In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

Abstract

Abstract. The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects. Key words: Blastocyst, Co-culture, Mouse, Pronuclear embryo, Sequential culture medium (J. Reprod. Dev. 54: [385][386][387][388][389][390] 2008) t has been suggested that the co-culture system exerts its beneficial effects by negative [1][2][3][4][5] or positive conditioning [4,[6][7][8][9][10][11] of the culture media, and by embryo-to-cell contact. Negative conditioning or detoxification is the ability of the feeder cells to remove deleterious components from the culture medium whereas positive conditioning is the ability of the feeder cells to modify it by releasing some components (embryotrophic factors) into the culture medium [12,13].There have been studies proposing that embryo-to-cell contact is necessary to allow cellular connections between the zona pellucida and feeder cells for cross-transfer of glycoproteins and other important metabolites [12]. In the cumulus oocyte complex, gap junctions have been shown to be present in the regions of contact between the cumulus cell projections and the oolemma, as well as between adjacent cumulus and granulosa cells [14,15]. Nevertheless, whether gap junctions are present ...

The role of co-culture systems on developmental competence of preimplantation mouse embryos against pH fluctuations

“…In contrast to our suggestion, some authors have proposed; addition of embryotrophic substances which are usually secreted by the somatic cells into the simple culture media, may improve embryo quality without the need of feeder cells [33,40]. However, recent studies have shown a direct contact between the embryo and feeder cells enhance embryo development better than the embryos cultured in presence of feeder cells without a direct contact [18]. Besides, this assumption may be true if the negative conditioning hypothesis was neglected.…”

“…The most ordinary methods are the improvement in culture components and conditions [10,11], using sequential culture media [12], and conditioned media [13,14], and often feeder cells [15,16]. Many investigators suggest coculture systems could improve embryo quality and in vitro cleavage rate efficiently [15,[17][18][19]. While, few studies have reported no improvement in the embryo quality and implantation rates following co-culture of embryos with somatic feeder cells [20,21].…”

Effects of Cumulus Cells on In Vitro Maturation of Oocytes and Development of Cloned Embryos in the Pig

“…2001; Johnson et al. 2008; Omar Farouk and Vlad 2008). Recently, CC co‐culture systems have been designed to support in vitro development of embryos in several species, including mice, cattle and humans (Parikh et al.…”

“…Recently, CC co‐culture systems have been designed to support in vitro development of embryos in several species, including mice, cattle and humans (Parikh et al. 2006; Omar Farouk and Vlad 2008; Goovaerts et al. 2009).…”

Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

“…We suspect that optimizing our technique would not only improve the percentage of two-cell embryos generated, but also show recognizable differences between using cryopreserved versus fresh gametes. One possible improvement to conventional IVF would be to maintain cumulus cell presence during culture, as this has been demonstrated to improve in vitro embryo development [26], [27] and fertilization efficiency [28]–[31]. One study found that the presence of an intact cumulus layer on oocytes increased the in vitro fertilizing ability of fresh capacitated mouse spermatozoa, with <10% of cumulus-free oocytes from GM female mice inseminated, compared to almost 40% two-cell embryos with COCs [28] Because cumulus cells must be removed for rapid and effective zona perforation, we decided to also remove cumulus cells for conventional IVF, to avoid this as a possible confounder.…”