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. 2015 Jun 17;14(17):2835–2843. doi: 10.1080/15384101.2015.1031438

RhoA-mediated FMNL1 regulates GM130 for actin assembly and phosphorylates MAPK for spindle formation in mouse oocyte meiosis

Fei Wang 1, Liang Zhang 1, Xing Duan 1, Guang-Li Zhang 2, Zhen-Bo Wang 2, Qiang Wang 3, Bo Xiong 1, Shao-Chen Sun 1,*
PMCID: PMC4614837  PMID: 26083584

Abstract

Formin-like 1 (FMNL1) is a member of Formin family proteins which are the actin nucleators. Although FMNL1 activities have been shown to be essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis, the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain. In this study, we investigated the functions of FMNL1 in mouse oocytes using specific morpholino (MO) microinjection and live cell imaging. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD). FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies. Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels. Cortical polarity was disrupted as shown by a loss of actin cap and cortical granule free domain (CGFD) formation, which was confirmed by a failure of meiotic spindle positioning. And this might be the reason for the large polar body formation. Spindle formation was also disrupted, which might be due to the abnormal localization of p-MAPK. These results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. Moreover, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130. Finally, we found that the small GTPase RhoA might be the upstream regulator of FMNL1. Taken together, our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130 pathway during mouse oocyte meiosis.

Key words: actin, FMNL1, golgi, polar body extrusion, spindle organization

Abbreviations

Formin-like 1

FMNL1

germinal vesicle breakdown

GVBD

cortical granule free domain

CGFD

germinal vesicle

GV

metaphase I

MI

anaphase/telophase I

ATI

metaphase II

MII

polar body

PB

Introduction

Mammalian oocyte meiotic maturation is characterized by a unique asymmetric division, during which oocytes extrude half of their chromosomes in a small daughter cell called the polar body. This asymmetric division is regulated by both microtubule and actin filaments.1 After germinal vesicle breakdown (GVBD), chromatin condenses and microtubules reorganize, which results in spindle formation. The chromosomes were arranged at an equatorial plate, and then actin drives spindle movement toward the cortex, which results in oocyte polarity and cytokinesis.2 Two main steps are involved in regulating this asymmetric meiotic division: spindle positioning, which involves spindle migration and anchoring, and polar body extrusion, which involves cortical polarity formation and cytokinesis.3,4

Formins are multi-domain proteins that are key regulators of actin and microtubule cytoskeletal dynamics and are involved in a wide range of actin-based cellular processes, including cell adhesion, cytokinesis, cell polarization, and migration.5,6 Formins comprise a diverse, large protein family, each of which has a characteristic formin homology 2 (FH2) domain of approximately 400 amino acids.7-10 The FH2 domain dimerizes into a stabilized “donut shape” to nucleate new actin filaments and binds to the barbed ends of filamentous actin (F-actin) during actin assembly.11-14 There are 15 known mammalian formins that are grouped into 7 different subclasses: formins (FMNs); diaphanous (DIA); formin-related proteins in leukocytes (FMNLs/FRLs); disheveled-associated activators of morphogenesis (DAAMs); inverted formins (INFs); formin homology domain proteins (FHODs); and delphilin.8 Formins are currently the broadest subset of known classes of actin nucleating proteins. In vitro biochemical assays have shown that mammalian formins exhibit actin-filament nucleating, polymerizing, bundling, and severing activities. 14-16 A recent study showed that actin-capping proteins promoted microtubule stability by antagonizing the actin activity of mDia1.17 Several previous studies also demonstrated that Formin-2 was required for spindle migration and cytokinesis by regulating actin filament assembly in mouse oocytes.1,18-20

The FMNL/FRL subclass of formins includes FMNL1 (FRL1), FMNL2 (FRL3), and FMNL3 (FRL2), all of which share a domain organization similar to that of diaphanous proteins.21 FMNL1 is a formin-related protein that is expressed predominantly in leukocytes, haematopoietic cells, spleen, and thymus.22-24 FMNL1 is involved in podosome dynamics, cell migration, phagocytosis, and cell adhesion.25-27 FMNL1 and mDia2 have been shown to mediate the F-actin formation and the bundling of pre-existing actin filaments,16,28 and FMNL1 was shown to sever actin filaments in vitro.29 Another study showed that FMNL1 accumulated as punctate structures near the microtubule-organizing center (MTOC) and regulated MTOC polarization in T cells.30 It has also been demonstrated that the dynamic remodeling of the actin cytoskeleton by FMNL1 was required for Golgi complex structural maintenance and that FMNL1 localized at Golgi complexes, where it interacted with actin barbed ends to maintain Golgi structure.31 However, previous work on FMNL1 primarily focused on mitotic cells, and its role during mammalian meiosis remains unknown.

Thus, we investigated the roles of FMNL1 during mouse oocyte meiotic maturation. Our study results provide information regarding actin filament and spindle organization regulation by FMNL1 in vitro and the signaling pathway that may be involved during mouse oocyte meiosis.

Results

FMNL1 expression during mouse oocyte meiotic maturation

Mouse oocytes were cultured for 0 h, 2 h, 8 h, 10 h, and 12 h, corresponding to the time points when most oocytes reached the germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase/telophase I (ATI), and metaphase II (MII) stages, respectively. We then examined FMNL1 expression during these stages of mouse oocyte meiotic maturation using immunofluorescent staining. As shown in Figure 1A, at the GV and GVBD stages, FMNL1 was distributed throughout the cytoplasm and appeared to accumulate around chromosomes in GVBD oocytes. After GVBD, FMNL1 accumulation was also found near the spindle poles at the MI and MII stages. As maturation progressed to the ATI stage, FMNL1 accumulated near chromosomes. To further prove the localization of FMNL1, we co-stained FMNL1 with α-tubulin, as shown in Figure 1B, FMNL1 localized at the poles of spindle. There was no signal of FMNL1 in the negative control oocytes (Fig. 1C). This dynamic localization pattern of FMNL1 indicates that it might be involved in spindle formation or spindle positioning during meiosis.

Figure 1.

Figure 1.

FMNL1 localization during mouse meiotic maturation. (A) FMNL1 antibody immunofluorescent staining was used to determine the subcellular localization of FMNL1 in mouse oocytes. After GVBD, FMNL1 accumulated at oocyte spindle poles. Green, FMNL1; blue, chromatin. Bar = 20 μm. (B) Co-staining of FMNL1 and α-tubulin. Green, α-tubulin; red, FMNL1; blue, chromatin. Bar = 20 μm. (C) No FMNL1 signal in the negative control oocytes. Green, FITC-conjugated goat-anti-rabbit IgG; blue, chromatin. Bar = 20 μm.

FMNL1 depletion results in abnormal polar body extrusion

To investigate possible roles of FMNL1 during mouse oocyte meiotic maturation, we knocked down FMNL1 expression by microinjection of FMNL1-specific morpholino (MO). As shown in Figure 2A, Western blot densitometry analysis showed that FMNL1 expression was significantly reduced after knockdown with FMNL1 morpholino (0.30 ± 0.01 vs. 0.04 ± 0.02; P < 0.05). After control or FMNL1 MO injection, oocytes were cultured for 21 h in M16 medium containing 2.5 μM milrinone to prevent the resumption of meiosis, and then were transferred to fresh M16 medium and cultured for an additional 12 h. These results showed that a large proportion of oocytes failed to extrude polar bodies or extruded abnormally large polar bodies after FMNL1 MO injection (Fig. 2B).

Figure 2.

Figure 2.

FMNL1 depletion disrupts first polar body extrusion and asymmetric division. (A) Western blot analysis for FMNL1 in FMNL1 MO treated oocytes and control oocytes. Relative intensity of FMNL1 or α-tubulin was assessed by densitometry. The molecular mass of FMNL1 is 122 kDa and that of α-tubulin is 55 kDa. *, significantly different (P < 0.05). (B) Images of eggs at the end of 12 h incubation. In control group, black arrow indicates oocyte with a normal size polar body. While in the KD group, black arrow indicates oocyte with symmetrical division. (C) Time lapse microscopy of maturing oocytes in control-MO injected oocytes and FMNL1-MO injected oocytes (red, chromatin, stained with Hoechst 33342). (D) Rates of polar body extrusion and large polar bodies in control-MO injected and FMNL1-MO injected oocytes. Results are mean percentages ± SEM's of 3 independent experiments. *, significantly different (P < 0.05)

Live cell imaging by time-lapse microscopy was used to confirm the dynamic changes that occurred in maturing oocytes after injection with FMNL1 MO. As shown in Figure 2C, in control MO-injected oocytes, the spindle moved toward the cortex and oocytes extruded polar bodies normally. In contrast, for FMNL1 MO-injected oocytes, 2 phenotypes were observed: 1) chromosomes segregated in the central cytoplasm but then re-joined together, and the polar body was not extruded; and 2) chromosomes separated in the central cytoplasm but the oocyte underwent symmetric division.

We also determined the rates of polar body extrusion and large polar bodies (PB) in control MO-injected and FMNL1 MO-injected oocytes. As shown in Figure 2D, for FMNL1 MO-injected oocytes, the polar body extrusion rate (39.1 ± 3.5%, n = 195) was significantly lower than that of control MO-injected oocytes (69.9 ± 1.5%, n = 138; P < 0.05). We defined the “large” as the polar body size that was larger than half of oocyte. In addition, the rate of large PB formation for FMNL1 MO-injected oocytes (44.5 ± 1.1%, n = 75) was significantly greater than that of control MO-injected oocytes (5.2 ± 1.0%, n = 96; P < 0.05).

FMNL1 depletion disrupts the actin filament distribution and oocyte cortical polarity

Actin filaments provide the main driving force for oocyte asymmetric division. Because FMNL1 depletion could result in abnormal polar body extrusion, the effect of FMNL1 on actin filament expression was analyzed. We first used time-lapse microscopy to examine actin distribution during oocyte meiosis. As shown in Figure 3A and Figure S1, in control MO-injected oocytes, spindles moved toward the cortex and the oocyte extruded a polar body, and actin filament signals were observed at the oocyte cortex. In contrast, in FMNL1 MO-injected oocytes, actin filament signals at the cortex gradually decreased.

Figure 3.

Figure 3.

FMNL1 depletion changes actin distribution and disrupts spindle migration. (A) Time lapse microscopy results of actin distribution in control MO-injected or FMNL1 MO-injected oocytes. In control MO-injected oocytes, spindles moved toward the cortex and extruded a polar body, and actin signals were observed strongly during this process. In contrast, in FMNL1 MO-injected oocyte, actin signals gradually decreased during this process. Red, chromatin; green, actin; white, DIC. Bar = 20 μm. (B) Actin distribution on oocyte membranes after treatment. During the MI stage, actin immunofluorescence staining intensity deceased at the membranes of FMNL1 MO injected oocytes. In addition, linear fluorescence intensity analysis showed that there was a peak in the actin cap regions of control oocytes, whereas the actin cap peaks were much lower in FMNL1 MO-injected oocytes (arrowhead shows an actin cap). Green, FMNL; blue, chromatin; red, actin. Bar = 20 μm. (C) Average actin immunofluorescence intensities for mouse oocyte membranes and cytoplasm were determined. Results are mean percentages ± SEM's of 3 independent experiments. (D) In control-MO injected oocytes, spindles formed and moved toward the cortex after culture for 9.5 h, but remained centrally located after injection with FMNL1 morpholino. Red, FMNL1; green, α-tubulin; blue, chromatin. Bar = 20 mm. (E) Rates of spindle localization after 9.5 h of culture in control-MO injected and FMNL1-MO injected oocytes. *, significantly different (P < 0.05).

To further investigate the relationship between FMNL1 and actin, we used immunofluorescent staining with FMNL1 antibody and Phalloidin-TRITC. As shown in Figure 3B, the confocal images showed the localization of FMNL1 disappeared in FMNL1-KD oocytes while FMNL1 localized to the spindle poles in the control oocytes. Actin fluorescence intensity on the membranes of FMNL1 MO-injected oocytes was significantly lower than that of control MO-injected oocytes (37.3 ± 2.9 vs. 60.6 ± 2.0, n = 135; P < 0.05) and was also lower in the cytoplasm (13.5 ± 1.9 vs. 20.3 ± 1.5, n = 135; P < 0.05) (Fig. 3B and C). Additionally, actin cap formation was disrupted: actin cap formed near the chromosome in control oocytes, whereas there was no actin cap formed in most FMNL1-MO oocytes. Since spindle migration is actin-related process, the decrease of actin might result in the failure of spindle migration, next we assessed this after FMNL1 knock down. We categorized spindle positioning of MI stage into 2 phenotypes: (1) Spindles that migrated to the cortex and one pole of spindles has been exposed to the cortex; we called this spindle position as MI cortex. (2) Spindles that localized in the center of cytoplasm and did not migrate to the cortex; we called this spindle position as MI center. As shown in Figure 3D, E, the fluorescent images showed the localization of FMNL1 disappeared in FMNL1-KD oocytes while FMNL1 localized to the spindle poles of the control oocytes. After 9.5 h of culture, most spindles in control oocytes had moved to the cortex (MI center: 8.3 ± 0.6%; MI cortex: 41.1 ± 2.4%; ATI/MII: 50.6 ± 0.5%; n = 159), while a large proportion of spindles in FMNL1 MO-injected oocytes were arrested in a central position (MI center: 36.8 ± 1.2%; MI cortex: 46.1 ± 2.1%; ATI/MII: 12.8 ± 2.3%; n = 147). Our results indicated that spindle migration was disrupted by FMNL1 depletion.

FMNL1 depletion causes abnormal spindle organization

Because FMNL1 became localized at the spindle poles after GVBD, we next examined involvement of FMNL1in spindle organization. As shown in Figure 4A, we co-stained with FMNL1, α-tubulin and Hoechst 33342. The fluorescent images showed that the localization of FMNL1 disappeared in FMNL1-KD oocytes while FMNL1 localized to the spindle poles of the control oocytes. In control MO-injected oocytes, spindles exhibited normal morphologies, whereas in FMNL1 MO-injected oocytes, various morphologically defective spindles were observed. As shown in Figure 4B, the rate of abnormal spindles (53.1 ± 1.6%, n = 93) in FMNL1 MO-injected oocytes was significantly higher than that in control MO-injected oocytes (19.0 ± 1.1%, n = 126; P < 0.05).

Figure 4.

Figure 4.

FMNL1 depletion causes abnormal spindle organization. (A) Oocytes that were microinjected with FMNL1 or control MO were collected at 9 h of culture in fresh M16 medium. In FMNL1 MO injected oocytes, various morphologically defective spindles were observed. These defects were divided into 4 types. Oocytes were double stained with FMNL1 (red), α-tubulin (green) and Hoechst (blue). Bar = 20 μm. (B) Percentages of oocytes with abnormal spindles in FMNL1-MO injected and control-MO injected oocytes. Results are mean percentages ± SEM's of 3 independent experiments. *, significantly different (P < 0.05). (C) Localization of p-MAPK after FMNL1 knock down. FMNL1 MO and control MO injected oocytes were cultured for 9 h, and then stained for p-MAPK (green) and DNA (blue). Bar = 20 μm. Western blot analysis of p-MAPK expression in FMNL1 MO and control oocytes. The molecular masses of p-MAPK are 42 kDa and 44 kDa and that of α-tubulin is 55 kDa.

To further confirm the involvement of FMNL1 in spindle formation, we examined the localization of phospho-p44/42 mitogen-activated protein kinase (p-MAPK), which also localizes at spindle poles and has been demonstrated to be required for proper spindle formation during oocyte meiosis. After culture for 9 h, p-MAPK was localized at spindle poles in control MO-injected oocytes, whereas in most FMNL1 MO-injected oocytes with abnormal spindles, p-MAPK had detached from spindle poles and had dispersed into the cytoplasm (Fig. 4C and Fig. S1). We also employed the Western blot to analyze the protein expression of p-MAPK. As shown in Figure 4C, p-MAPK had no expression in control GV stage oocyte but strongly expressed in control MI stage oocytes, while in FMNL1 knock down MI stage oocytes, the expression of p-MAPK decreased. The results demonstrated FMNL1-KD reduced p-MAPK expression and disrupted the localization of p-MAPK during mouse oocyte meiotic maturation.

FMNL1 functions in the RhoA-FMNL1-GM130 signaling pathway in mouse oocytes

Because Formin proteins are downstream effectors of small GTPases, we investigated a possible relationship between the small GTPase RhoA and FMNL1 expression. As shown in Figure 5A, Western blot densitometry analysis showed that FMNL1 expression was significantly reduced after treatment with a RhoA inhibitor (Rhosin) (0.82 ± 0.01 vs. 0.38 ± 0.04; P < 0.05).

Figure 5.

Figure 5.

(A) Western blot analysis for FMNL1 expression in control and Rhosin inhibited oocytes. Relative intensity of FMNL1 or α-tubulin was assessed by densitometry. The molecular mass of FMNL1 is 122 kDa and that of α-tubulin 55 kDa. (B) FMNL1 MO and control MO injected oocytes were cultured for 9 h, and then stained for GM130 (green) and DNA (blue). Bar = 20 μm. (C) Western blot analysis for GM130 expression in control and FMNL1 MO-injected oocytes. Relative intensity of GM130 or α-tubulin was assessed by densitometry. The molecular mass of GM130 is 140 kDa and that of α-tubulin is 55 kDa. (D) A diagram of the possible signaling pathway of FMNL1 during mouse oocyte meiosis. FMNL1 may regulate p-MAPK for spindle formation, and regulate GM130 for actin assembly, which further affects polar body extrusion of mouse oocyte. And the regulatory of FMNL1 may be depended on the activity of RhoA GTPase.

A previous study showed that FMNL1 was required for maintaining the structural integrity of Golgi complexes and that FMNL1 had similar localization pattern with GM130, which both were localized on spindle poles.31 Thus, we investigated the relationship between FMNL1 and GM130 expression during oocyte meiosis. As shown in Figure 5B, immunofluorescent staining revealed that GM130 was concentrated primarily at spindle poles in control MO-injected oocyte after culture for 9 h, when most oocytes reached late MI stage, similar to the localization pattern of FMNL1. In contrast, in FMNL1 MO-injected oocytes, GM130 was dispersed throughout the cytoplasm with no specific localization pattern. We also assessed whether GM130 expression changed after FMNL1 depletion. Western blot densitometry analysis showed GM130 expression was significantly reduced (0.36 ± 0.01 vs. 0.13 ± 0.02; P < 0.05; Fig. 5C). Because GM130 is a cis-Golgi marker protein, these results indicate that FMNL1 affects the distribution and expression of Golgi complexes in mouse oocytes. In addition, a recent study has demonstrated that a vesicle-based mechanism of actin network modulation is essential for the asymmetric positioning of meiotic spindles in mouse oocytes.32 Since the Golgi mediates the long-range transport of vesicles, we assume that FMNL1 might regulate the GM130 to affect polar body formation (Fig. 5D).

Discussion

In this study, we reported that FMNL1 was primarily localized to the spindle poles of oocyte. Loss of FMNL1 disrupted meiotic spindle assembly and actin-based asymmetric division. In addition, FMNL1 functioned in the Rho-FMNL1-GM130 pathway during mouse oocyte meioisis. Taken together, our data support a model in which FMNL1 regulates asymmetric division and cytokinesis by controlling actin assembly and spindle organization during mouse oocyte meiotic maturation.

FMNL1 is a member of the Formin family proteins and is an actin nucleator. A previous study has shown that FMNL1 is concentrated in the MTOC's in somatic cells.30 During oocyte meiotic maturation, we found that FMNL1 is primarily concentrated at oocyte spindle poles. Other molecules that are localized to spindle poles, such as γ-tubulin, MAPK, and GM130, have been shown to regulate meiotic spindle assembly and asymmetric division in mouse oocytes.33,34 This FMNL1 localization pattern prompted us to speculate that FMNL1 might be involved in oocyte meiosis.

To confirm this assumption, we used FMNL1 morpholino injection to investigate possible roles for FMNL1 during mouse oocyte meiotic maturation. Our results showed that knock down of FMNL1 could result in abnormal polar body extrusion. Living cell imaging result also showed that the chromosomes segregated but there was still no polar body extrusion, indicating that FMNL1 did not affect chromosome segregation but regulated cytokinesis. To investigate a possible mechanism for FMNL1 effects in oocyte polar body extrusion, we first examined actin filaments during oocyte meiosis, as previous studies showed that the actin network is the main driving force for cytokinesis and polar body extrusion.35,36 Immunofluorescent staining showed that actin expression decreased at the membrane after injecting oocytes with FMNL1 MO. Similar results has been previously observed in other members of the Formin family, such as Formin-2. Formin-2 or Spire depletion resulted in reduced cellular actin amounts during meiotic maturation and Formin-2 has been shown to regulate spindle migration and cytokinesis by regulating actin filament assembly in mouse oocytes.1,18–20 Thus, our results suggest that FMNL1 participates in polar body extrusion by regulating actin filaments.

We also examined oocyte cortical polarity, an earlier process for polar body extrusion. Our results showed that the actin cap formation, a feature of cortical polarity, was disrupted after FMNL1 depletion. And we also showed that FMNL1 depletion resulted in the arrest of spindles in a central location. A previous study showed that actin drove spindle migration and moved spindles toward the cortex, which resulted in oocyte asymmetric division.2 Because spindle migration is known to be actin-dependent, FMNL1 may regulate spindle migration through its effect on actin nucleation, which can further affect oocyte cortical polarity formation. And this might be the reason for the large polar body formation: the meiotic spindle failed to migrate to the cortex while the cleavage occurred.

In mammalian mitotic cells, FMNL1 localized at the MTOC.30 During mitosis, spindle formation relies on centrosomes, although a centrosome spindle assembly in mouse oocytes during meiosis primarily depends on MTOCs.37 FMNL1 accumulation is similar to what has been described for the mitotic spindles of somatic cells and suggests that FMNL1 may have played a role in spindle formation and assembly during mouse oocyte meiotic maturation. The centrosome-associated protein MAPK was previously shown to be required for proper spindle formation during oocyte meiosis.34,38–40 Recent study showed Rho GTPase RhoA RNAi reduced the expression of p-MAPK and resulted in aberrant spindle formation during mouse oocyte meiotic maturation.41 While our result showed FMNL1 depletion resulted in the disrupted localization and the decreased expression of p-MAPK, as Formin proteins are the effectors of Rho GTPases, our results indicated that similar with Rho GTPase RhoA, FMNL1 might regulate spindle formation during oocyte meiosis through its effects on p-MAPK expression. Moreover, our results also showed that FMNL1 was the downstream molecule of RhoA, we speculated a RhoA-FMNL1-MAPK signal pathway which involved into spindle formation during oocyte meiotic maturation. And these results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. The failure of polar body extrusion might be due to both aberrant actin and spindle formation, while the large polar body might be due to the aberrant actin-induced spindle migration.

GM130 is a cis-Golgi protein that maintains the structure of the Golgi apparatus in which almost all newly synthesized proteins from the endoplasmic reticulum are processed, sorted, and then sent to their final destinations.42 It has been shown that GM130 expression is enriched, particularly on spindle poles, both during mitotic progression and oocyte meiosis.33,43,44 A recent study has shown that FMNL1 is required for maintaining the structural integrity of Golgi complexes in HeLa cells, as FMNL1 co-localized with GM130 and is dispersed after treatment with brefeldin A (+BFA).31 Our results also showed that FMNL1 and GM130 all localized to mouse oocyte spindle poles. We found that FMNL1 depletion resulted in GM130 detachment from spindle poles and its dispersal in the cytoplasm. Moreover, GM130 protein expression was also significantly reduced. This indicates that FMNL1 is involved in regulating the distribution and expression of GM130 for the spindle formation in mouse oocytes.

Other studies have also shown that actin and associated proteins play a significant role in Golgi structure and function 45 and that FMNL1 may have regulated cellular F-actin levels to maintain the structural integrity of Golgi complexes.31 Different with previous studies in other cell types, an interesting phenotype is that F-actin increased in the cytoplasm in FMNL1-depleted cells.31 In addition, a recent study demonstrates that a vesicle-based mechanism of actin network modulation is essential for the asymmetric positioning of meiotic spindles in mouse oocytes.32 Because the Golgi mediates the long-range transport of vesicles, we assume that FMNL1 might regulate the Golgi apparatus marker GM130, which further modulates an actin network for asymmetric division in mouse oocytes.

To investigate a possible functional signaling pathway for FMNL1, we also examined its relationship to RhoA expression. Formin proteins are effectors of Rho GTPases. Recent studies reveal that FMNL1 is the effector protein of Rho GTPase Rac1 which is reported to regulate Drosophila oogenesis by actin filament and spindle anchoring; moreover, Ran, another small GTP-binding protein is found to regulate cortical actin dynamics to participate in oocyte meiotic division.46-48 Our results showed that inhibition of RhoA activity resulted in decreased FMNL1 expression, which indicates that RhoA may be the upstream regulator of FMNL1.

In conclusion, our results show that FMNL1, which functions in the RhoA-FMNL1-GM130 pathway, is required for actin and spindle assembly during mouse oocyte meiosis.

Materials and Methods

Ethics statement and oocyte culture

The care and use of 4–6 week old ICR mice complied with the Animal Research Committee guidelines of Nanjing Agricultural University. The animal facility had a license authorized by the experimental animal committee of Nanjing City. Mice were sacrificed by cervical dislocation. Germinal vesicle-intact oocytes were cultured in M16 medium under liquid paraffin oil at 37°C in an atmosphere of 5% CO2. After 9 h or 12 h of culture, oocytes were harvested for immunostaining, microinjection, and Western blot analysis.

Antibodies

Rabbit polyclonal anti-FMNL1 antibody, rabbit monoclonal anti-GM130 were from Abcam (Cambridge, UK). Rabbit polyclonal anti- phospho-p44/42 MAPK (Erk1/2) was from Cell Signaling Technology (CST, Beverly, MA). Phalloidin -TRITC, Alexa 488-Phalloidin, and mouse monoclonal anti-α-tubulin-FITC antibody were from Sigma (St Louis, MO). FITC-conjugated goat-anti-rabbit IgG, TRITC-conjugated goat-anti-rabbit IgG, and TRITC-conjugated goat-anti-mouse were from Zhongshan Jin Qiao (Beijing, China).

Immunofluorescent staining and confocal microscopy

Oocytes were fixed in 4% paraformaldehyde at pH 7.4 at room temperature for at least 30 min and then transferred to 0.5% Triton X-100 for 20 min. Oocytes were blocked with 1% BSA (in PBS) for 1 h and then incubated with rabbit anti-FMNL1 antibody (1:50), rabbit anti-p-MAPK antibody (1:200), rabbit anti-GM130 antibody (1:200), or anti-α-tubulin-FITC (1:100) at 4°C overnight. After three washes in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with FITC-conjugated goat-anti-rabbit IgG (1:100), TRITC-conjugated goat-anti-rabbit IgG (1:100), or TRITC-conjugated goat-anti-mouse IgG (1:100) at room temperature for 1 h and then washed 3 times. Oocytes were then co-stained with Hoechst 33342 (10 mg/ml in PBS). Stained oocytes were mounted on glass slides and examined with a confocal laser scanning microscope (Zeiss LSM 700 META, Germany).

Time-lapse microscopy

After microinjecting Alexa 488-Phalloidin (1 μM), oocytes were incubated in M16 medium that contained Hoechst 33342 (5 ng/ml; Sigma) to image actin and chromosome dynamics during oocyte maturation using a Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System. The exposure time was set to between 200 and 800 ms, depending on the Phalloidin-FITC fluorescence level. Digital time-lapse images were acquired under the control of IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software. Confocal images of actin in live oocytes were acquired with a 10x objective on a spinning disk confocal microscope (Perkin Elmer).

Microinjection of FMNL1 or control morpholino

To knockdown FMNL1 expression in mouse oocytes, FMNL1 morpholino (MO) 5′-CTC CAG CCT CGG AGA TCC AGT TTT C-3′ (Gene Tools, LLC) was diluted with water to give a stock concentration of 1 mM. Each GV oocyte was microinjected with 5–10 pl of FMNL1 morpholino using an Eppendorf FemtoJet (Eppendorf AG, Hamburg, Germany) under an inverted microscope (Olympus IX71, Japan). After injection, these oocytes were cultured for 20–24 h in M16 medium that contained 2.5 μM milrinone, and then washed 4 times (2 min each) in fresh M2 medium. These oocytes were then transferred to fresh M16 medium and cultured under paraffin oil at 37 degree in an atmosphere of 5% CO2 in air. Control oocytes were microinjected with MO standard control 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′, and the subsequent steps were the same used for treated oocytes.

RhoA inhibition

A solution of RhoA inhibitor (Rhosin) (Merck Millipore, Germany) in DMSO (50 mM) was diluted in M16 medium to concentrations of 50 μM. Oocytes were then cultured in this medium for 9 h of time and used for western blot. Control oocytes cultured in M16 added the same concentration of DMSO but no Rhosin contained.

Western blot analysis

A total of 100 mouse oocytes were collected in SDS sample buffer, then heated at 100°C for 5 min and rapidly frozen, then subjected to 12% SDS-PAGE. Separated proteins were transferred to a PVDF membrane. Membranes were blocked with TBS (containing 0.1% Tween 20 and 5% non- fat dry milk) for 1 hour and then incubated with primary antibodies, followed by incubation at 4°C overnight with a first antibody (1:1,000) and a rabbit monoclonal anti-tubulin antibody (1:2,500). After washing 3 times (10 min each) with PBST, the membranes were incubated at 37°C for 1 h with a horseradish peroxidase-conjugated secondary antibody (1:10,000). Protein bands were visualized using the ECL Plus Western Blotting Detection System Tanon-5500. To quantify Western blot results, band intensity values were determined using ImageJ software.

Fluorescence intensity analysis

To perform fluorescence intensity analysis, the oocytes in the control group and treatment group were all mounted on the same glass slide. Image J software was used to define a region of interest (ROI), and the average fluorescence intensity per unit area within the ROI was determined. Independent measurements using identically sized ROIs were taken for the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities between control and treatment groups.

Statistical analysis

At least 3 replicates were done for each treatment used. Group results were expressed as means ± SEM's. Statistical comparisons of group results were made using analysis of variance (ANOVA), followed by Student-Newman-Keuls test using SPSS software (SPSS Inc., Chicago, IL). A p-value of < 0.05 was considered significant.

Supplementary Material

1031438_Supplemental_figure.tif

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the National Basic Research Program of China (2014CB138503), the Natural Science Foundation of Jiangsu Province (BK20140030), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130097120055).

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