Gene expression differences induced by equimolar low doses of LH or hCG in combination with FSH in cultured mouse antral follicles

Abstract

In a natural cycle, follicle growth is coordinated by FSH and LH. Follicle growth stimulation in Assisted Reproductive Technologies (ART) requires antral follicles to be exposed to both FSH and LH bioactivity, especially after GNRH analog pretreatment. The main aim was to detect possible differences in gene expression in granulosa cells after exposing the follicle during antral growth to LH or hCG, as LH and hCG are different molecules acting on the same receptor. Effects of five gonadotropin treatments were investigated for 16 genes using a mouse follicle culture model. Early (day 6) antral follicles were exposed to high recombinant FSH combined or not with equimolar concentrations of recombinant LH (rLH) or recombinant hCG (rhCG) and to highly purified human menopausal gonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression differences were tested for genes involved in steroidogenesis: Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1, Nr4a1, and Timp1; final granulosa differentiation: Lhcgr, Oxtr, Pgr, Egfr, Hif1a, and Vegfa; and cytokines: Cxcl12, Cxcr4, and Sdc4. Lhcgr was present and upregulated by gonadotropins. Nr4a1, Cxcl12, and Cxcr4 showed a different expression pattern if LH bioactivity was added to high FSH in the first hours after exposure. However, no signs of premature luteinization were present even after a 3-day treatment as shown by Cyp19a1, Oxtr, Pgr, and Egfr and by estrogen and progesterone measurements. The downstream signaling by rhCG or rLH through the LHCGR was not different for this gene selection. Granulosa cells from follicles exposed to HP-hMG showed an enhanced expression level for several genes compared with recombinant gonadotropin exposure, possibly pointing to enhanced cellular activity.