Reproduction Abstracts

The onset of mammalian follicle development is marked by granulosa cell (GC) proliferation, GC shape change, and oocyte growth. Despite their critical role in female fertility the factors and mechanisms underlying these cellular changes remain largely unknown. The transforming growth factor beta (TGFβ) superfamily has been strongly implicated in early follicle growth. Disruption of the function or expression of members of the TGFβ superfamily, receptors or key components of the downstream SMAD signalling pathway induces profound effects on the number and developmental potential of preantral follicles. This study aimed to examine the role of TGFβ2 on follicle activation and preantral follicle growth.

Postnatal day 4 (PND4) mouse ovaries (C57BL/6) were treated with TGFβ2 (1, 10, and 100 ng/ml) in a whole-ovary culture system. Cultured ovaries were fixed for immunohistochemical localization of vasa and laminin and caspase-3. Image analysis was performed to quantify follicle proportions and abundance of caspase-3 staining. TGFβ2 suppressed follicle activation in the whole ovary as demonstrated by a smaller proportion of growing follicles in TGFβ2 treated ovaries compared to vehicle treated (n=3–6 ovaries, logistic regression; 1 ng/ml (P<0.05) and 100 ng/ml (P<0.01)). No qualitative differences in caspase-3 staining were demonstrated between treatment groups (i.e. no evidence of TGFβ2 toxicity).

Preantral follicles were isolated from PND15 mouse ovaries and cultured with TGFβ2 (0.01, 0.1, and 1 ng/ml) or TGFβ2 neutralizing antibody (100 and 10 ng/ml). Follicle area was measured at 0, 24, 48, and 72 h (ImageJ). TGFβ2 promoted increased growth of isolated follicles, relative to vehicle, at all concentrations after 24, 48, and 72 h (n=6 ovaries, two-way ANOVA; P<0.0001). The TGFβ2 antibody suppressed the effect of exogenously applied TGFβ2. However, at the doses used, the antibody did not supress follicle growth below that of the vehicle suggesting it is not affecting any endogenous TGFβ2. In conclusion, our results highlight differential effects of TGFβ2 in two different models of preantral follicle growth.