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 present study was conducted to evaluate the effect of cumulus cells on the in vitro maturation (IVM) and glutathione (GSH) synthesis of porcine oocytes cultured in the presence or absence of cysteamine under different oxygen tensions, and on their subsequent male pronucleus formation after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 45 h in modified TCM-199 supplemented with or without 150 microM cysteamine under a humidified atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2. When cultured in medium supplemented with cysteamine under 20% O2 tension, the rates of COC maturation to the metaphase II (MII) stage were significantly higher than those of DOs (P<0.05). Regardless of the addition of cysteamine and oxygen tension, the rates of male pronucleus formation in COCs after IVM and IVF were significantly higher than in DOs (P<0.05). The GSH content of oocytes was significantly increased by the addition of cysteamine to the maturation medium (P<0.05), with significantly higher GSH content in COCs than in DOs (P<0.05). However, the GSH content of COCs and DOs was not significantly different when cultured in medium without cysteamine. These results indicate that cumulus cells play an important role in nuclear maturation to MII, GSH synthesis in porcine oocytes cultured in the presence of cysteamine, and subsequent male pronucleus formation after IVF.

The aim of this study was to identify the presence of functional oxytocin (OT) receptors on bovine granulosa cells. Freshly prepared bovine granulosa cells from small (3-5 mm in diameter) or preovulatory (mature) follicles were examined for OT receptors by a radioreceptor assay. Scatchard analysis revealed that both binding capacity and affinity in granulosa cells from small follicles were significantly higher than those in granulosa cells from mature follicles (p < 0.01). With use of a reverse transcriptase polymerase chain reaction analysis, expression of OT receptor mRNA was detected in granulosa cells obtained from both small and mature follicles. When the granulosa cells obtained from small follicles were cultured in Dulbecco's Modified Eagle's Medium and Ham's F-12 medium (1:1 [v:v]) with 10% calf serum up to 72 h, as the period of culture was prolonged, the concentration of OT receptor decreased with increases of progesterone and OT release in the medium. However, the binding affinity was not changed during culture for 72 h. When bovine follicular oocytes with cumulus oophorus were cultured for 24 h in tissue culture Medium-199 with 10% fetal calf serum and OT (0-10 nM), the percentages of oocytes reaching maximum cumulus expansion were significantly increased at 0.5, 1, and 10 nM OT, although nuclear maturation in oocytes surrounded by compact cumulus cells was not affected by the addition of OT. Coexposure with OT antagonist blocked the stimulatory effect of OT on cumulus expansion, confirming the specificity of the effect. Furthermore, anti-OT rabbit serum inhibited the percentages of oocytes with expanded cumulus compared to those supplemented with normal rabbit serum (p <0.05). The overall results indicate the presence of functional OT receptors in bovine granulosa cells and support the hypothesis that OT plays a role (or roles) in regulating the function of granulosa cells as an autocrine factor during follicular growth.

Abstract. The present study was undertaken to examine the effects of recombinant bovine growth hormone (rbGH) on nuclear and cytoplasmic maturation of bovine oocytes in vitro and subsequent embryonic development after in vitro fertilization. When cumulus-oocyte complexes (COCs) and cumulus-free oocytes were cultured in modified Medium 199 supplemented with 0, 0.1, 1, 10, 100 or 1000 ng/ml rbGH for 24 h, no cumulus expansion was observed in most COCs in the presence of any of the tested concentrations of rbGH. The efficacy of nuclear maturation of oocytes to metaphase II was promoted by rbGH only in COCs showing higher proportions at 10-1000 ng/ml (77-78%) than at 0-1 ng/ml (57-64%). When COCs were cultured in the presence or absence of 10 ng/ml rbGH for 0-9 h, the proportions of oocytes that reached prometaphase I and metaphase I after 6 and 9 h of culture, respectively, were higher in the presence (53% and 51%, respectively) than in the absence (34% and 30%, respectively) of rbGH. After in vitro maturation and fertilization, no differences were observed in the penetration rate (82-91%), pronuclear formation (27-45%) and polyspermy (23-29%) at 8 h post-insemination among cumulus-enclosed oocytes cultured in the presence of different concentrations (0-1000 ng/ml) of rbGH. When starting 8 h after insemination, the oocytes were cultured for 136 h in a chemically defined medium, 21-25% of oocytes developed to the morula stage or beyond without difference among different concentrations of rbGH during oocyte maturation. However, at 192 h post-insemination higher proportions of oocytes developed to the blastocyst stage when COCs were matured in the presence (13-15%) than in the absence (6%) of 10-1000 ng/ml rbGH. These results indicate that a stimulatory effect of rbGH on bovine oocyte maturation is dependent on the cumulus cells. Presence of rbGH in the media improved maturation rate and subsequent embryonic development to the blastocyst stage.

Abstract:The oxygen environment in cell culture has a significant impact on the health and performance of cells. Here, we compared the effects of reduced (5%) and ambient (20%) oxygen concentrations on bovine oocyte-granulosa cell complexes, each containing a growing oocyte 90-102 µm in diameter, cultured for 14 days. Both oxygen concentrations showed some advantages and disadvantages; in 5% oxygen, the survival rate of oocytes was significantly higher than in 20% oxygen, but the resulting oocytes were significantly smaller, which was a serious disadvantage. During the first 4 days of culture, the growth and viability of oocytes were satisfactory using 5% oxygen. This observation led us to examine the effect of changing the oxygen concentration from 5% to 20% on Day 4 in order to minimize the expected disadvantages of constant 5% and 20% oxygen. The largest population of fully grown oocytes was obtained from cultures in which the oxygen concentration was changed in this way, which also led to higher oocyte viability than in constant 20% oxygen. A similar tendency was found in the frequency of oocytes becoming blastocysts after in vitro fertilization. Surviving oocytes eventually became located within an enlarged dome-like structure, and although the 5% oxygen environment may have been appropriate for oocyte growth in the early stages, 20% oxygen may have been necessary for the growth of oocytes in the dome-like structure. These results indicate an effective way of modulating oxygen concentration according to the growth of oocyte-granulosa cell complexes in vitro. Key words: Bovine, Granulosa cell, Growing oocyte, In vitro, Oxygen concentration (J. Reprod. Dev. 58: [204][205][206][207][208][209][210][211] 2012) M ost of the growing oocytes stored in the mammalian ovary are eventually lost, and in order to be able to utilize them, it is important to develop culture systems that effectively support oocyte growth and the acquisition of developmental competence. We have previously described a 2-week culture system [1] in which bovine oocytes obtained from early antral follicles are able to complete the growth phase. However, the resulting oocytes are on average smaller than those grown in follicles in vivo. Further improvement of the culture system is therefore necessary.Our previous work comparing bovine oocyte growth in reduced (5%) and ambient (20%) oxygen concentrations [2] suggests the importance of oxygen optimization in determining culture conditions. The oxygen environment has significant effects on cell activities in culture in various cell types, such as somatic cells [3,4], embryos [5,6] and stem cells [7]. Although a universal optimal oxygen concentration for cell culture has not been determined, many studies on embryos [8][9][10], preantral follicles [11], testicular germ cells [12] and other cells [13][14][15][16][17] have suggested that a reduced oxygen concentration gives better results than an ambient oxygen concentration. A partial explanation for this would be that there is less cell damage asso...