Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility.

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0-80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.

Membrane raft-mediated regulation of glucose signaling pathway leading to acrosome reaction in chicken sperm†.

Despite knowledge that glucose metabolism is essential for the regulation of signaling cascades in the sperm that are pre-assembled into specific areas and function at multistage for fertilization, the physiological roles of glucose in avian sperm are poorly understood. Accumulated results of studies conducted in our laboratory and others indicate that sperm possess membrane microdomains, or membrane rafts, which play important roles in several processes, including the induction of acrosome reaction (AR). When characterizing proteomes associated with chicken sperm rafts, we observed marked enrichment of glucose transporter 3 (GLUT3). Here we show that glucose uptake is mediated by membrane rafts and stimulates AR induction by activating AMP-activated protein kinase (AMPK). Using a specific antibody, we observed that GLUT3 is localized to the entire flagellum and acrosome region and highly associated with membrane rafts. The addition of glucose stimulated AR in a dose-dependent manner without affecting sperm motility. AR and glucose uptake assays were performed using both inhibitors and activators, and demonstrated that glucose-dependent AR results from the activity of a glucose transporter located in membrane rafts and associated with AMPK. To better understand the mechanism of AMPK activation by glucose, we evaluated localization and phosphorylation status of AMPKα, showing that glucose uptake stimulates AMPKα phosphorylation, leading to its complete activation. Together, these results lead us to propose a novel mechanism by which glucose uptake stimulates the AMPK signaling pathway via membrane rafts, resulting in maximal acrosomal responsiveness in avian sperm as migrating upward to a fertilization site.

Membrane rafts regulate sperm acrosome reaction via cAMP-dependent pathway in chickens (Gallus gallus domesticus).

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-ß-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.

Membrane-Mediated Regulation of Sperm Fertilization Potential in Poultry.

Fertilization requires successful completion of molecular events taking place at different spatiotemporal scales. Transcriptionally and translationally inactive sperm need to rely on pre-assembled pathways modulated by extracellular signals that traverse the plasma membranes. However, species differences in how sperm respond to them delay the progress toward a comprehensive understanding of how activation of the signaling cascades is coordinated in poultry sperm. In chickens, recent studies have found that membrane rafts are present on the sperm surface and play important roles in regulating multistage fertilization. In this review, we focus on three steps in which membrane alteration plays a key role. The first is post-testicular maturation, in which bird sperm acquire fertilization functions through biochemical changes. The second part of this review concerns membrane regulation of sperm-egg binding and the acrosome reaction. Finally, we extend our discussion to the translation of membrane raft theory into a technical principle for the commercial production and genetic preservation of poultry.

Functional difference of ATP-generating pathways in rooster sperm (Gallus gallus domesticus).

Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity.

Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus).

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.