Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

Abstract

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called "FLI medium," improves nuclear maturation of oocytes in cumulus-oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus-oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.cumulus cell | embryo development | genetic modification | in vitro fertilization | MAPK signaling

Effect of cumulus cells and sperm concentration on fertilization and development of pig oocytes

“…One possible reason may be gilt oocyte's smaller size (Pedersen et al., ) or mitochondrial content (Pedersen, Løvendahl, Larsen, Madsen, & Callesen, ). Another explanation could relate to the CCs in gilt oocytes, as adding cytokines during IVM improve CC maturation and embryonic development (Yuan et al., ).…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Also, results from RT‐PCR analysis showed that activation of the mitogen‐activated protein kinase (MAPK) pathway in cumulus cells seems to have an important role in cumulus expansion and meiotic maturation of porcine COCs. Based on this, Yuan et al () supplemented the maturation medium with three cytokines (FGF2, LIF, and IGF1), named “FLI,” and observed a fourfold increase in the efficiency of piglet production. Supplementation of the IVM medium with FLI enhanced nuclear maturation and led a twofold increase in blastocyst production after IVF.Deep sequencing analysis of IVV or IVC porcine blastocysts identified 1,170 differentially expressed transcripts between these two conditions with 588 of these transcripts exhibiting at least a twofold difference (Bauer et al, ).…”

“…Based on this, Yuan et al (2017) supplemented the maturation medium with three cytokines (FGF2, LIF, and IGF1), named "FLI," F I G U R E 1 Advancements in in vitro culture system protocols to improve development of porcine embryos. Selection of adequate external conditions and supplementation with amino acids, cytokines, and different small molecules during in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) improved development of in vitro-produced porcine embryos.…”

“…Besides the importance of controlling culture external conditions, supplementation in vitro to correct specific molecular deficiencies has been reported to increase the efficiency of assisted reproduction techniques (Chen et al, ; Heras et al, ; Moussa, Shu, Zhang, & Zeng, ; Redel et al, ; Yuan et al, ). Recent studies have explored the potential of specific growth factors and amino acids to optimize maturation and embryo culture media (Chen et al, ; Krisher & Prather, ; Redel et al, ; Redel, Tessanne, Spate, Murphy, & Prather, ; Yuan et al, ). However, due to the different chemical and biological composition of supplements, strategies to enable more efficient use of these additives by preventing premature degradation, increasing their bioavailability and facilitating their delivery are desired.…”

Circular RNA profiling in the oocyte and cumulus cells reveals thatcircARMC4is essential for porcine oocyte maturation

“…Pigs are increasingly being used as an ideal animal model for humans in reproductive medicine research because they share many similarities in the aspects of physiology (aneuploidy rate), developmental timing (oocyte maturation and early embryo development), and genetics with humans (Mordhorst and Prather, 2017). Although many efforts have been made to improve the oocyte maturation in vitro in pigs, its meiotic and developmental capacity is still suboptimal relative to that under the physiological states (Nagai, 2001; Yuan et al, 2017). This may be due to the imperfectness of in vitro culture conditions currently used that cannot achieve the optimal maturational outcomes, and there is inadequate information regarding the unique molecular mechanisms of porcine oocyte meiotic maturation (Sun and Nagai, 2003; Prather et al, 2009).…”