Characterization of gonadotropins and their receptors in a chondrichthyan, Scyliorhinus canicula, fills a gap in the understanding of their coevolution.

In Gnathostomes, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis, with the involvement of the pituitary gonadotropic hormones (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which activate their cognate receptors, FSHR and LHR, expressed in gonads. Each GTH consists of a common α subunit and of a specific FSHß or LHß subunit. Chondrichthyes (holocephalans and elasmobranchs) is a sister group of bony vertebrates. This position is highly favorable for the understanding of the evolution of endocrine regulations of reproduction among gnathostomes. Surprisingly, the characterization of gonadotropins and their receptors is still limited in chondrichthyes. In the present study, GTH and GTHR sequences have been identified from several chondrichthyan genomes, and their primary structures were analyzed relative to human orthologs. 3D models of GTH/GTHR interaction were built, highlighting the importance of the receptor hinge region for ligand recognition. Functional hormone-receptor interactions have been studied in HEK cells using the small-spotted catshark (Scyliorhinus canicula) recombinant proteins and showed that LHR was specifically activated by LH whereas FSHR was activated by both FSH and LH. Expression profiles of GTHs and their receptors were explored by real-time PCR, in situ hybridization and immunohistochemistry during spermatogenesis, along the male genital tract and other tissues, as well as in some female tissues for comparison. Tissue-expression analyses showed that the highest levels were observed for fshr transcripts in testis and ovary and for lhr in specific extragonadal tissues. The two receptors were expressed at all stages of spermatogenesis by both germ cells and somatic cells, including undifferentiated spermatogonia, spermatocytes, spermatids, somatic precursors and Sertoli cells; differentiated Leydig cells being absent in the testis of S. canicula. Receptors were also expressed by the lymphomyeloid epigonal tissue and the testicular tubules. These results, suggest a wide range of gonadotropin-regulated functions in Elasmobranchs, as well as functional redundancy during spermatogenesis. These extended functions are discussed in an evolutionary context in which the specificity of gonadotropin signaling must have contributed to the evolution of gonadal cells' morphology and function.

Highly-Sensitive In Vitro Bioassays for FSH, TSH, PTH, Kp, and OT in Addition to LH in Mouse Leydig Tumor Cell.

We demonstrate here that highly sensitive in vitro bioassays for FSH, TSH, and PTH can be set up in mouse Leydig Tumor Cells (mLTC), in addition to the normal LH/CG bioassay, after they were transfected with expression vectors encoding the corresponding Gs Protein-Coupled Receptors (GsPCR), such as FSHR, TSHR, or PTHR. Although the ß2 adrenergic receptor is also a GsPCR, its expression in mLTC led to a significant but very low cAMP response compared to those observed with FSH, TSH, or PTH. Similarly, after transfection of the GiPCR MT1 melatonin receptor, we did not observe any inhibitory effect by melatonin of the LH or hCG stimulation. Interestingly, after transfection of mLTC with the human kisspeptin receptor (hKpR), which is a GqPCR, we observed a dose-dependent synergy of 10-12-10-7 M kisspeptin variants with a fixed concentration of 0.3 nM LH or hCG. Without any exogenous receptor transfection, a 2 h preincubation with OT or AVP led to a dose-dependent cAMP response to a fixed dose of LH or hCG. Therefore, highly sensitive in vitro bioassays for various hormones and other GPCR ligands can be set up in mLTC to measure circulating concentrations in only 3-10 µL of blood or other body fluids. Nevertheless, the development of an LHRKO mLTC cell line will be mandatory to obtain strict specificity for these bioassays to eliminate potential cross-reaction with LH or CG.

Effect of Soluble Adenylyl Cyclase (ADCY10) Inhibitors on the LH-Stimulated cAMP Synthesis in Mltc-1 Leydig Cell Line.

In contrast to all transmembrane adenylyl cyclases except ADCY9, the cytosolic soluble adenylyl cyclase (ADCY10) is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions. In the present paper, we focus on ADCY10 localization and a kinetic analysis of intracellular cAMP accumulation in response to human LH in the absence or presence of four different ADCY10 inhibitors (KH7, LRE1, 2-CE and 4-CE) in MTLC-1 cells. ADCY10 was immuno-detected in the cytoplasm of MLTC-1 cells and all four inhibitors were found to inhibit LH-stimulated cAMP accumulation and progesterone level in MLTC-1 and testosterone level primary Leydig cells. Interestingly, similar inhibitions were also evidenced in mouse testicular Leydig cells. In contrast, the tmAC-specific inhibitors ddAdo3' and ddAdo5', even at high concentration, exerted weak or no inhibition on cAMP accumulation, suggesting an important role of ADCY10 relative to tmACs in the MLTC-1 response to LH. The strong synergistic effect of HCO3- under LH stimulation further supports the involvement of ADCY10 in the response to LH.

Fluoxetine affects cytosolic cAMP, ATP, Ca2+ responses to forskolin, and survival of human ovarian granulosa tumor COV434 cells.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, exhibits various other mechanisms of action in numerous cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The aim of this study was to determine the off-target effects of the anti-depressant drug FLX, on the human ovarian granulosa tumor COV434 cells stimulated by forskolin (FSK), by measuring the real-time kinetics of intracellular cyclic AMP (cAMP), ATP level, cytoplasmic calcium ([Ca2+]cyt) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.6 and 10 µM) induces a decrease in intracellular cAMP response to FSK, a drop in ATP content and stimulates cytoplasmic Ca2+ accumulation in COV434 cells. Only the highest concentrations of FLX (5-10 µM) diminished cell viability. The present report is the first to identify an action mechanism of FLX in human tumor ovarian cells COV434 cells and thus opening the way to potential use of fluoxetine as a complementary tool, in granulosa tumor treatments.

Cell Communications among Microorganisms, Plants, and Animals: Origin, Evolution, and Interplays.

Cellular communications play pivotal roles in multi-cellular species, but they do so also in uni-cellular species. Moreover, cells communicate with each other not only within the same individual, but also with cells in other individuals belonging to the same or other species. These communications occur between two unicellular species, two multicellular species, or between unicellular and multicellular species. The molecular mechanisms involved exhibit diversity and specificity, but they share common basic features, which allow common pathways of communication between different species, often phylogenetically very distant. These interactions are possible by the high degree of conservation of the basic molecular mechanisms of interaction of many ligand-receptor pairs in evolutionary remote species. These inter-species cellular communications played crucial roles during Evolution and must have been positively selected, particularly when collectively beneficial in hostile environments. It is likely that communications between cells did not arise after their emergence, but were part of the very nature of the first cells. Synchronization of populations of non-living protocells through chemical communications may have been a mandatory step towards their emergence as populations of living cells and explain the large commonality of cell communication mechanisms among microorganisms, plants, and animals.

Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.

We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells.

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-oFSHß or anti-oLHß 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 αß 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.

Single-chain human follicle-stimulating hormone with a di-N-glycosylated linker.

The classic way to produce single-chain (sc) glycoprotein hormones is to fuse their two subunits through the carboxy-terminal peptide (CTP) from human Choriogonadotropin (hCG). The CTP confers a longer half-life to single-chain hormones thanks to its four O-glycosyl side chains. However, unlike syncytiotrophoblastic cells, most cells used for recombinant protein production do not transfer O-glycosyl chains efficiently. We thus choose to fuse the hFSH subunits with a linker comprising two N-glycosyl side chains (sc-hFSH LNN) or none (sc-hFSH L0N), that were generated using two expression systems, HEK293 and CHO K1 cells. Their production levels and biological activities were tested and compared. Both expression systems successfully produced biologically active sc-hFSH, but, in our hands, CHO K1 cells yielded about 30-fold higher amounts of recombinant protein than HEK293 cells. Moreover, sc-hFSH L0N was considerably less expressed than sc-hFSH LNN in both cell types. Our data show that sc-hFSH L0N and sc-hFSH LNN produced from both cell lines stimulate cAMP and progesterone production in mLTC cells expressing hFSH receptors and exhibit similar B/I (in vitro Bioactivity/Immuno activity) ratios. Finally, the ratio of in vivo/in vitro bioactivities for sc-hFSH LNN relative to natural pituitary heterodimeric hFSH increased 8-fold, most likely because of a longer half-life in the blood.

Undissociable chemically cross-linked and single-chain gonadotropins.

Undissociable gonadotropins can be obtained either by chemical cross-linking of the natural heterodimeric hormones or by expressing recombinant single-chain molecules through the fusion of their α and ß polypeptide sequences. These undissociable hormones are not more active than their natural heterodimeric counterparts indicating that the ß-subunit seatbelt embracing the α-subunit ensures the αß heterodimer stability in physiological conditions. The main interests of single-chain gonadotropins are that 1/only one single plasmid is required to produce an active recombinant hormone, 2/the two subunits' domains are constantly present in equal amounts and 3/they remain in close proximity even at low concentration for forming the hormone bioactive 3D structure. These undissociable gonadotropins have been shown to exhibit excellent stability and activity but they have not yet been commercialized probably because of immunogenicity risks and cost of production. Nevertheless, they might be used as a basis for the development of chemically simpler and cheaper ligands of LH and FSH receptors.

Membrane Hormone Receptors and Their Signaling Pathways as Targets for Endocrine Disruptors.

The endocrine disruptors are mostly small organic molecules developed for numerous and very diverse industrial applications. They essentially act through nuclear receptors with small and hydrophobic endogenous ligands. Nevertheless, potential adverse effects through membrane hormone receptors cannot be ruled out, and have indeed been observed. The present paper reviews how orthosteric and allosteric binding sites of the different families of membrane receptors can be targets for man-made hydrophobic molecules (components of plastics, paints, flame retardants, herbicides, pesticides, etc.). We also review potential target proteins for such small hydrophobic molecules downstream of membrane receptors at the level of their intracellular signaling pathways. From the currently available information, although endocrine disruptors primarily affect nuclear receptors' signaling, membrane receptors for hormones, cytokines, neuro-mediators, and growth factors can be affected as well and deserve attention.

Chicken white egg chemerin as a tool for genetic selection for egg weight and hen fertility.

Embryo mortality rate, which can reach up to 40% in avian species, is a major issue for breeding. It is therefore important to identify new embryo development biomarkers for genetic selection to improve reproductive performances. We have recently shown that chemerin is expressed in the oviductal hen magnum, accumulates in egg white, is correlated with embryo survival and could thus be used as a molecular marker of embryo development. Eggs from seven hen breeds (n = 70) were collected during five successive days at the end of the laying period. After weighing eggs, yolk and albumen, an egg white sample from each egg was collected and a blood sample was taken from each hen. Chemerin concentrations in albumen and blood samples were measured by a specific home made ELISA assay. Hen's plasma and egg's albumen chemerin levels were found to be correlated with reproductive parameters such as fecundity, fertility, embryo mortality, hatchability and laying rates. The inter-hen chemerin level variability in albumen was higher than intra-hen except for one breed (R+). We observed significantly different levels of chemerin in egg white between breeds. However, chemerin concentrations in egg white were not significantly associated to variations of hen plasma chemerin levels. Interestingly, we observed negative correlations between albumen chemerin concentrations and egg weight (r = -0.43, p = 0.001), between albumen weight (r = -0.40, p = 0.002), and between yolk weight (r = -0.28, p = 0.03). We also showed negative correlations between egg white chemerin concentrations and fecundity (r = -0.32, p = 0.011) and fertility (r = -0.27, p = 0.04) whereas no significant correlation was observed with the laying rate. Taken together, these results suggest that egg white chemerin concentration might be a good biomarker for genetic selection for egg weight and fertility in hens, provided these data are confirmed on a larger scale.

Potentiation of the reductase activity of protein disulphide isomerase (PDI) by 19-nortestosterone, bacitracin, fluoxetine, and ammonium sulphate.

Protein disulphide isomerase (PDI) in the endoplasmic reticulum catalyzes the rearrangement of disulphide bridges during folding of secreted proteins. It binds various molecules that inhibit its activity. But here, we looked for molecules that would potentiate its activity. PDI reductase activity was measured in vitro using di-eosin-oxidized glutathione as substrate. Its classical inhibitor bacitracin was found to exert a biphasic effect: stimulatory at low concentrations (∼10(-6) M) and inhibitory only at higher concentrations (∼10(-4)-10(-3) M). The weak oestrogenic molecule bisphenol A was found to exert a weak inhibitory effect on PDI reductase activity relative to the strong oestrogens, ethynylestradiol, and diethylstilbestrol. Like 19-nortestosterone, fluoxetine was found to exert a potentiating effect on PDI reductase activity and their potentiating effects could be reversed by increasing concentrations of oestrogens. In conclusion, this paper provides the first identification of potentiators of PDI activity that are potential pharmaceuticals against pathologies affecting protein folding such as Alzheimer's disease.

Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma.

In previous studies, we had shown the synergistic effect of 10-5 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10-12-10-6 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10-12-10-6 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK to optimize the assay down to a sensitivity compatible with the detection of the circulating concentrations of these hormones in various species. Finally, the optimization relies on three independent phenomena: (1) the inhibition of nucleotide phosphodiesterase by IBMX (3-isobutyl-1-methylxanthine) to avoid cAMP degradation; (2) the strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-h luminescence measurement; (3) the stimulatory effect of 10-8M OXT on the amplitude of transfected cAMP-sensitive luciferase response. By doing this, the detectable concentrations are at the 1-10 pg/well (pM range) for the LHs and CGs from various species. The bioactivities of circulating LHs and CGs in blood or urine are therefore expected to be measurable in 10 µL-plasma samples from mammalian species and maybe others. Indeed, a preliminary study with equine and donkey plasma samples shows that the measured bioactivity was fully inhibited by a specific MAB against the receptor-binding region of equine LH (eLH) and equine CG (eCG), thus eliminating a possible response due to interfering substances other than eLH or eCG. From these data, it is expected that the bioactivity profiles of these hormones will be measurable in the blood of human, equine, and ovine species and very likely in rodents, ruminants, and hopefully in most other mammalian species. LAY SUMMARY: Luteinizing hormone (LH) plays a central role in controlling ovary and testicle functions in many animals, including humans. The highly sensitive method, known as an assay, described in this paper, measures the biological activity of LH in the blood of mammals. The assay is performed in culture of cells derived from mouse testicles in the presence of factors that diminish the detection threshold for LH. The knowledge of the bioactive LH concentration dynamics in the blood is very informative about the reproductive status of male and female mammals. This new in vitro bioassay provides a powerful tool to get this information.

Comparative structure analyses of cystine knot-containing molecules with eight aminoacyl ring including glycoprotein hormones (GPH) alpha and beta subunits and GPH-related A2 (GPA2) and B5 (GPB5) molecules.

BACKGROUND: Cystine-knot (cys-knot) structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH) superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS) bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), Thyroid-Stimulating Hormone (TSH) and Chorionic Gonadotropin (CG) are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa) rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins), 9 (cerberus, DAN), 10 (GPA2, GPB5, GPHalpha) and 12 (GPHbeta) cysteine residues in their sequence. DISCUSSION: The comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization resembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH beta-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation. SUMMARY: The 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0.1 microM: this would be consistent with a short-term paracrine role but not with an endocrine role after dilution in circulation. Consequently, GPA2 and GPB5 could exert separate endocrine roles either during development and/or during adult life of both vertebrates and invertebrates.

Comparative Overview of the Mechanisms of Action of Hormones and Endocrine Disruptor Compounds.

Endocrine Disruptor Compounds (EDCs) are synthetic or natural molecules in the environment that promote adverse modifications of endogenous hormone regulation in humans and/or in wildlife animals. In the present paper, we review the potential mechanisms of EDCs and point out the similarities and differences between EDCs and hormones. There was only one mechanism, out of nine identified, in which EDCs acted like hormones (i.e. binding and stimulated hormone receptor activity). In the other eight identified mechanisms of action, EDCs exerted their effects either by affecting endogenous hormone concentration, or its availability, or by modifying hormone receptor turn over. This overview is intended to classify the various EDC mechanisms of action in order to better appreciate when in vitro tests would be valid to assess their risks towards humans and wildlife.

Inhibition by fluoxetine of LH-stimulated cyclic AMP synthesis in tumor Leydig cells partly involves AMPK activation.

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor (SSRI), has been shown to exhibit other mechanisms of action in various cell types. Consequently, it might have unexpected adverse effects not related to its intended use, possibly in the endocrine regulation of reproduction. We show in the present report that after a 1-hour preincubation of MLTC-1 Leydig cells with FLX, the intracellular cyclic adenosine monophosphate (cAMP) responses to Luteinizing Hormone (LH) and forskolin (FSK) are reduced through AMPK-dependent and -independent pathways respectively. FLX at low concentrations (12.5µM and 25µM) induced this inhibition without triggering AMPK phosphorylation, while higher FLX concentrations (50µM and 100µM) induced AMPK phosphorylation and lowered ATP concentration similarly to Metformin. Pretreatment with the specific AMPK inhibitor Compound C (CpdC), significantly diminished the inhibition of cAMP synthesis caused by high concentration of FLX. Moreover, as expected FLX also caused a decline of steroidogenesis which is under the control of cAMP. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent and occurs in MLTC-1 cells through two mechanisms, AMPK-independent and AMPK-dependent, at low and high concentrations, respectively. FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells and mouse testicular Leydig cells, suggesting similar mechanisms in both cell types.

Estrogenic Compounds or Adiponectin Inhibit Cyclic AMP Response to Human Luteinizing Hormone in Mouse Leydig Tumor Cells.

Mouse Leydig Tumor cells (mLTC), transiently expressing cAMP-dependent luciferase, were used to study the influence of sexual steroids and of adiponectin (ADPN) on the cAMP response to luteinizing hormones (LH). While testosterone and progesterone had no significant effect, several molecules with estrogenic activity (17ß-estradiol, ethynylestradiol, and bisphenol A) provoked a decrease in intracellular cyclic AMP accumulation under 0.7 nM human LH stimulation. Adiponectin exhibited a bimodal dose-effect on LH response: synergistic between 2-125 ng/mL and inhibitory between 0.5-5 µg/mL. In brief, our data indicate that estrogens and ADPN separately exert rapid (<1 h) inhibitory and/or synergistic effects on cAMP response to LH in mLTC-1 cells. As the inhibitory effect of each estrogenic molecule was observed after only 1-h preincubation, it might be mediated through the G protein-coupled estrogen receptor (GPER) membrane receptor, but this remains to be demonstrated. The synergistic effect with low concentrations of ADPN with human Luteinizing Hormone (hLH) was observed with both fresh and frozen/thawed ADPN. In contrast, the inhibitory effect with high concentrations of ADPN was lost with frozen/thawed ADPN, suggesting deterioration of its polymeric structure.

Choice of protocol for the in vivo bioassay of equine Chorionic Gonadotropin (eCG / PMSG) in immature female rats.

Equine Chorionic Gonadotropin (eCG) previously known as Pregnant Mare Serum Gonadotropin (PMSG) has been used for decades in regulating reproduction in various domestic animal species. Its use necessitates a good measurement of its bioactivity in commercial preparations. The EUROPEAN PHARMACOPEIA (EP 7.0) recommends 5-6 subcutaneous injections in immature female rats for the in vivo bioassay for eCG as in the case for measurement of FSH bioactivity in the Steelman & Pohley assay (1953). This recommendation is in marked contrast with the classical and long-time used PMSG assay of Cole & Erway (1941) that includes only one subcutaneous injection, 3 days before measurement of ovarian weight. As this difference introduces much confusion in the determination of eCG/PMSG bioactivity in commercial sources, we have performed parallel assays of several PMSG preparations, with both protocols. The single-injection protocol takes into account the long half-life of eCG in bloodstream and provokes an ovarian stimulation at lower concentrations than the multiple-injection protocol. As the single-injection protocol also mimicks the protocol used in cattle, it is preferable to the multiple-injection protocol of the current EP.

Long-term exposure of male rats to low-dose ethinylestradiol (EE2) in drinking water: effects on ponderal growth and on litter size of their progeny.

Male rats were exposed to 17alpha-ethinylestradiol (EE2) in their drinking water either at concentrations of 1-1000 ng/mL (1 ppb to 1 ppm) for 3 weeks immediately after weaning from post-natal day 22 (PND 22) to PND 43 or at a concentration of 10 ng/mL from PND 5 onwards. Although given temporarily between PND 22 and PND 43, the 0.1 and 1 ppm doses of EE2 were found to significantly affect the ponderal growth of the animals. No male was found to be sterile neither in both exposed groups nor in control groups. Nevertheless, the males exposed to 10 ng/mL EE2 in drinking water from PND 5 onwards conceived a significantly higher proportion (25%) of small litters (one to five pups) than control males (0-3%). When exposed to 1 microg/mL EE2 but only from PND 22 to PND 43, the males produced an intermediate proportion (15%) of small litters in their progeny but this might be consecutive to the observed retardation in weight gain.

Endocrine Disruptor Compounds (EDCs) and agriculture: The case of pesticides.

A number of pesticides are suspected or proved to act as endocrine disruptor compounds (EDCs). In the present survey of the literature, we try to define the main issues to be considered to classify individual pesticides as EDC or not.