Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Characterization of apoB, E receptor function in the luteinized ovary

Recent findings from this laboratory have led to the suggestion that the hormone-producing cells of the rat luteinized ovary in situ may obtain a large share of low density lipoprotein (LDL) cholesterol without actually internalizing the intact lipoprotein particles. We have shown that the lipoproteins are trapped at the surface of the luteal cells in a rich network of "microvillar channels" and have theorized that these channel membranes, with their large surface area for interacting with lipoprotein particles, may function in the cholesterol transfer process. In the current study, we try to establish what proportion of the human (h)LDL-cholesterol transfer in the in situ perfused tissue occurs by a classical apoB, E receptor-mediated process versus a surface extraction process. We examine the tissue for the presence of apoB, E receptors, and characterize the structural/functional interaction of hLDL with the apoB, E receptor utilizing a variety of modified hLDL particles as probes. Then, using nonmetabolizable radiolabels for both the protein and cholesteryl ester moieties of these LDL probes, we attempt to quantify the extent to which apoB, E receptors in the ovary contribute to the uptake of hLDL-cholesterol during steroidogenesis. Our experiments show that although the luteinized ovary contains apoB, E receptor protein, hLDL interacts with the tissue atypically. That is, despite modifications of LDL amino acid residues to prevent interaction with the apoB, E receptor, the modified ligands continue to contribute cholesterol for luteal cell internalization and/or steroidogenesis. We conclude, therefore, that in this tissue much of the LDL-cholesterol is not delivered by the apoB, E receptor pathway.     

Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.     

Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum

The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the 125I-hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum 125I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of 125I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were then characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14. These results suggest that the decrease in 125I-hCG binding activity in rat ovarian membranes from day 7 to day 14 of pseudopregnancy is due to a decrease in receptor concentration rather than a change in the receptor subunit structure.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Characterization of newly cloned variant of rat glycine receptor alpha1 subunit

Responses to glycine, a major inhibitory neurotransmitter within the nervous system, are mediated by glycine receptors (GlyRs). Here, we report the cloning and analysis of a novel splicing variant of the GlyRalpha1 subunit. This variant, named GlyRalpha1del, has a truncated cytoplasmic region between transmembrane domains (TM)3 and TM4, and compared to other variants, the truncation is contributed by a different acceptor site in exon 9. We transfected GlyRalpha1 or GlyRalpha1del into HEK293 cells, and then examined the glycine-activated currents using a whole-cell patch-clamp recording technique. Maximal currents and current-voltage relationships showed no clear difference between GlyRalpha1del and GlyRalpha1. Moreover, dose-response curves indicated that the EC50 values for glycine differed significantly between the two GlyRalpha1 derivatives, although their Hill coefficients were similar. When present with other isoforms, GlyRalpha1del might alter the response to glycine or to other agonists, as this variant expands the potential heterogeneity among glycine receptors.     

Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation.     

Evidence for the existence of gonadotropin receptors in the nuclei isolated from rat ovary

Specific binding of radiolabeled human chorionic gonadotropin (hCG) to nuclei isolated from pseudopregnant rat ovaries was studied. Incubation of cultured luteal cells or isolated nuclei with fluorescein isothiocyanate conjugated hCG showed concentration of fluorescence in the nuclear region. Isolated nuclei exhibited saturable high affinity binding of radiolabeled hCG with an apparent Kd of 3.42 X 10(-10) M. The binding was inhibited by increasing concentrations of unlabeled hCG. Under dissociating conditions, the bound hCG was dissociated from the nuclei. However, unlike the plasma membranes, the hCG bound to nuclei was not degraded before dissociation. Radiolabeled hCG bound to the nuclei could also be dissociated by brief exposure to MgCl2 or acidic incubation medium. The bound hCG was not extractable with 4M KCl or 2% Triton X-100. The available evidence suggest that nuclear receptors are distinct from plasma membrane receptors for hCG.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of ovarian gonadotropin receptor. Monomer and associated form of the receptor

We have purified luteinizing hormone/human choriogonadotropin (hCG) receptor from rat ovary by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose chromatography. The purified receptor, previously identified as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Kusuda, S., and Dufau, M.L. (1986) J. Biol. Chem. 261, 16161-16168), was further characterized by radioiodination with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril, and column chromatography on wheat germ lectin-Sepharose. Autoradiography of SDS-PAGE analysis under reducing conditions showed a single radiolabeled band of Mr = 80,000. The radioiodinated receptors treated with peptide:N-glycosidase F migrated at Mr = 54,000. Treatment with neuraminidase alone caused only a minor reduction in molecular weight, and subsequent treatment with endo-alpha-N-acetyl-D-galactosaminidase had little further effect on the receptor. When the radioiodinated receptor was analyzed by fast protein liquid chromatography, a single broad peak was eluted with Mr of approximately 350,000. The higher Mr of radioiodinated receptors than that of native receptors (Mr = 190,000 dimeric form) could be due to the aggregation of labeled molecules. These complexes dissociated into the monomeric form in the presence of SDS. To determine whether the monomers can bind hormone, the purified unlabeled receptors resolved with SDS were electroblotted to nitrocellulose membranes and incubated with 125I-hCG. Autoradiograms of the blots showed a band of monomer (Mr = 78,000) as well as one of dimer (Mr approximately 150,000). These studies have demonstrated that the luteinizing hormone/hCG receptors are predominantly N-linked glycosylated and suggest that the native receptor is a dimer of identical hormone binding subunits associated by noncovalent interactions. Although the individual subunits can bind hormone, it is conceivable that the dimeric form is necessary for signal transduction.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

The effect of gonadotropin on glucose transport and apoptosis in rat ovary

Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1-4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake.     

Divergent regulation of gonadotropin subunit mRNA levels by androgens in the female rat.

To examine the effects of gonadal steroids on the pretranslational regulation of the gonadotropin subunits in the female, adult female rats, beginning 7 or 28 days after ovariectomy, received daily injections of testosterone propionate (T), dihydrotestosterone propionate (D), or estradiol benzoate (E) for 7 days. Intact cycling females and ovariectomized rats that received vehicle served as controls. Serum was obtained for LH and FSH levels to assess changes in gonadotropin secretion. Total RNA from individual rats was recovered and analyzed by blot hybridization with specific radiolabeled cDNA probes for the alpha, LH beta, and FSH beta subunits. Autoradiographic bands were quantitated and standardized to mRNA levels in the intact animals. Ovariectomy resulted in a rise in serum gonadotropin levels and all three gonadotropin subunit mRNA levels. Estrogen replacement resulted in suppression of alpha, LH beta, and FSH beta mRNAs whether given at 7 or 28 days after ovariectomy. In contrast, whereas androgen replacement decreased alpha and LH beta mRNAs, D or T did not consistently suppress FSH beta mRNAs. We conclude that chronic estrogen administration to the castrated female rat uniformly suppresses all three gonadotropin subunit mRNA levels. In female rats, as in male rats, chronic androgen administration fails to negatively regulate FSH beta mRNAs.     

Presence of immunoreactive gonadotropin releasing hormone (GnRH) and its receptor (GnRHR) in rat ovary during pregnancy

The present study aims at quantification of gonadotropin releasing hormone (GnRH) by radioimmunoassay, relative expression of its mRNA by real-time PCR accompanied by its cellular localization in the rat ovary by immunonohistochemistry (IHC) during different time points of pregnancy. To determine the involvement of endogenous ovarian GnRH in receptor mediated local autocrine/paracrine functions within the ovary, the cell specific localization of the classical receptor for GnRH (GnRHR) in the ovary by IHC and expression pattern of its mRNA were studied during pregnancy. Receptor expression during each time point within the ovary was reconfirmed by Western blot analysis accompanied by densitometric analysis of the signal intensity. Results reveal that the content of ovarian GnRH reaches its maximum on Day 20. The densitometric analysis of GnRHR receptor expression from Western blot study exhibits a decreasing trend by Day 20. Presence of GnRH and GnRHR mRNA in the ovary indicates the local synthesis of both ligand and receptor in the rat ovary. Differential expression of GnRH/GnRHR in the corpus luteum throughout pregnancy strengthens the hypothesis of the involvement of ovarian GnRH in local ovarian functions by receptor-mediated mechanisms. The expression of GnRH and GnRHR in the atretic antral follicles is indicative of the possible involvement of this decapeptide in processes like follicular atresia. The expression of GnRH/GnRHR in the nonatretic antral follicles and their oocytes requires further in-depth investigation. Collectively, this study for the first time reveals the presence of endogenous ovarian GnRH/GnRHR supporting their possible involvement in local autocrine/paracrine functions during pregnancy.     

Stimulation of the formation of 13,14-dihydroprostaglandin F2 alpha by gonadotropin in rat ovary

Effects of pregnant mare serum gonadotropin and human chorionic gonadotropin on the formation of 13,14-dihydroprostaglandin F2 alpha, a biologically active compound, were investigated in rat ovarian homogenate. The mass number of the compound, which was formed prostaglandin F2 alpha via 13,14-dihydro-15-ketoprostaglandin F2 alpha in rat ovarian homogenate but was not produced in rat homogenate, accorded with that of the authentic 13,14-dihydroprostaglandin F2 alpha by negative ion chemical ionization mass spectrometry. In the present experiment, the radioactivity of [3H]prostaglandin F2 alpha added to ovarian homogenate was decreased linearly and immediately until the incubation time of 10 min. The formation of 13,14-dihydroprostaglandin F2 alpha was increased up to 60 min. The formation of 13,14-dihydroprostaglandin F2 alpha from prostaglandin F2 alpha was markedly increased by pregnant mare serum gonadotropin and human chorionic gonadotropin. However, there was no additive or synergistic effect of these hormones. The formation of 13,14-dihydroprostaglandin F2 alpha from 13,14-dihydro-15-ketoprostaglandin F2 alpha weas also greatly stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. The formation of 13,14-dihydro-15-ketoprostaglandin F2 alpha steeply declined until 24 h after treatment with human chorionic gonadotropin in pregnant mare serum gonadotropin-primed rats. In contrast, the formation of 13,14-dihydroprostaglandin F2 alpha was markedly increased until 24 h after human chorionic gonadotropin treatment, and the level was about 2.5-fold higher than that at 0 h, 48 h after injection of pregnant mare serum gonadotropin.