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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, ß-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on ß-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.

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. The reversibility of this stimulation was studied by removing the hormone from the culture medium after 10 min of incubation. The ratios of kinetics AUC after removing or not the hormone were used to evaluate the stimulation reversibility of each hormone. Natural and recombinant hLHs and hCGs were found to exhibit slowly reversible activation compared to pituitary rat, ovine, porcine, camel and equine LHs, serum-derived eCG (PMSG) and recombinant eLH/CGs. Carbohydrate side chains are not involved in this phenomenon since natural and recombinant homologous hormones exhibit the same reversibility rates. It is still unknown whether only one human subunit, α or ß, is responsible for this behaviour or whether it is due to a particular feature of the hLH and hCG quaternary structure.

[AMPK, regulator of sperm energy and functions]. / L'AMPK, régulateur de l'énergie et des fonctions des spermatozoïdes.

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. AMPK is involved in the regulation of sperm quality by its action on the proliferation of Sertoli cells. AMPK also directly controls the quality of sperm by its involvement in the regulation of motility and acrosome reaction. It is also involved in the management of lipid peroxidation and gametes antioxidant enzymes. Thus, AMPK appears as a key signaling protein for sperm and male fertility control.

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). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction.

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 presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and ß), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKß was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and ß) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry in the cells.

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. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.