Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

NANOS3 is an evolutionarily conserved gene expressed in primordial germ cells that is important for germ cell development. Germ cell deletion by NANOS3 knockout has been reported in several mammalian species, but its function in pigs is unclear. In the present study, we investigated the germline effects of NANOS3 knockout in pigs using CRISPR/Cas9. Embryo transfer of CRISPR/Cas9-modified embryos produced ten offspring, of which one showed wild-type NANOS3 alleles, eight had two mutant NANOS3 alleles, and the other exhibited mosaicism (four mutant alleles). Histological analysis revealed no germ cells in the testes or ovaries of any of the nine mutant pigs. These results demonstrated that NANOS3 is crucial for porcine germ cell production.