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가토 난포난자(卵胞卵子)의 체외성숙 유도에 관한 연구

Title
 가토 난포난자(卵胞卵子)의 체외성숙 유도에 관한 연구
Other Titles
 Studies on the maturation in vitro of rabbit follicular oocytes
Issue Date
1973
Publisher
 연세대학교 대학원
Description
의학과/박사
Abstract
[한글] Studies on the Maturation In Vitro of Rabbit Follicular Oocytes Sung Sun Hong, M.D. Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hyun Mo Kwak, M.D.) Pincus and Enzmann (1935) attempted to culture rabbit follicular oocytes in vivo and in vitro. Chang (1955) attempted to induce the maturation process in vitro using blood serum and Ringer's solution. Since that time many biological media have been developed and used with good results for maturation. Biggers et al. (1967), Donahue (1968), and Cross and Brinster (1970) succeeded in the induction of mouse oocyte maturation in the chemically defined media of modified Krebs-Ringer's solution with a good maturation rate. In the culture of mouse one-cell embryos, pyruvate and oxaloacetate are very important as an energy source for maturation (Biggers et al., 1977). In the rabbit fertilized ova, however, pyruvate is not essentially important and the amino acids are considered to be the major providing substrates (Kane and foote, 1970). Considering such difference in substrate requirements, one may theorize that rabbit follicular oocytes might have different metabolic pathyways, different cytoplasmic enzyme systems or metabolism, and different cytoplasmic organelles that those Biggers et al. (1957) found in their study of mouse follicular oocytes. At this time is introduced the results of an investigation on the maturation of the rabbit follicular oocyte in the presence of nutrient substances such as bovine serum, bovine serum albumin, and amino acids in the culture media, in order to clarify the in vitro effects of various supplements. Materials and Methods The animals used in the present study were Korean virgin rabbits weighing from 1.2 kg to 1.8 kg. Both ovaries were removed through a flank incision after anesthesia with seconal solution (0.85% saline : absolute alcohol=1 : 1). The removed ovaries were washed four times in basic medium and each of them was cut into small pieces with scissors, vertically and lengthwise. Under a stereo-dissecting microscope, graffian follicles were dissected out with a sharp end pin into the basic medium. Oocyte collection and culture set up Oocytes released into the basic medium had a thick mucous layer around them and several layers of cumulus cells. They were washed in the basic medium by sucking and releasing the oocytes through finely drawn pasteur pipettes. The mucous layer could be nearly entirely removed by suction and release, but small amounts of cumulus cells remained, The oocytes were finally introduced into a small drop of culture media suspended under about 10 ㎖ of mineral oil in a plastic culture dish. The culture procedure was developed y Brinster (1963). The oocytes were incubated in the incubator at 37℃, and supplied continuously with moist gas of 95% air and 5% carbon dioxide for 24 hours. Culture media and supplements The components of the standard egg culture medium (SECM) and modified egg culture medium appear in Table 1. The basic medium was a standard egg culture medium without supplement of bovine serum albumin. Bovin serum was prepared by clotting bovine blood taken from a slaughter house. The serum was poured into a beaker and then it was centrifuged for 30 minutes at 3,000 r.p.m. and inactivated at 60℃. Amino acids used were arginine, histidine, Iysine, tryptophane, methionine, phenylalanine, leucine, valine, threonine and serine. The concentrations of each amino acid are listed in Table 2. Each culture medium was sterilized by syringe filteration before use, and the hydrogen ion concentration of basic medium and culture medium was kept within the range of pH 7.1∼7.4. All instruments and glassware were sterilized in a hot air sterilizer or autoclave just before use. At the end of cultivation, the cumulus cells adhering to the oocytes were removed mechanically by shaking the oocytes vigorously in 10 ml of culture medium and the oocytes before fixation, were washed twice in a embryological watch glass containing basic medium and then were fixed with acid-alcohol for 6 to 24 hours and stained with 0.5% acetolacmoid for microscopic examination. Results As in Table 3, of basic control mouse oocytes cultured in standard egg culture medium, 84 per cent resumed their maturation division, producing a metaphase I chromosome or extruding a polar body and only 7. 1 per cent of the same group remained the same or showed early prophase and in 8.9 per cent a degenerating nuclear phase resulted. The results were obtained in the present experiments of rabbit oocytes, using standard egg culture medium (0.308 osmole) and modified standard egg culture medium (0.278 osmose). In standard egg culture medium (SECM), 24.4 per cent of oocytes resumed their maturation division, producing metaphase Ⅰ or Ⅱ chromosomes, 37.8 per cent remained at dictyate stage and another 37.8 7er cent showed degenerating nuclei. In modified standard egg culture medium, no oocytes resumed their maturation division (M Ⅱ). However, degeneration rate was as low as 5.9 per cent. When 1.5∼3.0 per cent of BSA was added to the basic medium, 32.1 per cent remained at the dictyate stage and 52.4 per cent underwent degeneration. Only 15.5 Per cent resumed their maturation division, producing a metaphase I chromosome or extruding a polar body. When the oocytes were cultured in the basic medium supplemented with 10 per cent bovine serum, a very high proportion (70.4%) showed degenerating nuclei, 25.9 percent remained at dictyate or late prophase and only 3.7 per cent of them showed maturation division. This medium was thought to be not appropriate even for maintaining the oocytes. In the medium supplemented with BSA and BS, a high per cent (63.0%) of them remained at dictyate or late prophase as before, and only 10.9 of them showed maturation division. When oocytes were cultured in the medium supplemented with amino acids, 61.5 per cent of them showed maturation division (M Ⅰ -M Ⅱ) while the proportion of degenerating nuclei decreased to 10.5 per cent. Summary When rabbit follicular oocytes were cultured by the Brinster's technique in a chemically defined medium supplemented with different amounts of bovine serum albumin and bovine serum or 10 amino acids, the following results were obtained. 1. Standard egg culture medium is not adequate for the maturation of rabbit follicular oocytes even though it is adequate for the maturation in vitro of mouse follicular oocytes. 2. When 5∼30 per cent of bovine serum was used to supplement the modified standard egg culture medium, the bovine serum seemed to be no good energy source for the maturation and increased the number of degenerated oocytes. 3. When the oocytes were cultured in the standard egg culture medium supplemented with ten amino acids (arginine, histidine, leucine, lysine, methionine, tryptophane, valine, phenylalanine, serine and threonine), 61.5 per cent of oocytes showed maturation division.
[영문] Pincus and Enzmann (1935) attempted to culture rabbit follicular oocytes in vivo and in vitro. Chang (1955) attempted to induce the maturation process in vitro using blood serum and Ringer's solution. Since that time many biological media have been developed and used with good results for maturation. Biggers et al. (1967), Donahue (1968), and Cross and Brinster (1970) succeeded in the induction of mouse oocyte maturation in the chemically defined media of modified Krebs-Ringer's solution with a good maturation rate. In the culture of mouse one-cell embryos, pyruvate and oxaloacetate are very important as an energy source for maturation (Biggers et al., 1977). In the rabbit fertilized ova, however, pyruvate is not essentially important and the amino acids are considered to be the major providing substrates (Kane and foote, 1970). Considering such difference in substrate requirements, one may theorize that rabbit follicular oocytes might have different metabolic pathyways, different cytoplasmic enzyme systems or metabolism, and different cytoplasmic organelles that those Biggers et al. (1957) found in their study of mouse follicular oocytes. At this time is introduced the results of an investigation on the maturation of the rabbit follicular oocyte in the presence of nutrient substances such as bovine serum, bovine serum albumin, and amino acids in the culture media, in order to clarify the in vitro effects of various supplements. Materials and Methods The animals used in the present study were Korean virgin rabbits weighing from 1.2 kg to 1.8 kg. Both ovaries were removed through a flank incision after anesthesia with seconal solution (0.85% saline : absolute alcohol=1 : 1). The removed ovaries were washed four times in basic medium and each of them was cut into small pieces with scissors, vertically and lengthwise. Under a stereo-dissecting microscope, graffian follicles were dissected out with a sharp end pin into the basic medium. Oocyte collection and culture set up Oocytes released into the basic medium had a thick mucous layer around them and several layers of cumulus cells. They were washed in the basic medium by sucking and releasing the oocytes through finely drawn pasteur pipettes. The mucous layer could be nearly entirely removed by suction and release, but small amounts of cumulus cells remained, The oocytes were finally introduced into a small drop of culture media suspended under about 10 ㎖ of mineral oil in a plastic culture dish. The culture procedure was developed y Brinster (1963). The oocytes were incubated in the incubator at 37℃, and supplied continuously with moist gas of 95% air and 5% carbon dioxide for 24 hours. Culture media and supplements The components of the standard egg culture medium (SECM) and modified egg culture medium appear in Table 1. The basic medium was a standard egg culture medium without supplement of bovine serum albumin. Bovin serum was prepared by clotting bovine blood taken from a slaughter house. The serum was poured into a beaker and then it was centrifuged for 30 minutes at 3,000 r.p.m. and inactivated at 60℃. Amino acids used were arginine, histidine, Iysine, tryptophane, methionine, phenylalanine, leucine, valine, threonine and serine. The concentrations of each amino acid are listed in Table 2. Each culture medium was sterilized by syringe filteration before use, and the hydrogen ion concentration of basic medium and culture medium was kept within the range of pH 7.1∼7.4. All instruments and glassware were sterilized in a hot air sterilizer or autoclave just before use. At the end of cultivation, the cumulus cells adhering to the oocytes were removed mechanically by shaking the oocytes vigorously in 10 ml of culture medium and the oocytes before fixation, were washed twice in a embryological watch glass containing basic medium and then were fixed with acid-alcohol for 6 to 24 hours and stained with 0.5% acetolacmoid for microscopic examination. Results As in Table 3, of basic control mouse oocytes cultured in standard egg culture medium, 84 per cent resumed their maturation division, producing a metaphase I chromosome or extruding a polar body and only 7. 1 per cent of the same group remained the same or showed early prophase and in 8.9 per cent a degenerating nuclear phase resulted. The results were obtained in the present experiments of rabbit oocytes, using standard egg culture medium (0.308 osmole) and modified standard egg culture medium (0.278 osmose). In standard egg culture medium (SECM), 24.4 per cent of oocytes resumed their maturation division, producing metaphase Ⅰ or Ⅱ chromosomes, 37.8 per cent remained at dictyate stage and another 37.8 7er cent showed degenerating nuclei. In modified standard egg culture medium, no oocytes resumed their maturation division (M Ⅱ). However, degeneration rate was as low as 5.9 per cent. When 1.5∼3.0 per cent of BSA was added to the basic medium, 32.1 per cent remained at the dictyate stage and 52.4 per cent underwent degeneration. Only 15.5 Per cent resumed their maturation division, producing a metaphase I chromosome or extruding a polar body. When the oocytes were cultured in the basic medium supplemented with 10 per cent bovine serum, a very high proportion (70.4%) showed degenerating nuclei, 25.9 percent remained at dictyate or late prophase and only 3.7 per cent of them showed maturation division. This medium was thought to be not appropriate even for maintaining the oocytes. In the medium supplemented with BSA and BS, a high per cent (63.0%) of them remained at dictyate or late prophase as before, and only 10.9 of them showed maturation division. When oocytes were cultured in the medium supplemented with amino acids, 61.5 per cent of them showed maturation division (M Ⅰ -M Ⅱ) while the proportion of degenerating nuclei decreased to 10.5 per cent. Summary When rabbit follicular oocytes were cultured by the Brinster's technique in a chemically defined medium supplemented with different amounts of bovine serum albumin and bovine serum or 10 amino acids, the following results were obtained. 1. Standard egg culture medium is not adequate for the maturation of rabbit follicular oocytes even though it is adequate for the maturation in vitro of mouse follicular oocytes. 2. When 5∼30 per cent of bovine serum was used to supplement the modified standard egg culture medium, the bovine serum seemed to be no good energy source for the maturation and increased the number of degenerated oocytes. 3. When the oocytes were cultured in the standard egg culture medium supplemented with ten amino acids (arginine, histidine, leucine, lysine, methionine, tryptophane, valine, phenylalanine, serine and threonine), 61.5 per cent of oocytes showed maturation division.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/117381
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 박사
Yonsei Authors
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