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.

Human oocyte maturation in vitro

“…In some IVM protocols, assuming that CC can respond to LH, hCG is used as a substitute. However, such a choice does not consider that LH and hCG are not identical, the latter eliciting a different cell response, as shown by recent gene expression studies (Segers et al, 2012).…”

Influence of gonadotropins on ovarian follicle growth and developmentin vivoandin vitro

“…LH is always present in the body, and by stimulating folliculogenesis in vivo it is possible to obtain concentrations that are sufficient for follicle maturation (Gal et al ., 2014). LH has not always been considered a necessary component of the culture medium, but currently the question of its positive influence on follicular growth in vitro is being actively discussed (Segers et al ., 2012; Park et al ., 2013).…”

Global analysis of FSH‐regulated gene expression and histone modification in mouse granulosa cells

“…As mentioned above, eCG treatment induced Cyp19a1 , which is known to increase in response to FSH and is a rate‐limiting enzyme for estrogen synthesis in granulosa cells of multiple species (Hillier, 1994; Parakh et al, 2006; Stocco, 2008; Verbraak et al, 2011). We also found that eCG‐induced Cyp11a1 , which catalyzes the conversion of cholesterol to pregnenolone (Segers, Adriaenssens, Wathlet, & Smitz, 2012; Wigglesworth, Eppig, Lee, Emori, & Sugiura, 2015). It was shown, using microarray analysis, in cultured bovine granulosa cells that FSH induced both CYP19A1 and CYP11A1 at 4 h after treatment (Nivet et al, 2018).…”

“…We also found an increase in Lanosterol synthase ( Lss ) at 48 h eCG (2.5‐fold). Lss functions as a catalyst in cholesterol biosynthesis and is induced by FSH (Fan et al, 2009; Segers et al, 2012). The promoter of Lss , like Fshr also had H3K4me3 enrichment although this was not significantly regulated by FSH.…”