HighlightsLH and PKA induce HSL phosphorylation at Ser563and Ser660 HSL is required for optimal LH-induced P4 synthesis LH stimulates translocation of HSL to lipid droplets and mitochondria LH stimulated trafficking of cholesterol from lipid droplets to mitochondria AbstractThe 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.