Optimal Milieu for Culturing Porcine Sertoli Cell

Md, Anower Jabed,Tania Kamel,Byung-Ki Kim 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.3

The purpose of the present study was to establish culture conditions for the in vitro study of the neonatal piglet Sertoli cell. Isolation for the culture of Sertoli cell was established using collagenase and pancreatin digestion of testicular tissues. The effects of various culture media, fetal bovine serum (FBS), follicular stimulating hormone (FSH), epidermal growth factor (EGF) and insulin-transferrin-sodium selenite (ITS) on growth of neonatal piglet Sertoli cells were investigated. The mitogenic effects of Dulbecco’s modified Eagle’s medium + Ham’s F-12 medium was higher than other media used in this experiment. The addition of 1% FBS in cultures was necessary for attachment of Sertoli cell clusters. However, except FBS and EGF, FSH and ITS did not stimulate Sertoli cell proliferation. When Sertoli cells isolated from neonatal piglets were cultured in Dulbecco’s modified Eagle’s medium + Ham’s F-12 medium supplemented with 1% FBS, FSH, EGF and ITS, the yield and plating efficiency of Sertoli cells were largely increased. Confluency of Sertoli cells was reached as early as 4 days of culture. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate and culture of Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.

In Vitro Maturation of Round Spermatids Using Porcine Oviduct Epithelial Cell Monolayer Condition Medium

Md. Anower Jabed,Tania Kamal,Seung-Min Lee,Byung Ki Kim 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & developmental biology Vol.29 No.4

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that 20% of round spermatid cultured were matured into elongating spermatid after 24 h, and about 10% of round spermatid cultured showed complete elongation (elongated spermatid) within 24~48 h of in vitro culture. No further development was observed within 50~72 h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

In Vitro Maturation of Round Spermatids Using Porcine Oviduct Epithelial Cell Monolayer Condition Medium

Jabed Md. Anower,Kamal Tania,Lee Seung-Min,Kim Byung Ki The Korean Society of Animal Reproduction 2005 Reproductive & developmental biology Vol.29 No.4

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

Optimal Milieu for Culturing Porcine Sertoli Cell

Jabed Md. Anower,Kamal Tania,Kim, Byung-Ki The Korean Society of Animal Reproduction 2006 Reproductive & developmental biology Vol.30 No.3

The purpose of the present study was to establish culture conditions for the in vitro study of the neonatal piglet Sertoli cell. Isolation for the culture of Sertoli cell was established using collagenase and pancreatin digestion of testicular tissues. The effects of various culture media, fetal bovine serum(FBS), follicular stimulating hormone(FSH), epidermal growth factor(EGF) and insulin-transferrin-sodium selenite(ITS) on growth of neonatal piglet Sertoli cells were investigated. The mitogenic effects of Dulbecco's modified Eagle's medium+Ham's F-12 medium was higher than other media used in this experiment. The addition of 1% FBS in cultures was necessary for attachment of Sertoli cell clusters. However, except FBS and EGF, FSH and ITS did not stimulate Sertoli cell proliferation. When Sertoli cells isolated from neonatal piglets were cultured in Dulbecco's modified Eagle's medium+Ham's F-12 medium supplemented with 1% FBS, FSH EGF and ITS, the yield and plating efficiency of Sertoli cells were largely increased. Confluency of Sertoli cells was reached as early as 4 days of culture. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate and culture of Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.

Stimulatory Effect of Porcine Epididymal Fluid on In Vitro Maturation of Porcine Immature Oocytes

Yim, Cha-Ok,Lee, Seung-Min,Kim, Hye-Rim,Jabed Md. Anower,Lee, Chin-Bum,Kim, Byung-Ki The Korean Society of Animal Reproduction 2006 Reproductive & developmental biology Vol.30 No.4

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%, 51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

Stimulatory Effect of Porcine Epididymal Fluid on In Vitro Maturation of Porcine Immature Oocytes

Cha Ok Yim,Seung Min Lee,Hye Rim Kim,Md. Anower Jabed,이진범,김병기 사단법인 한국동물생명공학회 2006 Reproductive & developmental biology Vol.30 No.4

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48 hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%,51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.