The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

The ultrastructure of bovine embryos developed from in vitro‐matured and ‐fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum‐free medium (IVMD101) or in a serum‐containing medium (TCM199+CS) was compared. Embryos up to the eight‐cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight‐cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well‐developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome‐like vesicles were partially filled with electron‐dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome‐like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome‐like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)‐cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome‐like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM‐cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum‐free and serum‐supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos. Mol. Reprod. Dev. 53:325–335, 1999. © 1999 Wiley‐Liss, Inc.

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.

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Precise reaction cross sections (oR) for 24_38M g on C targets at energies around 240 M eV /nucleon have been measured at the Radioactive Isotope Beam Factory at RIKEN. The oR for 36-38 Mg have been measured for the first time. An enhancement o f oR compared to the systematics for spherical stable nuclei has been observed, especially in the neutron-rich region, which reflects the deformation of those isotopes. In the vicinity of the drip line the aR for 37Mg is especially large. It is shown by analysis using a recently developed theoretical method that this prominent enhancement of oR for 37Mg should come from the p-orbital halo formation breaking the N = 28 shell gap.Since the early years of the study of atomic nuclei, the nuclear shell model has been the basic framework for understanding nuclear structure. The high stability of nuclei with certain numbers of neutrons (or protons) observed in stable nuclei indicates the existence of the shells filled at certain so-called "magic numbers." Studies in the last few decades have revealed that those magic numbers are sometimes broken or changed in unstable nuclei [1], The breakdown of the N = 20 shell gap between the sd and f p shells has been extensively studied since the irregularities in binding energies and 2+ excitation energies were observed in neutron-rich nuclei around N = 20 [2-6]. The term "island of inversion" was applied to this region [6] and deformed nuclear structures related to the changing of shell structures have been reported in this region [7]. The vanishing of the N = 28 shell closure has been also extensively studied, starting from neutronrich S-Ar isotopes [8][9][10][11][12][13][14]. The development of deformation observed in those nuclei could be interpreted as degeneracy of the f p shell, which induces strong quadrupole deformation [9][10][11][13][14][15][16][17][18]. Such deformation has been reported also for Si isotopes [19,20], and studies have recently indicated that this * takechi @ np.gs .niigata-u. ac .jp PACS number(s): 21.10.Gv, 25.60.Dz phenomenon could be seen even in a very neutron-rich Mg region [21].The purpose of our present study is to elucidate the changes of nuclear structures, such as a development of deformation, a breakdown of the magic numbers and possible halo formation in Mg isotopes, from the stability line to the vicinity of the neutron drip line. For this purpose, precise measurements of reaction cross sections for 24_38Mg have been performed at the Radioactive Isotope Beam Factory (RIBF) at RIKEN. The reaction cross section aR or interaction cross section ay reflects the nuclear size, and has been a powerful probe in searching for halo formation since the first study by Tanihata et al. [22], Recently, measurements of o, for Ne isotopes performed at RIBF [23] have successfully revealed the halo structure of 3lNe in which the sd-pf shell inversion associated with nuclear deformation causes the formation of a halo [23][24][25]. Moreover, theoretical studies on those data have shown that a precise data set on crR is v...