Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment

Abstract

Of all the major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Until recently, traditional thinking was that female mammals are provided with a non-renewable pool of oocyte-containing follicles at birth that are depleted during postnatal life to exhaustion, driving ovarian failure. However, a growing body of evidence, including the isolation of germline stem cells (GSC) from adult mouse ovaries that produce developmentally-competent oocytes, has challenged this belief. In addition, rare germline stem-like cells capable of generating oocytes in vitro that undergo parthenogenesis to form blastocyst-like structures have recently been identified in postmenopausal human ovaries. Here we show that the germline-specific meiosis-commitment genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted in azoospermia-like (Dazl), are highly expressed in aged mouse ovaries. However, histological and marker analyses fail to demonstrate the presence of oocytes, supporting that Stra8 and Dazl are expressed in premeiotic germ cells that do not undergo further differentiation. Through the use of aged germline-specific GFP-expressing transgenic mice, we further show that these germ cells can generate GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and form follicles when grafted into young adult wild-type female hosts. Thus, aged mouse ovaries possess a rare population of premeiotic germ cells that retain the capacity to form oocytes if exposed to a young host environment.

Keywords: Stra8; aging; germ cell; meiosis; oocyte; oogenesis; ovary; stem cell.

Figure 1.. Premeiotic germ cells persist in aged atrophic mouse ovaries.

(A) Analysis of germline marker gene expression in ovaries of young adult (2-month) and aged (20-month) female mice. Results from all 4 mice per age group are shown (β-actin, housekeeping gene used as a sample loading control). (B) In-vivo blockade of premeiotic DNA replication by HU in ovaries of young adult mice results in enhanced levels of Stra8 expression, consistent with premeiotic germ cell accumulation. (C) Immunofluorescence analysis of STRA8 expression (green, cytoplasm) in ovaries of aged or HU-treated young adult female mice. (D) Control immunofluorescence analysis of STRA8 expression (green, cytoplasm) in testes of young adult wild-type or Stra8-null male mice (a representative cross-section of seminiferous tubule is shown for each.). C, D: scale bar = 10 μm; DAPI counterstain, blue (nucleus).

Figure 2.. Young adult female mice support oocyte formation from germ cells in aged mouse ovaries.

(A-F) Dual immunofluorescence analysis of GFP (green) and NOBOX (red, nucleus) expression in ovaries of young adult wild-type female mice (A, B), young adult TgOG2 female mice (C, D), and young adult wild-type recipient mice 6 weeks after proximal intrabursal grafting of aged TgOG2 ovarian tissue (E, F) (scale bar = 10 μm; DAPI, blue, nucleus). (G) Immunohistochemical detection of GFP (brown; upper), or dual immunofluorescence analysis of GFP (green) and NOBOX (red, nucleus) expression (middle; DAPI, blue, nucleus), along with numbers of non-follicle-enclosed GFP-positive germ cells and follicle-enclosed GFP-positive oocytes, in ovaries of wild-type young adult recipients treated with vehicle or TSA (recipient mouse #1-#3 or #4-#6, respectively) after proximal intrabursal grafting of aged TgOG2 ovarian tissue (scale bar = 10 μm).

Figure 3.. Assessment of the influence of age on the ovarian follicle reserve.

(A) Immature follicle numbers in ovaries of young adult (2-month-old) female mice 7 weeks after parabiotic joining with either young adult (3-month-old) or aged (24-month-old) female mice (mean ± SEM, n = 5 mice per group). (B) Immature follicle numbers in young adult (2-month-old) mouse ovaries 3 weeks after grafting under the kidney capsules of young adult (3-month-old), middle-aged (12-month-old) or aged (24-month-old) female mice (mean ± SEM, n = 4 mice per group).

Figure 4.. Induction of ovarian Stra8 expression in adult female mice is correlated with oocyte renewal.

(A) Number of non-atretic immature follicles in ovaries of 2-month-old mice at the indicated times following a single intraperitoneal injection of DXR (mean ± SEM, n = 4 mice per group). (B) RT-PCR analysis of Stra8 expression in contralateral ovaries of 2-month-old mice at the indicated times following DXR injection (β-actin, house-keeping gene used as a sample loading control). (C, D) Examples of STRA8-immunopositive cells (green) in the surface epithelial layer of ovaries of mice 30 hours after DXR injection. Scale bar = 5 μm.