Fig. 1. The establishment of leptin resistance in the ovary of diet induced obese mice. (A) Experimental design: animals were maintained on chow diet (CD) or high fat diet (HFD) for 4 weeks (wk) or 16 wk. Protein abundance of components of the leptin signalling pathway in ovarian extracts analysed by Western blot or real-time PCR (RT-PCR). Abundance of (B) SOCS3 protein, (C) phosphorylation of STAT3, (D) leptin receptor (ObR), (E) phosphorylation of tyrosine 985 of leptin receptor, (F) phosphorylation of Janus kinase 2, (G) phosphorylation of STAT5. (H) Heatmap showing fold change in expression of mRNA of leptin signalling components measured in whole ovary or theca and stroma enriched (TC) fraction determined by RT-PCR. Immunohistochemical localisation of SOCS3 protein during follicle development in ovaries of mice subjected to diet-induced obesity (4 wk and 16 wk). Positive staining in brown, counterstaining with heamatoxylin. Negative control stained with polyclonal rabbit IgG (I) 4 wk CD and (J) 4 wk HFD, localisation of SOCS3 in primary follicle (K) 4 wk CD and (L) 4 wk HFD, antral follicles (M) 16 wk CD and (N) 16 wk HFD. Staining is present in oocyte, granulosa and theca cells. Oval-headed arrow indicates oocyte; large-headed arrow indicates granulosa cells and small-headed arrow indicates theca cells. Scale bars represent 100 µm. The staining was confirmed by immunofluorescent localisation of SOCS3 (Q-T). Positive staining in orange, nuclear counterstaining with DAPI in blue. (O-P) negative control 16 wk CD performed with polyclonal rabbit IgG, SOCS3 localised in (Q-R) primordial follicles 16 wk CD, (S-T) primary follicles 16 wk CD. Images are representatives of 3 biological replicates. Inserts in left top corners are magnifications of granulosa cells. mRNA expression of Rpl37 and protein expression of β-actin were used to normalize the expression data. Each bar represents the mean ± SD. Differences between groups were analysed by Mann-Whitney test. N=4-8 for immunoblots and N=8 for RT-PCR analysis. * p<0.05; ** p<0.01; ***p<0.001; + p=0.06 or p=0.09.