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Bovine mesenchymal-derived cells support long-term survival and growth of bovine preantral follicles in vitro
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Theriogenology BOVINE MESENCHYMAL-DERIVED CELLS SUPPORT LONG-TERM SURVIVAL AND GROWTH OF BOVINE PREANTRAL FOLLICLES IN VITRO T. Itoh, H. Abe, S. Yamashita, Y. Sendai, T.Satoh and H. Hoshi Research Institute for the Functional Peptides 4-3-32, Shimojo, Yamagata 990-0823, Japan
Successful culture of preantral follicles would be a great advantage for the production of large quantities of oocytes for embryo transfer and recipient cells for production of cloned embryos. The aim of this study was to determine the effect of various types of somatic cells on long-term survival and initial growth of bovine preantral follicles in vitro. Preantral follicles (58.99+18.42/~m) surrounded by an intact basement membranewere efficientlyisolated from Ovaries of slaughtered cattle by a mechanical method. Bovine ovary mesenchymal cells (BOM) and fetal bovine fibroblasts (FBF) were isolated from adult ovarian cortex and fetal skin (2 months), respectively. These somatic cells were placed in 24-well culture plates containing basal HP-M199 medium (IFP, Japan) supplemented with 10% FBS (ICN Biochemicals, CA) and then cultured until confluency at 37°C in 5% CO2/95% air. Twenty-four hours prior to the onset of co-culture, the medium was changed to 0.5 ml of fresh serum-free medium (HP-M199+0.1% BSA+ITS+25/~g/ml hypoxanthine+l ug/ml aprotinin). Control cultures of preantral follicles without somatic cell support were prepared in type-1 collagen-coated culture plates. Follicular morphology, follicular diameters and oocyte viability under different culture conditions were determined during 30 days in culture. Morphology of each follicle was obselwed under an inverted microscope and follicular diameter was measured with an image processor (ARGUS-10, Hamamatsu Photonics, Japan). Oocyte viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258. Survival rates of preantral follicles in control culture dramatically decreased following with an extended culture period and no morphologically normal follicles (0/76; 0%)and viable oocytes (0/33; 0%) were seen after 30 days in culture. In contrast, the higher proportions of normal follicles and viable oocytes were obtained in co-culture with BOM (57/98; 58.0% and 8/43; 18.9%) and FBF (39/79; 49.3% and 6/35; 17.2%) respectively. Furthermore, mean diameter of preantral follicles in the presence of BOM (77.13__12.97/~m) and FBF (83.38+21.42ktm) on day 30 was slightly larger than follicles present on day 1 (65.83_+13.26ktm). This culture system may be useful, not only for studying the mechanisms of early folliculogenesis, but also for obtaining a large number of oocytes for practical uses such as embryo transfer, cloning, and production of transgenic cows.
Theriogenology BOVINE MESENCHYMAL-DERIVED CELLS SUPPORT LONG-TERM SURVIVAL AND GROWTH OF BOVINE PREANTRAL FOLLICLES IN VITRO T. Itoh, H. Abe, S. Yamashita, Y. Sendai, T.Satoh and H. Hoshi Research Institute for the Functional Peptides 4-3-32, Shimojo, Yamagata 990-0823, Japan
Successful culture of preantral follicles would be a great advantage for the production of large quantities of oocytes for embryo transfer and recipient cells for production of cloned embryos. The aim of this study was to determine the effect of various types of somatic cells on long-term survival and initial growth of bovine preantral follicles in vitro. Preantral follicles (58.99+18.42/~m) surrounded by an intact basement membranewere efficientlyisolated from Ovaries of slaughtered cattle by a mechanical method. Bovine ovary mesenchymal cells (BOM) and fetal bovine fibroblasts (FBF) were isolated from adult ovarian cortex and fetal skin (2 months), respectively. These somatic cells were placed in 24-well culture plates containing basal HP-M199 medium (IFP, Japan) supplemented with 10% FBS (ICN Biochemicals, CA) and then cultured until confluency at 37°C in 5% CO2/95% air. Twenty-four hours prior to the onset of co-culture, the medium was changed to 0.5 ml of fresh serum-free medium (HP-M199+0.1% BSA+ITS+25/~g/ml hypoxanthine+l ug/ml aprotinin). Control cultures of preantral follicles without somatic cell support were prepared in type-1 collagen-coated culture plates. Follicular morphology, follicular diameters and oocyte viability under different culture conditions were determined during 30 days in culture. Morphology of each follicle was obselwed under an inverted microscope and follicular diameter was measured with an image processor (ARGUS-10, Hamamatsu Photonics, Japan). Oocyte viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258. Survival rates of preantral follicles in control culture dramatically decreased following with an extended culture period and no morphologically normal follicles (0/76; 0%)and viable oocytes (0/33; 0%) were seen after 30 days in culture. In contrast, the higher proportions of normal follicles and viable oocytes were obtained in co-culture with BOM (57/98; 58.0% and 8/43; 18.9%) and FBF (39/79; 49.3% and 6/35; 17.2%) respectively. Furthermore, mean diameter of preantral follicles in the presence of BOM (77.13__12.97/~m) and FBF (83.38+21.42ktm) on day 30 was slightly larger than follicles present on day 1 (65.83_+13.26ktm). This culture system may be useful, not only for studying the mechanisms of early folliculogenesis, but also for obtaining a large number of oocytes for practical uses such as embryo transfer, cloning, and production of transgenic cows.