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.

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin.

OBJECTIVE: To identify amino-acids in the alpha-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) alpha-subunit combined with an equid beta-LH subunit. DESIGN: Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey alpha-subunit at positions 70, 85, 89, 93 and 96 (alphadk5xmut), 18 (alphadkK18E) or 78 (alphadkI78A). METHODS: These different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH and beta-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. RESULTS: alphap-dk or alphadk-p exhibited FSH activity when co-expressed with beta-eLH but not with beta-dkLH. alphadkK18E or alphadkI78A gave hybrids with no FSH activity and important LH activity when expressed with beta-dkLH. alphadkI78A/betaeLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in alpha-dk had no effect on FSH bioactivity when co-expressed with beta-eFSH. CONCLUSIONS: Amino-acids present in both the first two-thirds and the last third of the alpha-subunit of equid LHs are involved in their heterologous biospecificity. Ile alpha78 exerts as strong an influence on it as the beta102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone.

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes.

Induced lactation with a dopamine antagonist in mares: different responses between ovariectomized and intact mares.

The aim of this study was to compare the effects of treatment with repeated injections of sulpiride (a dopamine D2 antagonist) on prolactin secretion and induced lactation in ovariectomized and intact adult mares and to verify if this induction was possible at the beginning and at the end of the birth season. Two experiments were carried out in September [experiment (expt) 1], and in March (expt 2), in France (48 degrees N). In expt 1, three groups of five mares were tested: intact-control, intact-treated and ovariectomized-treated mares. In expt 2, mares previously subjected to artificial photoperiod were assigned in two groups: four intact-control and five intact-treated mares. The cyclicity of intact mares was previously synchronized with PGF2alpha injections, then all the mares were in the follicular phase at the beginning of treatment. Sulpiride was intramuscularly injected (0.5 mg/kg of BW), twice a day. Mares were milked at 7:30, 11:45, 16:00 and 20:15 hours. Blood samples were collected every day during the treatment for progesterone, total oestrogen and prolactin assays. In the two experiments, only treated intact mares produced milk, with a large inter-animal variability. Prolactin increase after sulpiride treatment was not so great in the ovariectomized-treated mares as in the intact-treated mares. The total correlations between prolactin, progesterone, oestrogen plasma concentrations and daily milk production were significant (0.57, 0.25, 0.17 respectively). This induction of lactation can be performed during the entire birth season in intact mares, but not in ovariectomized mares, indicating that steroids are necessary for this induction in mares treated by dopamine D2 antagonist.

Conformational stability and in vitro bioactivity of porcine luteinizing hormone.

Temperature-dependent dissociation of porcine luteinizing hormone (pLH) and of two of its glycoforms was studied by a combination of SDS-PAGE and micro-scale size-exclusion HPLC in parallel with the study of co-operative folding by high-sensitivity differential scanning calorimetry (HS-DSC). The transition temperature of dissociation of pLH at pH 7.0 as quantified by SDS-PAGE, HPLC and residual activity in radioreceptor assay was found to match exactly the transition temperature of its unfolding as measured by HS-DSC. Free alpha- and beta-subunits did not exhibit any unfolding transition in the same conditions. The microcalorimetric data for two pLH isoforms exhibiting different glycosylations were identical to those of a preparation of non-separated isoforms. It is concluded that: (a) free subunits exhibit no co-operative folding (i.e. no stable three-dimensional structure) and co-operative folding occurs only in alphabeta heterodimers; (b) the co-operative folding is responsible for the stability of the association of subunits; and (c) the heterogeneity of carbohydrate chains does not affect the stability of folding and association of subunits. The fastening of the "seat-belt" of the beta-subunit embracing the alpha-subunit by the Cysbeta26-beta110 disulfide bridge had been postulated to play a role in the preservation of the dimeric structure of gonadotropins. The present work shows that dissociation of subunits is directly related to their loss of common co-operative folding.

beta-Subunit 102-104 residues are crucial to confer FSH activity to equine LH/CG but are not sufficient to confer FSH activity to human CG.

Horse LH/CG (eLH/CG) and donkey LH/CG (dkLH/CG) are strictly LH-specific in their respective homologous species. However, both bind to the FSH receptors from non-equid species, whereas the zebra hormone (zbLH/CG) does not. The FSH/LH ratio of eLH/CG and of the alphadkbetae hybrid is about tenfold higher than that of dkLH/CG and of the alphaebetadk hybrid, showing that the betae subunit contains the structural features responsible for the high FSH activity of eLH/CG. Only six amino acid positions (51, 94, 95, 102, 103 and 106) are unique to the betae subunit when compared with the betadk and betazb subunits. The Gly-Pro and Val-Phe sequences in positions 102-103 of betadk and betae respectively were swapped by site-directed mutations and the mutated beta-subunits cDNAs were cotransfected in COS cells with either alphae or alphadk subunit cDNA. Other mutations were also introduced in 102-103 dkLH/CG beta-subunit: Ala-Ala, Gly-Ala or Ala-Pro. These mutations with Ala-Ala, Gly-Ala or Ala-Pro in the 102-103 betadkLH/CG subunit did not change the FSH/LH ratio of dkLH/CG but the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation promoted a marked increase in the FSH/LH activity ratio. This was observed with the two heterodimers containing alphae or alphadk. Conversely, the Val(102)-Phe(103) mutation in betae led to a dramatic drop in FSH/LH activity ratio of eLH/CG, to a level similar to that of dkLH/CG. Since all FSHs possess a Gly residue at position 104, we introduced the Gly(102)-Pro(103)-Arg(104)-->Val(102)-Phe(103)-Gly(104) mutation in betadk with the expectation that the increase in FSH activity observed with the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation could be potentiated. In fact, the additional Arg(104)-->Gly(104) mutation was found to abolish the increase in FSH activity observed with Gly(102)-Pro(103)-->Val(102)-Phe(103). Mutations Gly(102)-Pro(103)-->Val(102)-Arg(103) or Gly(102)-Pro(103)-Lys(104)--> Val(102)-Arg(103)-Gly(104) were also introduced in human CGbeta (hCGbeta) to compare the impact of these amino acid changes in the well-studied gonadotrophin hCG. The betahCG mutants obtained, co-expressed either with the human or the horse alpha-subunit, did not display any FSH activity. In conclusion, the 102-104 sequence in eLH/CG beta-subunits appears to be of utmost importance for their binding to FSH receptors. However, these results obtained with equid beta-subunits are not transposable to other gonadotrophins as similar mutations in hCGbeta did not lead to any increase in FSH activity.

Stimulating effect of glycoprotein hormone free alpha-subunit and daily gonadotropin releasing hormone treatment on prolactin release from 50-day ovine foetal pituitary explants.

The aim of our study was to determine whether free alpha of glycoprotein hormones (free alpha) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free alpha, luteinzing hormone beta-subunit (LHbeta) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free alpha- and LHbeta-containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin-containing cells were first identified on day 42. Analysis of serial sections suggested that free alpha immunoreactive cells were also LHbeta-positive, indicating that free alpha was mainly synthesized by gonadotrophs. In early foetal stages, free alpha occurred in the antero-medio ventral region of the pituitary gland, whereas prolactin-containing cells were more dorsally and more caudally localized. The free alpha-, LHbeta- and prolactin-immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free alpha or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42-day-old ovine foetuses for 8 days with 10-9 or 10-7 M ovine free alpha did not affect prolactin release. By contrast, free alpha administration on pituitary explants from male and female 50-day-old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10-8 M GnRH had similar stimulatory effect to free alpha whereas a 'first day' treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free alpha at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free alpha on prolactin release.

Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge.

The C-terminal region of the beta subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (beta26-110 disulphide bridge), in vitro LH bioactivity (region beta102-110) and in in vivo LH bioactivity (beta CTP). Like the hCG beta, the equine eLH and eCG beta subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the beta subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the beta26-110 disulphide bond was mutated and the resulting mutated beta subunits were transiently co-expressed with common alpha subunit in COS7 cells. These regions were also deleted in a betaalphaeLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the betaCTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the beta26-110 disulphide bond in the betaalpha single chain had no effect on its production. However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG beta subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.

Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity.

OBJECTIVE: To test whether extension of the C-terminus of human chorionic gonadotropin (hCG) alpha-subunit (halpha) alters the bioactivity of the recombined alphabeta heterodimer. DESIGN: The stop codon of halpha was mutated to produce a 24 amino acid extension. METHODS: The extended halpha (alpha(+24)) was co-expressed with hCGbeta in COS-7 cells and the receptor binding and in vivo bioactivity of the secreted hormone was compared with its wild-type counterpart. RESULTS: This extension did not impair the binding of hCG to rat LH/CG receptors and provoked a sixfold reduction in its stimulatory activity of testosterone secretion in rat Leydig cells. CONCLUSIONS: The extension of alpha by itself does not lead to inhibition of the alphabeta heterodimer to LH receptors but the structure of the extension appears to play an important role. It is thus possible that one-chain hCG chimeras with the beta N-terminus fused to the alpha C-terminus might be active.

The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Two free isoforms of ovine glycoprotein hormone alpha-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries.

alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Humoral immune response to equine chorionic gonadotropin in ewes: association with major histocompatibility complex and interference with subsequent fertility.

In dairy ewes, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). In this report we show the presence of anti-eCG antibodies in plasma of treated ewes. The major histocompatibility complex (MHC) was involved in the individual variability of the humoral immune responses to eCG. We found significant associations between the anti-eCG response phenotype and some MHC class II alleles. The low immune response phenotype was associated with one MHC class II allele only in Lacaune ewes, and the high immune response phenotype was associated with one MHC class II allele both in Manech and in Lacaune ewes. In herds, the impact of residual anti-eCG antibodies on subsequent fertility after AI seems minimal because of an indirect elimination of high-responder ewes from AI breeding. Therefore, the true magnitude of the association between residual anti-eCG antibody concentration and fertility has been underestimated. An additional experiment without any high-responder female elimination showed a significant correlation between high residual antibody concentrations and lower lambing rate after AI at a fixed time, possibly because of a delayed preovulatory LH surge. The results suggest that anti-eCG antibody concentration is one risk factor for infertility after AI.

Cloning, sequencing and functional expression of zebra (Equus burchelli) LH.

Although donkey luteinizing hormone exhibits a very high degree of amino acid sequence identity with horse LH, its FSH activity in non-equine species is tenfold lower. The coding regions of the common zebra (Equus burchelli) glycoprotein hormone alpha-subunit and LH beta-subunit transcripts were cloned by reverse transcription-PCR from pituitary gland RNA to investigate more precisely the structure-function relationships of this gonadotrophin family. Zebra LH was then expressed in COS-7 cells and its LH and FSH activities were assessed in a rat Leydig cell bioassay (for LH) and in a cell line stably expressing the human FSH receptor bioassay (for FSH). The recombinant zebra LH, although displaying LH activity similar to that of recombinant donkey and horse LH, had no detectable FSH activity. The LH amino acid sequences of these three species are very similar, leaving only very few amino acids as potential candidates to explain the difference in their FSH activities. Moreover, according to the difference in FSH bioactivity and to the percentage identity between the sequences, the common zebra is phylogenetically closer to the donkey than it is to the horse.

The negative effect of repeated equine chorionic gonadotropin treatment on subsequent fertility in Alpine goats is due to a humoral immune response involving the major histocompatibility complex.

In dairy goats, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination. However, repeated eCG treatments are followed by decreased fertility in goats inseminated at a fixed time after treatment. In this report, we show the presence of anti-eCG antibodies in plasma of treated goats. A 500 IU eCG injection induces a humoral response, with variable concentrations of anti-eCG antibody being produced in individual goats. The analysis of successive anti-eCG immune responses over several years has demonstrated the existence of different populations of goats, defined as low, medium, and high responders. By the use of two caprine microsatellites located inside (OLADRB) and outside (BM1258) the major histocompatibility complex (MHC), a significant association (p < 0.05) between the anti-eCG antibody response and some MHC-DRB alleles was found. Goats with high antibody concentrations at the time of eCG injection (> 2.5 microg/ml) exhibited a much lower kidding rate than did other females (41.3% vs. 66.7%). Lower fertility of these goats, inseminated at a fixed time after eCG treatment, might be due to the observed delay in estrus occurrence and the preovulatory LH surge.

Beta2 adrenergic receptors mediate cAMP, tissue-type plasminogen activator and transferrin production in rat Sertoli cells.

FSH is the main regulator of Sertoli cell function. Nevertheless, several other effectors such as catecholamines can also stimulate these cells through the adenylyl cyclase transduction pathway. However, the expression of beta adrenergic receptors in Sertoli cells is a subject of controversy. The aim of the present study was to determine if there are physiologically functional beta adrenergic receptors in Sertoli cells and to which subtype(s) they belong. In freshly isolated Sertoli cells, isoproterenol, a non selective beta-adrenergic agonist, was found to stimulate cAMP production and tissue-type plasminogen activator secretion. Specific transcripts for the beta1 and beta2, but not beta3, subtypes were detected by RT-PCR analysis. Beta2 transcripts were the form expressed predominantly in Sertoli cells. Binding experiments carried out on freshly isolated and on cytospined Sertoli cells indicated that in both conditions, [125I]iodocyanopindolol binding was inhibited by a non-selective and a 2 selective antagonist, whereas a beta1 selective antagonist had no effect. Scatchard analysis of beta2 specific inhibition revealed a dissociation constant of 0.3 nM and a receptor density of 14000 sites per cell. In freshly isolated Sertoli cells, we observed that cAMP and tissue-type plasminogen activator were stimulated by isoproterenol and a beta2 selective agonist, but not by beta1 or beta3 selective agonists. Accordingly, the isoproterenol-stimulated tissue-type plasminogen activator responses were abolished by the beta2 selective antagonist only. In cultured Sertoli cells, the trend was the same: tissue-type plasminogen activator and transferrin secretions were increased by isoproterenol and beta2 but not by beta1 or beta3 selective agonists. We conclude that freshly isolated Sertoli cells express beta2 adrenergic receptors which are functionally coupled to adenylyl cyclase and that these characteristics are preserved in cell culture. For the tested parameters, catecholamines and FSH effects were similar, but response magnitudes were systematically lower with beta agonists than with FSH. As norepinephrine is normally present in physiologically-relevant amounts in the interstitial fluid, it can be suspected to play a role in the regulation of Sertoli cell function.

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.

High-level secretion of biologically active recombinant porcine follicle-stimulating hormone by the methylotrophic yeast Pichia pastoris.

An active recombinant glycoprotein hormone, porcine follicle-stimulating hormone (recFSH), has been produced for the first time in the methylotrophic yeast, Pichia pastoris. The yield of secreted recFSH (10 mg/l) was the highest ever reached. RecFSH displayed an apparent molecular mass of 41 kDa by SDS-PAGE and was found to bear only N-linked carbohydrates of the high-mannose type. Its in vitro binding and cell-stimulating activities were identical to those of pituitary porcine FSH. The large availability and the noncharged N-glycans of FSHrec should render it highly valuable for structural studies.

Deterioration of goat spermatozoa in skimmed milk-based extenders as a result of oleic acid released by the bulbourethral lipase BUSgp60.

The aim of the present study was to elucidate the mode of action of goat bulbourethral lipase (BUSgp60 lipase) previously identified as responsible for the deterioration of goat sperm viability in skimmed milk-based extenders. Milk fractions were purified by micro- and ultrafiltration and characterized by SDS-PAGE, thin layer chromatography, triglyceride quantitative analysis and by their ability to potentiate the lipase and the sperm-deteriorating activity of the bulbourethral lipase. Components in both the phosphocaseinate and soluble whey protein fractions enhanced the lipase activity of BUSgp60 but only the phosphocaseinate fraction, which contains triglycerides, promoted deterioration of spermatozoa in the presence of bulbourethral gland secretion. These data suggest that the sperm-deteriorating effect of bulbourethral gland secretion is due to the catalysis of triglyceride hydrolysis, and that proteins increase this activity. BUSgp60 hydrolysed milk triglycerides and triolein very effectively, and its lipase activity was enhanced by several highly purified milk proteins. The major cis-unsaturated fatty acid from milk (oleic acid) but not the major saturated fatty acid (palmitic acid) exhibited dose-dependent detrimental effects on goat spermatozoa. Therefore, the catalysis of oleic acid formation from residual milk triglycerides by BUSgp60 appears responsible for the deterioration of goat spermatozoa when unwashed semen is diluted in skimmed milk-based extenders. The precise mechanism of action of oleic acid remains to be elucidated but the drawbacks of washing buck semen might be avoided by inhibiting BUSgp60 or by depriving it of substrate.