RETRACTED ARTICLE: Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3

Abstract

BackgroundWe have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants ‘progenitors’ termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FSH-R3 have biological activity. Present study was undertaken to identify FSHR isoforms mediating FSH action on ovarian stem cells, using sheep OSE cells culture as the study model.MethodsCultures of sheep OSE cells (a mix of epithelial cells, VSELs, OGSCs and few contaminating red blood cells) were established with and without FSH 5IU/ml treatment. Effect of FSH treatment on self-renewal of VSELs and their differentiation into OGSCs was studied after 15 hrs by qRT-PCR using markers specific for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4). FSH receptors and its specific transcripts (R1 and R3) were studied after 3 and 15 hrs of FSH treatment by immunolocalization, in situ hybridization and qRT-PCR. FSHR and OCT-4 were also immuno-localized on sheep ovarian sections, in vitro matured follicles and early embryos.ResultsFSH treatment resulted in 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. Both FSHR and OCT-4 were immuno-localized in nuclei of stem cells, showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles.ConclusionsFSH modulates ovarian stem cells 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.

FSH–FSHR3–stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer

“…Variable expression levels (highest in OSE and contralateral ovaries with and without OETs; lowest in antral follicles, SBTs, and peritoneal implants associated with SBTs, increasing parallel to proliferation activity of OETs and decreasing level with advancing carcinoma grade) were observed Stewart et al (2004) Both gonadotropins and hormones increased OSE proliferation in rats Tan & Fleming (2004) Performed PCNA staining in normal mouse OSE and found PCNA-positive cells at sites distant from the site of follicle rupture Burdette et al (2006) Both PMSG and hCG increased OSE proliferation that could occur before and also after ovulation. No signs of apoptosis were evident in OSE post-ovulation in CD1 mice Bhartiya et al (2012) PMSG treatment results in increased FSHR expression in adult mouse OSE Patel et al (2013) FSHR expressed on the stem cells and germ cell nests in sheep OSE smears and the surrounding epithelial cells are negative…”

“…Targeted disruption of FSHR in FORKO mice resulted in abnormal gametogenesis and hormonal imbalance (Dierich et al 1998). As shown in Table 4, two studies clearly showed that FSH/pregnant mare serum gonadotropin (PMSG) treatment results in selective upregulation of Fshr3 compared with Fshr1 in mouse granulosa cells (Babu et al 2001) and in sheep OSE cells (Patel et al 2013). Sullivan et al (2013) studied FSHR transcripts by qPCR in sheep follicles segregated based on the size into small, medium, and large.…”

“…Oocytes get surrounded by granulosa cells, which possibly arise by epithelial-mesenchymal transition of the epithelial cells to assemble as a primordial follicle. This process of neo-oogenesis and PF assembly in adult ovary is regulated by FSH via FSHR3 (Patel et al 2013). Any alteration in the FSH-FSHR-stem cell interaction will result in POF.…”

“…FSHR3 functions as a growth factor receptor and acts via calcium signaling and the MAPK/ERK pathway. It is expressed on ovary surface epithelial cells (Li et al 2007) specifically on the stem cells present in the OSE (Patel et al 2013) and also in follicles at different stages of development (Patel et al 2013, Sullivan et al 2013). …”

Epithelial ovarian cancer stem cells: underlying complexity of a simple paradigm

“…The somatic stem cells in the OSE have been investigated in three distinct contexts: potential stem cells able to regenerate germ cells (Parte et al 2011, Patel et al 2013, Virant-Klun et al 2013a, potential stem cells that give rise to granulosa cells (Hummitzsch et al 2013), and more traditional somatic stem cells whose role is to replenish the OSE following ovulatory wounding (Szotek et al 2008, Motohara et al 2011, Usongo & Farookhi 2012, Auersperg 2013, Flesken-Nikitin et al 2013, Ng et al 2014. While this review focuses briefly on studies that have addressed somatic stem cells that are thought to be required for ovulatory wound repair, the relationship with the HGSEOC of the two former potential stem cell pools cannot be discounted.…”