Primordial Follicles in the Mouse Ovary Are Not Renewed after Depletion with Ovotoxins or Irradiation.

Abstract

The notion of a fixed, non-renewable pool of oocytes in the ovary around birth was questioned after reports of germline stem cells giving rise to new oocytes in the adult ovary. Our aims were to test the hypothesis that in the C57BL/6 mouse ovary, the primordial follicle pool can be restored de novo after treatment with ovotoxins or γ-irradiation, which substantially reduce the size of the primordial pool. Exp1 Mice (6 wk old) received doxorubicin (DXR;5mg/kg/100 μL saline) via tail iv and ovaries were collected at 1, 2 and 7 days later (7/group). Control ovaries (6 wk; 7/group; vehicle) were collected at 6 and 7 wk. Exp 2 Mice (6 wk old) received DXR (5mg/kg/100 μL saline) via tail iv and repeat doses at 7 and 8 wk, and ovaries collected at 10 wk. Control ovaries (6 wk; n=7; vehicle) were collected at 10 wk. Exp3 Mice (25 day old) received Trichostatin A (TSA;10mg/kg/100 μL DMSO sc). Ovaries were collected at 1 and 7 days (7-8/group). Control ovaries (day 25; 7/group; vehicle) were collected at days 1 and 7. Exp 4 Mice (5 days old) were whole-body gamma-irradiated with either 0.45Gy (n=6) or 4.5Gy (n=6) or untreated (controls; n=6) and ovaries collected at 10 days. Primordial and primary follicles per ovary (mean ± sem) in Exps 1-3 were estimated using the fractionator/optical disector technique. In randomly selected ovaries from Exp 4, follicles with an oocyte nucleus were counted in 3 selected 5μm sections per ovary and the area (μm2) of the sections measured with image analysis software. Representative follicle numbers per ovary and per group (mean ± sem) were expressed per 104 μm2 ovarian tissue area. At 1 and 2 days after DXR treatment, mean primordial follicle numbers per ovary declined significantly (P < 0.05) to 65% from their mean number in 6 wk control ovaries and declined further to 51% (P< 0.01) at 7 days after DXR treatment. Over the same treatment period mean primary follicle numbers per ovary were unchanged (P=0.89) in comparison to controls. Following multiple DXR treatments beginning at 6 wk, mean primordial follicle numbers per ovary at 10 wk had declined significantly (P< 0.001) to 28% in comparison to the age-matched controls but primary follicle numbers per ovary were not significantly different (P=0.91) compared with age-matched controls. Between 1 and 7 days after TSA treatment, mean primordial follicle numbers per ovary declined significantly (P < 0.01) to 51% and 61% compared to the mean number in day 25 control ovaries, whereas mean primary follicle numbers per ovary after TSA treatment were not significantly different (P =0.27) in comparison to control. Estimates of primordial and primary follicle numbers in 10 day ovaries after exposure to 0.45Gy or 4.5Gy gamma-irradiation on day 5 showed their respective total or near complete elimination (P< 0.001). There was no evidence in any of the treatments of a significant restoration of primordial numbers in the time frames examined. The results suggest that if primordial follicle numbers are experimentally reduced, there is no compensatory mechanism for full or partial renewal of the follicle pool. Supported by NH&MRC of Australia (#441101 & 241000). (poster)