Jacquelyn L. Nelson

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.

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.

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.

Overexpression of RPGR Leads to Male Infertility in Mice Due to Defects in Flagellar Assembly

Abstract Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.

Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa

Abstract The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3s pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1s lipid and sugar components and CaV2.3s α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Sequential Reactions of Surface- Tethered Glycolytic Enzymes

The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ-cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices.

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.

Biomimicry Promotes the Efficiency of a 10‐Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.