To date, there are no protocols optimized to the effective separation of spermatogonial stem cells (SSCs) from testicular cells derived from mouse testes, thus hindering studies based on mouse SSCs. In this study, we aimed to determine the most efficient purification method for the isolation of SSCs from mouse testes among previously described techniques. Isolation of SSCs from testicular cells derived from mouse testes was conducted using four different techniques: differential plating (DP), magnetic-activated cell sorting (MACS) post-DP, MACS, and positive and negative selection double MACS. DP was performed for 1, 2, 4, 8, or 16 h, and MACS was performed using EpCAM (MACSEpCAM), Thy1 (MACSThy1), or GFR α1 (MACSGFRα1) antibodies. The purification efficiency of each method was analyzed by measuring the percentage of cells that stained positively for alkaline phosphatase. DP for 8 h, MACSThy1 post-DP for 8 h, MACSGFRα1, positive selection double MACSGFRα1/EpCAM, and negative selection double MACSGFRα1/α-SMA were identified as the optimal protocols for isolation of SSCs from mouse testicular cells. Comparison of the purification efficiencies of the optimized isolation protocols showed that, numerically, the highest purification efficiency was obtained using MACSGFRα1. Overall, our results indicate that MACSGFRα1 is an appropriate purification technique for the isolation of SSCs from mouse testicular cells.

Microenvironments surrounded with various extracellular matrix (ECM) components can decide specifically the fate of spermatogonial stem cells (SSCs) and integrin heterodimers recognizing directly ECM proteins play an important role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrin heterodimers expressed functionally on the undifferentiated SSCs derived from mouse with hybrid strain remain unclear. Therefore, we tried to investigate systematically what kind of integrin heterodimers are expressed transcriptionally, translationally and functionally in the SSCs derived from testis of hybrid (B6CBAF1) mouse. For these, magnetic activated cell sorting (MACS) using Thy1 antibody was used for isolating SSCs from testis, and real-time PCR or fluorescence immunoassay was conducted for measuring transcriptional or translational level of integrin α and β subunits in the isolated SSCs. Subsequently, antibody inhibition assay was conducted for confirming functionality of presumed integrin heterodimers. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, 7 integrin α (α4, α6, α7, α9, αV, αL and αE) and 2 integrin β (β1 and β5) subunit genes showed significantly increased transcriptional up-regulation, compared to the other integrin subunit genes. In contrast, integrin α3, α5, α10 and α11, and integrin β2, β3, β4 and β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV, αL and αE) were measured, significantly strong translational up-regulation of integrin α6, α7, α9, αV and αL subunit genes were detected, whereas integrin α4 and αE subunit genes were weakly. In case of integrin β subunit, β1 evaluated more expression than β5. Based on these results, we speculated that the undifferentiated SSCs derived from B6CBAF1 mouse might express integrin α4β 1, α6β1, α7β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Subsequently, the hybrid strain SSCs showed significantly increased adhesion to fibronectin, laminin, tenascine-C and vitronectin and functional blocking of integrin α4β1, α6β1, α9β1, and αVβ1 or αVβ5 in SSCs significantly inhibited attachment to fibronectin, laminin, tenascin-C and vitronectin, respectively. Accordingly, we could identify that the hybrid (B6CBAF1) mouse-derived SSCs had integrin α4β1, α6β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Generally, fate of spematogonial stem cells (SSCs) can be determined specifically by microenvironments enclosed with various extracellular matrix (ECM) components and integrins recognizing directly ECM proteins play an pivotal role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrins expressed on the undifferentiated SSCs remain unclear. Therefore, we tried to investigate systematically what kind of integrin subunits are expressed transcriptionally or translationally in the SSCs derived from testis of hybrid B6CBAF1 mouse. For these, isolation of SSCs from testis were conducted by magnetic activated cell sorting (MACS) using Thy1 antibody. Subsequently, transcriptional and translational level of integrin α and β subunits in the isolated SSCs were measured by real-time PCR and fluorescene immunoassay, respectively. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, and integrin α4, α6, α7, α9, αV, αL and αE and integrin β1, β5 showed higher expression levels than other subunits. By contrast, integrin α3, α5, α 10 and α11 and integrin β2, β3, β4, β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV αL, and αE) were measured, integrin α6, α7, α9, αV and αL were higher than integrin α4 and αE. In case of integrin β subunit, β1 evaluated more expression than β5. From these results, we speculate that the undifferentiated SSCs derived from hybrid B6CBAF1 mouse may express integrin α4β1, α6β1, α7β1, α9β1, αVβ1 and/or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a useful tool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are resided in the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneously quantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs), but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCs derived from mouse with different strains. For these, proliferation and viability were measured and compared by culturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or 37℃. In case of ICR strain, primary TSCs cultured at 37℃ showed significantly higher proliferation and viability than those at 35℃ and significant increase of proliferation and viability in sub-passaged TSCs was detected in the 35℃ culture condition. Moreover, sub-passage of primary TSCs at 35℃ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2 strain, significantly improved proliferation were detected in the primary TSCs cultured at 35℃, which showed no significant difference in the viability, compared to those at 37℃. Furthermore, sub-passaged TSCs cultured at 37℃ showed no significant differences in proliferation and viability, compared to those at 35℃. However, with significant decrease of proliferation induced by sub-passage of primary TSCs at 35℃, no significant effects on proliferation and viability were resulted from sub-passage of primary TSCs at 37℃. From these results, culture temperature of primary TSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture.

Thermal processing of (+)-catechin was carried out at 121℃ for different reaction times (1, 2, 3, 6, and 12 h). The reacted products, compounds (1) and (2), were isolated and quantified via HPLC analysis. The antioxidant properties of processed (+)-catechin and its isolated compounds for different reaction time was measured via radical scavenging assays using DPPH and ABTS+ radicals. Additionally, the anti-obesity efficacy of the thermal treated (+)-catechin was evaluated via porcine pancreatic lipase assay. The reacted (+)-catechin for 3 h had a slightly higher antioxidant capacity than that the parent (+)-catechin. Products 1 and 2, which were isolated from the reacted mixture during 3 h, showed an antioxidant capacity, and these two compounds may be responsible for the antioxidant capacity of processed (+)-catechin. Simple thermal treatment of (+)-catechin can be used to produce (+)-epicatechin (1) and protocatechuic acid (2) with enhanced antioxidant and anti-adipogenic effects.

A new sprout-soybean cultivar, “Wonhwang” was developed at the Honam Agricultural Research Institute (HARI) in 2005. Wonhwang was selected from a cross between Camp and Myeongjunamulkong. The preliminary, advanced, and regional yield trials for evaluation and selection of Iksan45 were carried out from 2001 to 2005. This cultivar has a determinate growth habit with purple flower, grayish brown pubescence, grayish brow hilum, lanceolate leaflet shape and small seed size (10.0 grams per 100 seeds). The maturity date of Wonhwang is 7 days earlier than that of the check variety, Pungsan. It has good seed quality for soybean-sprout, and resistance to lodging. It has also been identified to have resistance to soybean mosaic virus (SMV) and necrotic symptom(SMV-N). The average yield of “Wonhwang” was 2.80MT/ha, which was higher by 4% than “Pungsannamulkong” at the regional yield trials.