D 4476 | Plx5622

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study was to evaluate the effect of Casein Kinase1 (CK1) inhibition D4476 on in vitro maturation(IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20μM) for 24h. After IVM and in vitro fertilization (IVF), through expansion average scores of cumulus cells (CCs), oocytes maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5μM D4476, the results of quantitative real-time PCR (QPCR) analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown (GVBD) and nuclear morphology, D4476 could significantly decrease CK1 and up-regulate TCF-4 in oocytes. Furthermore, Under without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β-catenin, TCF-4, Cx43, MAPK, PTGS-2, PTX-3, TGS-6, Bax and Bcl-2 in CCs. Western Blot analysis revealed that the addition of 5μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12h and a higher expression of Cx43 protein at 24h compared to the group without D4476. These results indicate that adding optimum D4476 (5μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.

1 | INTRODUCTION
In vitro maturation of mammalian oocytes has been one of basic and key technology in embryo engineering technology, such as somatic cell nuclear transfer and production of transgenic animals. Therefore, an important aspects of embryo engineering technology is obtaining high-quality oocytes. Although IVM of bovine oocytes had been established and made great progresses, the efficiency of oocytes IVM was still lower than that in vivo due to environmental difference existed in vivo and vitro (Smith et al., 2009). Under natural conditions, COCs separated from follicles can spontaneously recover meiosis in vitro. Therefore, cytoplasm maturation is commonly posterior to nuclear maturation for oocytes in vitro.
Oocytes meiosis is a complicated process regulated by sorts of proteins and signaling pathways. Casein Kinase 1 is a serine/threonine protein kinase. It was distributed in nuclear interior, cytoplasm and cell membrane, and participated in DNA reparation, growth regulation and cell division, and closely related to centromere function (Brockman et al., 1992; Graña et al., 1995). For the effect of CK1 on oocytes meiosis, it had been claimed that inhibiting the expression of CK1 would lead to the failure of meiosis (Wang et al., 2013). However, there was a report indicated that the lack of CK1 increased the duration of meiosis and did not stop meiosis (Qi et al., 2015).

In addition, CK1 is also a key protein kinase in the Wnt/β-catenin signaling pathway. Wnt/β-catenin signal would promote cell proliferation and differentiation by controlling timely transcription and translation of RNA as well as post-translational modification of proteins (Harwood et al., 2008). Many studies have reported that Wnt/β-catenin signal regulated oocytes maturation and embryonic development (Tanaka et al., 2011;Li et al, 2012). Therefore, high-quality oocytes can be obtained by increasing Wnt signal level during IVM.
Developmental competence of oocytes was gradually acquired through undergoing a series of maturity regulation (Mermillod et al., 1999; Gandolfi et al., 2001; Krisher, 2004; Sirard et al., 2006). Previous studies had demonstrated that CCs and its structural integrity around oocytes was essential for oocytes maturation (Ka et al., 1997; Prather et al., 1998; Zhuo et al., 2001; Salustri et al., 2004; Harris et al., 2009). Moreover, Gap junctions (GJ) between oocytes and CCs was bridges of signal transmission and nutrient transport (Mori et al., 2000; Su et al., 2003; Wongsrikeao et al., 2005; Atef et al., 2005). What,s more, GVBD was negatively correlated with gap junctions intercellular communication (GJIC) between CCs (Thomas et al., 2004). In gap junction channel, Cx43 protein occupied a dominant position (Salhab et al., 2013). The expression of CX43 in CCs around matured oocytes was significant higher than that of immature oocytes (Li et al., 2015). Furthermore, expression level of Cx43 regulated by Wnt/β-catenin signaling pathway (Heyden et al., 1998; Le et al., 2017; Jeong et al., 2017;Wang et al., 2017) was one of marked for oocytes maturation, and its function required the phosphorylation modified of CK1 (Cooper, et al. 2002).
Therefore, regulating CK1 expression has important research value for oocytes IVM. As an efficient, selective-cell and permeable CK1 inhibitors, D4476 was commonly used to study the role of CK1 in cells (Rena et al., 2004). In terms of oocytes, there was a report declared using 50μM D4476 resulted in the failure of meiosis and abnormal nuclear morphogenesis (Wang et al., 2013). However, the action mechanism of maturation failure had not been further studied. Therefore, in this study, D4476 was added into mature culture medium, and the effect of D4476 on efficiency of bovine oocytes IVM and its mechanism were discussed in order to provide a theoretical basis for improving the maturation and culture system of bovine oocytes in vitro.

2 | MATERIALS AND METHODS
2.1 | Reagents and media
Medium 199 (Earle’s Salts, powder) was purchased from Gibco Corporation. The Anti-Connexin 43 (ab11370) was purchased from Abcam, and D4476 (s7642) was purchased from Selleck.cn. Other unspecified reagents were purchased form Sigma Corporation.

2.2 | Collection and in vitro maturation of oocytes
The bovine ovaries were collected from a local commercial slaughter house and transported to laboratory at 37°C in physiological saline (0.9% NaCl) within 4h, then were disinfected with 75% ethanol and cleaned with physiological saline for 2-3 times. COCs were aspirated from ovarian follicles (2-6 mm) with an 18-gauge needle attached to a disposable 10mL syringe, then washed two times with cell cleaning medium(TCM-199 supplemented with 20mL fetal bovine serum (FBS), 60mg/L penicillin, 100mg/L streptomycin, 0.9g/L NaCl, 1.2g/L Hepes, 0.4g/L NaHCO3). COCs with at least two intact CCs layers were selected and randomly placed into maturation medium (TCM-199 supplemented with 6.5% FBS, 2.2g/L NaHCO3, 60mg/L penicillin, 100mg/L streptomycin, 100ng/mL follicle-stimulating hormone, 1.2g/L Hepes) at 38°C in a 100% humidified atmosphere of 5% CO2 for 24h.

2.3 | Sperm preparation
High activity bovine sperms were collected by using swim-up method. 2ml fertilization medium (modified Tyrode’s medium, containing 2.5mM caffeine, 50mg/L heparin, 60mg/L penicillin, 100mg/L streptomycin) was putted into a 10ml sterile test tube and pre-heated for 30 min at 38°C in a 100% humidified atmosphere of 5% CO2. After thawing in 38°C water bath, bovine frozen semen (Improvement Station of Livestock Breed in Guangxi Area) was immigrated slowly into bottom of test tube to swim up for 30 min. Subsequently, 1.5ml supernatantfluid was transferred to another test tube. After centrifugation, the bottom sediment was used to IVF, and the density of sperms was adjusted to 1.0 x 106 sperms/ml.

2.4 | In vitro fertilization and in vitro embryo culture
After maturation, the COCs were gently blown away and denuded with a pipette gun, and were washed in fertilization medium. After washing, denuded oocytes with first polar body (PB1) were placed into fertilization culture medium droplets (Each droplet have 15 oocytes at most) to IVF for 24h. After fertilization, the zygotes were washed with embryo culture medium (containing TCM-199, 1.2g/L Hepes, 2.2g/L NaHCO3, 3% FBS, 60mg/L penicillin, 100mg/L streptomycin), and subsequently were co-incubated with CCs (secreting some cytokines for the development of zygotes) in embryo culture medium droplets (Each droplet have 15 oocytes at most) covered with mineral oil at 38.5°C in a 100% humidified atmosphere of 5% CO2. Half of medium of each group was exchanged every 48h within 8 days.

2.5 | RNA extraction, reverse transcription (RT) and real-time PCR (qPCR)
RNA extraction and reverse transcription were reference to a report (Torres et al., 2018). qPCR were performed by Fast SYBR Green Master Mix kit (TaKaRa) according to the manufacturer’s instructions. Each sample was repeated for threetimes, and qPCR specificities were determined by examining the melting curves and amplicon sizes on an agarose gel. qPCR was run in the following condition: 95°C for 2 min, 35 cycles of 94°C for 15 s, 56°C for 30 s and 72°C for 15 s. The quantitative specific primers were designed by Premier 5 and Oligo 6, and internal reference gene was β-actin. The gene names, primer sequences and amplicon sizes were listed in Table1.

2.6 | Experimental Design
2.6.1 | Experiment 1. Effects of D4476 on expansion of CCs, maturation efficiency and embryonic development
The aim of this experiment was to select optimum treated concentration of D4476.
COCs with at least two intact CCs layers were randomly placed into maturation medium containing different concentrations D4476 (0, 2, 5, 10, 20μM) for 24h, then expansion average score of CCs, PB1rate and blastocyst rate were assessed to select optimum treated concentration of D4476.
2.6.2 | Experiment 2. Effects of D4476 on CK1 expression, GVBD and nucleus morphology of oocytes
The objective of this experiment was to explore the effect of D4476 on bovinoocytes meiosis. COCs with at least two intact CCs layers were randomly placed into maturation medium containing different concentrations of D4476 (0, 5μM). After culture for 8h, oocytes were collected, then Lichen red staining was used to count number of GVBD in oocytes. After culture for 24h, oocytes were collectedto qPCR analysis and PI staining. Lichen red staining and PI staining was conducted as previously reported (Mood et al., 2004; Shen et al., 2015; Torres et al., 2018).
2.6.3 | Experiment 3. Effects of D4476 on Wnt/β-catenin signaling, cell proliferation, expansion, apoptosis and Cx43 protein
The aim of this experiment was to explore the mechanism that D4476 improved maturity efficiency and developmental competence of bovine oocytes. COCs randomly placed into maturation medium containing different concentrations of D4476 (0, 5μM). After maturation, the oocytes and CCs were respectively collected to qPCR analysis and Western Blot. Western Blot was conducted as previously reported (Shen et al., 2015).

2.7 | Statistical analysis
All results were analyzed by ANOVA. Probability values less than 0.05 were considered to be statistically significant difference. Each experiment was repeated at least three times and all data was analyzed by IBM SPSS 19 software.

3 | RESULTS
3.1 | Effects of D4476 on expansion of CCs, maturity efficiency and embryonic development
After maturation, the expansion of CCs of each treated group was classified by the method of previously reported (Kobayashi et al., 1994). The full expansionof CCs is recorded as three points (Figure 1A), part expansion is recorded as two points (Figure 1D), and no extension is recorded as one point (Figure 1E). The results showed (Figure 1 and Table 2) that there were no significant differences in the expansion average score of CCs of the control group, 2μM group and 5μM group (2.57±0.05 、2.67±0.06 vs 2.68±0.05 ， P > 0.05). However, expansion average score of CCs in 10μM group and 20μM group was significantly lower than that of the control group (1.92±0.06、1.26±0.06 vs 2.68±0.05， P < 0.05).
As expected, D4476 improved the PB1 rate of oocytes. Moreover, with the increase of concentrations of D4476 in maturation medium, the trend of PB1 rate increased first and then decreased. The results showed (Table 3) that the PB1 rate in 5μM group and 10μM group was significantly higher than that in control group (70.48%±0.09、73.94%±0.08 vs 53.75%±0.11，P < 0.05), but there was no significant difference between 5μM group and 10μM group. Furthermore, the PB1 rate in 2μM group and 20μM group was no significant difference compared to control group (62.77%±0.11、67.85%±0.04 vs 53.75%±0.11，P > 0.05).
Then, the treated oocytes were fertilized in vitro. The results (Table 4) revealed that no significant difference in cleavage rate was found in 2μM group, 5μM group and the control group (70.90%±0.24、73.52%±0.34 vs 69.33%±0.16， P > 0.05), however, cleavage rate in 10μM group and 20μM group was significantly lower than that in control group (59.17%±0.22、48.57%±0.23 vs 69.43%±0.17， P < 0.05). We also found that blastocyst rates in 2μM group, 5μM group and 10μMgroup was significantly higher than that in control group (20.05%±0.83 、28.00%±0.98 、19.72%±0.56 vs 16.35%±0.54 ，P < 0.05), but no significant difference in blastocyst rates was found between 20μM group and control group (14.71%±0.45 vs 16.35%±0.54，P > 0.05).

3.2 | Effects of D4476 on meiosis process
5μM D4476 was add into maturation medium to explore the effect on bovine oocytes meiosis process. The results (Figure 2A) showed that expression level of CK1 in oocytes of 5μM group was significantly lower than that in control group. Concerning this study, we postulated that D4476 would inhibit spontaneous meiosis in oocytes, but results (Figure 2B) displayed D4476 had no significant effect on GVBD in oocytes. Subsequently, PI staining of mature oocytes showed that D4476 also had no effect on nucleus morphology of oocytes (Figure 2C).

3.3 | Effects of D4476 on Wnt/β-catenin signaling, cell proliferation, expansion, apoptosis and Cx43 protein
When 5μM D4476 was added into maturation medium, Wnt/β-catenin signaling related genes β-catenin and Cx43 was no significantly up-regulation, but TCF-4 was up-regulated in oocytes (Figure.3A). Furthermore, β-catenin, TCF-4 and Cx43 in CCs also were up-regulated (Figure.3B). These indicated D4476 would simulate Wnt/β-catenin signaling to regulate proliferation, expansion and apoptosis of CCs. Therefore, qPCR analysis showed that under without influencing the levelof apoptosis gene Bad and proliferation gene CTSB , D4476 could significantly increase the expression of proliferation gene MAPK, expansion genes PTGS-2, PTX-3 and TGS-6, apoptosis gene Bax, and anti-apoptosis gene Bcl-2 (Figure.3C). Western Blot analysis revealed that the addition of 5μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12h and a higher expression of Cx43 protein at 24h compared to the group without D4476. (Figure.3 D).

4 | Discuss
This study investigated for the first time the influence of D4476 supplementation in the maturation medium on maturation efficiency and developmental competence of bovine oocytes. In the present study, we observed the effect of D4476 on efficiency of bovine oocytes IVM and subsequent embryonic cleavage rate and blastocyst rate. Results showed that high concentration of D4476 would not improve maturity efficiency of oocytes, and the results of the present study were -to some extent- consisted with those of Wang et al, but there were also many differences (for example applied concentration of D4476)(Wang et al., 2013). Furthermore, we also found CCs expansion in the 10μM group and 20μM group was inhibited, and the reason might be due to the accumulation of large amounts of β-catenin protein in CCs. β-catenin would form a complex with E-cadherin in cell membrane, and the complex resulted in homotypic cell adhesion (Plancha et al., 1994; Pokutta et al., 2000; Brunet et al., 2005; Galli et al.,2012) to inhibit proliferation and expansion of CCs (Veeman et al., 2003). However, the expansion of cumulus cells was the basis of oocyte maturation (Yue et al., 2005). Concerning this study, we guessed that the reasons that high concentration of D4476 leaded to the decrease of maturation efficiency and developmental competence of oocytes were that the spindle did not evenly pull chromosomes during meiosis, and cell adhesion inhibited diffusion of paracrine secretion of CCs. Consequently, according to the test results, we used 5μM D4476 to explore the action mechanism of affecting maturation and developmental competence of oocytes.
D4476 is an efficient, selective-cell and permeable CK1 inhibitors. Therefore, in the study of the action mechanism of D4476, we found the results that D4476 significantly inhibited the expression of CK1 in oocytes was consistent with previous studies (Hämmerlein et al, 2005; Cheong et al., 2011). In addition, this study also showed D4476 did not significantly reduce the incidence rate of GVBD, and not affect the nuclear morphology of mature oocytes. Therefore, we had come to the conclusion the lack of CK1 does not result in the failure of oocytes meiosis and just increase the duration of GVBD, and speculated that D4476 improved the maturation efficiency and quality of oocytes by delaying the spontaneous meiosis in vitro.
It has been reported that WNTs family played an important role in contributing to follicle formation, growth, ovulation and luteinization (Vainio et al., 1999; Heikkilä et al., 2002; Jeaysward et al., 2003). Moreover, the Wnt/β-cateninsignal was also closely related oocytes IVM and embryonic development(Tanaka et al., 2011; Li et al., 2012). CK1 is one of the key kinases in Wnt/β-catenin signaling pathway. D4476 will affect maturity efficiency of oocytes by regulating Wnt/β-catenin signal. Results that D4476 had no significant effect on expression of β-catenin and Cx43, but up-regulated TCF-4 in oocytes were consistent with a report that the accumulation of transcription cofactor and proteins was major activities during oocytes maturation (Fair et al., 1995). In addition, we also found that compared to control group, 5μM D4476 up-regulated β-catenin, TCF-4 and Cx43 in CCs. This result indicated that D4476 significantly simulated Wnt/β-catenin signal in CCs. Therefore, we explored the effect of D4476 on the genes of proliferation, expansion and apoptosis of CCs. The results showed that 5μM D4476 significant inhibited cumulus cell apoptosis, and promoted cumulus cell proliferation and expansion. Furthermore, expansion genes PTGS-2, PTX-3 and TGS-6 were marker genes for oocytes maturation quality (Salustri et al., 2004; Wisniewski et al., 2004; Maina er al., 2009; Marei et al., 2014). Consequently, we speculated that D4476 improved developmentalcompetence of oocytes by promoting proliferation and expansion of CCs.
Moreover, among the many gap junction proteins, Cx43 protein was the most abundant and most important gap junction protein, and played an important role in cell growth, proliferation and differentiation during many tissues (Gellhaus et al., 2004). Cx43 protein was one of key proteins during the process of embryonic densification, and also was a downstream target gene of theWnt/β-catenin signaling pathway (Heyden et al., 1998; Le et al., 2017). The expansion of cumulus cells was the basis of oocyte maturation (Yue et al., 2005), and the expansion and proliferation of CCs would break GJ between cells (Thomas et al., 2004). In addition, the effect of high cAMP levels in CCs on oocytes meiotic was similar to the lack of CK1 in oocytes (Thomas et al., 2004). Consequently, we postulated reduction of Cx43 protein at 12h also was conducive to expansion of CCs and mitigate the inhibition effect of D4476 on meiosis. With the proliferation of CCs, we speculated that D4476 imitated Wnt/β-catenin signal to increase transcription of Cx43 protein at maturation late in order to establish intercellular communication to provide nutrients and signals for oocytes.
In conclusion, our research showed that adding appropriate concentration of D4476 (5μM) to maturation medium was beneficial to IVM and developmental competence of bovine oocytes. This effect of D4476 might be through delaying spontaneous meiosis of oocytes, increasing transcriptional cofactor TCF-4 in the oocytes, simulating Wnt/β-catenin signal to promote proliferation and expansion of CCs and regulating Cx43 protein.

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