Autophagic activation in porcine oocytes is independent of meiotic progression.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

Brachygnathia Inferior in Cloned Dogs Is Possibly Correlated with Variants of Wnt Signaling Pathway Initiators.

Abnormalities in animals cloned via somatic cell nuclear transfer (SCNT) have been reported. In this study, to produce bomb-sniffing dogs, we successfully cloned four healthy dogs through SCNT using the same donor genome from the skin of a male German shepherd old dog. Veterinary diagnosis (X-ray/3D-CT imaging) revealed that two cloned dogs showed normal phenotypes, whereas the others showed abnormal shortening of the mandible (brachygnathia inferior) at 1 month after birth, even though they were cloned under the same conditions except for the oocyte source. Therefore, we aimed to determine the genetic cause of brachygnathia inferior in these cloned dogs. To determine the genetic defects related to brachygnathia inferior, we performed karyotyping and whole-genome sequencing (WGS) for identifying small genetic alterations in the genome, such as single-nucleotide variations or frameshifts. There were no chromosomal numerical abnormalities in all cloned dogs. However, WGS analysis revealed variants of Wnt signaling pathway initiators (WNT5B, DVL2, DACT1, ARRB2, FZD 4/8) and cadherin (CDH11, CDH1like) in cloned dogs with brachygnathia inferior. In conclusion, this study proposes that brachygnathia inferior in cloned dogs may be associated with variants in initiators and/or regulators of the Wnt/cadherin signaling pathway.