Yves Combarnous

Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity

Abstract A method for the simultaneous purification of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from equine pituitaries is briefly described. Different forms of each hormone were obtained. The total yield of LH was 24.2 mg·kg −1 with a recovery of 22% and the yield of FSH was 26 mg·kg −1 with a recovery of 34%. The specific activities of both hormones, measured in homologous equine radio-receptor assays are equal to or higher than those of the preparations described so far. In all species studied so far the acid-dissociation curves of LH and FSH are similar; this is an agreement with the view that the binding of the common α-subunit and the specific β-subunits involves polypeptide regions which are identical in both hormones. In contrast, the acid-dissociation p K a of equine LH was found to be considerably lower (3.9) than that of equine FSH (5.8). The equine gonadotropins exhibit a much lower specificity with receptors of a porcine testicular fraction compared with an equine fraction. Equine LH exhibited a binding activity on FSH receptors from a porcine testicular fraction equal to 20% that of equine FSH instead of only 1% for an equine binding fraction. Similarly, all the equine FSH preparations tested exhibited a five-fold higher binding-activity on porcine LH receptors than on equine LH receptors. In the porcine system, pregnant mare serum gonadotropin behaved like equine LH towards LH and FSH receptors. In contrast, on equine binding fraction, pregnant mare serum gonadotropin was only 4% as active as equine LH and was devoid of FSH activity. All the data we have obtained are consistent with the ‘negative specificity’ model we proposed recently.

Topography of equine chorionic gonadotropin epitopes relative to the luteinizing hormone and follicle-stimulating hormone receptor interaction sites

In order to localize the epitopes of equine chorionic gonadotropin (eCG) involved in interaction with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) receptors, we used 14 monoclonal anti-eCG antibodies (mAbs). Different effects of these mAbs on the bioactivities of eCG were observed in in vitro bioassays, but the effects of each mAb on the two bioactivities were similar for all but four mAbs. All mAbs were found to inhibit the binding of eCG to LH receptors except 3A3 mAb, in radioreceptor assay. Six mAbs, which were strong inhibitors of eCG binding to LH receptors and of both bioactivities, recognized the same area on the alpha subunit of eCG. All others, except 3A3, recognized epitopes close to the former, and close to each other. 3A3 mAb had a hyperstimulatory effect on FSH bioactivity, and was the only mAb that did not inhibit binding. It appeared to recognize a different epitopic area. These observations suggest that there is a main antigenic area on eCG, which corresponds to the interaction site of eCG with both receptors. It mostly involves the alpha subunit and to a lesser extent the beta subunit.

Study of the superactivity of equine follicle-stimulating hormone in in vitro stimulation of rat sertoli cells

We have previously shown that equine follicle-stimulating hormone (FSH) stimulates plasminogen activator secretion in Sertoli cells at much lower concentrations than would be expected from its relative binding activity. We have introduced the term superactivity to designate this particular behavior. In the present study, we show that equine FSH triggers a long-lasting (20 h) plasminogen activator secretion, whereas rat, porcine and ovine FSH as well as equine LH and equine choriogonadotropin (CG) provoke a short-term response (2.5 h). Moreover, equine FSH was also shown to be superactive in the stimulation of estradiol secretion and cyclic AMP production. This indicates that the step responsible for the long-term stimulation by equine FSH is not located beyond cAMP accumulation. Equine and porcine FSH were found to be equally stable during incubation with the cells demonstrating that equine FSH superactivity was not due to higher stability. Besides, phosphodiesterase inhibition led to a similar increase in the responses to both hormones. This rules out the possibility that equine FSH superactivity is due to less stimulation of phosphodiesterase activity. All these data strongly suggest that equine FSH exhibits superactivity in rat Sertoli cells by stimulating adenylate cyclase activity for a much longer period of time than do all other gonadotropins. The molecular mechanism of this outstanding behavior remains to be elucidated.

Rapid in vitro desensitization of the testosterone response in rat Ley dig cells by sub-active concentrations of porcine luteinizing hormone

We have studied in rat Leydig cells, the effect of sub‐active concentrations of porcine LH on the subsequent stimulation of the cAMP and testosterone production by a sub‐maximal concentration of pLH or hCG. We found that extremely low concentrations of pLH (0.01–2.0 ) were able to induce rapidly a partial but highly significative desensitization of the testosterone response without affecting the cyclic AMP response. These data indicate that desensitization of the steroidogenic response might be due to some lesion beyond cAMP formation or at the level of one discrete compartment of cyclic AMP, directly involved in the control of steroidogenesis. Moreover, our data strongly suggest that the basal circulating concentrations of LH can exert an inhibitory control on the testosterone response to LH pulses in vivo.

Purification and characterization of luteinizing hormone from the dromedary (Camelus dromedarius).

Luteinizing hormone (LH) has been purified from 150 dromedary pituitaries and its partial physicochemical, biological and immunological characterization has been achieved. Purification of the hormone was monitored by a porcine LH radioreceptor assay (RRA). In this system, the final camLH preparation exhibited an activity 0.6-fold that of highly purified porcine LH. The acid half-dissociation of camLH at equilibrium was observed at pH 4.2. A homologous camLH RRA was developed using the testicular plasma membrane fraction from prepubertal camels and radioiodinated, highly-purified camLH. Pituitary and chorionic gonadotropins (CG) from several mammalian species were compared to camLH in this system. The equine gonadotropins eLH and eCG were shown to be 6 times less potent in the camel RRA than in the porcine RRA, whereas the LH from other species exhibited similar activities in both systems. This particularity of camel LH receptors offers a new tool for the study of structural features of gonatropin interactions with their receptors.

Role of sialic acid residues in the in vitro superactivity of human choriogonadotropin (hCG) in rat Leydig cells

The binding activity (B) of porcine Luteinizing Hormone (pLH) to rat LH receptor as well as its stimulating activity (S) of testosterone secretion by rat Leydig cells in vitro are similar to those of the homologous hormone rat LH (S/B = 1). By contrast, the human Chorionic Gonadotropin (CG) and hLH exhibit stimulating activities relative to rat LH that are considerably higher than their relative binding activities (S/B > 100) indicating that they have an abnormally high transducing efficiency (superactivity) after receptor binding. The heterologous hybrid alpha pLH x beta hCG is as superactive as native hCG and recombined alpha hCG x beta hCG whereas alpha hCG x beta pLH exhibits no superactivity, like native pLH and alpha pLH x beta pLH demonstrating that hCG superactivity is due to its beta-subunit. The removal of sialic acid residues with neuraminidase dramatically diminished hCG stimulating activity without impairing its receptor binding activity but the S/B ratio for asialo-hCG never reached values lower than 1. Similar treatments had no effect on the S/B ratios of non-superactive gonadotropins, pLH and equine CG. Sialic acid residues in the Asn beta 30 carbohydrate chains of hLH and hCG appear to be responsible for their superactivity in the in vitro stimulation of testosterone secretion by rat Leydig cells.

Effect of Pharmaceutical Potential Endocrine Disruptor Compounds on Protein Disulfide Isomerase Reductase Activity Using Di-Eosin-Oxidized-Glutathion

Background Protein Disulfide Isomerase (PDI) in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC) could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. Methodology/Principal Findings Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathion (DiE-GSSG), we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH). Our data show that estrogens (ethynylestradiol and bisphenol-A) as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. Conclusions The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainely be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

Use of the immature rat uterotrophic assay for specific measurements of chorionic gonadotropins and follicle-stimulating hormones in vivo bioactivities

The uterine weight growth stimulation by equine Chorionic Gonadotropin (eCG/PMSG) was found to occur at much lower eCG concentrations than ovarian growth. Human Chorionic Gonadotropin (hCG) which has only LH activity, was found to be as active as eCG in the uterotrophic assay whereas equine Luteinizing Hormone (eLH) which has dual LH+FSH activities like eCG, exhibited a much lower potency. In contrast to hCG, porcine and ovine LH as well as pFSH and oFSH exhibited no uterotrophic activity indicating that only gonadotropins with both LH activity and long half-lives are active alone in this assay. The FSH preparations were nevertheless found to trigger a dose-dependent response, but only in the presence of a subactive dose of hCG. The uterotrophic activity of hCG was found to be suppressed in ovariectomized immature rats and to be diminished after injection of GnRH antagonist suggesting an indirect pathway implicating the hypothalamo-pituitary complex. The data in this report together with the analysis of literature suggest that choriogonadotropins exert their stimulatory role on uterine growth by an indirect mechanism involving an increase in ovarian FSH receptors and FSH release by the pituitary. At the lowest concentrations of hCG, the increase in ovarian FSH receptors without endogenous FSH release is thought to be responsible for the sensitivity of the uterotrophic assay to exogenous FSHs. In conclusion, the immature rat uterotrophic assay is a sensitive and convenient assay for eCG and hCG as well as for FSHs in the presence of a sub-active dose of hCG.

Straightforward isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) and ubiquitin from bovine testis by hydrophobic-interaction chromatography (HIC)

Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS-PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions.

Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures

Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHβ or anti-oLHβ antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin αβ heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined.