Alexander J. Travis

The facilitative glucose transporter GLUT3: 20 years of distinction.

Glucose metabolism is vital to most mammalian cells, and the passage of glucose across cell membranes is facilitated by a family of integral membrane transporter proteins, the GLUTs. There are currently 14 members of the SLC2 family of GLUTs, several of which have been the focus of this series of reviews. The subject of the present review is GLUT3, which, as implied by its name, was the third glucose transporter to be cloned (Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers MG, Shows TB, Bell GI. J Biol Chem 263: 15245-15248, 1988) and was originally designated as the neuronal GLUT. The overriding question that drove the early work on GLUT3 was why would neurons need a separate glucose transporter isoform? What is it about GLUT3 that specifically suits the needs of the highly metabolic and oxidative neuron with its high glucose demand? More recently, GLUT3 has been studied in other cell types with quite specific requirements for glucose, including sperm, preimplantation embryos, circulating white blood cells, and an array of carcinoma cell lines. The last are sufficiently varied and numerous to warrant a review of their own and will not be discussed here. However, for each of these cases, the same questions apply. Thus, the objective of this review is to discuss the properties and tissue and cellular localization of GLUT3 as well as the features of expression, function, and regulation that distinguish it from the rest of its family and make it uniquely suited as the mediator of glucose delivery to these specific cells.

The role of cholesterol efflux in regulating the fertilization potential of mammalian spermatozoa

Following spermatogenesis and spermiogenesis, mammalian spermatozoa leaving the testis appear to be morphologically mature but clearly are immature from a functional standpoint; that is, they have acquired neither progressive motility nor the ability to fertilize a metaphase II‐arrested egg. Although progressive motility is acquired and signaling pathways mature during sperm transit through the epididymis, complete fertilization capacity in vivo is conferred only during residence in the female reproductive tract. Similar observations have been made using a variety of in vitro assays, suggesting that a series of events, some initiated by environmental cues, confer on sperm the ability to fertilize the egg. This acquired capacity to fertilize was first observed by Austin (1) and Chang (2), who demonstrated that freshly ejaculated sperm cannot fertilize eggs until they reside in the female reproductive tract for a finite period of time. All of the cellular events that allow the ejaculated sperm to fertilize an egg were subsumed into a single phenomenon that was termed “capacitation.” The ability to capacitate sperm in vitro has been of great importance to both scientists and clinicians, who have used it to study the basic biology of fertilization and to develop various assisted reproductive technologies for humans and other species. Work by many investigators has established that the process of fertilization, not surprisingly, represents a series of elegant intercellular communication and cellular activation events (3‐5). Sperm functions such as motility and capacitation in the female reproductive tract are likely modulated by environmental cues in the luminal fluid, as well as by interactions with oviductal epithelium or other female tissues (6). When sperm arrive in the oviduct and encounter the ovulated, metaphase II‐arrested egg enclosed in its cumulus cell matrix, a complex series of cell-cell and cell-ECM interactions ensues, initiating cellular signaling events that permit the fusion of the sperm and egg plasma membranes. Several of these cell-matrix and cell-cell interactions involve novel gamete surface proteins and matrices. Signal transduction events leading to gamete activation, in particular sperm acrosomal exocytosis and egg cortical granule secretion, share some features with signaling events described in somatic cells. Sperm membrane cholesterol efflux contributes to one such novel signaling mechanism that controls sperm capacitation, and the details of this effect are now beginning to be understood at the molecular level. Knowledge of how cholesterol efflux occurs in these cells, as well as how this efflux is integrated with transmembrane signaling to regulate sperm function, may reveal much about the fertilization process and may also provide insights into the role and dynamics of membrane cholesterol efflux in somatic cell function. Here, we offer a short overview of the role of cholesterol efflux in regulating sperm capacitation, with an aim toward identifying areas of future investigation that may ultimately pro

On Biodiversity Conservation and Poverty Traps

This paper introduces a special feature on biodiversity conservation and poverty traps. We define and explain the core concepts and then identify four distinct classes of mechanisms that define important interlinkages between biodiversity and poverty. The multiplicity of candidate mechanisms underscores a major challenge in designing policy appropriate across settings. This framework is then used to introduce the ensuing set of papers, which empirically explore these various mechanisms linking poverty traps and biodiversity conservation.

Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog.

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.

Stem cells in veterinary medicine

The stem cell field in veterinary medicine continues to evolve rapidly both experimentally and clinically. Stem cells are most commonly used in clinical veterinary medicine in therapeutic applications for the treatment of musculoskeletal injuries in horses and dogs. New technologies of assisted reproduction are being developed to apply the properties of spermatogonial stem cells to preserve endangered animal species. The same methods can be used to generate transgenic animals for production of pharmaceuticals or for use as biomedical models. Small and large animal species serve as valuable models for preclinical evaluation of stem cell applications in human beings and in veterinary patients in areas such as spinal cord injury and myocardial infarction. However, these applications have not been implemented in the clinical treatment of veterinary patients. Reviews on the use of animal models for stem cell research have been published recently. Therefore, in this review, animal model research will be reviewed only in the context of supporting the current clinical application of stem cells in veterinary medicine.

Segregation of micron-scale membrane sub-domains in live murine sperm.

Lipid rafts, membrane sub‐domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside GM1 localized to a micron‐scale membrane sub‐domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub‐domain maintenance. GM1 segregation was maintained in live sperm incubated under non‐capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of GM1 diffusion to the post‐acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, GM1 dramatically redistributed several microns from the acrosomal sub‐domain to the post‐acrosomal, non‐raft sub‐domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid–lipid interaction model for sub‐domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin‐1 maintained segregation of both sterols and GM1, arguing against a role for lipid shells surrounding caveolin‐1 in sub‐domain maintenance. Scanning electron microscopy of sperm freeze‐dried without fixation identified cytoskeletal structures at the sub‐domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of GM1, we found that disulfide‐bonded proteins played a significant role in sub‐domain segregation. Together, these data provide an example of membrane sub‐domains extreme in terms of size and stability of lipid segregation, and implicate a protein‐based membrane compartmentation mechanism.

Community Markets for Conservation (COMACO) links biodiversity conservation with sustainable improvements in livelihoods and food production

In the Luangwa Valley, Zambia, persistent poverty and hunger present linked challenges to rural development and biodiversity conservation. Both household coping strategies and larger-scale economic development efforts have caused severe natural resource degradation that limits future economic opportunities and endangers ecosystem services. A model based on a business infrastructure has been developed to promote and maintain sustainable agricultural and natural resource management practices, leading to direct and indirect conservation outcomes. The Community Markets for Conservation (COMACO) model operates primarily with communities surrounding national parks, strengthening conservation benefits produced by these protected areas. COMACO first identifies the least food-secure households and trains them in sustainable agricultural practices that minimize threats to natural resources while meeting household needs. In addition, COMACO identifies people responsible for severe natural resource depletion and trains them to generate alternative income sources. In an effort to maintain compliance with these practices, COMACO provides extension support and access to high-value markets that would otherwise be inaccessible to participants. Because the model is continually evolving via adaptive management, success or failure of the model as a whole is difficult to quantify at this early stage. We therefore test specific hypotheses and present data documenting the stabilization of previously declining wildlife populations; the meeting of thresholds of productivity that give COMACO access to stable, high-value markets and progress toward economic self-sufficiency; and the adoption of sustainable agricultural practices by participants and other community members. Together, these findings describe a unique, business-oriented model for poverty alleviation, food production, and biodiversity conservation.

BIOCHEMICAL CHARACTERIZATION OF MEMBRANE FRACTIONS IN MURINE SPERM: IDENTIFICATION OF THREE DISTINCT SUB-TYPES OF MEMBRANE RAFTS

Despite enormous interest in membrane raft micro‐domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components—sterols, phospholipids, and proteins—or additional raft‐associating lipids such as the ganglioside, GM1. Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in GM1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent‐based approaches, we utilized a non‐detergent‐based method, separating membrane fractions that were reproducibly distinct based on sterol, GM1, phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub‐types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub‐types, but that multiple membrane micro‐domain sub‐types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either “raft” or “non‐raft” oversimplifies the actual biochemical complexity of cellular membranes. J. Cell. Physiol. 218: 537–548, 2009.

Characterization of the Proteomes Associating with Three Distinct Membrane Raft Sub-types in Murine Sperm

Mammalian sperm are transcriptionally and translationally inactive. To meet changing needs in the epididymis and female tract, they rely heavily on post‐translational modifications and protein acquisition/degradation. Membrane rafts are sterol and sphingolipid‐enriched micro‐domains that organize and regulate various pathways. Rafts have significance in sperm by transducing the stimulus of sterol efflux into changes in intracellular signaling that confer fertilization competence. We recently characterized three biochemically distinct sub‐types of sperm rafts, and now present profiles for proteins targeting to and associating with these sub‐types, along with a fraction largely comprised of “non‐raft” domains. Proteomics analysis using a gel‐based LC‐MS/MS approach identified 190 strictly validated proteins in the raft sub‐types. Interestingly, many of these are known to be expressed in the epididymis, where sperm membrane composition matures. To investigate potential roles for rafts in epididymal protein acquisition, we compared the expression and localization of two different sterol‐interacting proteins, apolipoprotein‐A1 (apoA1) and prominin‐1 (prom1) in sperm from different zones. We found that apoA1 was gradually added to the plasma membrane overlying the acrosome, whereas prom1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft‐associating proteins, demonstrate functional similarities and differences among raft sub‐types, and provide insights into raft‐mediated epididymal protein acquisition.

Mechanisms underlying the micron-scale segregation of sterols and GM1 in live mammalian sperm

We demonstrate for the first time that a stable, micron‐scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, GM1, localizes to this same domain in live sperm. Interestingly, the GM1 undergoes an unexplained redistribution upon cell death. We now demonstrate that GM1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing GM1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB‐bound endogenous GM1 and exogenous BODIPY–GM1 in live murine sperm demonstrate that the sub‐acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol‐ and GM1‐enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or GM1 that can be separated biochemically. J. Cell. Physiol. 218: 522–536, 2009.