretion of various hormones and growth factors and cytokines that are involved in signalling pathways of folliculogenesis and oogenesis. It is a vibrant organ lined by a solitary layer of cuboidal surface germinal epithelial cells which is generally less defferentiated and uncommitted and under typical conditions express epithelial and mesenchymal markers. OSE plays role in ovulation, arrival of the mature oocyte, ensuing ovarian redesigning, and repair of follicle dividers and thus turns into a spasmodic layer in the event of anovulatory cycles anovulatory cycles, polycystic ovarian disorder and amid menopause and in sclerotic ovaries [6].

First confirmation for the presence of ovarian stem cells in OSE was given by Tilly's group [2] when they demonstrated MVH and BrdU coexpressing cells in the OSE alongside meiotic markers (Scp3, Spo11, and Dmc1) and that on uniting wild type ovary in GFP mice prompted development of follicles with GFP oocyte encased by wild-type granulosa cells. From that point different research groups xame forward to involve and examine ovarian stem cells with the assistance of diverse methodologies like immunomagnetic antibody and flow cytometry based cell arranging procedures (MACS and FACS), in vitro culture and differentiation of ovarian stem cells, genetic linkage tracing and transplantation tests, proposing that the follicle pool is certainly not a static however to be sure a dynamic populace of differentiating and regressing structure in adult mice and human ovary.A detailed report on stem cells in adult mammalian ovaries was first time given by Tilly’s and Bukovsky’s groups . Both the groups found a favourable influence of bone marrow cells on ovarian function. Whereas Bukovsky’s team found that OSE cells gives rise to structures somewhat similar to the oocytes in vitro at the same time Tilly’s and Virant-Klun’s groups reported the presence of stem cells in POF and menopausal women.. Tilly’s team successfully validated PF niche in ovarian cortical tissue samples in vitro by using PGC’s, germ cells, and primordial oocyte specific markers. [21].

Virant-Klun’s group has reported the presence of spherical, very small 4 ????m cells which express pluripotent and PGC specific markers [23]. Johnson et al. [18] reported the presence of PGCs (Stella, Fragilis, and Nobox) and germ cells (Oct4, Mvh, and Dazl) specific transcripts in bone marrow. Bhartiya team with the help of Bukovsky reported the presence of stem cells in sheep, monkey, rabbit and human OSE and for the first time they demonstrated that the OSE is the home to two distinct types of stem cells including (i) spherical cells which were smaller than RBCs in agreement with Virant-Klun’s observations and (ii) a slightly bigger population of “progenitors.” Immunolocalization studies showed that the smaller cells were pluripotent and expressed nuclear OCT-4, whereas the bigger ones expressed cytoplasmic OCT-4. An extensive review of literature revealed that Professor Ratajczak’s group had reported similar cells termed very small embryonic-like stem cells (VSELs) in various adult tissues [24]. The VSELs are the smaller cells with nuclear OCT-4 markers and the cells with cytoplasmic OCT-4 were the germ stem cells (OGSCs) and resembles the oogonial stem cells (OSCs) described by Tilly’s group. The exhaustive procedures to study these cells (VSELs, OGSCs, and GCN) by mechanical scraping of bigger sized mammalian ovaries and after enzymatic digestion of mouse OSE were recently described by us [12]. Presence of germ cell markers in bone marrow and expression of PGC markers on these stem cells hints to the presence of a common population of VSELs in bone marrow/peripheral blood and ovary as suggested by Ratajczak’s group [25]. Presence of stem cells and GCN in adult ovary contradicts the report by Lei and Spradling [26] and technical reasons resulting in the discrepancy have been discussed [22]. Existence of stem cells in mammalian ovary has not yet been all around acknowledged; rather there are groups who have produced evidence against the presence stem cells in adult mammalian ovary. This obviously suggests that further research is required in the field. Above survey of writing demonstrate that foundational microorganisms do exist in the OSE and it presently ends up to see how these stem cells function and add to postnatal oogenesis in ordinary adult mammalian ovaries.

FSHR presence and FSH action on Ovarian Stem CellsThe present belief in reproductive biology suggests that in the ovary only granulosa cells harbour the FSHR and the initial growth of follicle is gonadotropin independent. Various scientists have already pulished reports on various concepts regarding the FSHR existence and action in ovarian stem cells. Sairam’s group has reported that an alternative splicing of ovarian and testicular FSHR of sheep produces 4 distinct isoforms of which FSHR1 and FSHR3 have biological roles [28]. Babu et al. [29] stated that when mouse ovary is exposed to PMSG treatment then FSHR isoforms are formed with varied expression. Both isoforms FSHR1 and FSHR3 were detected by RT-PCR in normal ovary and it was found that the expression of FSHR3 was selectively increased after 24 and 48 h of PMSG treatment. Western blotting confirmed the presence and upregulation of FSHR3 in ovary after PMSG treatment by using a FSHR3 specific peptide IgG,. Sullivan et al. [30] have studied relative mRNA expression for alternately spliced FSHR transcripts (FSHR1, FSHR2, and FSHR3) and LHR [14].

These outcomes have significance particularly in light of the way that no critical affiliation has been seen between transformations or single nucleotide polymorphisms (SNPs) in the undoubted FSHR1 with premature ovarian failure and infertility. Bartiya group in their review have delibrated about the probable role of FSHFSHR3-stem cells association in the OSE bringing about ovarian cancers, POF, and menopause and how the scientists have been deceived by screening for changes in FSHR1 with an emphasis on exon 10 though FSHR3 may have a more huge part (has exon 11 and needs exons 9 and 10) in this manner clarifying the amassed negative information on absence of transformations in FSHR in women with POF and cancer [31].The fact findings by this team propose a unique activity of FSH on the stem cells situated in the OSE and call for outlook change in the field of reproductive biology. A recent study describes presence of gonadotropin receptors on human bone marrow hematopoietic progenitors including VSELs [32] supporting a developmental link between hematopoiesis and the germline. It turns out to be to a great degree bewildering with respect to how FSH acts on the ovary when an infertility expert treats a woman in the clinic to collect eggs or assisted reproduction. Is FSH really only playing a survival role on ovarian follicles, avoiding cell demise of a cohort of eggs when they begin developing or is it that FSH treatment applies coordinate activity on the ovarian stem cells preventing cell demise of a cohort of eggs when they start growing or is it that FSH treatment exerts direct action on the ovarian stem cells and an altogether new cohort of follicles assembles and starts growing starting from the stem cells! We need better means to interpret these well-kept mysteries of Mother Nature on the surface of the ovary. In humans the process of initiation of maturation of the follicles from the primordial pool is totally gonadotropins independent. Though FSH is a chief factor regulating folliculogenesis, the “initial recruitment” of human PF is mostly controlled by factors synthesized in the ovaries [1].

In general FSH is secreted in high levels at mid cycle (preovulatory surge) but there is another smaller peak which occurs during late luteal phase and is termed the “intercycle peak” in humans or the “proestrus peak” (secondary surge) in rodents and is understood to be associated with recruitment of follicles for the next cycle. Rani and Moudgal [60] showed that rather than the “preovulatory” FSH peak, the “proestrus” peak affects follicular growth and blocks ovulation in the next cycle. It is probably this intercycle peak of FSH that triggers stem cell activity in the OSE, resulting in PF assembly [15] and these follicles then rapidly grow and mature. But more carefully planned studies need to be undertaken to generate more evidence to support this preliminary observation. Germline Stem Cell Niche in Adult OvariesA characteristic property of the stem cell activity found in adult mammalian ovaries is the location of cysts germ cell nests) in it [26]. In the recent years Zhang et al. [27] while using 3 genetically modified adult mice models gave strong evidence criticising the activity of stem cell and postnatal oogenesis.

Their researches propose that there is no production of oocytes from stem cells in adult ovary and somatic cells don’t get recruited to aid primordial follicle assembly with de novo regenerated oocytes. As against to their experiment where genetic manipulations are done to answer a biological question, Bhartiya team used a more technical approach to give compelling answers to the same question of postnatal oogenesis [16]. Firstly they characterized the stem cells in enzymatically separated OSE cells and used flow cytometry to study LIN−/CD45−/SCA+ VSELs in normal (0.02 + 0.01%) adult ovaries and chemoablated (0.03 + 0.017%) mouse ovary. The VSELs survive chemotherapy and 48 h of PMSG treatment to chemoablated ovary resulted in increase in their numbers (0.08 + 0.03%) accompanied by initiation of neo-oogenesis in the OSE layer. The process was modulated by FSH both in culture of mechanically isolated OSE cells and adult chemoablated intact ovary culture. Successful formation of germ cell nests was observed and the manuscript was accepted for publication after a very strict review process. Oocytes can be obtained from endogenous VSELs/OSCs or iPS cells Horan and Williams [36] in their review suggested the probable use of induced pluripotent stem cells (iPS) cells replacing the OSCs to obtain oocytes in future. However, these cells have the capability of epigenetic memory retention of the somatic cells from which they originate and also show genomic and mitochondrial DNA mutations thus restricting their clinical use [14,17,19]. Hence, an aged infertile couple cannot be suggested to use autologous adult fibroblasts to obtain iPS cells which in turn could be differentiated into oocytes. Even for other clinical conditions, it is being advocated to use allogeneic rather than autologous iPS cells [20]. Certain growth factors and cytokines are derived from transplanted cells and are responsible for the differentiation of persisting VSELs in chemoablated testes into sperm. Experiments in the similar manner could not be performed in mice ovaries because of their small size which reduces further after chemotherapy. In this regard mesenchymal cells (MSCs) transplantations would prove beneficial to improve ovarian function reviewed recently [23].

It is interesting that transplanting MSCs replenishes normal ovarian function/morphology (but do not themselves form oocytes) and rather endogenous oogenesis is restored (possibly from the VSELs that survive cytotoxic insult). Similarly, Tilly’s group reported that transplanting bone marrow or peripheral blood cells in chemoablated ovary resulted in restoration of function and formation of oocytes from endogenous stem cells [18]. Taking all the interesting data collectively from various laboratories into consideration, it can be stated that a quiescent population of VSELs in adult mammalian ovary which survive oncotherapy should be appreciated and further exploited to restore ovarian function. Positive relationship between VSELs and PCOS was shown by Bhartiya group [49], but needs to be investigated in detail. Zhang et al. [27] further stated that rather than the ovarian stem cells, embryonic and induced pluripotent stem cells (iPS) have a promising future to generate oocytes for fertility treatment. Research efforts to make oocytes using mES cells are ongoing for last 30 years and recent efforts for using hES cells are ongoing for more than 15 years. Similarly iPS cells have also been used to make gametes in vitro but as concluded recently obtaining gametes from these stem cells remains a distant dream [33]. Bhartiya team recently discussed and compared the ovarian/testicular VSELs with ES/iPS cells and why VSELs (isolated from adult human, sheep, monkey, and rabbit OSE) and from chemoablated mice ovaries spontaneously differentiate into oocytes [16, 63]. Also for the first time, they succeeded to spontaneously differentiate testicular VSELs enriched from chemoablated mouse testis into sperm [35]. It is difficult to convert ES/iPS cells into PGCs as they have very distinct epigenetic status which cannot be replicated easily in a culture dish. On the other hand, VSELs are considered equivalent to PGCs and show excellent ability to differentiate into gametes.Germ Stem Cells and Ovarian CancerIt is clinically imperative to deliberate the probable contribution of ovarian somatic stem cells, [37-38] or recently discovered GSCs in the pathogenesis of ovarian cancer [12]. Compelling evidence supports the role of stem cells in ovarian cancer.

The high rate of chemoresistance recurrence observed in ovarian cancer correlates with the property of stem cells to remain in a quiescent state, rendering stem cells resistant to cytotoxic drugs that target mitotic cells [39]. It is known that stem cells share many characteristics with cancer cells. They both are able to self-renew and proliferate for a long period of time under specific conditions[12,40,41] Analogous to normal stem cells, cancer cells are thought to possess the capacity for unlimited self-renewal through symmetric cell division, the ability to give rise to progeny cells through asymmetric division, and an innate resistance to cytotoxic therapeutics.[40] While the process of differentiation initiated by a normal stem cell ultimately results in a specialized progeny with no proliferative potential, a cancer cell gives rise to progeny that do not undergo terminal differentiation but instead exhibit uncontrolled proliferation.[42] The normal interplay between somatic cells and stem cells is crucial for maintaining normal stem cell function.[43] A disturbance in the balance between these two compartments may lead to abnormal stem cell behaviour, eventually leading to cancer.[42] Understanding the possible involvement of somatic ovarian stem cells or GSCs in the pathogenesis of ovarian cancer may provide new therapeutic strategies for patients with infertility problem.A further understanding and potential manipulation of adult female GSCs may provide these answers.

Conclusion and Future Perspective

To conclude, data collected from numerous laboratories and citing literature clearly suggests the existence of stem cells in adult ovary surface epithelium. In addition, a recurrent question concerns the physiological role of GSCs in the adult mammalian ovary. Nonetheless, the isolation and characterization of GSCs from human ovaries open new perspectives to design protocols for producing mature, competent oocytes in vitro for clinical application in assisted reproduction techniques (ART). It has been shown that stem cells in the OSE comprise smaller VSELs and slightly larger progenitors (OGCSs/OSCs) equivalent to SSCs in the testes. They have the ability to undergo postnatal oogenesis, differentiate into oocytes, and undergo PF assembly under the influence of FSH which acts through alternatively spliced FSHR3 isoform. This increasing understanding of ovarian stem cell biology led to initial success of restoration of function in chemoablated adult mice ovary. Further studies are warranted to confirm this amazing success achieved so far by various groups around the globe in the field.