Biomedical subjects

H Ribbes

Publications and source records attributed to H Ribbes.

5 recordsLinked to original sources

PubMed1990

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture.

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.

Cells, Cultured↗

PubMed1987

Phospholipase C from human sperm specific for phosphoinositides.

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.

Humans↗

PubMed1987

Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187.

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.

Acrosome↗

PubMed1987

High density lipoprotein and low density lipoprotein utilization by human granulosa cells for progesterone synthesis in serum-free culture: respective contributions of free and esterified cholesterol.

Human preovulatory granulosa cells cultured in serum- and gonadotropin-free medium secreted progressively less progesterone as time elapsed. Addition of purified high density lipoproteins (HDL) as well as low density lipoproteins [very low density (VLDL) plus low density lipoproteins (LDL)] restored optimal synthesis of progesterone, and HDL was as effective as VLDL + LDL. The use of cholesterol doubly labeled lipoproteins allowed calculation of the proportions of free and esterified cholesterol converted into progesterone. Granulosa cells used either free or esterified cholesterol from VLDL + LDL. In contrast, HDL-esterified cholesterol was a poor substrate for progesterone synthesis, while HDL-free cholesterol was used preferentially. LH increased the use of both kinds of lipoproteins without changing the way in which they were used. Pretreatment of HDL by purified phospholipase A2 increased the conversion of free cholesterol into progesterone. Similar treatment of VLDL + LDL had little effect on progesterone secretion. We conclude that HDL as well as VLDL + LDL can provide cholesterol to human preovulatory granulosa cells and that utilization of HDL-cholesterol may depend on gonadotropin (LH) and enzymatic (phospholipase) regulation.

Cells, Cultured↗

PubMed1985

Lipoprotein and phospholipid distribution in human follicular fluids.

Human preovulatory follicular fluids, obtained in the course of stimulated cycles, were analyzed for their lipid and protein compositions. By combining different methods, a single class of lipoprotein, high-density lipoprotein, was detected in all cases. The phospholipid distribution revealed an accumulation of lysophosphatidylcholine. These data are discussed in view of the possible roles of the follicular fluid during fertilization and in relation to the high levels of estradiol and progesterone measured in those fluids.

Body Fluids↗