Prepubertal primordial follicle loss in mice is not due to classical apoptotic pathways

Abstract

More than half of the primordial follicles that are formed by Day 6 of postnatal life in the mouse will be eliminated from the ovary by the time of puberty. Apoptosis, a form of programmed cell death, is one mechanism by which these follicles could be actively lost. To investigate whether apoptosis is responsible for the loss of primordial follicles, follicular atresia was examined during the prepubertal period, when follicles die and are cleared from the ovary at an extremely high rate. Four hallmarks of classical apoptosis were measured in follicles present in prepubertal ovaries. The primordial follicle cohort was not positively associated with nuclear condensation or cell shrinkage, activation of caspase 3, cleavage of poly(ADP ribose) polymerase 1 (PARP1), or fragmentation of DNA. These data are consistent with a nonapoptotic pathway that is responsible for small follicle death.

FIG. 1.

Mathematical model of primordial follicle dynamics. Modeling based on empirical counts provides predictions for (A) loss dynamics and (B) total remaining primordial follicle number. C) Primordial follicles can leave the initial follicle pool by dying (L) or through transition to the primary stage (T).

FIG. 2.

Apoptotic pathway effectors are not activated in primordial or primary follicles (P). A) Assay of activity shows significant increase of active caspase 3 or 7 only at Day 19. B) An antibody against the cleavage product of caspase 3 localizes to granulosa cells of secondary (S) and antral (A) follicles across the entire prepubertal period. Different lowercase letters indicate a significant difference between groups. Symbol indicates that the group marked is not significantly different from any unmarked groups. Error bars are + SEM. Representative (C) secondary/antral and (D) primordial follicles (yellow asterisks) are shown. Bars = 100 μm (C) and 25 μm (D).

FIG. 3.

Primordial and primary follicles do not exhibit classic apoptotic pathway endpoints. DNA fragmentation as indicated by TUNEL staining can be recognized in antral follicles (A) from Day 26, but not in primordial follicles (B). DNAse treatment serves as a clear positive control (C), and the image of an entire Day 10 ovary demonstrates global absence of DNA fragmentation at this day (D). Trend shown in A and B is true across the period (C). The same results can be seen for the cleavage of PARP1 (F–H). Western blots show that cleaved PARP1 increases significantly at Day 19 (I). Different lowercase letters indicate a significant difference between groups. Yellow asterisks indicate representative primordial follicles. Error bars are + SEM. P, primordial or primary follicles; S, secondary follicles; A, antral follicles. Bars = 100 μm (A, C, D, F) and 25 μm (B, G).

FIG. 4.

Morphological endpoints of apoptosis are absent in primordial and primary follicles. A) Large secondary and antral follicles show extensive nuclear condensation and cell shrinkage (pyknosis; red arrowheads) in granulosa cells. Bar = 100 μm. B) Pyknosis is not detected in primordial or primary follicles (representatives marked with yellow asterisks). Bar = 20 μm. Quantification is shown in C. D) Animal model with supraphysiological follicle loss exhibits no significant increase in pyknosis in primordial follicles. Different lowercase letters and asterisks (***) indicate a significant difference between groups. Error bars are + SEM. Prim, primary; Sec, secondary; Ant, antral.

FIG. 5.

Lack of apoptosis visualization is not due to rapid clearance. Using an antibody against cleaved PARP1, positive primordial and primary follicles were counted across a 24-h period on Postnatal Day 10 (D10). Error bars are + SEM.

FIG. 6.

Follicle shedding does not explain prepubertal primordial follicle loss. Cortical follicles were categorized as either normal (A), subepithelial (B), intraepithelial (C), or intrabursal. Isolated cells were seen in the intrabursal space (D). To ascertain whether these cells were actually degenerated oocytes, DDX4 antibody was used to label oocytes. E) Intrabursal cells and stained oocytes in primordial follicles (yellow asterisks). Black arrows indicate ovarian surface epithelium, and red arrowheads indicate the bursa. Quantification of above results is shown in F. Error bars are + SEM. Bar = 2 μm.