Kalpana Sriraman

Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries

BackgroundFollicle stimulating hormone (FSH) exerts action on both germline and somatic compartment in both ovary and testis although FSH receptors (FSHR) are localized only on the somatic cells namely granulosa cells of growing follicles and Sertoli cells in the seminiferous tubules. High levels of FSH in females are associated with poor ovarian reserve, ovarian hyper stimulation syndrome etc. and at the same time FSH acts as a survival factor during in vitro organotypic culture of ovarian cortical strips. Thus a further understanding of FSH action on the ovary is essential. We have earlier reported presence of pluripotent very small embryonic-like stem cells (VSELs express Oct-4A in addition to other pluripotent markers) and their immediate descendants ‘progenitors’ ovarian germ stem cells (OGSCs express Oct-4B in addition to other germ cell markers) in ovarian surface epithelium (OSE) in various mammalian species including mice, rabbit, monkey, sheep and human. Present study was undertaken to investigate the effect of pregnant mare serum gonadotropin (PMSG) on adult mice ovaries with a focus on VSELs, OGSCs, postnatal oogenesis and primordial follicle assembly.MethodsOvaries were collected from adult mice during different stages of estrus cycle and after 2 and 7 days of PMSG (5 IU) treatment to study histo-architecture and expression for FSHR, pluripotent stem cells , meiosis and germ cell specific markers.ResultsPMSG treatment resulted in increased FSHR and proliferation as indicated by increased FSHR and PCNA immunostaining in OSE and oocytes of primordial follicles (PF) besides the granulosa cells of large antral follicles. Small 1–2 regions of multilayered OSE invariably associated with a cohort of PF during estrus stage in control ovary were increased to 5–8 regions after PMSG treatment. This was associated with an increase in pluripotent transcripts (Oct-4A, Nanog), meiosis (Scp-3) and germ cells (Oct-4B, Mvh) specific markers. MVH showed positive immuno staining on germ cell nest-like clusters and at places primordial follicles appeared connected through oocytes.ConclusionsThe results of the present study show that gonadotropin (PMSG) treatment to adult mouse leads to increased pluripotent stem cell activity in the ovaries, associated with increased meiosis, appearance of several cohorts of PF and their assembly in close proximity of OSE. This was found associated with the presence of germ cell nests and cytoplasmic continuity of oocytes in PF. We have earlier reported that pluripotent ovarian stem cells in the adult mammalian ovary are the VSELs which give rise to slightly differentiated OGSCs. Thus we propose that gonadotropin through its action on pluripotent VSELs augments neo-oogenesis and PF assembly in adult mouse ovaries.

Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation

This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin−/CD45−, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.

Very Small Embryonic-Like Stem Cells Survive and Restore Spermatogenesis after Busulphan Treatment in Mouse Testis

Study Objectives: Adult mammalian testes harbor a novel population of quiescent, pluripotent, very small embryonic-like stem cells (VSELs) along with spermatogonial stem cells (SSCs). Present study was undertaken to (i) characterize testicular VSELs (ii) investigate differential effect of chemotherapy on VSELs and SSCs and (iii) to restore the differentiation ability of surviving VSELs by providing healthy somatic microenvironment. Methods: Effect of busulphan (25 mg/Kg) was studied on mouse testes. Syngenic Sertoli cells(105 cells per testis) and bone marrow derived mesenchymal cells (104 cells per testis) were transplanted separately through intertubular route. Effect of niche reconstruction was studied two months later by histology and immuno-localization of germ cell markers MVH and PCNA. Caudal sperm were evaluated for their ability to fertilize oocytes in vitro. Results: VSELs were 2-6 μm in size, SCA-1+/CD45-/LIN-, had high nucleo-cytoplasmic ratio and comprised 0.03% of testicular cells whereas SSC specific marker GFRa localized on a distinct, larger cell population. Busulphan selectively destroyed SSCs and other germ cells however, nuclear OCT-4A, Nanog, Sox-2 and SCA-1 positive VSELs (0.06%) survived. Persisting VSELs were unable to differentiate possibly because chemotherapy also affected the ‘niche’ comprising Sertoli cells. Complete restoration of spermatogenesis was observed two months post transplantation of Sertoli and mesenchymal cells. Transplanted cells formed neo-tubules in the vicinity of surviving tubules and were possibly a source of growth factors essential for VSELs proliferation and differentiation. Both MVH and PCNA showed increased staining in the transplanted group. qRT-PCR studies revealed existence of a meiotic block in busulphan treated testis which was overcome after transplantation. Resulting sperm progressed to epididymis, showed normal motility and ability to fertilize in vitro. Conclusion: Results show that VSELs survive chemotherapy and can restore spermatogenesis in germ cells depleted mice. Results have direct relevance to address fertility issues of cancer survivors.

Stem cell interaction with somatic niche may hold the key to fertility restoration in cancer patients.

The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs), and slightly larger “progenitor” ovarian germ stem cells (OGSCs). Besides compromising the somatic niche, oncotherapy destroys OGSCs since, like tumor cells, they are actively dividing; however VSELs persist since they are relatively quiescent. BMT or transplanted ovarian cortical tissue may help rejuvenate the ovarian niche, which possibly supports differentiation of persisting VSELs resulting in neo-oogenesis and follicular development responsible for successful pregnancies. Postnatal oogenesis in mammalian ovary from VSELs may be exploited for fertility restoration in cancer survivors including those who were earlier deprived of gametes and/or gonadal tissue cryopreservation options.

Isolation and characterization of stem cells in the adult mammalian ovary.

Female mammals are born with a fixed pool of germ cells, which does not replenish during adult life. However, this has been recently challenged and adult ovaries produce oocytes throughout adult life just like sperm in the testes. Evidence is accumulating on the presence of ovarian stem cells, but the need for robust protocols to isolate, identify, further characterize, and subject them to various functionality tests is essential. Knowledge about the function and potential of ovarian stem cells is well demonstrated by various groups, but their true identity remains elusive because of the variability in the approaches used to identify them by different groups. In order to address this we have made attempts to compile our protocols to isolate, identify, characterize, and culture the stem cells using different animal models including human. Two distinct populations of stem cells exist in adult mammalian ovary, including very small embryonic-like stem cells (VSELs) and the progenitors termed ovarian germ stem cells (OGSCs). VSELs are relatively quiescent and undergo asymmetric cell division to give rise to OGSCs, which divide rapidly, occasionally form germ cell nests and undergo meiosis and differentiation into oocytes, which are surrounded by granulosa cells to assemble as primordial follicles.

Pluripotent Very Small Embryonic-like Stem Cells in Adult Mammalian Gonads

The presence of very small embryonic-like stem cells (VSELs) in adult mammalian gonads is set to disrupt several existing paradigms in the field of reproductive biology. Being pluripotent, VSELs are present at the top of hierarchy among the tissue-specific stem cells. In the testis, they exist as a sub-population of small spherical cells with high nucleo-cytoplasmic ratio among the spermatogonial stem cells (SSCs) along the basement membrane of the seminiferous tubules. They undergo asymmetric cell division to self-renew, give rise to the SSCs and may also be responsible for the embryonic stem (ES) cell-like colonies observed on culturing testicular biopsy. The SSCs undergo rapid division (clonal expansion as chains), meiosis, and further differentiate into sperm. In the ovary, VSELs are lodged in the ovary surface epithelium (OSE) along with immediate progenitors termed ovarian germ stem cells (OGSCs). Ovarian VSELs also undergo asymmetric cell division to give rise to OGSCs which undergo clonal expansion to form nests (cysts) and further differentiate into oocytes which assemble as primordial follicles below the OSE. Stem cell function in the adult mammalian ovary is modulated by follicle stimulating hormone (FSH) via a novel FSH receptor isoform R3. These results are in contradiction to the existing paradigm that initial primordial follicle growth is independent of FSH and that it acts on the granulosa cells of growing follicles through a G protein-coupled FSH receptor. Being relatively quiescent in nature, VSELs survive oncotherapy in both testis and ovary but are unable to differentiate because of a compromised niche. Their functionality may be restored by providing a healthy niche. This newer understanding of VSELs biology in mammalian gonads will provide deep insight in various fields of reproductive health.