Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1, 2-propanediol.

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.