A TSPO ligand is protective in a mouse model of multiple sclerosis.

Local production of neurosteroids such as progesterone and allopregnanolone confers neuroprotection in central nervous system (CNS) inflammatory diseases. The mitochondrial translocator protein (TSPO) performs a rate-limiting step in the conversion of cholesterol to pregnenolone and its steroid derivatives. Previous studies have shown that TSPO is upregulated in microglia and astroglia during neural inflammation, and radiolabelled TSPO ligands such as PK11195 have been used to image and localize injury in the CNS. Recent studies have shown that modulating TSPO activity with pharmacological ligands such as etifoxine can initiate the production of neurosteroids locally in the injured CNS. In this study, we examined the effects of etifoxine, a clinically available anxiolytic drug, in the development and progression of mouse experimental autoimmune encephalomyelitis (EAE), an experimental model for multiple sclerosis (MS). Our results showed that etifoxine attenuated EAE severity when administered before the development of clinical signs and also improved symptomatic recovery when administered at the peak of the disease. In both cases, recovery was correlated with diminished inflammatory pathology in the lumbar spinal cord. Modulation of TSPO activity by etifoxine led to less peripheral immune cell infiltration of the spinal cord, and increased oligodendroglial regeneration after inflammatory demyelination in EAE. Our results suggest that a TSPO ligand, e.g. etifoxine, could be a potential new therapeutic option for MS with benefits that could be comparable to the administration of systemic steroids but potentially avoiding the detrimental side effects of long-term direct use of steroids

Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3+-13.0 vs. 44.9+-30.8%) and positive Spermac staining (51.9+-14.5 vs. 7.5+-14.4%), in addition to higher total volume (135.1+-89.6 vs. 88.8+-73.1 ml) and lower sperm concentration (473.0+-511.2 vs. 1313.8+-764.7 x 106 cells ml-1) compared to ejaculates exhibiting poor sperm motility (≤10%; P\u3c0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ~80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P\u3c0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species

Membrane Organization and Dynamics in Mammalian Sperm

In somatic cells, membrane rafts are dynamic, existing in various time and space scales. The transient nature of these membrane microdomains and the use of detergents or multivalent probes/cross-linkers to isolate or visualize them have incited controversy in this field. In the following studies, I demonstrate for the first time in live spermatozoa, the presence of a micrometer-scale, stable domain in the plasma membrane enriched in sterols and the glycosphingolipid GM1 at physiological temperature. This sterol-enriched membrane platform is positioned to regulate the acquisition of sperm fertilization competence. I examine the properties of this domain and the mechanism behind its segregation and maintenance. I also conduct in-depth functional studies on FABP9, which was a strong candidate for tethering membrane lipids to underlying cytoskeletal elements in this region. My studies showed that the maintenance of this stable segregation in the sperm head was at least in part due to a specialized diffusion barrier at a region called the sub-acrosomal ring. I found that FABP9 did not play a role in this membrane segregation. Interesting dynamics of GM1 bound to cholera toxin subunit B (CTB) revealed that complex membrane interactions occur in this region. This led to the finding that the acrosomal vesicle that immediately underlies this domain is a GM1-enriched organelle. The dynamics of CTB-bound GM1 also resulted in our ability to distinguish different functional changes in sperm membrane properties occurring in response to capacitating stimuli. Based on these stimulus specific changes seen with CTB-bound GM1 distribution, we found that sperm could respond to bicarbonate ions or mediators of sterol efflux independently, thereby refining existing models of capacitation. The applicability of CTB-bound GM1 dynamics as a diagnostic tool to assay sperm response to stimuli for capacitation is potentially of significant clinical importance. These studies set the stage for exploring both capacitation-related changes in the microheterogeneities within this domain and ensuing signaling events in response to stimuli for capacitation. Furthermore, my data have led to a complex model that involves several complementary mechanisms of lipid segregation acting at different spatial scales. Testing this model will be the subject of continuing investigations

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility