Optimization of Oxygen Concentration for Growing Bovine Oocytes <i>In Vitro</i>: Constant Low and High Oxygen Concentrations Compromise the Yield of Fully Grown Oocytes

Abstract

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...

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

“…It is known that angiogenesis in physiological conditions occurs markedly during progress of ovarian development (Fraser 2006). Several groups reported that modulating oxygen concentration was effective in follicle culture (Heise et al 2009, Hirao et al 2012. IUFD, intrauterine fetal death; bw, body weight (g); pw, placenta weight (g).…”

Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

“…This tension is higher than the tension that is observed in the female reproductive tract and follicular fluid, which ranges between 1.3 and 8.5% O 2 (BANWELL et al, 2007). Oxygen tension has an important role in cell activity in many cell types, such as oocytes, somatic cells, stem cells and embryos (HIRAO et al, 2012). Some studies demonstrated that lower oxygen tension (5%) during IVM resulted in higher embryo production (HASHIMOTO et al, 2000(HASHIMOTO et al, , 2009MILLER;RORIE, 2000;BERMEJO-ALVAREZ et al, 2010), whereas other studies reported detrimental effects under the same conditions (CASTRO E PAULA; HANSEN, 2007;PINYOPUMMINTR;BAVISTER, 1995).…”

<i>In Vitro</i> Growth and Maturation of Vitrified-Warmed Bovine Oocytes Collected from Early Antral Follicles

“…The complexes consisting of oocytes and cumulus/granulosa cells were withdrawn from the follicles using previously described methods [16]. The collection medium comprised a modified minimum essential medium (MEM, pH 7.2; Nissui Pharmaceutical, Tokyo, Japan) that was buffered with 20 mM HEPES and 5 mM sodium bicarbonate.…”

“…Because viable oocytes were obtained after cryopreservation in these previous studies, the present study addressed issues related to in vitro oocyte growth with respect to the development of maturational competence. We previously reported a 14-day culture system for bovine oocytes in which full size was achieved from approximately the late mid-growth phase [16]. The present study aimed to establish a combined technology to produce mature bovine oocytes: vitrification-warming of growing oocytes and subsequent culture of the recovered oocytes.…”