The online version of this article (https://doi.org/10.1186/s13048-017-0377-5) contains supplementary material, which is available to authorized users.
Stem cells in the ovary comprise of two distinct populations including very small embryonic-like stem cells (VSELs) and slightly bigger progenitors termed ovarian stem cells (OSCs). They are lodged in ovary surface epithelium (OSE) and are expected to undergo neo-oogenesis and primordial follicle (PF) assembly in adult ovaries. The ovarian stem cells express follicle stimulating hormone (FSH) receptors and are directly activated by FSH resulting in formation of germ cell nests (GCN) in vitro. Present study was undertaken to further characterize adult sheep OSCs and to understand their role during neo-oogenesis and PF assembly.
Stem cells were collected by gently scraping the OSE cells and were characterized by H&E staining, immuno-localization, immuno-phenotyping and RT-PCR studies. Expression of FSH receptors and markers specific for stem cells (OCT-4, SSEA-4) and proliferation (PCNA) were studied on stem/progenitor cells in OSE culture and on adult sheep ovarian cortical tissue sections. Effect of FSH on stem cells was also studied in vitro. Asymmetric cell division (ACD) was monitored by studying expression of OCT-4 and NUMB.
Additional evidence was generated on the presence of two populations of stem cells in the OSE including VSELs and OSCs. FSHR expression was observed on both VSELs and OSCs by immuno-localization and immuno-phenotyping studies. FSH treatment in vitro stimulated VSELs that underwent ACD to self-renew and give rise to OSCs which divided rapidly by symmetric cell divisions (SCD) and clonal expansion with incomplete cytokinesis to form GCN. ACD was further confirmed by differential expression of OCT-4 in VSELs and NUMB in the OSCs. Immuno-histochemical expression of OCT-4, PCNA and FSHR was noted on stem cells located in the OSE in sheep ovarian sections. GCN and cohort of PF were observed in the ovarian cortex and provided evidence in support of neo-oogenesis from the stem cells.
Results of present study provide further evidence in support of two stem cells populations in adult sheep ovary. Both VSELs, OSCs and GCN express FSH receptors and FSH possibly regulates their function to undergo neo-oogenesis and primordial follicle assembly.
Additional file 1: Figure S1. The presence of two stem cell population in OSE by flow cytometry analysis is shown. Table S1. List of antibodies used in the study. Table S2. List of primers used in the study. Figure S2. H & E staining of freshly isolated sheep OSE smears (arrows represent small putative VSELs smaller then RBCs and asterisk represent OSCs and cell doublets (circled) show dividing stem cells). Figure S3. RT-PCR on freshly isolated OSE cells and granulosa cells surrounding oocytes. Figure S4. FSHR expression on ovarian stem cells. Figure S5a. Z stack of OCT-4 expressing germ cell clusters in FSH treated OSE cell culture. Figure S5b. Z stack of FSHR expressing germ cell clusters in FSH treated OSE cell culture. Figure S6. Co-expression of FSHR and SSEA-4 on ovarian stem cells. Figure S7. FSHR expression on proliferating and dividing ovarian stem cells cultured in vitro within OSE cells on FSH treatment. Figure S8. H&E staining of ovarian sections (A) shows a distinct layer of OSE cells (B-C) PCNA immuno-localization on sheep ovarian sections showed few cells in OSE positive for PCNA, cluster of oocytes/germ cell cyst, individual primordial and primary follicles located in cortical region of ovary showed strong nuclear PCNA with faint stain in ooplasm. Figure S9a. Negative control for ICC to show specificity of staining (i) Negative for VASA (ii) Negative for SSEA-4 and PCNA (iii) Negative for FSHR and OCT-4. Figure S9b. Negative control by peptide blocking of FSHR antibody to show specificity of staining obtained using FSHR. (PDF 1911 kb)13048_2017_377_MOESM1_ESM.pdf
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