Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

[1]  J. Veldhuis,et al.  Concerted Regulation of Steroidogenic Acute Regulatory Gene Expression by Luteinizing Hormone and Insulin (or Insulin-Like Growth Factor I) in Primary Cultures of Porcine Granulosa-Luteal Cells** This work was supported by NIH Grants R01-HD-16393, HD-16806, HD-38945, and U54-HD-96008 Specialized Coo , 2000, Endocrinology.

[2]  L. Jarett,et al.  Ultrastructural basis for chloroquine-induced increase in intracellular insulin in adipocytes: alteration of lysosomal function. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[3]  E. K. White,et al.  Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells* , 2000, The Journal of Biological Chemistry.

[4]  H. Bose,et al.  Cholesterol-mediated conformational changes in the steroidogenic acute regulatory protein are essential for steroidogenesis. , 2013, Biochemistry.

[5]  D. Gospodarowicz,et al.  Modulation of low density lipoprotein metabolism in bovine granulosa cells as a function of their steroidogenic activity. , 1981, The Journal of biological chemistry.

[6]  J. Geiger,et al.  Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons. , 2012, Life sciences.

[7]  T. Osborne,et al.  Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a. , 2001, Endocrinology.

[8]  A. Evans,et al.  Decline in Circulating Estradiol During the Periovulatory Period Is Correlated with Decreases in Estradiol and Androgen, and in Messenger RNA for P450 Aromatase and P450 17α-Hydroxylase, in Bovine Preovulatory Follicles1 , 2001, Biology of reproduction.

[9]  T. Osborne,et al.  Oxysterol Regulation of Steroidogenic Acute Regulatory Protein Gene Expression , 1998, The Journal of Biological Chemistry.

[10]  Noa Sher,et al.  Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein β confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models , 2006, Molecular and Cellular Endocrinology.

[11]  M. Wiltbank,et al.  The Role of Luteinizing Hormone in Regulating Gene Expression During Selection of a Dominant Follicle in Cattle1 , 2011, Biology of reproduction.

[12]  S. Macedo-Ribeiro,et al.  Molecular and computational analyses of genes involved in mannose 6‐phosphate independent trafficking , 2015, Clinical genetics.

[13]  S. King,et al.  Transcriptional Regulation of Steroidogenic Genes: STARD1, CYP11A1 and HSD3B , 2009, Experimental biology and medicine.

[14]  D. Stocco,et al.  Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene. , 2007, Journal of molecular endocrinology.

[15]  I. Swanston,et al.  Gonadotrophin and steroid concentrations in bovine follicular fluid and their relationship to follicle size. , 1982, Journal of reproduction and fertility.

[16]  D. Gospodarowicz,et al.  Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A reductase in progesterone production by cultured bovine granulosa cells. , 1982, Endocrinology.

[17]  M. Nowik,et al.  Stimulatory effect of LH, PGE2 and progesterone on StAR protein, cytochrome P450 cholesterol side chain cleavage and 3beta hydroxysteroid dehydrogenase gene expression in bovine luteal cells. , 2005, Prostaglandins & other lipid mediators.

[18]  Raluca Rusovici,et al.  Epidermal Growth Factor-Mediated Inhibition of Follicle-Stimulating Hormone-Stimulated StAR Gene Expression in Porcine Granulosa Cells Is Associated with Reduced Histone H3 Acetylation1 , 2005, Biology of reproduction.

[19]  J. Paul Luzio,et al.  Lysosomes: fusion and function , 2007, Nature Reviews Molecular Cell Biology.

[20]  D. Stocco,et al.  Regulation of Porcine Granulosa Cell Steroidogenic Acute Regulatory Protein (StAR) by Insulin-Like Growth Factor I: Synergism with Follicle-Stimulating Hormone or Protein Kinase A Agonist1. , 1997, Endocrinology.

[21]  R. Grummer,et al.  Progesterone production by cultured luteal cells in the presence of bovine low- and high-density lipoproteins purified by heparin affinity chromatography. , 1992, Journal of animal science.

[22]  M. Waterman,et al.  Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle. , 1987, Molecular endocrinology.

[23]  S. Dieleman,et al.  Changes in oestradiol, progesterone and testosterone concentrations in follicular fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone. , 1983, The Journal of endocrinology.

[24]  M. Waterman,et al.  Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle. , 1986, Endocrinology.

[25]  J. E. Fortune,et al.  Gonadotropin Surge Induces Two Separate Increases in Messenger RNA for Progesterone Receptor in Bovine Preovulatory Follicles1 , 2002, Biology of reproduction.

[26]  M. J. Chapman.  Animal lipoproteins: chemistry, structure, and comparative aspects. , 1980, Journal of lipid research.

[27]  H. Garverick,et al.  Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves: a review. , 1998, Journal of animal science.

[28]  B. Murphy,et al.  Use of low-density and high-density lipoproteins in undifferentiated porcine granulosa cells. , 1989, Biology of reproduction.

[29]  R. Ivell,et al.  Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells. , 2000, Journal of molecular endocrinology.

[30]  B. Murphy,et al.  Steroidogenic acute regulatory protein in bovine corpora lutea. , 1996, Biology of reproduction.

[31]  Takashi Shimizu,et al.  Histone H3 acetylation of StAR and decrease in DAX-1 is involved in the luteinization of bovine granulosa cells during in vitro culture , 2009, Molecular and Cellular Biochemistry.

[32]  P. Leung,et al.  TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells. , 2014, The Journal of clinical endocrinology and metabolism.

[33]  M. Waterman,et al.  Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity. , 1983, The Journal of biological chemistry.

[34]  N. Sugino,et al.  Changes in histone modification and DNA methylation of the StAR and Cyp19a1 promoter regions in granulosa cells undergoing luteinization during ovulation in rats. , 2013, Endocrinology.

[35]  Takashi Shimizu,et al.  BMP-4 suppresses progesterone production by inhibiting histone H3 acetylation of StAR in bovine granulosa cells in vitro , 2011, Molecular and Cellular Biochemistry.

[36]  Xiaobo Li,et al.  Cholesterol, LDL, and 25-hydroxycholesterol regulate expression of the steroidogenic acute regulatory protein in microvascular endothelial cell line (bEnd.3). , 2006, Biochemical and biophysical research communications.

[37]  N. Morrell,et al.  The lysosomal inhibitor, chloroquine, increases cell surface BMPR-II levels and restores BMP9 signalling in endothelial cells harbouring BMPR-II mutations , 2013, Human molecular genetics.

[38]  R. Meidan,et al.  Delayed effect of heat stress on steroid production in medium-sized and preovulatory bovine follicles. , 2001, Reproduction.

[39]  B. Murphy.  Models of Luteinization1 , 2000, Biology of reproduction.

[40]  B. Loftus,et al.  CALL FOR PAPERS NextGen Sequencing Technology-based Dissection of Physiological Systems Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis , 2012 .

[41]  G. L. Williams,et al.  Regulatory roles of high-density and low-density lipoproteins in cellular proliferation and secretion of progesterone and insulin-like growth factor I by enriched cultures of bovine small and large luteal cells. , 1997, Journal of animal science.

[42]  K. Okuda,et al.  Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture. , 2012, Cytokine.

[43]  B. J. Clark,et al.  Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter. , 2005, Endocrinology.

[44]  G. Rothblat,et al.  Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[45]  E. K. White,et al.  Oxysterols regulate expression of the steroidogenic acute regulatory protein. , 2004, Journal of molecular endocrinology.

[46]  R. Grummer,et al.  A review of lipoprotein cholesterol metabolism: importance to ovarian function. , 1988, Journal of animal science.

[47]  Xian-long Wang,et al.  Follicle-stimulating Hormone Regulates Pro-apoptotic Protein Bcl-2-interacting Mediator of Cell Death-Extra Long (BimEL)-induced Porcine Granulosa Cell Apoptosis* , 2012, The Journal of Biological Chemistry.

[48]  M. Brown,et al.  Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. , 1975, The Journal of biological chemistry.

[49]  J. Veldhuis.  Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells. , 1988, Endocrinology.

[50]  J. A. Arosh,et al.  Expression of Cyclooxygenases 1 and 2 and Prostaglandin E Synthase in Bovine Endometrial Tissue During the Estrous Cycle1 , 2002, Biology of reproduction.

[51]  F. Ross,et al.  Disruption of the Man‐6‐P Targeting Pathway in Mice Impairs Osteoclast Secretory Lysosome Biogenesis , 2011, Traffic.

[52]  H. LaVoie,et al.  GATA4 reduction enhances 3',5'-cyclic adenosine 5'-monophosphate-stimulated steroidogenic acute regulatory protein messenger ribonucleic acid and progesterone production in luteinized porcine granulosa cells. , 2008, Endocrinology.

[53]  M. Waterman,et al.  Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells. , 1984, The Journal of biological chemistry.

[54]  M. Brown,et al.  Receptor-mediated endocytosis: insights from the lipoprotein receptor system. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[55]  R. Meidan,et al.  Hormonal regulation of messenger ribonucleic acid expression for steroidogenic factor-1, steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage in bovine luteal cells. , 1999, Biology of reproduction.