Podophyllotoxin is used as medical cream which is widely applied to genital warts and molluscum contagiosum. Although previous study showed that podophyllotoxin had minimal toxicity, it was forbidden to use during pregnancy since it might be toxic to the embryos. In present study we used mouse as the model and tried to examine whether podophyllotoxin exposure was toxic to oocyte maturation, which further affected embryo development. Our results showed that podophyllotoxin exposure inhibited mouse oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on oocytes was dose-depended. Further studies showed that the meiotic spindle formation was disturbed, the chromosomes were misaligned and the fluorescence signal of microtubule was decreased, indicating that podophyllotoxin may affect microtubule dynamics for spindle organization. Moreover, the oocytes which reached metaphase II under podophyllotoxin exposure also showed aberrant spindle morphology and chromosome misalignment, and the embryos generated from these oocytes showed low developmental competence. We also found that the localization of p44/42 MAPK and gamma-tubulin was disrupted, which further confirmed the effects of podophyllotoxin on meiotic spindle formation. In all, our results indicated that podophyllotoxin exposure could affect mouse oocyte maturation by disturbing microtubule dynamics and meiotic spindle formation.

LIM kinases (LIMK1/2) are LIM domain‐containing serine/threonine/tyrosine kinases that mediate multiple cellular processes in mitosis. In the present study, we explored the functional roles and potential signaling pathway of LIMK1/2 during mouse oocyte meiosis. Disruption of LIMK1/2 activity and expression significantly decreased oocyte polar body extrusion. Live‐cell imaging revealed that spindle migration was disturbed after both LIMK1 and LIMK2 knock down, and this might be due to aberrant distribution of actin filaments in the oocyte cytoplasm and cortex. Meanwhile, our results demonstrated that the function of LIMK1 and LIMK2 in actin assembly was related to cofilin phosphorylation levels. In addition, disruption of LIMK1/2 activity significantly increased the percentage of oocytes with abnormal spindle morphologies, which was confirmed by the abnormal p‐MAPK localization. We further, explored the upstream molecules of LIMK1/2, and we found that after depletion of ROCK, phosphorylation of LIMK1/2 and cofilin were significantly decreased. Moreover, RhoA inhibition caused the decreased expression of ROCK, p‐LIMK1/2, and cofilin. In summary, our results indicated that the small GTPase RhoA regulated LIMK1/2‐cofilin to modulate cytoskeletal dynamics during mouse oocyte meiosis.

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6‐month‐old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.

Front Cover: The cover image is based on the Original Research Article Loss of PKC mu function induces cytoskeletal defects in mouse oocyte meiosis by Yu Zhang et al., DOI: https://doi.org/10.1002/jcp.28487.