The humunosuppressant rapamycin (RAP) has been demonstrated to specifically inhibit the activity of p70 S6 kinase (p700) and subsequent phosphorylation of ribosomal S6 [3HJThymidine and 3H-Labeled Ami Acid ('H-Amino Acid) Incorporation. DNA synthesis was evaluated by incorporation of [3H]thymidine into cells as described (3). General protein synthesis was evaluated by incorporation of 3H-amino acid mixture (Amersham) into cells. After labeling cells with 5 KCi (1 Ci = 37 GBq) ofthe 3H-amino acid mixture per ml, cells were washed twice with ice-cold phosphatebuffered saline (PBS) and then lysed in PBS containing 1% SDS followed by addition of 7% trichloroacetic acid/1% pyrophosphate. The precipitates were loaded on GF/A filters and washed extensively with 7% trichloroacetic acid/1% pyrophosphate. Radioactivity was measured by scintillation counting.Kinase Assay of p70W. Specific activity of p706k was determined by 32p incorporation into S6 peptide in the immune complex as described (4).[ Abbreviations: RAP, rapamycin; p7086k, p70 S6 kinase; eEF-2, elongation factor 2; eEF-la, elongation factor la; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNP, mRNA-ribonucleoprotein complex; PCNA, proliferating cell nuclear antigen; DHFR, dihydrofolate reductase. tTo whom reprint requests should be addressed. 11477The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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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.

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