Bovine preimplantation embryo development is affected by the stiffness of the culture substrate

Abstract

A recent study (Kolahi et al., 2012) demonstrated that mouse embryo development in vitro may be affected by the stiffness of the substrate on which the embryos are placed during culture. Those results showed that embryos had a developmental advantage, and more of them hatched from their zona pellucidae when cultured on a collagen gel matrix (stiffness = 1 kPa—the expected stiffness of uterine epithelium) compared to those cultured on polystyrene Petri dishes (stiffness = 1 GPa; Kolahi et al., 2012). For our study, we hypothesized that the use of a collagen gel substrate during culture would be advantageous for bovine embryo development when compared to the commonly used polystyrene substrate. On three separate occasions, bovine oocytes were matured for ∼22 hr. After maturation, cumulus oocyte complexes were co-cultured with Percoll gradient-purified spermatozoa (a mixture from three bulls) for 18 hr (Rivera et al., 2003). Putative zygotes were stripped of their cumulus cells by vortexing, and then cultured in KSOM-BE1 (Rivera et al., 2003) at 38.5°C in a humidified atmosphere containing 5% O2, 5% CO2, and 90% N2. After 24 hr of culture, all embryos at or beyond the two-cell stage (cleavage = 82 ± 2.1%) were harvested and pooled. Embryos were equally divided and cultured on either polystyrene Petri dishes or collagen gels at a density of 30 embryos/50 µl microdrop, 90 embryos per treatment/day. The collagen gel (rat tail collagen, high concentration type-1; BD biosciences, San Jose, CA) was prepared to a final concentration of 3 mg/ml following the manufacturer's specifications (i.e., Alternate Gelation Procedure for BD™ Collagen I, Rat tail) and used to coat the Petri dish surface. Gelled collagen was rinsed three times with KSOM-BE1 prior to embryo culture. Embryos were cultured for 9 days post-fertilization, and development was assessed on Days 7–9. Embryos were divided into six categories, namely: (1) early blastocyst (embryos without an obvious inner cell mass, but with discernible pockets of fluids within); (2) blastocyst (embryo containing a blastocoelic cavity and an obvious inner cell mass, but of similar size as an oocyte); (3) expanding blastocyst (enlarging blastocyst with a thinning zona pellucida); (4) expanded blastocyst (large blastocyst with an extremely thin, yet intact, zona pellucida); (5) hatching blastocysts (blastocyst escaping the zona pellucida); or (6) hatched blastocysts (embryos without a zona pellucida). ANOVA was used to determine differences between treatments. Our results show that more of the embryos cultured on the collagen gel developed to the blastocyst stage when compared to their polystyrene-cultured counterparts (P < 0.001; Fig. 1). In addition, blastocysts were more developmentally advanced in the collagen gel-cultured group than in the polystyrene-cultured group (P < 0.006; Fig. 1). Lastly, embryos tended to have an increased hatching frequency (P = 0.08) when cultured in collagen (i.e., 0 ± 0, 2.6 ± 1.3, 10 ± 1.3 vs. 0 ± 0, 0.74 ± 0.7, 5.2 ± 1.3, Days 7–9 for collagen or polystyrene substrates, respectively). Thus, similar to murine embryos, bovine embryos benefit from being cultured on a collagen gel when compared to the commonly used polystyrene Petri dish. Bovine embryo development to the blastocyst stage. Embryos were randomly selected at the two- to four-cell stage, and cultured on either a collagen gel (1 kPa surface stiffness) or the commonly used polystyrene Petri dish (1 GPa surface stiffness). The experiment was repeated on three different days. Development was assessed on Days 7–9 after fertilization. n = 270 embryos/treatment. Data are represented as mean ± standard error mean.