Present study demonstrates that pluripotent stem cells VSELs and progenitors OGSCs reported earlier by our group in adult mice, rabbit, sheep, monkey and human ovary
[32, 39] are possibly regulated by FSH and are responsible for postnatal oogenesis and follicular assembly during adult reproductive life. Normal adult mice ovaries were studied during different stages of estrus cycle and after 2 and 7 days of PMSG treatment by histology, immunolocalization (FSHR, PCNA, OCT-4, SCP3 and MVH), and RT-PCR/ Q-PCR for various gene transcripts including pluripotent (Oct-4A, Nanog); early germ cell (total Oct-4, Stella, Fragilis, Mvh), oocyte-specific (Nobox & HoxA10); pre-meiosis (Stra8) and meiosis (Scp3, Dmc1) specific markers. The process of ovarian stem cell differentiation and meiosis giving rise to new oocytes, which get enclosed by the granulosa cells to assemble as PFs in the OSE appears to be a normal feature of adult ovary (Figure
2). The granulosa cells arise by epithelial-mesenchymal transition of OSE as suggested earlier by us based on in vitro evidence
. Present study provides in vivo evidence in favor of our earlier observations that OSE cells may be involved in the formation of granulosa cells (Figure
6B,C, E & F). PMSG treatment further augments the process of postnatal oogenesis and PF assembly resulting in increased PFs by D7 (Figure
PMSG is known to exert its actions on mammalian ovary through FSH-FSHR interaction. In the present study, besides granulosa cells of bigger follicles, FSHR was detected in both OSE and oocytes of PF in agreement with earlier reports
[15–17, 19, 27, 28] and an up-regulation was observed after PMSG treatment at both protein by immunohistochemistry (Figure
7) and transcript (Figure
10) level. Presence of FSHR in locations like oocytes and OSE suggests that the various diverse functions of FSH are probably not exerted only through the classical FSHR present on the granulosa cells of growing follicles. Sairam’s group has earlier reported that FSH may exert diverse actions through alternatively spliced FSHR isoforms based on their studies from sheep and mice ovaries
In the current study, upregulation of FSHR after PMSG treatment correlated well to the proliferation observed in OSE. OSE proliferation and multilayer appearance which were normally observed in control ovaries at few focal areas were increased after PMSG treatment (Figure
4). Ovarian sections on 2D of PMSG treatment showed the presence hyperplastic epithelial cells and small protuberances bulging from the ovarian surface (Figure
3) which subside by 7D (Figure
6). PF cohorts normally observed with hyper-proliferative OSE regions in control ovaries, increased significantly after 7D of PMSG treatment in the ovarian cortex (Figure
5). This correlated well with increased mRNA expression for HoxA10- a primordial follicular oocyte marker
 and Nobox that is expressed abundantly in primordial and primary follicular oocytes
 post PMSG treatment. Occurrence of meiosis was supported by expression of pre-meiotic (Stra8) and meiotic (Scp3 and Dmc 1) markers. All the three markers were detected in control as well as all PMSG treated ovarian samples. A distinct up regulation of Scp3 was noted after PMSG treatment, both at the protein (Figure
8 H-J) and mRNA transcript level (Figure
10). As reported earlier
, SCP3 staining was observed throughout the nuclei suggesting that the oocytes are in prophase I of meiosis and was gradually lost in more developed oocytes. The PF at birth are generally arrested in diplotene stage of prophase 1 of meiosis. SCP3 expression is restricted to earlier stages i.e. zygotene and pachytene stage. Thus expression of Scp3 and pre-meiotic marker Stra8 in adult ovary are considered direct evidence in support of postnatal oogenesis and have been reported earlier by other groups also
It is intriguing to point out that OSE which expresses FSHR also lodges VSELs as observed in rabbit, sheep, monkey, human
 and also mouse ovaries
. The VSELs with nuclear OCT-4 differentiate into ‘progenitors’ ovarian germ stem cells (OGSCs) with cytoplasmic OCT-4 and undergo spontaneous differentiation into oocyte-like structures in vitro
[32, 37]. Possibly, in the present study, the pluripotent VSELs were activated by PMSG treatment (increased expression of Oct-4A, Nanog Figure
10), undergo proliferation (increased PCNA staining and Oct-4A expression, Figure
8) and differentiation (increased expression of stella, fragilis, total Oct-4, Vasa and MVH Figure
10) resulting in meiosis (Stra-8, Scp3 and Dmc1 Figure
10) and formation of oocyte nests with prominent intercellular cytoplasmic bridges that assemble to form PF in the OSE (Figure
6). These nests later shift to the cortex and are visualized as small cohorts on 7D after PMSG treatment. This data demonstrates the mechanism of oogenesis occurring in situ in adult ovary for the first time in OSE similar to well-studied spermatogenesis in the testicular seminiferous tubules where the VSELs are lodged at the basement membrane
[67, 68]. The process is regulated by FSH and is in contradiction to the existing paradigm that initial PF growth is gonadotropin independent
[69, 70]. The results of the present study support the model for postnatal oogenesis proposed by us earlier
Messinis et al.
 have reviewed a huge body of literature and summarized the current understanding of the role played by FSH during folliculogenesis. According to existing understanding, the ‘intercycle peak’ of FSH is crucial and responsible for ‘cyclic recruitment’ of follicles. Ovary experiences waves of folliculogenesis and a cohort of follicles start growing in each cycle, however, only one becomes dominant and others undergo atresia
. On treating with gonadotropins, FSH window gets widened and multiple follicles get selected and start to grow. Whether more follicles get recruited due to the treatment or FSH prevents atresia in the already recruited cohort of PF is still controversial. Results of the present study add another dimension to this by demonstrating that FSH also exerts action on the OSE and induces neo-oogenesis and PF assembly from the stem cells residing in the OSE.
The prevailing notion of extrusion of atretic oocytes from the ovary surface
[71–73] or that the PF may get pushed to the ovarian surface because of the growing follicles in response to the PMSG treatment (and thus increased number of PF are visualized after treatment in the present study) may not be valid since this reasoning cannot account for distinct increased pattern of pluripotent and meiotic markers in the developing follicles (Figure
9) observed in the present study. Also the earlier published reports of BrdU incorporation in OSE
[34, 36] may not be due to mtDNA synthesis – rather it may be due to stem cell activity that gets augmented by PMSG.
The extensive germ cell nest formation, initiation of meiosis, PF assembly etc. are well documented and studied during fetal ovarian development in the existing literature
. It is interesting to point out that the stage of fetal development when PF assembly occurs is actually associated with several folds higher levels of FSH in females
[2–6, 74]. Thus FSH is directly related to PF assembly and regular intercycle peak of FSH may be stimulating the VSELs to result in differentiation and PF assembly. Lei et al.
 reported that the high levels of circulatory FSH in perinatal period and increased FSHR mRNA in mouse ovaries facilitate germline nest breakdown and PF assembly.