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Y Combarnous

Publications and source records attributed to Y Combarnous.

At least 37 records · Page 2Linked to original sources
PubMed1998

International collaborative calibration of a preparation of equine chorionic gonadotrophin (eCG NZY-01) proposed as a new standard.

A batch of partially purified equine chorionic gonadotrophin (eCG NZY-01) was freeze-dried in vials of 500 iu and characterized in a large number of in vivo and in vitro assays. These assays were performed in eight laboratories in seven countries (Argentina, Belgium, England, France, Germany, Holland and USA). Four of these laboratories were in universities or government research organizations and four were those of pharmaceutical companies. Good correlation between in vivo and in vitro assays was found for unknown samples and for commercial preparations from different sources when performed against eCG NZY-01 as the standard. This result suggests that, in contrast to the WHO IRP2 international standard, the eCG NZY-01 preparation contains all eCG isoforms in proportions roughly similar to those found in serum and commercial preparations. The consistency of the data when using eCG NZY-01 as the reference in all types of in vivo and in vitro assays validates the wide use of in vitro assays which are cheaper, quicker and ethically preferable to in vivo assays. Vials of 500 iu eCG NZY-01 are available to researchers and manufacturers.

Animals↗
PubMed1997

Binding of rat and ovine epididymis-specific prealbumins (PES) to rat spermatozoa without effect of heterologous immunization on rat fertility.

The epididymis, under control of testosterone, secretes proteins which bind to the membrane of the spermatozoa during their passage through the lumen. One such class is termed PES (prealbumin epididymal specific). Injection of heterologous oPES (ovine PES) into male rats caused antibody production but failed to induce sterility, unlike results previously obtained when rat PES was injected into male rats. This suggests that only very restricted species-specific epitopes of PES might be useful for causing immunocontraception. Despite this, the sperm binding properties of PES purified from the rat (rat PES) and from the ram (oPES) were shown to be similar. When either rat PES or oPES, conjugated with a fluorescent probe (dimethylamino-fluorescein), was incubated with washed rat spermatozoa originating from the caput, corpus or cauda epididymis, results of flow cytometric analysis showed: (1) the number of spermatozoa bound to isologous or heterologous fluorescent PES, and (2) the binding-affinity of spermatozoa for PES was greater for sperm collected from more distal sites in the epididymis.

Animals↗
PubMed1997

Deterioration of goat sperm viability in milk extenders is due to a bulbourethral 60-kilodalton glycoprotein with triglyceride lipase activity.

The aim of this work was to purify, identify, and characterize the component of goat bulbourethral gland secretion (BUS) responsible for goat sperm deterioration in skim milk extender. BUS extracts promote a decrease in the percentage of motile spermatozoa, deterioration in the quality of movement, breakage of acrosomes, and cellular death of goat spermatozoa diluted in skim milk. A 55- to 60-kDa monomeric glycoprotein (BUSgp60) was purified by cation-exchange, concanavalin A, and heparin-affinity chromatography and was identified as the only BUS component responsible for the deterioration of spermatozoa in milk. The BUSgp60 was shown to display triacylglycerol hydrolase activity, and partial sequences (37 amino acids in all) exhibited 50-70% homology with sequences of various types of pancreatic lipases (PLs), especially PL-related protein 2 (PL-RP2). In addition, porcine PL produced deterioration of goat sperm properties in milk as effectively as BUS and BUSgp60. These results support the preliminary identification of the goat BUSgp60 as a bulbourethral lipase belonging to the PL-RP2 family. Since seminal plasma also contains factors favorable for sperm survival, it is envisaged that, for the best preservation of goat semen, specific inhibitors of this family of lipase could be added into milk-based extenders without eliminating seminal plasma.

Amino Acid Sequence↗
PubMed1997

Cloning, sequencing and in vitro functional expression of recombinant donkey follicle-stimulating hormone receptor: a new insight into the binding specificity of gonadotrophin receptors.

Among all mammalian FSH receptors (FSH-R; including donkey (dk) FSH-R), only horse (hs) FSH-R does not bind hsLH/chorionic gonadotrophin (CG). In order to delineate the structural origin of hsFSH-R specificity precisely, we have cloned dkFSH-R cDNA from donkey testis mRNA by RT-PCR. Transiently expressed dkFSH-R endowed COS-7 cells with both hsLH/CG- and FSH-binding activity, as well as FSH-induced cAMP production. The deduced dkFSH-R amino acid sequence shares 96% identity with the hsFSH-R: notably, in the hormone-binding domain, the specificity of hsFSH-R may be ascribed to only four divergent amino acids: Thr 173, Asp 202, Asn 268 and Pro 322. Interestingly, hsAsn 268 could bear an additional N-glycosylation. According to receptor negative specificity, these amino acids could be implicated in preventing LH/CG binding to FSH-R.

Amino Acid Sequence↗
PubMed1997

Expression of horse and donkey LH in COS-7 cells: evidence for low FSH activity in donkey LH compared with horse LH.

Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs in heterologous species, recombinant eLH (r.eLH/CG) and recombinant dkLH (r.dkLH/CG) were expressed, for the first time, in COS-7 cells. Their LH activities were assessed in a rat Leydig cell bioassay, and their FSH activities were estimated in a bioassay using Y1 cells stably expressing the human FSH receptor. Human CG (hCG) was expressed (r.hCG) and analysed in the same system. The results showed that, whereas r.dkLH/CG was about twice as active as r.eLH/CG in the LH bioassay, it was five times less active than r.eLH/CG in the FSH bioassay; r.hCG was about three times less active than r.eLH/CG in the LH bioassay but was completely inactive in the FSH bioassay. These results confirm that dkLH/CG possesses significant FSH activity in heterologous species that is not attributable to contamination with FSH.

Animals↗
PubMed1997

Evidence that the alpha-subunit influences the specificity of receptor binding of the equine gonadotrophins.

Horse LH/chorionic gonadotrophin (eLH/CG) exhibits, in addition to its normal LH activity, a high FSH activity in all other species tested. Donkey LH/CG (dkLH/CG) also exhibits FSH activity in other species, but about ten times less than the horse hormone. In order to understand the molecular basis of these dual gonadotrophic activities of eLH/CG and dkLH/CG better, we expressed, in COS-7 cells, hybrids between horse and donkey subunits, between horse or donkey alpha-subunit and human CG beta (hCG beta), and also between the porcine alpha-subunit and horse or donkey LH/CG beta. The resultant recombinant hybrid hormones were measured using specific FSH and LH in vitro bioassays which give an accurate measure of receptor binding specificity and activation. Results showed that it is the beta-subunit that determines the level of FSH activity, in agreement with the belief that it is the beta-subunit which determines the specificity of action of the gonadotrophins. However, donkey LH/CG beta combined with a porcine alpha-subunit exhibited no FSH activity although it showed full LH activity. Moreover, the hybrid between horse or donkey alpha-subunit and hCG beta also exhibited only LH activity. Thus, the low FSH activity of dkLH/CG requires an equine (donkey or horse) alpha-subunit combined with dkLH/CG beta. These results provide the first evidence that an alpha-subunit can influence the specificity of action of a gonadotrophic hormone.

Animals↗
PubMed1997

[Application prospects of structure-function studies of gonadotropins].

The study of the respective roles of the polypeptide and oligosaccharide parts of gonadotropins in their biological activities has been undertaken for many years through chemical and enzymatic modifications of natural hormones, expression of wild-type and mutated recombinant hormones and synthesis of active peptides. The present paper proposes a short summary of the future application prospects of these studies as undertaken in our laboratory.

Gonadotropins↗
PubMed1996

Effect of indole-3-acetic acid (plant auxin) on the preservation at 15 degrees C of boar semen for artificial insemination.

In order to extend the duration of boar sperm survival at 15 degrees C for artificial insemination, we tested the effect of indole-3-acetic acid (IAA), which appeared to be the main sperm protective substance present in the Coco nucifera endosperm (coconut water). Two IAA concentrations (10 and 100 ng/mL) in Beltsville extender (BTS) were studied for their in vitro effects. The motility, the percentage of motile spermatozoa and the acrosome morphology of sperm were recorded each day over 13 days of storage at 15 degrees C, after 5 min and 3 h of incubation at 39 degrees C. The IAA effect on sperm preservation was also studied in vivo at a concentration of 10 ng/mL in BTS by inseminating groups of females twice at 24 h intervals either at D0 (day of sperm collection) and D1 (D0/1) or at D5 and D6 (D5/6). At D0/1, the two groups of females (control and IAA) were inseminated with a total of 6.3 x 10(9) spermatozoa (3.15 x 10(9) at D0 and the same dose at D1) whereas at D5/6, on IAA group was inseminated with a total of 6.3 x 10(9) spermatozoa and another one with 12.6 x 10(9) spermatozoa. The animals in the D5/6 control group were inseminated each with a total of 12.6 x 10(9) spermatozoa. For each group of females (n = 106-140), fertility rate (% farrowing) and prolificacy rate (litter size) were recorded. No effect of IAA in vitro on the motility rate and on the percentage of motile spermatozoa was observed over a 13 day storage. However, IAA (10 ng/mL) had a significant positive effect on the percentage of living spermatozoa with intact acrosomes after 13 days (66 vs 54%, P < 0.05). The fertility and prolificacy rates after 5-6 days of sperm preservation in BTS extender alone did not differ significantly between D5/6 and D0/1 but the total number of inseminated spermatozoa was 12.6 x 10(9) at D5/6 instead of 6.3 x 10(9) at D0/1. When the spermatozoa were stored in the presence of 10 ng/mL IAA for 5-6 days at 15 degrees C, the fertility and prolificacy of the females inseminated with only 6.3 x 10(9) spermatozoa were identical to those of the females inseminated with an equal number of spermatozoa at D0/1 in the presence or absence of IAA.

Acrosome↗
PubMed1995

[Autoimmune asthenospermia induced by an epididymal protein, ovine prealbumin (oPES)].

The motility of ejaculated spermatozoa (% of progressive spermatozoa) has been evaluated visually on 26 adult rams. Half the animals were immunized with a secretory epididymal protein (prealbumin epididymal-specific (PES) ovine) injected according to immunizing protocols (Freund or Hunter adjuvant) intraperitoneally or intradermally (Freund or Hunter adjuvant) or intramuscularly (Hunter adjuvant or PES only). The other animals received the same product without the protein (controls). The product resulted in a strong asthenospermia which parallels the transient presence of anti-PES antibodies in the seminal plasma. A study with 64 ewes showed that the fertility of 10 immunized rams that had recovered sufficient forward motility to ensure fertilization, did not differ from that of control rams.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Protein kinases and protein synthesis are involved in desensitization of the plasminogen activator response of rat Sertoli cells by follicle-stimulating hormone.

Tissue-type plasminogen activator (tPA) secretion is a specific response of Sertoli cells to follicle-stimulating hormone (FSH), which is lower after preincubation of the cells with low FSH concentrations because of FSH receptor/Gs protein uncoupling. In this report, we present evidence that this desensitization induced by the lowest FSH concentrations is suppressed by specific peptidic inhibitors of endogenous PKA and PKC in permeabilized Sertoli cells. In contrast, desensitization promoted by slightly higher FSH concentrations is not mediated through PKA or PKC activation but is dependent on protein neosynthesis.

Animals↗
PubMed1994

Structure-function relationships and mechanism of action of pituitary and placental gonadotrophins.

Data from the author's laboratory on relationships between structure and function of equine luteinizing hormone, follicle-stimulating hormone and choriogonadotrophin as well as their mechanisms of action are reviewed and compared with their human counterparts. Polymorphism of these hormones and problems associated with their purification are discussed as well as the association and dissociation of their alpha- and beta-subunits. The affinity of receptor binding, the superactivity of membrane transduction and homologous desensitization of target cells by non-stimulatory doses of the gonadotrophins are also reviewed.

Chorionic Gonadotropin↗
PubMed1993

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.

Animals↗
PubMed1993

Involvement of cyclic adenosine monophosphate-dependent protein kinase isozymes in tissue plasminogen activator secretion by rat Sertoli cells stimulated with follicle-stimulating hormone in vitro.

This study was undertaken to investigate, in freshly isolated rat Sertoli cells, the physiological function of the type I and type II cyclic adenosine monophosphate (cAMP)-dependent protein kinase isozymes in tissue-type plasminogen activator secretion and the regulation of this cAMP process by follicle-stimulating hormone (FSH). Follicle-stimulating hormone-induced tissue-type plasminogen activator secretion depends upon intracellular cAMP levels. The changes in cAMP amounts required to activate maximally the tissue-type plasminogen activator secretion are extremely small, a cAMP threshold having to be reached for triggering the tissue-type plasminogen activator output. Intact Sertoli cells were incubated with combinations of cAMP analogs specific for each cAMP-dependent protein kinase type and complementary in their cAMP binding site on the cAMP-dependent protein kinase regulatory subunits: 8-aminohexylamino-cAMP = type 1, site 1; 8-thiomethyl-cAMP = type II, site 1 and N6-benzoyl-cAMP = types I/II, site 2. This allowed us to activate selectively each cAMP-dependent protein kinase type in a synergistic manner and then to evaluate their respective influence in the specific tissue-type plasminogen activator response. We establish that both of the cAMP-dependent protein kinase types are present and functional; the activity of the type I isozyme is preponderant (60%) in the cAMP-dependent tissue-type plasminogen activator secretion. Likewise, when these cAMP analogs were coupled with endogenously generated cAMP by FSH or forskolin, both of the cAMP-dependent protein kinase types were involved in the tissue-type plasminogen activator production.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

Induction of ovulation and superovulation in mares using equine LH and FSH separated by hydrophobic interaction chromatography.

Pharmacological control of reproduction in mares requires the use of equine gonadotrophins to avoid induced immunological resistance. Crude equine gonadotrophins (CEG) have been used but the presence of equine luteinizing hormone (eLH) and follicle-stimulating hormone (eFSH) in CEG has led to disappointing results in superovulation studies. Separation of eLH and eFSH activities from CEG is necessary to overcome this problem. The hydrophobic properties of the two hormones were sufficiently different to permit their separation by hydrophobic interaction chromatography (HIC) on a phenyl Sepharose matrix. Good yields of separate FSH and LH fractions were readily obtained by stepwise elution and the method was adapted for large scale preparations of enriched fractions of eLH and eFSH. Two experiments were performed in vivo to evaluate the biological activity of the HIC fractions. Experiment 1 showed that biological activity of the LH fraction in inducing ovulation of preovulatory follicles was similar to that obtained with CEG, indicating that LH bioactivity was not altered by HIC. Experiment 2 demonstrated that biological activity of the FSH fraction was identical (as far as rate of ovulation was concerned) to that of CEG in superovulating mares, indicating that FSH activity was also not altered by HIC. Although we have not obtained better results with the separate equine gonadotrophins than with CEG, it is potentially advantageous to use preparations with single activity to obtain a controlled balance of FSH and LH activity. The HIC technique was chosen because it could easily be scaled up to provide the large amounts of the separate hormones needed for the treatment of a large number of mares.

Animals↗
PubMed1993

[Binding specificity and stimulation efficacy of gonadotropins].

Gonadotropins are made up of two glycoproteic subunits bound by non-covalent linkages. One subunit, named alpha, is common in all glycoprotein hormones; the second subunit, named beta, is specific for each hormone. This review present our recent results on dependence of activity on quaternary structures, on affinity and specificity of the binding of gonadotropins to their receptors and stimulation efficiency of gonadotropins.

Amino Acid Sequence↗
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↗