2. Abstract
Establishment and maintenance of pregnancy is dependent on progesterone synthesized by luteal tissue in the ovary. Our objective was to identify the characteristics of lipid droplets (LDs) in ovarian steroidogenic cells. We hypothesized that LDs are a major feature of steroidogenic luteal cells and store cholesteryl esters. Bovine luteal tissue was used for whole tissue analysis. Further analyses were performed on isolated ovarian steroidogenic cells: granulosa and theca cells of the follicle, and small/large luteal cells. Isolated luteal LDs were collected for lipid/protein analyses. Luteal tissue contained perilipins 2/3/5, hormone-sensitive lipase and abhydrolase domain containing 5. Luteal tissue was enriched in TGs compared to other tissues, except of adipose tissue. Large and small luteal cells were distinguished from follicular cells by the presence of LDs and LD-associated proteins. Furthermore, LDs from large luteal cells were numerous and small; whereas, LDs from small luteal cells were large and less numerous. Isolated LDs contained nearly all of the TGs and cholesteryl esters present in luteal tissue. Isolated luteal LDs were composed primarily of TG, with lesser amounts of cholesteryl esters, diglyceride and other phospholipids. Bovine luteal tissue LDs are distinct from LDs in other bovine tissues, including follicular steroidogenic cells.
Footnotes
Abbreviations: ABHD5, Abhydrolase Domain Containing 5; ANOVA, analysis of variance; ATGL, adipose triglyceride lipase; BSA, Bovine serum albumin; CE, cholesteryl ester; Cer, Ceramide; CL, corpus luteum; CYP11A1, cholesterol side-chain cleavage enzyme, mitochondrial; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified eagle medium; FFA, Free Fatty Acid; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GC, granulosa cell; GlucCer, Glucosylceramide; HPLC, high-performance liquid chromatography; HPTLC, High Performance Thin Layer Chromatography; HSD3B, Hydroxy-Delta-5-Steroid Dehydrogenase, 3 Beta- And Steroid Delta-Isomerase 1; HSL (LIPE), hormone-sensitive lipase; IHC, immunohistochemistry; LacCer, Lactosylceramide; LD, lipid droplet; LPC, Lysophosphatidylcholines; LPE, Lysophosphatidylethanolamine; LPG, Lysyl-phosphatidylglycerol; LPI, Lysophosphatidylinositol; LPS, Lysophosphatidylserine; MS/MS, tandem mass spectrometry; OTC, optimal cutting temperature compound; PC, Phosphatidylcholines; PE, Phosphatidylethanolamines; PG, Phosphatidylglycerol; PGF2α, prostaglandin F2 alpha; PI, Phosphatidylinositol; PLIN, perilipin; PNS, post-nuclear supernatant; PNS-LD, post-nuclear supernatant depleted of lipid droplet; PS, Phosphatidylserine; SEM, standard error of the mean; SM, Sphingomyelin; SB, sphingoid bases; SOAT1, Sterol O-acyltransferase; STAR, steroidogenic acute regulatory protein; TC, theca cell; TEM, Transmission electron microscopy
Funding: This work was supported by the Agriculture and Food Research Initiative from the USDA National Institute of Food and Agriculture (NIFA) [2018-67012-29531 to MRP, 2014-67011-22280 Pre-doctoral award to HT, 2011-67015-20076; 2017-67015-26450 and 2013-67015-20965 to ASC, JRW and JSD]; USDA Hatch grants [NEB26-202/W2112 to ASC, eNEB ANHL 26-213 to ASC and JRW, NEB 26-206 to ASC and JRW]; NIH grants HD087402 and HD092263 to JSD, the VA Nebraska-Western Iowa Health Care System, Department of Veterans Affairs, Office of Research and Development Biomedical Laboratory Research and Development funds [JSD]; and The Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE [JSD]; National Institute for General Medical Science (NIGMS) [INBRE - P20GM103427-14, COBRE - 1P30GM110768-01 to University of Nebraska Microarray Core and the Bioinformatics and Systems Biology Core]; and The Fred & Pamela Buffett Cancer Center Support [P30CA036727 to University of Nebraska Microarray Core and the Bioinformatics and Systems Biology Core].