Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs

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

Why there is no such thing as colostrum quality

Ultimately, we believe that that our results are exciting because they indicate producers do not need to measure colostrum Brix scores, or have to worry about having enough colostrum above a “cut-off” Brix score. We now know such cut offs are quite arbitrary. For the first meal we recommend just feeding four liters of colostrum. It is much better for neonatal calves than any replacement formula. Colostrum, irrespective of its Brix score, is still the finest choice for feeding calves.This research was funded by the Northeast Sustainable Agriculture Research and Education (NE-SARE) grant GNE19-220-33243, the Cornell CALS Charitable Trust Research Fund, and startup funds from Cornell CALS

Transition milk has a lot of immunoglobulins

Antibodies in the dam’s colostrum, specifically immunoglobulins G and A, are the reason why newborn calves are able to immediately combat bacterial and viral pathogens in the environment. Based on early research, the predominant recommendation to improve calf survival rates and health has been to feed newborn calves first-milking colostrum to obtain maternal IgG.This research was funded by the Northeast Sustainable Agriculture Research and Education (NE-SARE) grant GNE19-220-33243, the Cornell CALS Charitable Trust Research Fund, and startup funds from Cornell CALS

Developmental expression of translocator protein/peripheral benzodiazepine receptor in reproductive tissues.

Translocator protein (TSPO) present in the outer mitochondrial membrane has been suggested to be critical for cholesterol import, a rate-limiting step for steroid hormone biosynthesis. Despite the importance of steroidogenesis in regulating reproductive functions, the developmental profile of TSPO expression in the gonads and accessory sex organs has not been completely characterized. As a first step towards understanding the function of TSPO, we studied its expression in male and female murine reproductive organs. We examined testes and ovaries at embryonic days 14.5 and 18.5, and postnatal days 0, 7, 14, 21 and 56 of development. In the adult testis, TSPO was expressed in both Leydig cells and Sertoli cells. In the developing testes TSPO expression was seen in immature Sertoli cells, fetal Leydig cells and gonocytes. In the ovary, TSPO was expressed in the ovarian surface epithelium, interstitial cells granulosa cells and luteal cells. Corpora lutea of ovaries from pregnant mice showed strong expression of TSPO. In the developing ovary, TSPO expression was seen in the squamous pregranulosa cells associated with germ line cysts, together with progressively increasing expression in interstitial cells and the ovarian surface epithelium. In adult mice, the epithelia of other reproductive tissues like the epididymis, prostate, seminal vesicle, oviduct and uterus also showed distinct patterns of TSPO expression. In summary, TSPO expression in both male and female reproductive tissues was not only restricted to steroidogenic cells. Expression in Sertoli cells, ovarian surface epithelium, efferent ductal epithelium, prostatic epithelium, seminal vesiclular epithelium, uterine epithelium and oviductal epithelium suggest either previously unknown sites for de novo steroidogenesis or functions for TSPO distinct from its well-studied role in steroid hormone production