Low-Concentration Essential Amino Acids in PZM-3 Improve the Developmental Competence of Porcine Embryos Produced by Handmade Cloning.

Essential amino acids (EAA) of inappropriate concentration have been reported to compromise the development of embryo. This study aimed to investigate the effect of EAA on the developmental competence of porcine embryos produced by either handmade cloning (HMC) or parthenogenetic activation (PA). In experiment 1, we examined the in vitro developmental competence of PA embryos after culture in PZM-3 containing different concentrations (v/v) of EAA (0%, 1%, and 2%). The results indicated that reducing the concentration of EAA from 2% to 1% significantly improved the blastocyst formation (36% vs. 54%), while 0% would compromise the blastocyst formation rate (54% vs. 38%). In experiment 2, we further investigated the effect of EAA concentration (1% and 2%) on the in vitro developmental competence and gene expression of HMC embryos. Blastocyst rate significantly increased by reducing concentration of EAA (41% vs. 53%) and those genes upregulated were enriched in oxidative phosphorylation, PPAR signaling pathway, and metabolism-related pathways. In experiment 3, the in vivo developmental competence of HMC embryos cultured in the medium supplemented with 1% EAA was examined. Embryos derived from both non-gene-modified fetal fibroblasts (FFs) and gene-modified fetal fibroblasts (GMFFs) were transferred to recipients. The pregnancy rates were 83% and 78% separately. Out of the pregnancies, 5 (FFs) and 6 (GMFFs) were successfully developed to term. Our study indicates that supplementing EAA to embryo culture medium at a concentration of 1% can improve the in vitro developmental competence of porcine HMC embryos and the blastocyst obtained can successfully develop to term, which could be beneficial for the production of gene-modified piglets.

Mice lacking the B1 subunit of H+ -ATPase have normal hearing.

Acid-base homeostasis of endolymph is thought to be essential for normal inner ear function. This assumption was supported by clinical data from individuals affected by autosomal recessive distal renal tubular acidosis with sensorineural hearing loss. This recessive syndrome was recently demonstrated to be due to mutations in the gene encoding the B1 subunit of H(+)-ATPase (ATP6B1). To examine the potential roles of H(+)-ATPase B1 subunit in inner ear development and function, we defined its spatial and temporal expression patterns in the developing mouse inner ear and examined the morphologic and physiologic effects of loss of its function. Standard in situ hybridization was used for the expression study with routine morphologic and physiologic assessments of hearing and balance in H(+)-ATPase B1 subunit (Atp6b1) null mutant mice. Atp6b1 mRNA was first detected at embryonic day 11.5 (E11.5) in the endolymphatic duct epithelia. From E16.5 onward, Atp6b1 was also observed in the presumptive interdental cell layer of the spiral limbus in the cochlea. Auditory brainstem response tests revealed normal hearing in mice lacking Atp6b1. The inner ears of these mice develop normally and show no overt morphological abnormalities. Our data demonstrate that Atp6b1 is not critical for normal inner ear development or normal inner ear function in mice and suggest that other proton-transporting mechanisms or pH buffering systems must allow the mouse inner ear to compensate for lack of normal Atp6b1 activity.