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Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17beta-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.

molecular human reproduction

Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases