Earticle

The aims of this study were to establish a simple and effective method for isolating male germline stem cells (GSCs), and to test the possibility of using these cells as a new approach for male infertility treatment. Testes obtained from neonatal and adult mice were manually decapsulated. GSCs were collected from seminiferous tubules by a two-step enzyme digestion method and plated on gelatin-coated dishes. Over 5-7 days of culture, GSCs obtained from neonates and adults gave rise to large multicellular colonies that were subsequently grown for 10 passages. During in vitro proliferation, oct-4 and two immunological markers (Integrin ${\beta}1,\;{\alpha}6$) for GSCs were highly expressed in the cell colonies. During another culture period of 6 weeks to differentiate to later stage germ cells, the expression of oct-4 mRNA decreased in GSCs and Sertoli cells encapsulated with calcium alginate, but the expression of c-kit and testis-specific histone protein 2B(TH2B) mRNA as well as the localization of c-kit protein was increased. Expression of transition protein (TP-l) and localization of peanut agglutinin were not seen until 3 weeks after culturing, and appeared by 6 weeks of culture. The putative spermatids derived from GSCs supported embryonic development up to the blastocyst stage with normal chromosomal ploidy after chemical activation. Thus, GSCs isolated from neonatal and adult mouse testes were able to be maintained and proliferated in our simple culture conditions. These GSCs have the potential to differentiate into haploid germ cells during another long-term culture.

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.

본 연구는 유산간균 및 유산구균으로부터 plasmid DNA를 신속하고 간편하게 분리하기 위한 방법에 관한 것이다. 세포벽 형성 억제 인자인 glycine을 0.5% 첨가한 TCM 배지에서 유산균을 배양하였고 plasmid DNA는 mutanolysin을 처리한 cells로부터 alkaline-detergent lysis 법으로 분리되었다. 유산간균은 효소 처리 때 mutanolysin의 농도를 30$\mu\textrm{g}$/$m\ell$로 하고 37$^{\circ}C$에서 5-10분간 반응되었을 때 plasmid DNA가 아주 잘 추출되었다. 유산구균의 경우는 그 최적 조건이 조금 달랐다. 본 방법은 L. casei, L. acidophilus, L. helveticus, S. lactis, S. faecalis, S. faecium과 S. cremoris 균주로부터 plasmid DNA를 신속하게 분리하는데 사용할 수 있었다. 본 방법을 이용하여 배양액 $1.5m\ell$로부터 분리된 plasmidsDNA가 gel상에서 쉽게 확인될 수 있었다.

A simple procedure for rapid isolation of plasmid DNA from lactobacillus species and streptococcus species is described. Lactic acid bacteria were cultured in the TCM broth containing 0.5％ glycine and plasmid DNA was isolated from cells treated with mutanolysin by alkaline-detergent lysis method. Good results for releasing and isolating plasmid DNA from lactobacillus species were obtained by treatment of cells with 30$\mu\textrm{g}$ of mutanolysin per ml at 37$^{\circ}C$ for 5 to 10 min. For the streptococcus species, the optimum conditions were slightly different. The procedure could be used for rapid characterization of plasmid DNA in Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus lactis, Streptococcus faecalis, Streptococcus faecium, and Streptococcus cremoris strains. Using this procedure, plasmids isolated from $1.5m\ell$ cultures could readily be visualized in agarose gel.