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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation.

ABSTRACT: LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation.

SUBMITTER: Jia RX

PROVIDER: S-EPMC5068415 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation.

Jia Ru-Xia RX Duan Xing X Song Si-Jing SJ Sun Shao-Chen SC

PeerJ 20161012

LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the ...[more]

PMID: 27761340

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Project description:BackgroundThe ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.ResultsCyclic AMP-degrading PDE activity was detected in the cumulus-oocyte complex (COC) and was partially inhibited by a specific PDE3 inhibitor, cilostamide. When measured only in the denuded oocyte, PDE activity was almost completely inhibited by cilostamide, suggesting that cAMP-PDE3 activity is the major cAMP-PDE in porcine oocytes. PDE3A mRNA was detected by RT-PCR. PDE3 activity did not vary significantly during the early hours of IVM, but a maximum was observed at 13 hours. In cumulus-oocyte complexes, meiosis resumed after 20.81 hours of culture. PDE3 inhibition no longer maintained meiotic arrest if sustained beyond 17.65 hours of IVM, 3 hours prior to resumption of meiosis. Thereafter, PDE3 inhibition progressively lost its efficacy in GVBD. When the protein phosphatase 1 and 2A inhibitor okadaic acid was continuously or transiently (3 hours) present during IVM, meiosis resumed prematurely; PDE3 inhibition was unable to prevent GVBD. However, PDE3 inhibition in COC treated with OA for 3 hours significantly delayed meiosis at the intermediate stage.ConclusionThe present investigation has demonstrated that PDE3A is the major cAMP-degrading PDE in the oocyte. It regulates the resumption of meiosis until 3 hours prior to GVBD and transiently affects meiotic progression.

Project description:In vitro generation of porcine embryos is an indispensable method in the realms of both agriculture and biomedicine. Nonetheless, the extant procedures encounter substantial obstacles pertaining to both the caliber and efficacy of the produced embryos, necessitating extensive research to in vitro maturation (IVM), the seminal commencement phase. One potentially fruitful approach may lie in refining the media and supplements composition utilized for oocyte maturation. Fibroblast growth factor-7 (FGF7), alternatively termed keratinocyte growth factor, is a theca-derived cytokine integral to folliculogenesis. This study aimed to examine the ramifications of supplementing FGF7 during the IVM phase. To determine the FGF7 location and its receptor in porcine ovaries, immunohistochemistry was executed based on follicle size categories (1-2, 3-6, and 7-9 mm). Regardless of follicle size, it was determined that FGF7 was expressed in theca and granulosa cells (GCs), whereas the FGF7 receptor was only expressed in the GCs of the larger follicles. During the IVM process, the maturation medium was supplied with various concentrations of FGF7, aiming to mature porcine cumulus-oocyte complexes (COCs). The data indicated a significant augmentation in the nuclear maturation rate only within the group treated with 10 ng/mL of FGF7 (p < 0.05). Post-IVM, the oocytes diameter exhibited a significant expansion in all groups that received FGF7 supplementation (p < 0.05). Additionally, all FGF7-supplemented groups exhibited a substantial elevation in intracellular glutathione levels, coupled with a noticeable reduction in reactive oxygen species levels (p < 0.05). With respect to gene expressions related to apoptosis, FGF7 treatment elicited a downregulation of pro-apoptotic genes and an upregulation of anti-apoptotic genes. The expression of genes associated with antioxidants underwent a significant enhancement (p < 0.05). In terms of the FGF7 signaling pathway-associated genes, there was a significant elevation in the mRNA expression of ERK1, ERK2, c-kit, and KITLG (p < 0.05). Remarkably, the group of 10 ng/mL of FGF7 demonstrated an appreciable uptick in the blastocyst formation rate during embryonic development post-parthenogenetic activation (p < 0.05). In conclusion, the FGF7 supplementation during IVM substantially augments the quality of matured oocytes and facilitates the subsequent development of parthenogenetically activated embryos. These results offer fresh perspectives on improved maturation and following in vitro evolution of porcine oocytes.

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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation.

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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation.

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