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.

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 β2 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.

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 β polypeptide sequences. These undissociable hormones are not more active than their natural heterodimeric counterparts indicating that the β-subunit seatbelt embracing the α-subunit ensures the αβ 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.

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.

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.

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

Unlabelled: In previous studies, we had shown the synergistic effect of 10 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 10-10 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 10-10 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.

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.

Fluoxetine affects cytosolic cAMP, ATP, Ca 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 ([Ca]) and survival of COV434 cells. We show that incubating COV434 cells with FLX (between 0.

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.

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 (17β-estradiol, ethynylestradiol, and bisphenol A) provoked a decrease in intracellular cyclic AMP accumulation under 0.7 nM human LH stimulation.

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.

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.

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.

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.

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.

Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors.

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown.

Low reversibility of intracellular cAMP accumulation in mouse Leydig tumor cells (MLTC-1) stimulated by human Luteinizing Hormone (hLH) and Chorionic Gonadotropin (hCG).

In order to study the intracellular cAMP response kinetics of Leydig cells to hormones with LH activity, we used MLTC-1 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be followed using oxiluciferin luminescence produced from catalyzed luciferin oxidation. The potencies of the different LHs and CGs were evaluated using areas under the curves (AUC) of their kinetics over 60 min stimulation. All mammalian LHs and CGs tested were found to stimulate cAMP accumulation in these cells.

[AMPK, regulator of sperm energy and functions].

The 5'-AMP-activated protein kinase, AMPK, is a key protein kinase in the metabolism of the cell that regulates many metabolic pathways. The involvement of cell metabolism in sperm ability to fertilize is well established, but only a few studies have focused on the role of AMPK in the control of male fertility. This article summarizes the known role of AMPK in this area.

Calcium channels in chicken sperm regulate motility and the acrosome reaction.

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1).

Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway.

The 5'-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5'-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism.

Structure-function relationships of glycoprotein hormones and their subunits' ancestors.

Glycoprotein hormones (GPHs) are the most complex molecules with hormonal activity. They exist only in vertebrates but the genes encoding their subunits' ancestors are found in most vertebrate and invertebrate species although their roles are still unknown. In the present report, we review the available structural and functional data concerning GPHs and their subunits' ancestors.

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

Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M).