Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries?

Abstract

BackgroundFollicle stimulating hormone (FSH) exerts action on both germline and somatic compartment in both ovary and testis although FSH receptors (FSHR) are localized only on the somatic cells namely granulosa cells of growing follicles and Sertoli cells in the seminiferous tubules. High levels of FSH in females are associated with poor ovarian reserve, ovarian hyper stimulation syndrome etc. and at the same time FSH acts as a survival factor during in vitro organotypic culture of ovarian cortical strips. Thus a further understanding of FSH action on the ovary is essential. We have earlier reported presence of pluripotent very small embryonic-like stem cells (VSELs express Oct-4A in addition to other pluripotent markers) and their immediate descendants ‘progenitors’ ovarian germ stem cells (OGSCs express Oct-4B in addition to other germ cell markers) in ovarian surface epithelium (OSE) in various mammalian species including mice, rabbit, monkey, sheep and human. Present study was undertaken to investigate the effect of pregnant mare serum gonadotropin (PMSG) on adult mice ovaries with a focus on VSELs, OGSCs, postnatal oogenesis and primordial follicle assembly.MethodsOvaries were collected from adult mice during different stages of estrus cycle and after 2 and 7 days of PMSG (5 IU) treatment to study histo-architecture and expression for FSHR, pluripotent stem cells , meiosis and germ cell specific markers.ResultsPMSG treatment resulted in increased FSHR and proliferation as indicated by increased FSHR and PCNA immunostaining in OSE and oocytes of primordial follicles (PF) besides the granulosa cells of large antral follicles. Small 1–2 regions of multilayered OSE invariably associated with a cohort of PF during estrus stage in control ovary were increased to 5–8 regions after PMSG treatment. This was associated with an increase in pluripotent transcripts (Oct-4A, Nanog), meiosis (Scp-3) and germ cells (Oct-4B, Mvh) specific markers. MVH showed positive immuno staining on germ cell nest-like clusters and at places primordial follicles appeared connected through oocytes.ConclusionsThe results of the present study show that gonadotropin (PMSG) treatment to adult mouse leads to increased pluripotent stem cell activity in the ovaries, associated with increased meiosis, appearance of several cohorts of PF and their assembly in close proximity of OSE. This was found associated with the presence of germ cell nests and cytoplasmic continuity of oocytes in PF. We have earlier reported that pluripotent ovarian stem cells in the adult mammalian ovary are the VSELs which give rise to slightly differentiated OGSCs. Thus we propose that gonadotropin through its action on pluripotent VSELs augments neo-oogenesis and PF assembly in adult mouse ovaries.

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…”

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

“…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).…”

“…35 Similarly, to study the effect of PMSG on VSELs in chemoablated ovary, 5 IU PMSG (National Hormone & Peptide Program, Harbor-UCLA Medical Center, California) was injected subcutaneously 1 month after animals were treated with busulfan and cyclophosphamide. Ovaries were collected after 48 hours of PMSG treatment for analysis by flow cytometry to study effect of PMSG on SCA-1þ/LinÀ/CD45À VSELs.…”

“…Paraffin sections were used to carry out immunohistochemical studies using methods described earlier. 35 Briefly, sections were dehydrated in graded methanol series, endogenous peroxide was blocked in methanol, and antigen retrieval was performed using microwave in citrate buffer with a pH of 6. These steps were followed by blocking, primary antibody incubation, and detection using appropriate Vecta ABC elite detection kits from Vector Laboratories.…”

“…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. 6,[36][37][38] Intriguingly, the ovarian stem cells are present in ovaries of perimenopausal women (including a 60-year-old woman) with no naturally present follicles or oocytes 2,6,36 and aged mouse.…”