Pseudopregnancy-dependent changes in rat ovarian LH/hCG receptors in relation to membrane lipid fluidity

The specific binding of [125I] hCG to ovarian membrane preparations as well as membrane fluidity have been investigated in immature rats during hormonally-induced pseudopregnancy. Membrane fluidity was monitored either by fluorescence polarization analysis of 1,6-diphenyl-1,3,5-hexatriene or by electron spin resonance of 16-, 12-, 5-doxyl stearic acid and CAT 16. A significant positive correlation was found between membrane lipid rigidity and the number of LH/hCG receptors. Luteinization of the ovary induced mobility of molecules in the hydrophobic membrane part at about the C16 carbon level. The changes in rigidity of membrane lipid were the apparent result of alterations in the cholesterol to phospholipids ratio. The results suggest that the increased rigidity of membrane lipid during pseudopregnancy may maximally expose ovarian LH/hCG receptors maintained in a cryptic form.     

Identification of LH/hCG receptors in rabbit uterus

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.     

The effects of neuraminidase and gangliosides on ovarian LH/hCG receptors during rat development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6-8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (KD) of about 10(-9) M. The KD of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10(-10) M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptor in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Effects of photoperiod and hCG on the regulation of testicular LH/hCG receptors in Syrian hamsters (Mesocricetus auratus)

The regulation of testicular LH/hCG receptors was studied in Syrian (golden) hamsters with testicular atrophy induced by exposure to short photoperiod (5L:19D) and in gonadally active hamsters kept in a long photoperiod (14L:10D). By 24 h after injection of hCG, long-photoperiod hamsters showed a dose-related decrease in the number of testicular LH/hCG receptors. At 48 and 72 h, there was a recovery from this 'down-regulation'. The recovery was much faster than has been reported for the rat and mouse, and it resulted in elevation of testicular LH/hCG receptor concentrations above basal values. Hamsters with short photoperiod-induced testicular atrophy showed an increase in testicular LH/hCG receptors after injection of hCG, except for animals injected with a very high dose. The hCG-induced increase in testicular LH/hCG binding in these animals was associated with reappearance of testosterone responses to subsequent hCG stimulation. Response of testicular LH/hCG receptors to hCG in prepubertal hamsters resembled that measured in animals with short photoperiod-induced gonadal atrophy.     

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.     

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.     

The lipid fluidity of rat liver membrane subfractions

1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.r. techniques. 2. The sinusoidal (blood-facing) plasma-membrane subfraction was the most fluid of the three plasma-membrane regions. Fractions originating from the bile-canalicular and contiguous (lateral) regions were most rigid. Endocytic fractions isolated (endosomes and diacytosomes) were of a similar fluidity to fractions originating from the sinusoidal plasma-membrane region. By far the most fluid fractions examined were derived from the Golgi-apparatus complex. 3. The three plasma-membrane subfractions each showed a different response to the bilayer-fluidizing effect of benzyl alcohol. 4. Arrhenius-type plots of the order parameter S and outer hyperfine splitting, 2T, identified lipid-phase separations in the plasma-membrane subfractions.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (K_a) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×10⁹; II, (3.0±0.8)×10⁹; III, (3.2±0.9)×10⁹; IV, 5.5±0.7×10⁹ lm⁻¹]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The K_a in group I was 10 times greater (P<0.05) than K_a in groups II and III, (I, 3.1±0.9×10¹⁰ lm⁻¹; II, 3.4±0.3×10⁹ lm⁻¹; III, 3.3±1.1×10⁹ lm⁻¹). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.     

Labeling of LH/hCG receptor in rat ovaries.

Human chorionic gonadotropin (hCG) was found to stimulate the incorporation of [¹⁴C] N-acetyl-D-glucosamine in rat ovary in vitro. Subcellular fractionation of the ovarian tissue revealed that the plasma membranes were stimulated maximally to the extent of 200 to 300% by the hormone indicating the stimulation of the synthesis of plasma membrane glycoproteins. In addition, and appreciable amount of the radioactivity was incorporated in the cell surface LH/hCG receptor. The evidence in support of the labeling of the receptor was derived from the behavior of the detergent solubilized receptor on Sepharose 6B column and on hCG-Sepharose affinity adsorbent. The labeled receptor thus purified showed binding affinity for [¹²⁵I] hCG. Thus, the hormone stimulates the synthesis of cell surface glycoproteins as well as the LH/hCG receptor.     

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.     

Bovine cumulus/oocyte complex: quantification of LH/hCG receptors

LH/hCG receptors of the bovine cumulus/oocyte complex were quantified, and their maximum binding capacities and affinity constants were determined by Scatchard analysis. Specific binding of these gonadotropins to receptors in follicles of different sizes was also determined by radiolabeling techniques. A greater number of receptors was observed to be bound to LH than to hCG (P < 0.05); however, affinity constants did not significantly differ (P > 0.05). The results of specific binding of the gonadotropins presented differences in relation to follicle size. Differences in the specific binding values of LH and hCG were verified (P < 0.05), but when submitted to linear regression analysis, presented similar behaviors in relation to follicle size. It is concluded that receptors of bovine cumulus/oocyte complex cells bind specifically to LH/hCG, that binding capacity is inversely proportional to follicle size, and that the behavior of hCG is similar to that of LH, suggesting that hCG can also promote the maturation of bovine oocytes when used in concentrations greater than LH.     

Regulation of LH/hCG receptor by gonadotropins in rat ovary

To investigate the regulation of the LH/hCG receptor gene by gonadotropins, we examined the effect of PMSG and hCG on the expression of LH/hCG receptor in immature rat ovary. Northern blot analysis of ovarian RNA revealed a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300, and 1200 nucleotides, and PMSG treatment slightly increased the intensity of all LH/hCG receptor messengers. Subsequently, hCG treatment decreased the number of LH/hCG receptor by day 2 and mRNA levels by 12h after injection. The level of mRNA recovered and increased 5-fold of control by day 6, then returned to control levels by day 10, followed by slower decline in LH/hCG receptor in plasma membrane. These studies demonstrate that the effects of PMSG and hCG on the number of LH/hCG receptor are closely related to the actions of these hormones on LH/hCG receptor messenger levels.     

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.     

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.     

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.     

Postnatal maturation of rat intestinal membrane: lipid composition and fluidity.

1. We studied the lipid composition and the fluidity of small intestine brush border membrane (BBM) of rats of different age: 'very young' (5-7 weeks old), 'young' (9 weeks old), 'adult' (30 weeks old) and 'old' (85 weeks old). 2. Fluorescence anisotropy, as assessed by 1,6-diphenyl-1,3,5-hexatriene probe (DPH), was increased from very young to adult rats. 3. In agreement with these results the lipid composition in adult animals showed a lower lipid/protein ratio (derived mainly from a lower content of total polar lipids) and an increase of cholesterol esters and sphingomyelin (SM) saturation index. 4. A marked decrease of the order parameter was observed in the 'old' group, accompanied by a decreased cholesterol/phospholipid ratio. 5. The percentage distribution of membrane phospholipids significantly changed during development, but the modifications were not correlated with the anisotropy of DPH.     

Metabolism of doubly-labeled chylomicron cholesteryl esters in the rat

Chylomicrons labeled in vitro with doubly-labeled cholesteryl esters were injected intravenously into fasted rats, and the tissue distribution and chemical form of each isotope were observed for 24 hr. The use of doubly-labeled cholesteryl esters provided information about the metabolism of both the sterol and the fatty acid moieties. Similar results were obtained with doubly-labeled cholesteryl palmitate, oleate, and linoleate. In each instance, most (80-90%) of the chylomicron cholesteryl ester was removed from the plasma by the liver; small amounts were also taken up by all other tissues examined. There was no hydrolysis during uptake. In the liver the newly absorbed cholesteryl esters underwent slow hydrolysis (60% after 1 hr and 85-90% after 3.5 hr); the rate of reesterification of the liberated cholesterol was still slower. After 24 hr only 20-28% of the labeled cholesterol present in the animal was found in the liver. Labeled fatty acid disappeared from the liver, and was redistributed among other tissues, much more rapidly than the labeled cholesterol. Most of the labeled fatty acid apparently underwent oxidation, since only 15-20% of the injected labeled fatty acid was present in the animal after 24 hr. At this time the three fatty acids were differently distributed between and within the tissues. These differences reflected some known differences of fatty acid concentration and lipid composition in the various tissues.     

Effects of butylated hydroxytoluene on membrane lipid fluidity and freeze-thaw survival in mammalian cells

Butylated hydroxytoluene (BHT) increases the fluidity of membrane lipids in the hydrocarbon but not the polar regions, as measured by electron spin resonance spin label probes. BHT also sensitizes nucleated mammalian cells to freeze-thaw damage as measured by colony formation survival assays. Furthermore, the membranes of BHT-exposed cells are more susceptible to physical stress, as reflected by the BHT-induced sensitization to hypotonic stress. Since others have shown that BHT induces hexagonal phase lipids in lipid bilayers, this phenomenon may also influence the above survival results.