Onuma A et al. (2019), Expression and function of exportin 6 in full-g...

XB-ART-56056

J Reprod Dev 2019 Oct 23;655:407-412. doi: 10.1262/jrd.2019-040.

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Expression and function of exportin 6 in full-grown and growing porcine oocytes.

Onuma A , Fujioka YA , Fujii W , Sugiura K , Naito K .

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Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.

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Species referenced: Xenopus
Genes referenced: actb rpl19 xpo1 xpo6

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	Fig. 1. Expression and turnover of endogenous XPO6 in porcine growing (GO) or full-grown (FGO) oocytes. (A) The XPO6 protein expression was examined via immunoblotting. Fifteen oocytes were used without culture (GV) and 15 were used after 48 h of maturation culture (MII). A representative result is presented in the upper panel. Beta-actin (ACTB) was used as a loading control. The XPO6 expression levels are presented as the ratio to ACTB in the lower panel (mean ± SD, * P < 0.05 compared with GV of FGOs). (B) The mRNA expression of XPO6 was analyzed via RT-PCR. RPL19 was used as a loading control. (C) The turnover of endogenous XPO6 in FGOs was analyzed via immunoblotting. Meiotic resumption and protein synthesis were inhibited by roscovitine (Ros) and cycloheximide (CHX), respectively. Fifteen oocytes were loaded in each lane.
	Fig. 2. Overexpression and nuclear-export function of XPO6 in porcine growing (GO) and full-grown (FGO) oocytes. (A) Oocytes with or without XPO6 or XPO1 mRNA injection were cultured for 24 h and the expression of XPO6 (left panel) or XPO1 (right panel) was examined via immunoblotting. Fifteen oocytes were used for each lane. Beta-actin (ACTB) was used as a loading control. (B) Calculation method for the nuclear localization index of ACTB. A rectangle (broken line) was drawn on the oocyte diameter including the nucleus with the fixed lengthwise using ImageJ software as presented in the upper part of the image. The obtained plot represented in the lower part indicates the average fluorescence intensity of the lengthwise dimension of the rectangle. The nuclear and cytoplasmic averages of the fluorescence intensity were calculated as the quotient of their area (gray, nucleus (N); stripe, cytoplasm (C)) divided by their width. The part of the nucleolus was excluded for calculating the nuclear average. The ratio of fluorescence intensity in the nucleus to that in the cytoplasm was obtained from each oocyte and used as the nuclear localization index. (C) Representative localization of ACTB with and without XPO6 overexpression was examined via immunocytochemistry. DNA was stained by 4’,6-diamidino-2-phenylindole. Scale bar, 50 µm. (D) The nuclear localization index of ACTB was calculated as the ratio of fluorescence intensity in the nucleus to that in the cytoplasm as described in (B). More than eight oocytes were analyzed in each group (mean ± SEM, * P < 0.05).
	Fig. 3. Effect of XPO6 on meiotic resumption of porcine full-grown oocytes. (A) The GVBD rate of the XPO6 mRNA-injected oocytes was examined at the indicated culture periods (* P < 0.05). The oocytes into which only EGFP mRNA was injected were used as a control. The number of oocytes examined in every experimental group was indicated in each bar. Results were shown as mean ± SEM. (B) The XPO6 expression in oocytes with or without XPO6 antisense RNA (asXPO6)-injection was analyzed via immunoblotting. The oocytes were cultured for 48 h with roscovitine (Ros) for GV-stage maintenance. Fifteen oocytes were used for each lane. (C) The asXPO6-injected oocytes were precultured for 48 h with Ros to decrease endogenous XPO6. After the preculture, the oocytes were cultured without Ros and the GVBD rate was examined at the indicated culture periods (* P < 0.05). The oocytes into which only EGFP mRNA was injected were used as the control. The number of oocytes examined in every experimental group was indicated in each bar. Results are presented as mean ± SEM.

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