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Biomedical subjects

Y Combarnous

Publications and source records attributed to Y Combarnous.

At least 19 recordsLinked to original sources
PubMed1992

Immunochemical study of equine chorionic gonadotropin (eCG/PMSG): antigenic determinants on alpha- and beta-subunits.

In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG and LH. The epitopes recognized by E10 and D7 mAbs appear to be very similar and are present on the beta-subunit of eCG and of LHs from all species tested, except hLH, as well as on porcine and equine FSHs. Attempts to specify the amino-acid residues involved in these epitopes suggest that ECG01 mAb might preferentially bind to residues around position 70 whereas the region around disulfide bridges Cys-88-Cys-90 might be involved in the epitopes recognized by D7 and E10 mAbs. Topographical relationships of epitopes show that ECG01 mAb never binds to eCG simultaneously with either D7 or E10 mAbs. Furthermore, simultaneous binding of D7 and E10 mAbs on eCG could not be achieved. Thus, these three epitopes appear to be closely located on the surface of eCG. Finally, ECG01 mAb inhibits eCG binding to LH and FSH receptors, suggesting that its antigenic site is closely related to hormone-receptor interaction site(s).

Animals↗
PubMed1992

Characterization of human follicle-stimulating hormone binding to human granulosa cells by an immunoenzymological method.

An original, nonradiometric method has been developed for studying the binding parameters of native follicle-stimulating hormone (FSH) to its specific receptors in human ovarian granulosa cells. After binding and washing of the cells, hFSH was desorbed from its receptors and quantitatively measured by a specific enzyme immunoassay (EIA) in which nonspecific binding was estimated in the presence of an excess of equine chorionic gonadotropin (eCG/PMSG), which binds to human FSH receptors but does not interfere in the hFSH EIA. This method makes use of native nonmodified hFSH molecules (in contrast to radiometric methods) and permits direct estimation of the binding parameters (Kd and total number of sites). The Kd of hFSH for its human granulosa receptors measured by this technique (4.8 +/- 0.3 x 10(-10) M) is close to that determined by other methods. However, we found a total number of specific FSH receptors per granulosa cell (1 to 6 x 10(4) higher than that reported by others by Scatchard analysis of competition dose-response curves in radioreceptor assays. The method is also sensitive enough to measure the in vivo occupancy of receptors by endogenous hFSH, which was found to be less than 6% in women undergoing hormonal treatment for in vitro fertilization.

Adult↗
PubMed1992

Enzyme immunoassay (EIA) for equine chorionic gonadotropin/pregnant mare serum gonadotropin (eCG/PMSG).

A simple, accurate, sensitive enzyme immunoassay (EIA) has been developed that permits the measurement of equine Chorionic Gonadotropin activity in pregnant mare plasmas or serums as well as in commercial and highly-purified preparations. This assay is specific for eCG and eLH which share the same polypeptide structure but differ in their oligosaccharidic chains. The more important result is that this EIA has been found to be give data in very close agreement with the in vivo assay. Therefore this very rapid and convenient assay can be used to measure the activity of eCG/PMSG in pregnant mares serums in in-field conditions as well as in crude or highly-purified preparations.

Animals↗
PubMed1992

Purification of ovine transferrin and study of the hormonal control of its secretion in enriched cultures of ovine Sertoli cells.

Ovine transferrin (o-transferrin) was purified from sheep serum by fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE trisacryl and finally by affinity chromatography on Affigel blue to remove albumin. Ovine transferrin was identified by its apparent molecular weight in sodium dodecyl sulphate polyacrylamide gel electrophoresis and by its N-terminal amino-acid sequence. The procedure presented in this report permits the preparation of highly purified o-transferrin with a good recovery (52% of initial total immunoactivity). An antiserum against o-transferrin was then raised in rabbits, using this highly purified preparation. A specific radioimmunoassay was set up using 125I-labelled o-transferrin. Its detection threshold (4 ng/ml) was low enough to measure o-transferrin in spent culture media of ovine Sertoli cells, which ranged between 15 and 600 ng/ml. Sheep seminiferous tubule cells, containing approximately 80% Sertoli cells, were cultured at a high density (1.5 x 10(6) cells/cm2) on a thin layer of reconstituted basement membrane. Kinetic studies showed that basal daily secretion of o-transferrin was reduced by half (-49%) between Day 1 and Day 2 of culture, and progressively decreased thereafter. Under FIRT (500 ng ovine follicle-stimulating hormone (FSH)/ml + 10 micrograms insulin/ml + 500 ng retinol/ml + 5 x 10(-7) mol/l testosterone) stimulation, the ratio of stimulated to basal secretions increased 11-fold between Day 1 (1.1) and Day 6 (12). When 10% fetal calf serum was added, mean o-transferrin secretion was a third of that in serum-free medium, suggesting that fetal calf serum contains factors that inhibit secretion of ovine Sertoli cell transferrin. In the presence of serum, the ratio of FIRT-stimulated to basal secretions doubled between Day 1 (1.0) and Day 4-6 (2.0). Between Days 2 and 4 of culture, insulin had a slight stimulatory effect on o-transferrin secretion (128% of control at 10 micrograms insulin/ml), as well as epidermal growth factor (124% of control at 50 ng/ml). Testosterone at up to 5 x 10(-7) mol/l had no effect; 500 ng retinol/ml doubled o-transferrin secretion (218% of control) as did 500 ng FSH/ml (220% of control). A combination of retinol and FSH increased the secretion 4-fold, indicating that maximal stimulation of o-transferrin secretion by ovine Sertoli cells requires the combined actions of mechanisms dependent and independent of cAMP.

Animals↗
PubMed1991

Determination of the primary and secondary structures of the dromedary (Camelus dromedarius) prolactin and comparison with prolactins from other species.

A non-glycosylated form of camel prolactin (camPRL), isolated from one-humped camel (Camelus dromedarius) pituitaries, was totally sequenced. A glycosylated form, separated by affinity chromatography on ConA-Sepharose, was partially sequenced. The comparison of the N-terminal amino acid sequences of the glycosylated and non-glycosylated forms showed that the only putative site of N-glycosylation (Asn-31) was indeed glycosylated. The far ultraviolet (UV) circular dichroism (CD) spectra of the two isohormones were identical, suggesting that the carbohydrate moiety had no effect on the global camPRL secondary structure. The far UV circular dichroism spectra of the two isohormones were analyzed in order to determine their relative proportions of periodic secondary structure, 60% of which was found to be in alpha-helix, as in prolactins of other species. The dromedary sequence was compared to those of other species and interpreted in term of evolutionary process. As already found for gonadotropins, the closest species to the dromedary was found to be the pig.

Amino Acid Sequence↗
PubMed1991

Equine follicle-stimulating hormone action in cultured Sertoli cells from rat, sheep and pig.

Using a suspension of seminiferous tubule cells, we had previously shown that equine FSH is superactive in the male rat, i.e. that it exhibits a higher biological potency than expected from its binding activity. In this work we investigated equine FSH superactivity in rat, pig and sheep, by comparing in each species the equine FSH with the homologous FSH, both for their binding activities (in a radioreceptor assay using a testicular membrane fraction) and for their in vitro biological potencies (in a plasminogen activator assay using a Sertoli cell-enriched population cultured on plastic). In the rat, the binding activity of equine FSH was identical to that of rat FSH, and the biological potency of equine FSH was 47 times higher than that of rat FSH. Hence, superactivity of equine FSH was confirmed in the rat. In the pig, equine FSH was not superactive, since it exhibited binding activity and biological potency identical to those of porcine FSH. In the sheep, the binding activity of equine FSH was 6 times higher than that of ovine FSH, and its biological potency was also higher (14 times). Therefore, equine FSH cannot be considered superactive in this species. In conclusion, equine FSH superactivity is closely related to the species from which Sertoli cells are isolated.

Animals↗
PubMed1991

Development of radioimmunoassay and enzyme immunoassays for luteinizing hormone in dromedaries (Camelus dromedarius).

Polyclonal antibodies against luteinizing hormone in dromedary (camLH) were raised in a rabbit and enabled the development of homologous immunoassays (radioimmunoassay, competitive enzymeimmunoassay (EIA) and sandwich EIA) for the measurement of circulating camLH in plasma. These assays were highly specific for camLH since neither dromedary follicle-stimulating hormone, growth hormone nor prolactin cross-reacted significantly. The lowest detection limit (0.08 ng/ml) was obtained with the sandwich EIA. In addition to its high specificity and sensitivity, this method does not require radiolabelled molecules or expensive laboratory facilities. It can be performed in the field using a portable, battery-powered plate reader.

Animals↗
PubMed1990

Purification and characterization of glycosylated and non-glycosylated forms of prolactin from the dromedary (Camelus dromedarius).

1. Camel prolactin (camPRL) was isolated from the insoluble residue left after extraction of the gonadotropins FSH and LH from a single batch of one-humped camels (Camelus dromedarius) pituitaries. 2. Several isoforms of camPRL were isolated and characterized. 3. Glycosylated and non-glycosylated forms of camPRL were separated by affinity chromatography on Con-A Sepharose. 4. The glycosylated form represented more than 20% of total camPRL and was found to be only 10-20% as active as the non-glycosylated form in its ability to bind to rabbit mammary gland PRL receptors and to stimulate the proliferation of Nb2 cells.

Amino Acid Sequence↗
PubMed1990

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

Camel growth hormone (camGH) was isolated from the insoluble residue left after extraction of the gonadotropins FSH and LH from a single batch of one-humped camel (Camelus dromedarius) pituitaries. Only one form of camGH was isolated and characterized; no glycosylated form of camGH could be evidenced. The isoelectric points (pI) of camGH was determined by chromatofocusing. The N-terminal amino-acid sequences of camGH was determined and compared to those of GHs from other species. The availability of this hormone and our better knowledge of its structure will permit to undertake the study of its structure-function relationships and of its physiological functions in this economically important species.

Amino Acid Sequence↗
PubMed1989

N-terminal amino-sequencing of camel (Camelus dromedarius) luteinizing-hormone alpha and beta-subunits.

The subunits of luteinizing hormone from the Camelus dromedarius (CamLH) have been separated by reverse phase HPLC after reduction and alkylation of their disulfide bridges. The N-terminal amino-acid sequencing of the alpha and beta subunits has been performed up to the 53rd and 67th residue respectively (i.e. more than half of each polypeptide chain). These sequences have been compared to those of LH from other mammalian species in order to estimate the phylogenetic divergence of LH in this species and in order to point out characteristic features of its primary structure that can be related to its physico-chemical properties.

Amino Acid Sequence↗
PubMed1987

Homologous desensitization of rat Sertoli cells by non-stimulating concentrations of follicle-stimulating hormone.

Pre-incubation of rat Sertoli cells with concentrations of follicle-stimulating hormone (FSH) too low to stimulate plasminogen activator (PA) secretion, provoked an inhibition of its subsequent stimulation by an effective dose of the hormone. A kinetic study of this desensitization was performed using equine FSH (which exhibits prolonged stimulation of PA secretion) and porcine FSH (which like all other FSH tested, provokes a transient response). Low non-stimulating concentrations of both hormones were shown to inhibit the subsequent PA response to each of them. Desensitization of rat Sertoli cells by low (non-stimulating) concentrations of FSH did not modify the typical time course (transient or prolonged) of PA secretion under subsequent stimulation by porcine or equine FSH, respectively. Only the intensity of the response to each hormone was dramatically reduced. Besides, the induction of desensitization by these non-stimulating concentrations of FSH was shown to be very rapid (10-15 min). The precise mechanism of this desensitization is not yet clear but its abolishment by the cyclic nucleotide phosphodiesterase (PDE) inhibitor MIX is consistent with the hypothesis that activation of PDE occurs at lower FSH concentration than adenylate cyclase activation.

Animals↗
PubMed1987

Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays.

A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations exhibiting discordant relative activities in the different assay systems. Its relative in-vivo activity was almost 50% lower than that found in the 3 other assays. This could be due to denaturation of the hormone during its preparation or to selection of isoform(s) not representative of the whole population of molecules. For standardization of horse CG preparations by in-vivo assay, IRP2 has proved to be a reliable standard. However, for the standardization of preparations by in-vitro methods, a standard giving consistent results in in-vivo and in-vitro assays must be used. The present report indicates that the NIH standard, but not IRP2, fulfils these requirements.

Animals↗
PubMed1987

Alternative solutions to hCG induction of ovulation in the mare.

Injection of hCG (2000-2500 i.u., i.v.) to mares when a follicle reaches 35 mm induces ovulation between 24 and 48 h. However, repeated injections induce antibodies against hCG. We report attempts to induce ovulation without this inconvenience. We called 'response' an ovulation between 24 and 48 h after treatment. The typical response to hCG was obtained in 73% (N = 145) of treated mares. After immunization against hCG, the response (0%, N = 10) was less than in nonimmunized controls (100%, N = 9). Simultaneous injection of dexamethasone and hCG resulted in induction of ovulation (71%, N = 14). However, simultaneous hCG + dexamethasone resulted in antibody formation similar to that induced by hCG alone, when injected repeatedly every 21 days. Neither GnRH (2 mg i.m.) nor partly purified pig LH (26 mg i.v. or s.c.) induced ovulation consistently (40%, N = 30 and 31%, N = 16). Crude horse gonadotrophin (60 mg i.v. or s.c.) induced ovulation (86%, N = 14). Fertility was not different from control (61%, N = 13 vs 40%, N = 10). Crude horse gonadotrophin also induced ovulation in mares previously immunized against hCG (78%, N = 9): 50 or 25 mg gave satisfactory response (86%, N = 29 and 57%, N = 40). We conclude that crude horse gonadotrophin is a good alternative to hCG for the induction of ovulation in mares.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells.

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.

Animals↗
PubMed1985

Rapid in vitro desensitization of the testosterone response in rat Leydig 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 ng/ml) 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.

Animals↗