Jaroslav Kolena

Alterations in steroid hormone production by porcine ovarian granulosa cells caused by bisphenol A and bisphenol A dimethacrylate.

We have investigated the effects of bisphenol A (BPA) and BPA-dimethacrylate (BPA-DMA), endocrine disruptors used as plasticizers, on steroid hormone production by porcine ovarian granulosa cells after 72 h incubation. BPA at 10(-8) M to 10(-5) M increased basal progesterone levels, while the same concentration range of BPA-DMA did not cause any changes. After FSH-stimulation of the cells, BPA-DMA showed a tendency to inhibit progesterone production. BPA, however, at 10(-7) M and 10(-6) M concentrations was even able to amplify FSH-stimulated progesterone synthesis. BPA as well as BPA-DMA inhibited FSH-induced estradiol production in the whole concentration range. LH-stimulated progesterone production was not altered by BPA in 10(-8) M to 10(-5) M, while BPA-DMA decreased progesterone levels in the cultured media. Significant inhibitory effect of both tested agents at 10(-4) M concentrations was observed specifically on progesterone production, basal as well as gonadotropin-stimulated. The results indicate that ovarian steroidogenesis might be one of the possible sites afflicted by the endocrine disrupting action of BPA and BPA-DMA.

Modulation of rat testicular LH/hCG receptors by membrane lipid fluidity.

The specific binding of [125I]hCG to rat testicular membrane preparations was investigated as a function of membrane fluidity changed by lipids. Membrane fluidity was measured either by fluorescence depolarization of diphenylhexatriene or ESR spectra of I(1,14), I(12,3) and CAT 16 incorporated into the membrane. Incubation of membrane with cholesteryl-hemisuccinate increased both the rigidity of membrane lipids and the specific binding of [125I]hCG. A similar rigidifying action of saturated fatty acids was, however, not accompanied by increased accessibility of LH/hCG receptors. Treatment of testicular membranes with unsaturated fatty acids enhanced membrane fluidity but specific binding of the gonadotropin disappeared. In spite of the increase of LH/hCG receptors in cell membranes treated with cholesteryl-hemisuccinate, Leydig cells showed decreased sensitivity to cAMP response to LH stimulation. The results indicate that newly exposed LH/hCG receptors are not coupled with the adenylate cyclase system.

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be free: its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.

Structure-stabilizing effect of albumin on rat ovarian LH/hCG receptors.

The stabilizing effect of albumin on structure-functional alteration of LH/hCG receptors was analyzed by thermal perturbation technique. On exposing the membranes to bovine serum albumin (BSA) the heat inactivation profile of hCG-binding sites was shifted to a temperature higher by about 5 degreesC (T50 values). The receptor destabilizing action of arachidonic and oleic acids incorporated into ovarian membranes and reversal of this effect when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. This presumption was corroborated by digestion of membranes with phospholipase A2 (PLA2). This enzyme exerted effects on the thermal stability of the receptor protein resembling those observed upon insertion of fatty acids. The membrane fluidization induced by arachidonic acid can be reversed by BSA. However, alterations of lipid fluidity in membranes were not found to be a necessary prerequisite for stabilization of the LH/hCG receptor structure. Fluorescence quenching studies indicated that incorporation of oleic acid or digestion of membrane phospholipids with PLA2 elevated the accessibility of fluorophores for acrylamide. BSA scavenging of free fatty acids approached the quenching rate of control membranes. Analysis of fluorescence of membranes bound to monodansylcadaverine probe revealed that the negative surface charge derived from free fatty acids resulted in destabilization of the receptor protein. The effects of free fatty acids on membranes suggest that altered lipid-protein interactions may directly affect the stability of the LH/hCG receptor structure.

Osmolytes improve the reconstitution of luteinizing hormone/human chorionic gonadotropin receptors into proteoliposomes.

Rat ovarian membrane luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio‐Beads SM‐2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, HCG+GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more 125I‐hCG (528 fmol/mg protein) than membrane‐bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.

Effects of partial delipidation of rat ovarian membranes on thermal stability of LH/hCG receptors

The role of lipids and of possible structure-functional properties of the LH/hCG receptor were analyzed by thermal perturbation techniques in its native membrane environment. A method for the reversible removal of lipids from membranes with a mild detergent Tween 20 was developed. The receptor was reactivated with phosphatidylcholine (PC) by its reconstitution into proteoliposomes. The heat inactivation profile of LH/hCG binding sites in delipidated membranes was shifted to a temperature lower by approx. 8 C degrees (T50 values). Thermal inactivation of the delipidated LH/hCG receptor was found to be a quick process. Occupation of receptor binding sites by the agonist before thermal perturbation induced stabilization of the receptor. Thermal inactivation of the receptor by delipidation was fully reversed by treatment with soybean PC, dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC), partly with phosphatidylethanolamine (PE) and sphingomyelin (SpM), but not with phosphatidylserine (PS), phosphatidylglycerol (PGl) or cholesterol. Delipidation modified the differential scanning calorimetric profile characteristic of control membranes. Delipidation of ovarian membranes also increased membrane lipid rigidity. The addition of PC, DOPC and PS to delipidated membranes decreased, that of DPPC and SpM increased, while PGl did not change the degree of fluorescence polarization of DPH, suggesting that membrane lipid fluidity was not involved in the stabilizing action of specific phospholipids against thermal inactivation of the ovarian LH/hCG receptor.

Involvement of membrane surface charge in thermal stability of the rat ovarian LH/hCG receptor.

Analysis of fluorescence of membrane-bound 8-anilino-1-naphthalene sulfonate and monodansylcadaverine probes revealed that a negative membrane surface charge derived from free fatty acids (FFA) resulted in destabilization of structure-functional properties of the rat ovarian LH/hCG receptor. Removal of FFA from rat luteal and porcine ovarian granulosa cells by BSA increased gonadotropin responsiveness of cells in cAMP formation.

Effect of phospholipids on the reconstitution and thermal stability of delipidated rat ovarian luteinizing hormone/human chorionic gonadotropin receptors in proteoliposomes

The role of lipids and a possible structure-functional alteration of delipidated LH/hCG receptor reconstituted into proteoliposomes was analyzed by thermal perturbation techniques. Delipidated receptor lost to a great extent its binding activity and thermal stability. The LH/hCG receptor was almost fully reactivated by the reconstitution into proteoliposomes with phosphatidylcholine (PC) and sphingomyelin (SpM) and partly with phosphatidylethanolamine (PE) and phosphatidic acid (PA). The heat inactivation profile of delipidated LH/hCG binding sites was shifted to a lower temperature by about 4 degrees C (T50 values). Thermal inactivation of the receptor by delipidation was entirely inverted by treatment with soybean PC, dioleoyl PC and dipalmitoyl PC and partially with SpM. The presence of negatively charged phospholipids, phosphatidylserine (PS), phosphatidylglycerol (PGl) and PA, did not change the heat-inactivation profile of the LH/hCG receptor modified the differential scanning calorimetric profile and the quenching of protein fluorescence characteristic for control proteoliposomes. Delipidation increased membrane lipid rigidity. Reconstitution of delipidated proteoliposomes with soybean PC, dioleoyl PC, PGl, PS and PA decreased, and that of dipalmitoyl PC, lysoPC, SpM and cholesterol increased the degree of fluorescence polarization of DPH of proteoliposomes. The different action of phospholipids on the reconstitution, thermal inactivation of the receptor and membrane lipid fluidity in proteoliposomes suggests that lipid fluidity is not related to the stabilizing action of phospholipids on the LH/hCG receptor.