Kwon Young Lee

Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes.

PurposeTo date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.MethodsWe confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.ResultsPetri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn’t and was detected, respectively.ConclusionsWe successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

Glucose metabolism and neurogenesis in the gerbil hippocampus after transient forebrain ischemia

Recent evidence exists that glucose transporter 3 (GLUT3) plays an important role in the energy metabolism in the brain. Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and mRNA levels rather than tissue levels. In the present study, we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia. In the sham-operated group, GLUT3 immunoreactivity in the hippocampal CA1 region was weak, in the pyramidal cells of the CA1 region increased in a time-dependent fashion 24 hours after ischemia, and in the hippocampal CA1 region decreased significantly between 2 and 5 days after ischemia, with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia. In a double immunofluorescence study using GLUT3 and glial-fibrillary acidic protein (GFAP), we observed strong GLUT3 immunoreactivity in the astrocytes. GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfusion. In a double immunofluorescence study using GLUT3 and doublecortin (DCX), we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia. GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgranular zone of the dentate gyrus. These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.

Decrease in glucose transporter 1 levels and translocation of glucose transporter 3 in the dentate gyrus of C57BL/6 mice and gerbils with aging

In the present study, we compared the cell-specific expression and changes protein levels in the glucose transporters (GLUTs) 1 and 3, the major GLUTs in the mouse and gerbil brains using immunohistochemistry and Western blot analysis. In both mouse and gerbils, GLUT1 immunoreactivity was mainly found in the blood vessels in the dentate gyrus, while GLUT3 immunoreactivity was detected in the subgranular zone and the molecular layer of the dentate gyrus. GLUT1-immunoreactivity in blood vessels and GLUT1 protein levels were significantly decreased with age in the mice and gerbils, respectively. In addition, few GLUT3-immunoreactive cells were found in the subgranular zone in aged mice and gerbils, but GLUT3-immunoreactivity was abundantly found in the polymorphic layer of dentate gyrus in mice and gerbils with a dot-like pattern. Based on the double immunofluorescence study, GLUT3-immunoreactive structures in gerbils were localized in the glial fibrillary acidic protein-immunoreactive astrocytes in the dentate gyrus. Western blot analysis showed that GLUT3 expression in the hippocampal homogenates was slightly, although not significantly, decreased with age in mice and gerbils, respectively. These results indicate that the reduction in GLUT1 in the blood vessels of dentate gyrus and GLUT3 in the subgranular zone of dentate gyrus may be associated with the decrease in uptake of glucose into brain and neuroblasts in the dentate gyrus. In addition, the expression of GLUT3 in the astrocytes in polymorphic layer of dentate gyrus may be associated with metabolic changes in glucose in aged hippocampus.

Dendropanax morbifera Léveille extract ameliorates D-galactose-induced memory deficits by decreasing inflammatory responses in the hippocampus

In the present study, we examined the effects of Dendropanax morbifera Léveille leaf extract (DML) on D-galactose-induced morphological changes in microglia and cytokines, including pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) and anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus. Administration of DML to D-galactose-treated mice significantly improved D-galactose-induced reduction in escape latency, swimming speed, and spatial preference for the target quadrant. In addition, administration of DML to D-galactose-treated mice significantly ameliorated the microglial activation and increases of IL-1β, IL-6, and TNF-α levels in the hippocampus. Administration of D-galactose significantly reduced IL-4 levels in the hippocampus, while administration of DML to D-galactose-treated mice significantly increased IL-4 level. However, we did not observe any significant changes in IL-10 levels in hippocampal homogenates. These results suggest that DML reduces D-galactose-induced mouse senescence by reducing pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as increasing anti-inflammatory cytokine IL-4.

Age-related change of Iba-1 immunoreactivity in the adult and aged gerbil spinal cord

In the present study, we examined change of ionized calcium-binding adapter molecule 1 (Iba-1) in the adult and aged gerbil spinal cords. Significant change of morphological feature and neuronal cell loss were not observed in both adult and aged spinal cords of gerbil after NeuN immunohistochemistry and Fluoro-Jade B histofluoresce staining. Iba-1–immunoreactive microglia broadly distributed in the spinal cord. Most of Iba-1–immunoreactive microglia showed ramified forms in the adult gerbil cervical and lumbar spinal cords. However, morphological changes of Iba-1–immunoreactive microglia were observed in the cervical and lumbar regions of the aged gerbil spinal cord. These microglia were showed a hypertrophied body with shortened swollen processes which was characteristic of activated microglia. In addition, Iba-1 protein level significantly higher in aged cervical and lumbar spinal cords than those in the adult gerbil. The present study showed an increase of activated forms of Iba-1–immunoreactive microglia and its protein level without marked changes in morphological features and neuronal loss in the aged spinal cord compared to those in the adult gerbil spinal cord. This result suggests that the increase of Iba-1 expression in the aged spinal cord may be closely associated with age-related changes in aged gerbil spinal cord.

Effects of Extracellular Matrix Protein-derived Signaling on the Maintenance of the Undifferentiated State of Spermatogonial Stem Cells from Porcine Neonatal Testis

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

Lack of evidence in neurite growth in the gerbil hippocampal CA1 region 15 days after transient forebrain ischemia

ABSTRACT Transient forebrain ischemia promotes a robust increase in neuroblast differentiation in the hippocampal dentate gyrus that peaks 7–15 days after the surgery. In this study, we compared the glucose transporter 3 (GLUT3)-dependent glucose utilization and the dynamin-1 (DNM1)-dependent neurite growth in the hippocampus of Mongolian gerbils 15 days after the induction of transient forebrain ischemia. The animals were subjected to a 5 min transient ischemia protocol and sacrificed 15 days after the surgery. Both doublecortin (DCX) immunoreactive neuroblasts and DCX total protein levels were abundantly increased in the ischemic group compared to the levels observed in the control group. In addition, animals in the ischemic group showed elevated GLUT3 immunoreactivity in the subgranular zone of the dentate gyrus compared to animals in the control group. Based on the double immunofluorescent study, increased DCX-immunoreactive neuroblasts were co-localized with GLUT3-immunoreactive components in the dentate gyrus. However, both the immunoreactivity and the total protein levels of DNM1 were significantly decreased in the dentate gyrus and hippocampal CA1 regions of the ischemic group. These results suggest that the regeneration process such as neurite growth is lacking in the hippocampus 15 days after ischemia/reperfusion although neuroblasts production and glucose utilization increased in the hippocampus.