Novel Action of FSH on Stem Cells in Adult Mammalian Ovary Induces Postnatal Oogenesis and Primordial Follicle Assembly

Table 1

Group	Year	Studies conducted	References
Jonathan L. Tilly	2004	They challenged the central dogma of fixed number of follicles. Cells in ovary surface epithelium expressed SCP3, MVH-BrdU.	Johnson et al. [2]
	2005	Bone marrow and peripheral blood were shown as a source of germ cells. Germ line markers were found in the bone marrow. Bone marrow and peripheral blood transplantation restored oocyte production in chemoablated (and also after total body irradiation) wild-type and mutant mice. Bone marrow transplantation in chemoablated ovaries resulted in formation of oocytes, but surprisingly from the endogenous cells.	Johnson et al. [18]
	2007	Aged mouse ovaries harbor stem cells expressing Stra8 and Dazl but no oocytes. These stem cells retain the ability to undergo neo-oogenesis when grafted in young wild type mice.	Lee et al. [19]
	2009	Germ line stem cells were isolated from adult mouse ovary and human cortical tissue by FACS using DDX4 as a marker. These cells could be expanded for months in vitro and spontaneously differentiated into 35–50 µm oocytes. Human cells from reproductive age group were tagged with GFP and injected into human cortical tissue and resulted in GFP positive oocytes in immunodeficient mice.	Niikura et al. [20]
	2012	They described and validated FACS based protocol to isolate rare mitotically active germline stem cells from adult mouse ovaries and human ovarian cortical tissue, which upon further passage could give rise to 35–50-µm oocytes in vitro validated by various methods, as well as generated oocytes in vivo upon xenotransplantation into immunodeficient female mice.	White et al. [21]
	2013	Gene expression profiles of Ddx4 sorted OSCs and cultured OSCs, ESCs, PGCs, and SSCs were compared. OSCs expressed germline markers but distinct signatures as compared to that of PGCs and SSCs. In vitro culture of OSCs triggered pluripotency gene expression similar to PGCs	Imudia et al. [22]
	2013	Bone morphogenetic protein 4 promotes mammalian oogonial stem cell differentiation and results in increased expression of meiosis specific markers (Stra8, Msx1, and Msx2) via Smad 1/5/8 activation	Park et al. [23]
Antonin; Bukovsky	2004	Mesenchymal cells in tunica albuginea are bipotent progenitors which can differentiate into both granulosa and germ cells. They studied adult human ovarian tissue and concluded that granulosa cells originate in the OSE by epithelial-mesenchymal transition.	Bukovsky et al. [24]
	2005	Cultured adult human OSE cells in medium without phenol red led to development of granulosa-like cells and epithelial and neural and mesenchymal type cells. When OSE cells were cultured in the presence of phenol red medium, it resulted in the formation of >180 µm oocyte-like structures which exhibited germinal vesicle breakdown, polar body, and surface expression of zona pellucida proteins.	Bukovsky et al. [25]
	2008	Described neo-oogenesis and follicular assembly in adult ovary. Observed expression of meiotic entry synaptonemal complex protein 3 (SCP3)—a marker for meiosis in OSE.	Bukovsky et al. [26]
	2012	Follicular renewal in rodents is initiated by bone marrow derived cells related to the immune system, which interact with OSE cells in normal adult rats or medullary sex cord cells in adult neonatally estrogenized rats lacking OSE.	Bukovsky and Caudle [27]
Irma Virant-Klun	2008	OSE cells from 20 postmenopausal and 5 women with premature ovarian failure were used to isolate putative stem cells. Small round cells with a bubble-like structure, 2 to 4 µm in size, were detected which expressed pluripotent markers SSEA4, Oct-4, Nanog, Sox-2, and c-kit.	Virant-Klun et al. [28]
	2009	Ovarian tissue from postmenopausal women was cultured to study oogenesis in vitro. Small round cells with a bubble-like structure and with a diameter from 2 to 4 μm were isolated from OSE. They expressed pluripotent markers including SSEA-4, Oct-4, Nanog, Sox-2, and c-kit. These cells on culture proliferated and formed embryoid body-like structures but did not form teratoma in SCID mice. Cells grew in size and reached a diameter of approximately 95 µm and expressed Oct-4, c-kit, VASA, and ZP2. Few expressed a zona pellucida-like structure, germinal vesicle, and polar body-like structures. Parthenote embryos were also observed which expressed Oct-4, Sox-2, and Nanog and were normal for chromosomes X, 13, 16, 18, 21, and 22.	Virant-Klun et al. [29]
	2011	Small, round SOX-2 positive stem cells were detected in the OSE of women with premature ovarian failure and high FSH and LH. On culture with follicular fluid to provide ovarian niche, primitive oocyte-like structures and cell clusters were observed which were alkaline phosphatase positive and expressed pluripotent markers SOX-2 and SSEA-4. Single oocyte-like cells expressed genes OCT-4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells.	Virant-Klun et al. [30]
	2013	Small putative SSEA-4 positive stem cells up to 4 µm in size were isolated by FACS and MACS from cultures of human cortical tissue. They expressed pluripotent markers but were relatively low compared to hES cells. In addition, they expressed genes of primordial germ cell lineages VASA, PRDM1, PRDM14, and DPPA3.	Virant-Klun et al. [31, 32]
	2013	Ovarian cell cultures could be established from cortical biopsies and the cells expressed pluripotent markers (alkaline phosphatase, SSEA-4, OCT-4, SOX-2, NANOG, LIN28, and STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, and STRO-1). These cells were SSEA-4 positive, spherical in shape, and small up to 4 µm in size. These cells could be differentiated into 3 germ layers but did not form teratoma in immunodeficient mice.	Stimpfel et al. [33]
Deepa; Bhartiya	2011	Two distinct populations of stem cells were detected in OSE isolated from adult rabbit, monkey, sheep, and human ovaries. Spherical cells with high N/C ratio were smaller than RBCs in size and expressed nuclear OCT-4 and SSEA-4 and the bigger cell population expressed cytoplasmic OCT-4 and minimal SSEA-4. Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 were detected in human and sheep OSE cells by RT-PCR. The stem cells underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition. Oocyte-like structures expressed c-KIT, DAZL, GDF-9, VASA, and ZP4.	Parte et al. [13]
	2012	PMSG (5IU) was observed to exert direct proliferative effect on OSE and increased expression of FSHR and PCNA. OSE appeared multilayered at several positions and MVH positive germ cell nests and cohort of newly formed PF were visualized along with increased expression of Oct-4A, Nanog, Scp-3, Oct-4B, and Mvh. Study provided first evidence that stem cells activity in OSE and neo-oogenesis is modulated by FSH in adult mammalian ovaries. Similar findings were also observed in normal estrus cycle coinciding with the proestrus peak of FSH and numbers of cohorts along the surface of the ovary were increased after PMSG treatment.	Bhartiya et al. [15]
	2013	Effect of FSH and bFGF was studied on human and marmoset ovarian cortical tissues in organ culture format. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. Both FSH and bFGF induced proliferation of OSE along with increased expression of gene transcripts specific for pluripotent stem cells (Oct-4A and Nanog) suggestive of VSELs and early germ cells (Oct-4, c-Kit, and Vasa) suggestive of OGSCs and follicular transition (oocyte-specific Gdf-9 and Lhx8, and granulosa cell specific Amh).	Parte et al. [17]
	2013	Effect of FSH was studied on sheep stem cells in OSE in vitro. FSH increased stem cells self-renewal and clonal expansion evident by the appearance of stem cell clusters. FSH receptors were expressed on ovarian stem cells whereas the epithelial cells were distinctly negative. An increase in R3 mRNA transcripts was noted after 3 hrs of FSH treatment and was reduced to basal levels by 15 hrs, whereas R1 transcript expression remained unaffected. FSHR and OCT4 were immunolocalized in nuclei of stem cells and showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles. Thus FSH appears to modulate ovarian stem cells activity via FSH-R3 to undergo potential self-renewal, clonal expansion as “cysts,” and differentiation into oocytes. OCT-4 and FSHR proteins (required initially to maintain pluripotent state of VSELs and for FSH action, respectively) gradually shift from nuclei to cytoplasm of developing oocytes and are later possibly removed by surrounding granulosa cells as the oocyte prepares itself for fertilization.	Patel et al. [14]
	2013	A genetic lineage tracing study which failed to detect stem cells and germ cell clusters in adult mouse ovary was challenged. Cysts were observed and confocal microscopy imaging confirmed cytoplasmic continuity amongst the cells comprising the cysts. Germ cell nests expressed PCNA and SSEA-4 suggestive of their germ cell characteristic and rapid mitotic division.	Bhartiya et al. [34]
	2014	Ovarian stem cells and germ cell clusters were enriched by immunomagnetic sorting using SSEA-4 and were further characterized. Differential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, and CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, and VASA), and germ cells (DAZL, GDF-9, and SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud), and exhibited characteristic cytoplasmic streaming.	Parte et al. [35]
	2015	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. VSELs in adult mouse ovary were SCA-1+/Lin−/CD45− and positive for nuclear OCT-4, Nanog, and SSEA-1. VSELs survived chemotherapy and OSE culture of chemoablated ovary OSE resulted in appearance of proliferating germ cell clusters and MVH and GDF9 positive oocyte-like structures spontaneously differentiated by day 6. FSH exerted a direct stimulatory action on the OSE and induced stem cell proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovary culture. PMSG treatment to chemoablated mice resulted in self-renewal of VSELs (LIN−/CD45−/SCA1+) that were 0.02 ± 0.008% in normal ovary and 0.03 ± 0.017% in chemoablated ovary and PMSG treatment to chemoablated ovary increased VSELs to 0.08 ± 0.03%.	Sriraman et al. [16]