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. 2021 Aug 1;36(9):2514–2528. doi: 10.1093/humrep/deab165

Figure 4.

Figure 4.

LH promoted DNA repair and cell survival and reduced the deleterious effects of chemotherapy on ovarian tissue. Alkylating agents were administered with or without LH and ovaries assessed 12 and 24 h after (A) phosphorylated H2AX histone (γH2AX) immunofluorescence (red) counterstained with DAPI (blue) of ovarian samples collected 12 h after treatments with alkylating agents with or without a low (1×) or high (5×) dose of LH (n = 3 in controls, and n = 4 in ChT, ChT+LH-1×, and ChT+LH-5× groups). Images on the left are at 20× (white scale bar = 100 µm) and on the right at 40× (yellow scale bar = 50 µm). The percentages of follicles showing γH2AX positive oocytes were quantified. The LH-treated groups had lower percentages of follicles with double-strand breaks (DSB) than the ChT group. (B) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (red) counterstained with DAPI (blue) was performed on ovarian sections 12 h after treatments. Magnifications and scale bars are as described in (A). Both LH-treated groups had a lower percentage of apoptotic follicles than the ChT group. (C) Representative western blots (WB) showing the levels of the anti-apoptotic protein B-cell lymphoma-2 (Bcl2) and the pro-apoptotic protein cleaved caspase-3 (CC3). Both doses of LH increased the Bcl2: CC3 ratio protecting the ovaries from ChT-induced cell death at 24 h. (D) Representative WB showing phosphorylated-extracellular signal-regulated Kinase 1 and 2 (pERK1/2) and ERK1/2 protein levels. ChT activated ERK1/2 signaling and LH treatments reduced this effect at 12 and 24 h. (E) Representative WB for ataxia-telangiectasia mutated kinase (ATM) and RAD51 recombinase (Rad51) proteins. LH-5×-treated ovaries expressed higher levels of ATM and Rad51 than ChT-treated ovaries at 12 and 24 h. (F) Representative WB for phosphorylated-serine/threonine-protein Kinase 1 (pAkt) and Akt proteins. Chemotherapy caused a notable increase of Akt activation at both time-points, and cotreatment with LH blocked this effect. All WBs were performed from at least two independent experiments from two pools of two ovaries each per group. (G) mRNA expression levels of the indicated DNA repair genes at 12 (left) and 24 h (right). At 12 h, the expression levels of all repair genes were higher in the ChT+LH-1× samples than in the ChT samples. At 24 h, Rad51 expression was higher in both LH groups than in the ChT group. Three independent ovarian fragments per group and time-point was analyzed, and expression levels were normalized to those of the control group. Scatter plots indicate individual data and means; bar charts display means and SDs. Statistical significance was determined by two-tailed Mann–Whitney U test; P-values <0.05 were considered statistically significant. *P < 0.05 or ap <0.05, bP <0.05 and cP <0.05 indicating statistical differences from the control, ChT, and ChT+LH-1× group, respectively.