Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture

Abstract

BackgroundCryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells.MethodsSerum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR.ResultsA prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. 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. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition.ConclusionThe present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

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

“…It is interesting to mention here our earlier report that human and marmoset ovarian cortical tissue besides being a source of primordial follicles is also an excellent source of ovarian stem cells. 32 …”

“…The results provide further support to the results obtained on intact ovary culture (with and without FSH, as discussed earlier) that FSH treatment leads to increase in mouse ovarian VSELs activity and our earlier published results. 32,35 To summarize, all the above-mentioned results (both OSE and whole ovary culture) suggest that the chemoablated mouse ovaries, which lack oocytes, can be reinstated to have oocytespecific differentiation from surviving stem cells. As only VSELs were observed to persist in chemoablated ovary, the differentiation to germ cells observed can be presumed to have initiated from surviving VSELs.…”

“…Many studies have shown that ovarian stem cells are present in aged mouse and in perimenopausal women. 2,6,32,36,39,51 Niikura et al 39 demonstrated that the stem cells from aged ovary retain differentiation potential and form oocytes when exposed to young ovarian environment. Other studies have independently reported that the stem cells from perimenopausal women retain potential to differentiate into oocyte-like structures and parthenotes in in vitro conditions in which the inhibitory factors that exist in vivo are overcome.…”

“…Second, as mentioned in Introduction section, we have reported that FSH modulates ovarian stem cells. 31,32,35 Ovaries were isolated from surrounding tissue in an sterile environment and rinsed 2 to 3 times sequentially in sterile Dulbecco PBS, a-minimum essential medium (aMEM; Life Technologies) containing 1Â penicillin-streptomycin (Pen-Strep; Life Technologies) and finally in serum-free culture medium aMEM containing 3 mg/mL bovine serum albumin (BSA; Sigma-Aldrich), 5Â insulin transferrin and selenium (Sigma-Aldrich), 2 mmol/L sodium pyruvate (Sigma-Aldrich), linoleic acid (Sigma-Aldrich), and 1Â PenStrep. Ovaries were cultured on 0.4-mm pore size and 12 mm diameter Millicell inserts (EMD Millipore, Billerica, Massachusetts) in culture medium with 10 mIU FSH (FSH-plus group) or without FSH (FSH-minus group).…”

“…30 Our group has shown that ovarian stem cells (1) spontaneously differentiate into oocyte-like structures in culture; 2 (2) exhibit process of cytoplasmic streaming, germ cell nest, and Balbiani body formation in vitro 17 ; (3) express follicle-stimulating hormone receptors (FSHRs) and are modulated by FSH and bFGF. 31,32 We have also shown that the alternative FSHR isoform-FSHR3 (a growth factor type 1 receptor) 33 is involved in the activation of ovarian stem cells by FSH and results in germ cell differentiation 31,34 and pregnant mare serum gonadotropin (PMSG-a FSH analog) treatment augments the process of neo-oogenesis and primordial follicle assembly in mice. 35 Other groups have also supported the presence of pluripotent stem cells in ovary and their ability to form oocytes in vitro.…”