Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

Trafficking of Cholesterol from Lipid Droplets to Mitochondria in Bovine Luteal Cells: Acute Control of Progesterone Synthesis1

The corpus luteum (CL) is a transient endocrine gland that synthesizes and secretes the steroid hormone, progesterone. Progesterone biosynthesis is a complex process, converting cholesterol via a series of enzymatic reactions, into progesterone. Lipid droplets in luteal cells store cholesterol in the form of cholesterol esters, which can be utilized for steroidogenesis. In small luteal cells, luteinizing hormone (LH) increases intracellular cAMP concentrations leading to activation of protein kinase A (PKA), which phosphorylates downstream proteins, such as hormone sensitive lipase (HSL). Phosphorylation of HSL at Ser563 leads to increased HSL activation and association with lipid droplets, events which theoretically release cholesterol, which can be used for progesterone synthesis. Bovine CL were obtained from a local abattoir, dispersed, and luteal cells were enriched for SLC via centrifugal elutriation. Our results reveal that LH, forskolin, and cAMP induce HSL phosphorylation at Ser563and Ser660. Moreover, inhibiting HSL activity attenuates LH-induced P4 synthesis. Confocal analysis revealed that LH stimulates translocation of HSL to lipid droplets and mitochondria. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria which was dependent on both PKA and HSL activation. These results demonstrate cholesterol stored in lipid droplets are utilized for LH-induced progesterone biosynthesis. Likewise, PKA-induced activation of HSL is required for release and trafficking of cholesterol from the lipid droplets to the mitochondria. Taken together, these findings support a role for a PKA/HSL signaling pathway in response to LH and demonstrate the dynamic relationship between PKA, HSL, and the lipid droplets in the synthesis of progesterone.HighlightsLH and PKA induce HSL phosphorylation at Ser563and Ser660HSL is required for optimal LH-induced P4 synthesisLH stimulates translocation of HSL to lipid droplets and mitochondriaLH stimulated trafficking of cholesterol from lipid droplets to mitochondria