Angiogenesis plays an important role in the development of the ovarian follicle and its subsequent transition into the corpus luteum. Accordingly, follicular fluid is a rich source of mitogenic and angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor secreted by granulosa cells. In the present study, we show that follicular fluid deprived of basic fibroblast growth factor or vascular endothelial growth factor by means of thermal denaturation or antibody neutralization retains its capacity to stimulate endothelial proliferation and angiogenesis. Mass spectrometric analysis of chromatographic fractions stimulating endothelial growth obtained from follicular fluid revealed that the heat-stable mitogenic activity is identical with the subfraction ␣ of high density lipoproteins purified from follicular fluid (FF-HDL). Further investigations demonstrated that sphingosine 1-phosphate (S1P), one of the lysophospholipids associated with HDL, accounts for the capacity of this lipoprotein to stimulate endothelial growth and the formation of new vessels. Activation of mitogen-activated protein kinase (p42/44 ERK1/2 ), protein kinase C, and protein kinase Akt represent signaling pathways utilized by FF-HDL and S1P to induce endothelial proliferation and angiogenesis. We conclude that FF-HDL represents a novel mitogenic and angiogenic factor present in follicular fluid and that S1P is one of the FF-HDL lipid components accounting for this activity.

The ability to assemble and secrete apolipoprotein (apo) B-containing lipoproteins has long been known for enterocytes as well as hepatocytes ( 1, 2 ). These cells use chylomicrons to secrete resorbed cholesterol and triglycerides (TG) in the case of enterocytes ( 3 ), and VLDL to export endogenously synthesized or internalized cholesterol and TG in the case of hepatocytes ( 4, 5 ). ApoB-containing lipoproteins are also secreted by the human placenta, supposedly to transfer lipids from the maternal circulation to the fetus ( 6 ). Recently, another cell type in the body has been recognized to be capable of producing apoB-containing lipoproteins, namely cardiomyocytes ( 7 ). In the heart, VLDL secretion is thought to represent a means of protecting the organ against toxicity associated with TG accumulation ( 8,9 ). In contrast, other peripheral cells utilize apoBcontaining lipoproteins to meet their supply with TG and cholesterol.Developing oocytes within ovarian follicles grow rapidly and require supply with energy and cholesterol ( 10 ). Oocytes are surrounded by follicular fl uid (FF) that in contrast to human plasma mainly contains HDL cholesterol, the smallest lipoprotein subclass ( 11,12 ). Although expression of the LDL receptor as well as of LDL receptor-related protein 4 has been reported for mammalian oocytes ( 13,14 ), most research at present has been focused on HDL within FF. Granulosa cells, the major estrogen-producing cell type of the follicle, line the follicle and are shielded against the blood compartment by a basal membrane Abstract Thus far, liver, intestine, heart, and placenta have been shown to secrete apolipoprotein (apo)B-containing lipoproteins. In the present study, we fi rst investigated lipoproteins in human follicular fl uid (FF), surrounding developing oocytes within the ovary, as well as in corresponding plasma samples (n = 12). HDL cholesterol within FF correlated well with plasma HDL cholesterol ( r = 0.80, P < 0.01), whereas VLDL cholesterol did not, indicating that VLDL in FF might originate directly from the granulosa cells producing FF. Primary human granulosa cells expressed apoB, microsomal triglyceride transfer protein, and apoE, but not the apoB-editing enzyme apobec-1. Using 3 H-leucine, we show that granulosa cells secrete apoB100-containing lipoproteins and that secretion can be stimulated by adding oleate to the medium (+83%). With electron microscopy, apoB-containing lipoproteins within the secretory pathway of human granulosa cells were directly visualized. Finally, we found a positive relationship between apoB levels in FF and improved fertility parameters in a population of 27 women undergoing in vitro fertilization. This study demonstrates that human granulosa cells assemble and secrete apoB100-containing lipoproteins, thereby identifying a novel cell type equipped with these properties. These results might have important implications for female infertility phenotypes as well as for the development of drugs targeting the VLDL production pathway. 20 April 2010. ...

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.

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