Anti-ATP5A1 monoclonal antibody, clone 8I21CE5G0

To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 p,1 medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

Whether the mutation rate differs between sexes has been a matter of discussion for years. Molecular analyses of mammals have indicated that males mutate more often than females, as manifested by the faster rate of neutral sequence evolution on the Y chromosome than on the X chromosome. However, these observations can as well be interpreted as specific reduction of the X chromosome mutation rate, which would be adaptive because of reducing the number of slightly deleterious recessive mutations exposed in hemizygote males. Recently, data from birds have suggested that vertebrate mutation rates may indeed be male-biased. In birds, females are the heterogametic sex (ZW), and analyses of the Z-linked CHD1Z gene have shown that it evolves faster than its W-linked and thus female-specific homologue, CHD1W. We have now studied the second avian gene known to exist in a copy on the nonrecombining regions of both the Z and the W chromosome, viz., the ATP synthase alpha-subunit (ATP5A1). In independent comparisons of three pairs of bird species from divergent lineages, intron sequences of the Z-linked copy (ATP5A1Z) were consistently found to evolve faster than the W-linked copy (ATP5A1W). From these data we calculated male-to-female mutation rate ratios (alpha) of 1.8, 2.3, and 5.0 in Galliform, Anseriform, and Ciconiiform lineages, respectively. Therefore, this study provides independent support for a male-biased mutation rate in birds.