RASD1 Knockdown Results in Failure of Oocyte Maturation

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1 Original Paper 2016 The Author(s) Published The Author(s) by S. Karger AG, Basel Published online: December 19, Published by S. Karger AG, Basel Accepted: November 09, 2016 This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) ( Usage and distribution RASD1 Knockdown Results in Failure of Oocyte Maturation Youngeun Lee a Kyeoung-Hwa Kim a Hyemin Yoon a Ok-Hee Lee a Eunyoung Kim a Miseon Park b Hoon Jang a Kwonho Hong c Hyuk Song d Jung Jae Ko a Woo Sik Lee b,e Kyung-Ah Lee a,b Eun Mi Chang b,e Youngsok Choi a,b a Department of Biomedical Science, CHA University, Seongnam-si, Gyeonggi-do, b Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul, c Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, d Department of Animal Biotechnology, Konkuk University, Seoul, e Department of Obstetrics and Gynecology, CHA University, Seoul, Republic of Korea Key Words RASD1 Oocyte maturation MI-MII transition Cytokinesis Spindle formation Abstract Background: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. Methods: real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturationrelated gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation. Results: RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown. Conclusion: be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation The Author(s) Published by S. Karger AG, Basel Youngsok Choi, Ph.D., Eun Mi Chang, M.D. Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, (Republic of Korea); Department of Obstetrics and Gynecology, Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul, (Republic of Korea); youngsokchoi@cha.ac.kr / emchang@cha.ac.kr

2 1290 Introduction The Ras dexamethasone-induced protein 1 (Rasd1 dexamethasone-inducible gene in the AtT-20 pituitary cell line [1]. Rasd1, also known as Dexras1, encodes a monomeric G-protein and belongs to the Ras superfamily of small guanosine triphosphatases (GTPases). RASD1 is an activator of G-protein signaling and acts as a direct nucleotide exchange factor for Gi-Go proteins [2, 3]. It is known that RASD1 functions as a cytoplasmic signal transducer in various intracellular signaling pathways governing iron homeostasis, growth hormone secretion and circadian rhythm [2, 4, 5]. Recently, RASD1 has also been detected in the nucleus, acting as a transcriptional repressor of glycogen synthase kinase 3 (GSK3 ) and as an inhibitor of the camp-dependent pathway [2, 6-8]. RASD1 interactivity further includes a neuronal nitric oxide adaptor protein (CAPON) [9], and the FE65 ligand of amyloid precursor protein in Alzheimer's disease [7]. Rasd1 expression and activation are regulated by diverse extracellular stimuli, notably dexamethasone in pituitary cells [1] and glucocorticoid or prolactin in pancreatic -cells [10]. Activated RASD1 signaling is pivotal in cytoplasmic signaling networks controlling gene expression and the proliferation, differentiation, and survival of cells [11]. Hence, reputed The complex developmental progression in oocytes is dynamically regulated throughout folliculogenesis. Follicular development is controlled by several factors such as follicle stimulation hormone, luteinizing hormone, and anti-müllerian hormone [12-14]. Within the primordial follicle, the oocyte is reactivated, achieving secondary oocyte maturation germinal vesicle breakdown (GVBD), undergoing further meiotic divisions until secondary meiotic arrest occurs at metaphase II (MII). When oocytes undergo meiotic division, chromosomes and meiotic spindle are positioned closely to cortical region. This asymmetric positioning establishes oocyte polarity which is induced by signaling networks including the small GTPases RAC and CDC42, and the cytoskeleton regulators, ARP2/3 complex [15-17]. maturation, the role of RASD1 in oocyte maturation has not been explored in detail so far. In this study, we determined whether RASD1 is involved in mouse oocyte maturation. Materials and Methods Animals All experiments described in this study were approved by Institutional Animal Care and Use Committee (IACUC) of the CHA University and performed in accordance with the guidelines presented by IACUC. Female ICR (Institute for Cancer Research, CD-1) mice were obtained from Orient Bio Company (Seongnam, Korea). Mice were housed in a temperature-controlled facility under 12 hour (h) light-dark cycle and fed with a normal diet and water. Collection of oocytes and follicular cells Three-week-old ICR female mice were intraperitoneally injected with 5 IU of gonadotropin and pregnant mare s serum (PMS-G; Sigma-Aldrich, St. Louis, MO, USA) to isolate germinal vesicle (GV) oocytes from preovulatory follicles. After 46 to 48 h, ovaries were removed and maintained in M2 medium (Sigma- Aldrich) including 0.2 mm 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich) to keep immature oocytes. Cumulus-enclosed oocyte complexes (COC) and granulosa cells (GC) were obtained by piercing ovaries pipette. Isolated oocytes, CCs, and GCs were used for further experiments. Microinjection of dsrna into oocyte and in vitro oocyte maturation To knockdown Rasd1 in the oocyte, we used double-strand RNA (dsrna) system as described in previous studies [18, 19]. The strategy of Rasd1 knockdown was shown in Fig. 3A. Partial Rasd1 cdna

3 1291 Table 1. Primer sequences and RT-PCR conditions for this study. (1) Primers were used for preparation of dsrna. (2) Primers were used for RT-PCR. (3) Primers were used for qrt-pcr Rasd1 dsrna primers (Table 1), and cloned into pgem-t Easy vector (Promega, Madison, WI, USA). pgem-t Easy vector containing Rasd1 cdna was linearized by SpeI treatment, and then single-stranded sense and anti-sense RNAs were in vitro transcribed with T7 polymerase using MEGAscript kit (Ambion, Austin, TX, USA). Each RNA strands were hybridized at 75 C and slowly cooled to 25 C. After treated with DNaseI and RNase to remove DNA and single strand RNA, picolitter of injection medium containing Rasd1 or gfp dsrna (32 ~ 35 pg) was microinjected into cytoplasm microinjected with the injection medium for control. Microinjected oocytes were cultured in M16 medium (Sigma-Aldrich) including 0.2 mm IBMX under 5% CO 2 at 37 C. After 8 h, GV oocytes were cultured in M16 medium containing 3 mg/ml bovine serum albumin (BSA, Sigma-Aldrich) for 16 h to induce in vitro oocyte (GV oocyte), a dissolved nuclear membrane (GVBD oocyte), a polar body (MII oocyte), or neither a germinal vesicle nor a polar body (MI oocyte). RNA isolation and reverse-transcriptase PCR (RT-PCR) To isolate total RNA from tissue, tissue was placed into a vessel and homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using homogenizer. After added with chloroform, lysate was centrifuged

4 1292 at 13,000 g for 20 minute (min) at 4 C. Supernatant was transferred to a 1.5 ml tube. Total RNA was RNase-free water. Messenger RNA (mrna) from GV, GVBD, MI, and MII oocytes was isolated using the Dynabeads mrna resuspended in lysis/binding buffer and incubated for 3 min at room temperature [RT]. Prewashed Dynabeads oligo (dt) 25 was mixed and incubated with the oocyte lysate. The beads/mrna complex was washed with washing buffer by placing on the magnet. The mrnas were eluted in 10 mm Tris-HCl (ph 7.5) at 70 C for 2 min and stored at -80 C until use. reaction was carried out as followed conditions: 60 min at 42 C and 2 min at 94 C. The cdna was used for sequences and PCR conditions are listed in Table 1. PCR products were analyzed with gel electrophoresis Quantitative real-time RT-PCR (qrt-pcr) Quantitative real-time RT-PCR was carried out in the icycler (Bio-Rad laboratories Inc., Hercules, CA, software. The reaction mixture was made up by adding cdna to 1 products. To quantify gene expression level, cycle threshold (CT) value was calculated. The CT value for each calculated using 2 method [20]. Expression of each mrna was normalized to that of Gapdh or H1foo mrna. Immunohistochemistry analysis rehydration, sections were treated with 3% hydrogen peroxide for 10 min and boiled in antigen retrieval buffer (10 mm sodium citrate, 0.05 % Tween-20, ph 6.0) using microwave for 10 min. Slides were washed with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) and blocked with PBS-T including 4 % BSA and 5 % normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Then, sections were incubated with primary antibody (anti-rasd1 rabbit polyclonal IgG, 1:200 dilution, Cat. No. ab78459, Abcam, Cambridge, Cambridgeshire, UK) in blocking solution at 4 C overnight. After washed, the slides were treated with secondary antibody (anti-rabbit antibody conjugated with HRP, Cat. No , used as a negative control. Anti-DEAD box helicase 4 (DDX4) antibody (1:500 dilution, ab27591, Abcam) kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instruction. The sections were counterstained with hematoxylin and dehydrated. Sections were mounted using Permount Mounting performed. Oocytes were washed two times in PBS including 0.1% polyvinyl alcohol (0.1% PVA-PBS) for 5 X-100 for 20 min at RT. After washing oocytes three times in 0.1% PVA-PBS for 5 min at RT, oocytes were incubated in 1% BSA-0.1% PVA-PBS at 4 C overnight. After treated with 3% BSA-0.1% PVA-PBS for 1 h at

5 1293 tubulin (1:100 dilution, Cat. No. T9026, Sigma-Aldrich) and anti-rasd1 antibody (1:100 dilution; Cat. No. ab171370, Abcam) at 4 C overnight. After washing, oocytes were incubated with Alexa F488 goat anti-mouse IgG (1:100, Cat. No. Cat. No. D3571, Life Technologies, Carlsbad, CA, USA) and F-actin was stained by phalloidin (1:200, Cat. No. A12379, Life Technologies) for 10 min at RT. Stained oocytes were mounted with Malinol mounting analyzed by confocal microscope of Leica laser scanning (Leica, Heidelberg, Germany). To quantify, images were analyzed by LAS AF Lite imaging software (Leica). Western blot analysis Rasd1 RNAi-treated oocytes at MI stage were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors. The lysates corresponding to 100 oocytes were boiled with gel loading buffer. The protein samples were The membranes were blocked with 5% skim milk in PBS-T and incubated with anti-rasd1 (1:1000, Abcam) visualized using Western Femto ECL kit (LPS Solution, Daejeon, Korea) and the protein expression was analyzed by the ChemiDoc XRS system (Bio-Rad Laboratories). Time lapse video microscopy Time lapse video microscopy was performed to analyze effect of Rasd1 knockdown on in vitro oocyte TM time-lapse microscope (Digital Bio, Seoul, Korea) was placed in an incubator with 5% CO 2 at 37 o C. A culture dish containing oocytes was placed on the microscope stage. Images were automatically captured every 5 minutes for 16 hours and then sequential time lapse Statistical analysis The experimental data were presented as the means ± standard error of mean (SEM). Real-time PCR data was analyzed using a one-way analysis of variance (ANOVA) or student t-test for statistical evaluation. Results Expression and localization of Rasd1 in mouse tissues To examine expression levels of Rasd1 transcript in various tissues, RT-PCR was performed, using respective total RNA extracts and an expressly designed Rasd1 primers (Table 1). The Rasd1 transcript was detected in most tissues assayed (Fig. 1A). Of note, expression of Rasd1 mrna was detected at high levels in reproductive organs of adult mice, such as ovary and testis (Fig. 1A and 1B). Cell types expressing RASD1 in mouse ovary were determined by immunohistochemistry, incubating ovarian tissue from 3-week old mice with anti-rasd1 antibody (Fig. 1B). RASD1 protein expression was seen only in oocytes transitioning from primordial to secondary follicles (Fig. 1C). The cellular localization of RASD1 was in cytoplasm of oocytes. RASD1 was not co-localized with LHX8 which is preferentially expressed in the nucleus of oocytes. Instead, RASD1 was detected in the oocyte cytoplasm surrounding the nucleus (Fig. 1D). These results suggest that RASD1 is mainly expressed in oocytes and may play role in regulation of signals that are involved in oocytes maturation. Differential expression of Rasd1 during oocyte maturation To examine Rasd1 expression during mouse oocyte maturation, total RNA from GV-, GVBD-, MI-, and MII-stage oocytes was analyzed by RT-PCR. High level of Rasd1 expression reduced at MII stage (Fig. 2A and B).

6 1294 Fig. 1. Expression of Rasd1 mrna and protein in mouse tissues. (A) RT-PCR analysis of Rasd1 mrna in samples; Gapdh, internal control). (B) qrt-pcr analysis of Rasd1 mrna in the tissues. Expression levels were normalized with Gapdh mrna, expressing data as mean ±SEM. (C) Immunohistochemical analysis of RASD1 in mouse ovary at 3 weeks old. PF, primary follicle; PrF, primordial follicle. (D) Co-immunostaining of RASD1 and the germ cell marker LHX8 in mouse ovary. DAPI was used for nuclei staining. Fig. 2. Differential expression of Rasd1 mrna during in vitro oocyte maturation. (A) RT-PCR analysis of Rasd1 mrna in mouse oocytes at differing stages of in vitro maturation. Germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were obtained. H1foo, oocy- Rasd1 mrna in GV and MII-stage oocytes, calculated from CT values. Expression levels were normalized with H1foo mrna, expressing data as mean ±SEM. Student t-test was applied to calculate p-value. *p < (C) MII-stage oocytes with anti-rasd1 antibody. DAPI and Phalloidin were used to stain nucleus and F-actin, respectively. Oocytes were analyzed using confocal microscopy. Consistent with the RT-PCR data, protein level of RASD1 was highly detected in the oocytes at GV, GVBD, and MI stages. As shown in Fig. 2C, an even distribution of RASD1 was

7 1295 Fig. 3. RNAi-mediated knockdown of Rasd1 in mouse oocytes. (A) Schematic diagram of mouse Rasd1 coding region. Double-strand RNA (Rasd1-dsRNA; location ) was used for knockdown of genes. Pri- Rasd1 (location ). (B) Diagram of experimental methods. GV oocytes were microinjected with Rasd1 dsrna, then cultured in M16 medium containing IBMX for 8 h, and incubated in plain M16 medium to induce in vitro maturation for further 16 h. (C) RNAi-mediated knockdown of Rasd1 H1foo, internal control. (D) Relative expression of Rasd1 p-value, *p < 0.05, **p mosomes, DAPI (Blue); and F-actin, Phalloidin (Green). Level of RASD1 was analyzed using confocal microscopy. The oocytes injected with injection medium were used as control. (F) Western blot analysis showing the knockdown effect of Rasd1 RNAi. Total proteins were isolated from control or Rasd1 RNAi-treated GV oocytes. The proteins equivalent to 100 oocytes were loaded in a SDS-PAGE gel and analyzed for RASD1 seen in the cytoplasm of GV-, GVBD-, and MI-stage oocytes. In MII-stage oocytes, however, RASD1 level was decreased. Rasd1 knockdown in oocytes A double-stranded RNA (dsrna)-mediated RNAi system (Fig. 3A) was implemented to knockdown the Rasd1 gene, thus allowing insight into the role of RASD1 during oocyte

8 1296 Fig. 4. Effect of Rasd1 knockdown on in vitro maturation of mouse oocytes. (A) Photographs of injected oocytes (control, gfp RNAi, or Rasd1 RNAi) after 16 h in vitro culture. (B) Oocyte maturation rates of control, gfp-rnai, or Rasd1-RNAi oocytes after 16 h of in vitro maturation. The percentage of oocytes in GV, MI- and MII-stage was determined. Experiments were conducted in triplicate, expressing data as mean ±SEM. The ANOVA was used to calculate p-value, *p < The number in bracket is total number of oocytes that were used in three experiments. Rasd1 reduced in both GV- and MI-stage oocytes that were microinjected with Rasd1 dsrna (Fig. Rasd1 dsrna compared to control or gfp- RNAi oocytes (Fig. 3E). Furthermore, Rasd1 RASD1 in oocytes (Fig. 3F). Inhibition of MI to MII transition and abnormal ooplasmic extrusion in Rasd1 RNAitreated oocytes The physiologic role of RASD1 protein in oocytes was explored using 483 GV oocytes that were injected with the injection medium (93 GV oocytes), gfp RNAi (114 GV oocytes), or Rasd1 RNAi (276 GV oocytes). Developmental status of these oocytes was then monitored until MII stage (Fig. 4A). Morphologic analysis showed that control and gfp-rnai oocytes Rasd1 RNAiinjected oocytes had arrested at MI stage (Fig. 4A). Also, many Rasd1-knockdown oocytes appeared distorted, with lumpy ooplasmic membranes (Fig. 4A). To investigate further, in vitro rates of oocyte maturation were analyzed (Fig. 4B). Maturation rates in control and gfp-rnai oocytes were 100% and 89%, respectively. However, only 28% of Rasd1-knockdown oocytes were fully mature. Most oocytes (70%) subjected to Rasd1 dsrna microinjection had arrested at MI stage. Having also monitored the process of oocyte maturation for 16 h by video system, we found that the cytokinesis was abnormally occurred in Rasd1 RNAi-treated oocytes. Most of control body within 8 h (Fig. 5). Although GVBD in Rasd1 RNAi-treated oocyte was normally induced Rasd1 RNAi (Fig. 5). Moreover, we found the abnormal oocytes with multiple cytoplasmic protrusion

9 1297 Fig. 5. Inhibition of MI to MII transition and abnormal cytokinesis of Rasd1 RNAi-injected oocytes. Oocytes (control, gfp RNAi, or Rasd1 RNAi) in M16 with IBMX were transferred to plain M16 medium and incubated for 16 h. Images at the indicated time were obtained from a time lapse video of maturing oocytes. at the intermediate time (10~13 h after in vitro maturation induction) and degeneration signaling in the transition of MI to MII stage and the regulation of cytokinesis during oocyte maturation. Spindles and chromosomal abnormality in MI oocytes by Rasd1 knockdown Asymmetric cytokinesis, during which a bipolar spindle takes shape in central cytoplasm and migrates to cell periphery, is characteristic of maturing oocytes. During the process, polar body is extruded as a mark of cytokinetic success. In an attempt to discern why MI to MII transition of Rasd1 knockdown oocytes were abnormally occurred, we performed immunostaining analysis using oocytes at MI stage. In control and gfp-rnai oocytes, chromosomes were well-aligned, with symmetric spindles forming peripherally, whereas Rasd1 dsrna-injected oocytes displayed abnormal spindling and chromosomal aggregation (Fig. 6A). Although some cells did progress to MII stage (Fig. 4), when immunostained, they were phenotypically similar to other Rasd1 RNAi-treated was apt to be larger than that of controls (Fig. 6B). It is thus evident that the RASD1 signal is of considerable importance in regulating spindle formation and chromosome alignment, leading to improper separation of chromosomes and MI arrest in its absence. Expression of oocyte maturation-related genes in Rasd1 knockdown GV oocytes To examine the downstream regulatory network of RASD1 signaling in mouse oocyte maturation, we determined the effect of Rasd1 RNAi on the expression of factors involved in MI arrest (Cops3, Cops5, Diva, Obox4, Tkt, and Zap70), cytokinesis (Arp2, Arp3, Aurora C, and Dcnt3), or small GTPase signaling (Arp1, Cdc42, Esr1, Nono, Pl3k, Rac1, Ran, and RhoA). Most genes implicated in MI arrest were unchanged, with exception of Obox4 (Fig. 7A). The transcript of Obox4 in Rasd1 RNAi-treated oocytes (Fig. 7B). With respect to cytokinesis, Arp2, Arp3, Dcnt3, and

10 1298 Fig. 6. Effect of Rasd1 knockdown on chromosomal alignment and spindle formation during in vitro maturation of oocy- staining of MI-stage oocytes. Dot circles show chromosome alignment and spindle forma- staining of MII-stage oocytes. Control, gfp-rnai, and Rasd1- RNAi oocytes were immunostained with anti- -tubulin antibody to examine the spindle formation. Chromosomes were detected with DAPI, and F-actin was stained with Phalloidin. Aurora C Arp2 and Arp3 in Rasd1 knockdown oocytes (vs controls) (Fig. 7B). Arp2 and Arp3 are components of the [23]. Finally, genes of other small GTPases and related proteins, such as Cdc42, RhoA, Rac1, Nono, Esr1, Ran, Arf1, and Pl3k, were explored (Fig. 7A). Among these, Cdc42, RhoA, and Rac1 were likely to show diminished expression in Rasd1 successful oocyte maturation.

11 1299 Fig. 7. Effect of Rasd1 knockdown on expression of oocyte maturation-related factors involved in MI arrest, cytokinesis, and small GTPase pathway. (A) RT-PCR analysis performed using total RNA from control (Con) and Rasd1 RNAi-injected GV oocytes. Factors in MI arrest include Cops3, Cops5, Diva, Obox4, Tkt, and Zap70. Cytokinesis-related factors include Arp2, Arp3, Aurora C, and Dcnt3. Factors in small GTPase signaling include Arp1, Cdc42, Esr1, Nono, Pl3K, Rac1, Ran, and RhoA. (B) Relative expression levels of Obox4, Arp2, and Arp3 by qrt-pcr. H1foo, internal control. Student t-test was applied to calculate p-value. *p < Discussion Oocyte maturation is a dynamic cellular process involving asymmetric meiotic division through various signaling pathways. The role of RASD1 signaling in oocyte maturation has not been studied well. The objective of this study was to investigate the regulation and expression of Rasd1 in mouse oocytes during their maturation sequence. RASD1 signaling has been investigated in various cell types, including cortical neurons [9], pituitary cells [1], and pancreatic -cells [10]. RASD1 is an activator of G-protein signaling nitric oxide synthase (nnos) adaptor protein, CAPON, leading to enhance NO signaling [9]. In pancreatic cells, Rasd1 expression is regulated by glucocorticoids and prolactin during pregnancy via the glucocorticoid receptor (GR)/STAT5 pathway, which is also involved in insulin secretion [10]. In the brain, RASD1 binds to FE65, an adaptor protein, forming a transcriptional complex with the amyloid precursor protein (APP) intracellular domain [7]. The FE65-RASD1-APP tripartite complex then plays a regulatory role in GSK3 expression [7]. maturation. Herein, we have demonstrated that RASD1 is differentially expressed in growing protein to partner with RASD1, as in neurons and in pancreatic cells. Other contributors, namely maturation-promoting factor (MPF) and cytostatic factor (CSF), participating in

12 1300 oocyte maturation (reviewed in [24]) are tightly regulated by phosphorylation of protein Rasd1 knockdown, showing that expression levels of Obox4, Arp2/3, and Rho family (Cdc42, Rac1, RhoA) genes decline in Rasd1 Obox4 was originally discovered in germ cells [25]. Obox4- and Rasd1-knockdown oocytes are quite close in phenotype, both displaying incomplete cytokinesis. Obox4 knockdown in oocytes also prevents MI-MII transition through maturation arrest [18]. In fact, Obox4 contains a homeobox domain for DNA binding and regulates numerous genes related to meiosis during oocyte maturation [26]. Further studies to delineate a relationship between RASD1 signaling and Obox4 target gene expression are needed. We also have established that transcripts of Arp2 and Arp3 in oocytes injected with Rasd1-RNAi. Arp2 and Arp3 form an Arp2/3 complex, which is required for regulation of both the initiation of actin polymerization and the organization asymmetric meiotic spindle positioning and F-actin shell assemblies [17, 28]. Blockade of the Arp2/3 complex by inhibitors or by dsrna injection impairs meiotic maturation [17, 29, 30]. These features closely resemble those of Rasd1-knockdown oocytes. Indeed, the parallels between RASD1 signaling and the Arp2/3 complex are supported by our analysis. Finally, we have observed that transcripts of other Rho GTPases (Cdc42, RhoA, and Rac1), also members of the Ras superfamily, are reduced in Rasd1-knockdown oocytes. These GTPases, well-known as actin nucleators, are critical for oocyte spindle positioning [31, 32] and cortical polarity during polar body extrusion in mouse oocytes [15, 33]. Rac1 inhibition blocks meiosis I reduction division, resulting in an elongated spindle and scattering of chromosomes, much like a phenotype encountered in Rasd1-knockdown oocytes. RhoA is a small GTP-binding protein [34] that engages in fundamental somatic cellular processes, including cytoskeletal organization, cell-cycle progression, survival, polarity, and migration [35]. RhoA appears to regulate cytoskeletal dynamics during oocyte maturation [36, 37]. The Cdc42 transcript was similarly reduced in Rasd1-knockdown oocytes. Cdc42 has proven pivotal in regulating cellular polarity, spindle migration/orientation, and asymmetric cell division in several systems, including the one-cell embryo, neuroblasts, and epithelial cells [38-41]. Recent studies indicate that Cdc42 promotes polar body protrusion and pathway orchestrates asymmetric cell division and proper chromosomal alignment by regulating various related downstream targets. In conclusion, our current efforts have shown that RASD1 is expressed differentially during oocyte maturation in mice, and that the RASD1 signaling pathway directly or indirectly regulates expression of Obox4, Arp2/3, Rac1, RhoA, and Cdc42 new light on the complexities of oocyte maturation, especially in terms of spindle assembly and asymmetric cytokinesis. Further studies are needed to map the molecular aspects of these regulatory mechanisms. Acknowledgements This research was supported by grants from the Ministry of Science, ICT & Future Planning (2014R1A1A ) and from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A ), and also supported by Next-generation BioGreen 21 Program,

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