Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway

Accepted Manuscript Title: Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway Authors: Wanjing Liu, Chunhua Zhan, Tongzhou Zhang, Xuezhen Zhang PII: DOI: Article Number:

S0166-445X(18)31067-1 https://doi.org/10.1016/j.aquatox.2019.105261 105261

Reference:

AQTOX 105261

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

6 December 2018 14 June 2019 16 July 2019

Please cite this article as: Liu W, Zhan C, Zhang T, Zhang X, Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway, Aquatic Toxicology (2019), https://doi.org/10.1016/j.aquatox.2019.105261 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway Wanjing Liu1, Chunhua Zhan1, Tongzhou Zhang, Xuezhen Zhang*

IP T

College of Fisheries, Huazhong Agricultural University, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China. The authors contributed equally to the work.

SC R

1

*

Corresponding author: Xuezhen Zhang, Ph.D. College of Fisheries, Huazhong Agricultural

U

University, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan

N

430070, China.

A

Phone and fax: +86-27-87282114

M

E-mail: [email protected]

ED

Highlights

Microcystin-LR exposure results in MAPK hyper-phosphorylation and elevated GVBD in the oocytes.

PT

Microcystin -LR disrupts the normal course of meiotic resumption by regulating the MC-PP2A-MAPK-OM pathway

CC E

ERK and JNK pathway play important roles during the process of microcystin

influencing oocyte maturation.

A

Abstract

Harmful cyanobacteria and their production of microcystins (MCs) exert

significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms

of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 μg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 μg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR

IP T

for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 μg/L MC-LR treatment of 4 h. In the oocytes

SC R

exposed to 100 μg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels.

U

Moreover, the MAPK inhibitors (10 μM) were applied to explore the role of MAPK

N

subtypes during MC-LR influencing OM and results showed that ERK inhibitor

A

U0126 and p38 inhibitor SB203580 mitigated the effects of 100 μg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes.

M

In conclusion, the present study indicates that microcystins disrupt the meiotic

PT

disorder in oocytes.

ED

maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation

CC E

Keywords: microcystin; meiosis; oocyte maturation; hyper-phosphorylation; MAPK

1 Introduction

Harmful cyanobacteria blooms, comprised of photoautotrophic bacteria, are

A

found in various water bodies worldwide and able to produce toxic secondary metabolites called cyanotoxins (Preece et al., 2017). Several researchers have reported the accumulation of cyanotoxins in fish and other aquatic organisms, and these toxins can pose health hazards to animals and humans through the food chain (Ibelings and Chorus, 2007; Magalhães et al., 2003). Hepatotoxic microcystins (MCs) are the most widespread and the most studied cyanotoxins, with more than 100 identified variants

(Puddick et al., 2014). Among them, microcystin-LR (MC-LR) is known to be the most frequent and studied variant (Gupta et al., 2003), and the concentration in surface waters often exceeds the proposed lifetime safe consumption level of 1 μg/L (WHO, 2004). With regard to fish, it has been documented MCs are eventually released into

IP T

water bodies during senescence and decomposition processes, which may seriously threaten the survival and reproduction of fishes, amphibians and water birds

(Alonso-Andicoberry et al., 2002; Jia et al., 2014; Malbrouck and Kestemont, 2006).

SC R

Within the past decades, the decline in fertility of animals and humans, which is

potentially related to environmental exposure of MCs, has drawn global attention (Chen et al., 2011; Pant et al., 2013). Plenty of studies showed bioaccumulation of

U

MCs in gonads and reproductive toxicity on fishes (Chen et al., 2009; Lei et al., 2008;

N

Qiao et al., 2013). Furthermore, our previous studies also demonstrated that MC-LR

A

could induce reproductive dysfunction by affecting oogenesis in female zebrafish

M

(Danio rerio) (Liu et al., 2016, 2014; Qiao et al., 2013). However, the toxic effects and potential molecular mechannisms of MCs on oocyte development (involving

ED

meiosis) of fish are still relatively insufficiently known. In teleosts, oocytes will naturally stop their meiotic cell cycle in prophase I (PI),

PT

during which they grow by the accumulation of substances, necessary for early embryonic development. Accompanied by the oocyte (stage Ⅲ) growth, the nucleus

CC E

also becomes big and is called germinal vesicle (GV). Under the stimulation of the maturation-inducing hormone (MIH), fully grown oocytes resume meiosis and proceed to metaphase II (MII), the process being termed oocyte maturation (OM) in

A

the field of biological sciences. In the majority of fish species (including zebrafish), MIH is a C21 steroid, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) (Nagahama and Yamashita, 2008). MIH action at the oocyte surface is transduced to the cytoplasm through membrane progestin receptor (mPR), finally leading to the formation and activation of maturation-promoting factor (MPF) (Zhu et al., 2003). Morphologically, resumption of meiosis is characterized by the disappearance of the nuclear membrane

of oocytes (stage Ⅳ), which is also called germinal vesicle breakdown (GVBD) (Zhang et al., 2009). Thus, zebrafish oocyte provides an excellent experimental model to investigate the effects of MC-LR exposure on the recovery of the first meiotic arrest of oocytes and thereby reveal the underlying mechanism of its reproductive toxicity.

IP T

After uptake, MCs inhibit serine/threonine protein phosphatase 2A (PP2A) activity specifically and strongly by interacting with its catalytic subunit (Zhou et al., 2015).

PP2A

inhibition

induces

disruption

of

protein

SC R

phosphorylation/dephosphorylation homeostasis, leading to the damage in many cellular processes such as mitogen-activated protein kinase (MAPK) signaling pathway. MAPK is a class of serine/threonine protein kinases and widely present in

U

eukaryotic cells (Dias et al., 2010; Zhu et al., 2005). Notably, MAPK can play a role

N

as a component of cytostatic factor to suppress DNA replication between meiosis I

A

and II; however, the need for MAPK activation for GVBD is uncertain and

M

species-specific in fish and amphibian models (Ferrell, 1999; Kajiura-Kobayashi et al., 2000; Khan and Maitra, 2013; Liang et al., 2007). MAPK activation during oocyte

ED

maturation was dependent mostly on Mos, which is a MAPK kinase kinase (MAPKKK) and also the upstream activator of MAPK (Dupré et al., 2011; Kosako et

PT

al., 1994). Sagata (1997) noted that Mos-induced MAPK activity may contribute only to amplification of MPF activity rather than initiation of MPF activation. In

CC E

MIH-treated goldfish oocytes, the appearance and accumulation of Mos occurred first, followed by activation of MAPK, and lastly by the activation of MPF (Kajiura-Kobayashi et al., 2000).

A

The aim of the present study is to investigate the potential role of the MAPK

pathway during MC-LR induced OM in zebrafish. In the in vitro experiment, oocytes (stage Ⅲ) were exposed to MC-LR (1, 10, 100 μg/L) for 2 and 4 h, and then the PP2A activity, MAPK activation, MPF activity and Cyclin B expression, and GVBD rates were detected. Furthermore, immature oocytes (in vitro) were pretreated with MAPK inhibitors (10 μM) for 30 min and then exposed to MC-LR for 4 h. GVBD rates and

phosphorylation levels of MAPK were measured. The present results indicate that MC-LR can disrupt the normal process of oocyte maturation and ERK and JNK pathway may play important roles during the process, and then provide a theoretical foundation to evaluate the reproductive toxicity of MCs on fish.

IP T

2 Materials and methods 2.1. Fish maintenance

SC R

Adult female zebrafish (AB-type, 4 months old) were obtained from zebrafish

breeding center in Institute of Hydrobiology, Chinese Academy of Sciences. Fish were maintained on a 14 h:10 h light/dark cycle according to standard conditions (28

U

± 1 °C) and fed three times a day with Artemia nauplii.

A

N

2.2 Toxin and Chemicals

1 mg MC-LR was purchased from Taiwan Algal Science Inc. (Taiwan, China)

M

with a purity of 95% and was dissolved in 1mL milliQ water to obtain a concentration

ED

of 1mg/mL. ERK1/2 inhibitor U0126, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 were purchased from ApexBio Technology Inc. (Houston, USA).

sources.

PT

All other reagents were of analytical grade bought from multifarious commercial

CC E

2.3 Oocyte preparation and exposure To obtain oocytes, gravid female zebrafish were anaesthetized with ice for 1 min

before the ovaries were removed. The ovaries were gently placed immediately into a

A

petri dish containing Hank’s solution and separated into single oocytes by repeated gentle pipetting (Guan et al., 2008). The oocytes were collected and pre-cultured at 28°C for 30 min to discard manually damaged oocytes. Healthy oocytes of stage Ⅲ (mean diameter about 550-600 μm) were randomly distributed into 12-well plates containing L-15 medium with 10 ng/ml DHP. Oocytes were exposed to 0, 1, 10, 100 μg/L MC-LR and cultured at 28 °C for 4 h. In addition, oocytes of stage Ⅲ were

pre-incubated in L-15 medium with ERK inhibitor U0126 (10 μM), p38 inhibitor SB203580 (10 μM) and JNK inhibitor SP600125 (10 μM), respectively. After 30 min, 10 ng/mL DHP and MC-LR (0, 1, 10, 100 μg/L) were added and oocytes were cultured at 28 °C for 4 h. 2.4 GVBD test

IP T

After culture for 4 h, the translucent oocytes without visible nucleus under a dissecting microscope, were counted as GVBD oocytes (also called mature oocytes).

2.5 PP2A and MPF enzymatic activity and VTG content

SC R

Then the percentages of GVBD were calculated in each group.

PP2A enzymatic activity was assayed according to the instruction of

U

Serine/Threonine Phosphatase Assay System (Promega, V2460; Madison, Wisconsin,

N

USA). Live oocytes were rinsed and homogenized in ice-cold PSB to obtain

A

homogenate. The homogenate was added into the provided spin columns with

M

Sephadex beads to remove endogenous phosphates. The supernatant was taken for detecting total soluble protein concentration using a bicinchoninic acid assay (BCA

ED

kit; Beyotime, Shanghai, China). The reaction was initiated by adding 15 μL of reaction premixes containing 5 μL of 1 mM phosphopeptide and 10 μL of PPase-2A

PT

5×reaction buffer. After reaction at 37 °C for 30 min, 50 μL of stop solution was added to each well. The data was read at 630 nm. The activity levels of MPF and the

CC E

contents of VTG were determined through the zebrafish MPF ELISA Kit (Mlbio, Shanghai, China) and the zebrafish VTG ELISA Kit (Huding, Shanghai, China), following the manufacturer’s protocol.

A

2.6 Immunohistochemistry analysis Oocytes were fixed in 3.7% formaldehyde/0.2 Triton X-100/0.5 M taxol in a

microtubule assembly buffer. The oocytes were incubated with primary antibody of Cyclin B (rabbit, 1:200; ABclonal, Wuhan, China), and then stained with secondary antibody and observed under an optical microscope.

2.7 Western blotting analysis Western blotting was performed according to the previously described method (Liu et al., 2016). Briefly, tissue samples were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor at 4 °C, and centrifuged at 12,000×g for 15 min. The supernatant was aliquoted and protein concentration was

IP T

determined by BCA kit (Beyotime, Shanghai, China). The following primary antibodies were used: ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK (rabbit, 1:500;

Cell Signaling Technology, Houston, USA). Anti-GAPDH antibody (rabbit, 1:1000;

SC R

Servicebio, Wuhan, China) was selected as internal reference. After primary antibody

incubation, the membranes were washed with TBST and incubated with goat anti-rabbit secondary antibody (LICOR, Nebraska, USA) for 2 h at room temperature.

U

The intensities of the bands were quantified using Quantity One (Bio-Rad, California,

N

USA) software.

A

2.8 Statistical analysis

M

The results were represented as the mean ± SEM. One-way ANOVA followed by

ED

the post hoc Tukey test was employed for multi-group comparison, a P value < 0.05

3 Results

PT

was considered to be statistically significant.

CC E

3.1 PP2A activity inhibition of MC-LR in zebrafish oocyte Changes in PP2A activity of zebrafish oocytes in vitro after MC-LR exposure for

A

2 and 4 h were shown in Fig. 1. No significant changes in PP2A activity were observed in 1 μg/L MC-LR treated oocytes at either 2 or 4 h, whereas the activity in oocytes exposed to 10 and 100 μg/L MC-LR was significantly decreased at 2 and 4 h (P < 0.05). 3.2 Effects of MC-LR on MAPK activation in zebrafish oocytes. As Fig. 2 showed, MC-LR induced changes in phosphorylation levels of MAPK

subtypes in oocytes. Compared with the control group, no significant difference was observed in any MC-LR treatment at 2 h. In the oocytes cultured for 4 h, 100 μg/L MC-LR upregulated the phosphorylation levels of ERK, p38 and JNK pathways (P < 0.01). 3.3 MPF activity changes in oocytes exposed to MC-LR.

IP T

The changes in MPF activity of oocytes treated with MC-LR for 4 h were summarized in Fig. 3. Compared with the control group, MPF level increased

SC R

dramatically in the oocytes exposed to 100 μg/L MC-LR (P < 0.05), while no significant difference was detected in other MC-LR-treated groups. 3.4 Expression and location of Cyclin B in oocytes.

U

The results (Fig. 4) showed the expression and location of Cyclin B in oocytes

N

treated with MC-LR for 4 h. After culture for 4 h, Cyclin B was expressed in the

A

cytoplasm and cell membrane of oocytes. The protein level of Cyclin B increased

M

significantly in the oocytes treated with 100 μg/L MC-LR.

ED

3.5. Effects of MC-LR on the GVBD rates of oocytes. The pictures in Fig. 5 exhibited the difference between GV oocytes (immature) and GVBD oocytes (mature), and the effects of MC-LR on GVBD rates of oocytes

PT

were showed in Fig. 6. The results indicated that low concentrations of 1 and 10 μg/L MC-LR had no effect on the GVBD rates, but the GVBD rates increased noticeably in

CC E

the zebrafish oocytes treated with 100 μg/L MC-LR (P < 0.05). 3.6. Effects of MC-LR on the VTG contents of oocytes.

A

The effects of MC-LR on the VTG contents of oocytes were exhibited in Fig. 7.

The results showed that the VTG contents decreased remarkably in the zebrafish oocytes treated with 10 and 100 μg/L MC-LR (P < 0.05), and low concentration of 1 μg/L MC-LR had no effect on the VTG content. 3.7 Effects of MC-LR and MAPK inhibitor on ERK, p38 and JNK phosphorylation in oocytes.

The changes of ERK, p38 and JNK activity in oocytes pretreated with MAPK inhibitors for 30 min and then with MC-LR for 4 h, were presented in Fig. 8. The phosphorylation of ERK was downregulated in U0126 + 1 μg/L MC-LR group and U0126 + 10 μg/L MC-LR group, but upregulated in 100 μg/L MC-LR group (P < 0.05). The activation of p38 MAPK decreased in SB203580 + 1 μg/L MC-LR group and SB203580 + 10 μg/L MC-LR group, while it was increased in 100 μg/L MC-LR

IP T

group (P < 0.05). Similarly, the levels of JNK phosphorylation were downregulated in

SP600125 + 1 μg/L MC-LR group and SP600125 + 10 μg/L MC-LR group, but

SC R

upregulated in 100 μg/L MC-LR group (P < 0.05).

3.8 GVBD rates changes in oocytes treated with MAPK inhibitors and MC-LR.

U

Fig. 9 showed the changes of GVBD rates in oocytes pretreated with MAPK inhibitors for 30 min and then with 100 μg/L MC-LR for 4 h. Compared with the

N

control group, 100 μg/L MC-LR treatment alone induced a significant increase of

A

GVBD rate in oocytes (P < 0.05). No significant change was observed in the U0126 +

M

100 μg/L MC-LR group and SP600125 + 100 μg/L MC-LR group, while the GVBD

PT

4 Discussion

ED

rate increased notably in oocytes treated with SB203580 and MC-LR (P < 0.05).

In recent years, the frequent outbreak of cyanobacterial blooms and the

CC E

production of microcystins is of an urgent environmental concern both at home and abroad. A large number of studies have reported that MCs have significant reproductive toxicity on fish (Ding et al., 2006; Lone et al., 2015; Wang et al., 2013).

A

Our previous studies also found that MC-LR induced reproductive dysfunction by disrupting the oogenesis in female zebrafish (Liu et al., 2016; Qiao et al., 2013). However, recent studies have focused on reproductive dysfunction and endocrine disruption, the effects of MCs on oogenesis, especially oocyte maturation in female fish is still unkown. On the other hand, during the outbreak of cyanobacteria blooms, the concentrations of MC usually range from 0.1 to 10 μg/L (Lahti et al., 1997), and

even reach dozens of micrograms per liter. WHO risk guideline categories provide a basis for evaluating the relative risk of MC concentrations: <1 µg/L very low risk, 1-10 µg/L low risk, 10-20 µg/L moderate risk, 20-2000 µg/L high risk (WHO, 2003). On the basis of our previous experimental experiences (Huang et al., 2015) and other reference reports (Hou et al., 2018; Rogers et al., 2011), three concentrations of 1, 10 and 100 µg/L MC-LR were used in exposures of zebrafish oocytes for investigating

IP T

the influences of MC-LR on resumption of meiotic arrest and the pathways involved in microcystin-induced toxicity.

SC R

Data from the present study demonstrated that PP2A activity decreased

remarkably in the oocytes exposed to 10 and 100 μg/L MC-LR at both 2 and 4 h. Plenty of evidence has proven that MC-LR inhibits PP2A activity specifically and

U

strongly (Honkanen et al., 1990; Liu and Sun, 2015), thus leading to

N

hyperphosphorulation of downstream proteins, which may related to its toxicity.

A

Furthermore, MAPK is a class of serine/threonine protein kinases, widely present in

M

eukaryotic cells, and regulated by PP2A (Junttila et al., 2008). The results of Western blot in the oocytes cultured for 4 h showed that 100 μg/L MC-LR upregulated the

ED

phosphorylation levels of ERK, p38 MAPK and JNK significantly, meaning the activation of MAPK pathway. Researches about other types of cells also reported

PT

similar results that MC-LR significantly inhibited cellular PP2A activity and activated MAPK signaling pathway (Komatsu et al., 2007; Meng et al., 2011, 2015; Zhang et

CC E

al., 2013). The implication of PP2A in the regulation of OM was initially suggested through experiments using okadaic acid (OA), a potent and selective inhibitor of PP2A (Goris et al., 1989; Picard et al., 1989). OA reversed the inhibitory effect of

A

cAMP on meiotic resumption and MAPK phosphorylation (Lu et al., 2001). Alternatively, in the zebrafish oocytes exposed to MC-LR for 2 h, there was no significant change in the phosphorylation levels of MAPK between treated groups and control group. The above results indicated that the activation of ERK, p38 and JNK pathway induced by MC-LR required a certain length of time. Previous researches have illustrated in detail the behavior of the components of

MPF, Cdc2 and Cyclin B during goldfish oocyte maturation. Cyclin B protein is newly synthesized from its mRNA stored in the oocyte during maturation. The newly synthesized Cyclin B binds to preexisting Cdc2 and forms active MPF after the phosphorylation of Cyclin B-bound Cdc2. In the present study, MPF activity increased significantly in oocytes treated with 100 μg/L MC-LR for 4 h. Immunohistochemical results displayed Cyclin B located in cytoplasm and cell

IP T

membrane; and a high concentration of MC-LR (100 μg/L) upregulated the

expression of Cyclin B in oocytes, in accordance with the increase of MPF level in

SC R

100 μg/L MC treatment.

GVBD and VTG are usually regarded as hallmarks of the progress of oocyte maturation and MPF is the substance acting most downstream of oocyte maturation.

U

Zambolla et al. (2008) even used GVBD as a test of functionality to evaluation

N

zebrafish oocyte viability. In the oocytes treated with 100 μg/L MC-LR for 4 h,

A

GVBD rate increased significantly accompanied by a decrease in VTG level,

M

suggesting that a high concentration of MC-LR could disrupt the normal process of GVBD in zebrafish oocytes in vitro. Studies on zebrafish in vivo also reported that

ED

MC-LR interfered gametogenesis in female (Hou et al., 2014; Liu et al., 2014; Zhao et al., 2015). VTG is taken into the oocyte by receptor-mediated endocytosis and the

PT

follicles become opaque. Yolk is produced upon cleavage of the VTG into lipovitellins and phosvitin during oocyte maturation, after which the egg usually

CC E

regains transparency (Nagahama et al., 1995; Khanand and Thomas, 1999; Okumuraetal., 2002). Therefore, the present results of GVBD rates and VTG contents were consistent with MPF activity changes induced by MC-LR exposure. Some

A

studies reported that in certain fish, the recovery of the first meiotic arrest of oocytes must depend on the regulation of MAPK (Fan and Sun, 2004; Tokumoto et al., 2011). In zebrafish oocytes in vitro, the inhibition of mitogen-activated extracellular signal-regulated kinase (MEK) could suppress insulin-stimulated meiotic maturation significantly and ERK1/2 activation by OA-induced GVBD response (Maitra et al., 2014). The present study indicates that MC-LR exerts reproductive toxicity by

inhibiting PP2A activity, activating MAPK and then disrupting oocyte maturation, which can be summed up in MC-PP2A-MAPK-OM pathway. To further explore the effects of MC-LR induced activation of MAPK signaling pathway during oocyte maturation, immature oocytes were pretreated with ERK1/2 inhibitor U0126, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125, and

IP T

then exposed to MC-LR for 4 h. Compared with control group, no significant change of ERK phosphorylation was observed in U0126 + 100 μg/L MC-LR group; similar results were also detected in p38 phosphorylation in SB 203580 + 100 μg/L MC-LR

SC R

group and in JNK phosphorylation in SP600125 + 100 μg/L MC-LR group. The present results indicated that MAPK inhibitors could alleviate the MC-LR-induced MAPK hyperphosphorylation to some extent. In addition, GVBD data showed that

U

GVBD rate in 100 μg/L MC-LR treatment was significantly higher than that of the

N

control group. However, there was no significant difference between U0126 + 100

A

μg/L MC-LR group or SP600125 + 100 μg/L MC-LR group and the control group,

M

indicating that the addition of ERK inhibitor or JNK inhibitor could alleviate MC-LR-induced oocyte maturation in 100 μg/L group. The present results suggested

ED

that ERK and JNK pathway may play important roles during the process of MC-LR influencing oocyte maturation, which is correspondent with the results reproted by

PT

Maitra et al (2014).

CC E

5 Conclusion

In summary, the present results showed that MC-LR triggered the

A

hyperphosphorylation of MAPK pathway through inhibiting PP2A activity; and caused early occurrence of GVBD in zebrafish oocytes (in vitro), meaning disorder of OM progress. Similar changes of upregulation were also detected in MPF activity and Cyclin B expression. In addition, the presence of MAPK inhibitors could reverse the effects of MC-LR on meiotic maturation in zebrafish oocytes, and ERK and JNK pathways might play important roles during the process of MC influencing oocyte

maturation. The present study indicates that MC-LR disrupts the normal course of meiotic resumption by regulating the MC-PP2A-MAPK-OM pathway, which will contribute to a better understanding of the mechanisms of MCs induced reproductive toxicity in fish.

IP T

Acknowledgements This study is supported by the National Natural Science Foundation of China

SC R

(31670520).

References

U

Alonso-Andicoberry, C., García-Villada, L., Lopez-Rodas, V., Costas, E., 2002.

N

Catastrophic mortality of flamingos in a Spanish national park caused by

A

cyanobacteria. Vet. Rec. 151, 706-707. https://doi.org/10.1136/VR.151.23.706 Chen, J., Zhang, D., Xie, P., Wang, Q., Ma, Z., 2009. Simultaneous determination of

M

microcystin contaminations in various vertebrates (fish, turtle, duck and water

blooms.

ED

bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis Sci.

Total

Environ.

407,

3317-3322.

https://doi.org/10.1016/j.scitotenv.2009.02.005

PT

Chen, Y., Xu, J., Li, Y., Han, X., 2011. Decline of sperm quality and testicular function in male mice during chronic low-dose exposure to microcystin-LR.

CC E

Reprod. Toxicol. 31, 551-557. https://doi.org/10.1016/j.reprotox.2011.02.006

Dias, E., Matos, P., Pereira, P., Batoréu, M.C., Silva, M.J., Jordan, P., 2010.

A

Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney-derived cell line Vero-E6. Toxicol. Vitr. An Int. J. Publ. Assoc. with Bibra 24, 1689-1695.

Ding, X.S., Li, X.Y., Duan, H.Y., Chung, I.K., Lee, J.A., 2006. Toxic effects of Microcystis cell extracts on the reproductive system of male mice. Toxicon 48, 973-979. https://doi.org/10.1016/j.toxicon.2006.07.039 Dupré, A., Haccard, O., Jessus, C., 2011. Mos in the oocyte: how to use MAPK

independently of growth factors and transcription to control meiotic divisions. J. Signal Transduct. 2011, 350412.Fan, H.-Y., Sun, Q.-Y., 2004. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization

in

mammals.

Biol.

Reprod.

70,

535-47.

https://doi.org/10.1095/biolreprod.103.022830 Ferrell, J.E., 1999. Xenopus oocyte maturation: new lessons from a good egg.

IP T

Bioessays News Rev. Mol. Cell. Dev. Biol. 21, 833-842.

Goris, J., Hermann, J., Hendrix, P., Ozon, R., Merlevede, W., 1989. Okadaic acid, a

SC R

specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett. 245, 91-4.

Guan, M., Rawson, D.M., Zhang, T., 2008. Development of a new method for

U

isolating zebrafish oocytes (Danio rerio) from ovary tissue masses.

N

Theriogenology 69, 269-275.

A

Gupta, N., Pant, S.C., Vijayaraghavan, R., Rao, P.V.L., 2003. Comparative toxicity evaluation of cyanobacterial cyclic peptide toxin microcystin variants (LR, RR, in

mice.

Toxicology

M

YR)

188,

285-296.

ED

https://doi.org/10.1016/S0300-483X(03)00112-4 Honkanen, R.E., Zwiller, J., Moore, R.E., Daily, S.L., Khatra, B.S., Dukelow, M., Boynton, L., 1990. Characterization of microcystin-LR, a potent inhibitor of type

PT

1 and type 2A protein phosphatases. J. Biol. Chem. 265, 19401-19404. Hou, J., Li, L., Xue, T., Long, M., Su, Y., Wu, N., 2014. Damage and recovery of the

CC E

ovary in female zebrafish i.p.-injected with MC-LR. Aquat. Toxicol. 155, 110-118.

A

Hou, J., Su, Y., Lin, W., Guo, H., Li, L., Anderson, D.M., Li, D., Tang, R., Chi, W., Zhang, X., 2018. Estrogenic potency of MC-LR is induced via stimulating steroidogenesis: in vitro and in vivo evidence. Environ. Pollut. 240, 615-622.

Huang, X., Chen, L., Liu, W., Qiao, Q., Wu, K., Wen, J., Huang, C., Tang, R., Zhang, X, 2015. Involvement of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK cells. Aquat. Toxicol. 165, 41-50.Ibelings, B.W., Chorus, I., 2007. Accumulation of cyanobacterial toxins in freshwater

‘seafood’ and its consequences for public health: A review. Environ. Pollut. 150, 177-92. https://doi.org/10.1016/j.envpol.2007.04.012 Jia, X., Cai, C., Wang, J., Gao, N., Zhang, H., 2014. Endocrine-disrupting effects and reproductive toxicity of low dose MCLR on male frogs (Rana nigromaculata) in vivo. Aquat. Toxicol. 155, 24-31. https://doi.org/10.1016/j.aquatox.2014.06.002 Junttila, M.R., Li, S.-P., Westermarck, J., 2008. Phosphatase-mediated crosstalk

IP T

between MAPK signaling pathways in the regulation of cell survival. FASEB J. 22, 954-965. https://doi.org/10.1096/fj.06-7859rev

SC R

Kajiura-Kobayashi, H., Yoshida, N., Sagata, N., Yamashita, M., Nagahama, Y., 2000.

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish.

U

Dev. Genes Evol. 210, 416-425. https://doi.org/10.1007/s004270000083

N

Khan, P.P., Maitra, S., 2013. Participation of cAMP-dependent protein kinase and

A

MAP kinase pathways during Anabas testudineus oocyte maturation. Gen. Comp. Endocrinol. 181, 88-97. https://doi.org/10.1016/j.ygcen.2012.10.016

M

Khan, I.A., Thomas, P., 1999. Ovarian cycle, teleost fish. In: Knobil, E., Neil, J.D.

ED

(Eds.), Fish Physiology. Academic Press Inc., New York, pp. 552-564. Komatsu, M., Furukawa, T., Ikeda, R., Takumi, S., Nong, Q., Aoyama, K., Akiyama, S.I., Keppler, D., and Takeuchi, T., 2007. Involvement of mitogen-activated

PT

protein kinase signaling pathways in microcystin-LR-Induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicol. Sci. 97,

CC E

407-416.

Kosako, H., Gotoh, Y., Nishida, E., 1994. Requirement for the MAP kinase

A

kinase/MAP kinase cascade in Xenopus oocyte maturation. EMBO J. 13, 2131-2138.

Lahti, K., Rapala, J., Färdig, M., Niemelä, M., Sivonen, K., 1997. Persistence of cyanobacterial hepatotoxin, microcystin-LR in particulate material and dissolved in lake water. Water Res. 31, 1005-1012. Lei, H.H., Xie, P., Chen, J., Liang, G.D., Dai, M., Zhang, X.Z., 2008. Distribution of toxins in various tissues of crucian carp intraperitoneally injected with

hepatotoxic

microcystins.

Environ.

Toxicol.

Chem.

27,

1167-1174.

https://doi.org/10.1897/07-522.1 Liang, C.G., Su, Y.Q., Fan, H.Y., Schatten, H., Sun, Q.Y., 2007. Mechanisms regulating ocyte meiotic resumption: roles of mitogen-activated protein kinase. Mol. Endocrinol. 21, 2037-2055. https://doi.org/10.1210/me.2006-0408 Liu, J., Sun, Y., 2015. The role of PP2A-associated proteins and signal pathways in toxicity.

Toxicol.

Lett.

236,

1-7.

IP T

microcystin-LR

https://doi.org/10.1016/j.toxlet.2015.04.010

SC R

Liu, W., Chen, C., Chen, L., Wang, L., Li, J., Chen, Y., Jin, J., Kawan, A., Zhang, X.,

2016. Sex-dependent effects of microcystin-LR on hypothalamic-pituitary-gonad axis and gametogenesis of adult zebrafish. Sci. Rep. 6, 22819.

U

Liu, W., Qiao, Q., Chen, Y., Wu, K., Zhang, X., 2014. Microcystin-LR exposure to

N

adult zebrafish (Danio rerio) leads to growth inhibition and immune dysfunction

A

in F1 offspring, a parental transmission effect of toxicity. Aquat. Toxicol. 155, 360-367. https://doi.org/10.1016/j.aquatox.2014.07.011

M

Lone, Y., Koiri, R.K., Bhide, M., 2015. An overview of the toxic effect of potential

ED

human carcinogen Microcystin-LR on testis. Toxicol. Reports. 2, 289-296. https://doi.org/10.1016/j.toxrep.2015.01.008 Lu, Q., Smith, G.D., Chen, D.Y., Yang, Z., Han, Z.M., Schatten, H., Sun, Q.Y., 2001.

PT

Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3’,5’-adenosine monophosphate, and protein phosphatase

CC E

modulators during meiosis resumption in rat oocytes. Biol. Reprod. 64, 1444-50. https://doi.org/10.1095/biolreprod64.5.1444

A

Maitra, S., Das, D., Ghosh, P., Hajra, S., Roy, S.S., Bhattacharya, S., 2014. High cAMP attenuation of insulin-stimulated meiotic G2-M1 transition in zebrafish oocytes: Interaction between the cAMP-dependent protein kinase (PKA) and the MAPK3/1

pathways.

Mol.

Cell.

Endocrinol.

393,

109-119.

https://doi.org/10.1016/j.mce.2014.06.008 Malbrouck, C., Kestemont, P., 2006. Effects of microcystins on fish. Environ. Toxicol. Chem. 25, 72-86. https://doi.org/10.1897/05-029R.1

Meng, G., Sun, Y., Fu, W., Guo, Z., and Xu, L., 2011. Microcystin-LR induces cytoskeleton system reorganization through hyperphosphorylation of tau and HSP27 via PP2A inhibition and subsequent activation of the p38 MAPK signaling pathway in neuroendocrine (PC12) cells. Toxicology 290, 219-230. Meng, G., Liu, J., Lin, S., Guo, Z., and Xu, L., 2015. Microcystin-LR-Caused ROS generation involved in p38 activation and tau hyperphosphorylation in

IP T

neuroendocrine (PC12) cells. Environ. Toxicol. 30, 366-374.

Nagahama, Y., Yamashita, M., 2008. Regulation of oocyte maturation in fish. Dev Differ

50

Suppl

1,

S195-219.

https://doi.org/DGD1019

SC R

Growth

[pii]\r10.1111/j.1440-169X.2008.01019.x

Nagahama, Y., Yoshikuni, M., Yamashita, M., Tokumoto, T., Katsu, Y., 1995.

U

Regulation of oocyte growth and maturation in fish. Curr. Top. Dev. Biol. 30,

N

103-145.Magalhães, V.F., Marinho, M.M., Domingos, P., Oliveira, A.C., Costa,

A

S.M., Azevedo, L.O., Azevedo, S.M.F.O., 2003. Microcystins (cyanobacteria hepatotoxins) bioaccumulation in fish and crustaceans from Sepetiba Bay (Brasil,

M

RJ). Toxicon 42, 289-295. https://doi.org/10.1016/S0041-0101(03)00144-2

ED

Okumura, H., Todo, T., Adachi, S., Yamauchi, K., 2002. Changes in hepatic vitellogenin mRNA levels during oocyte development in the Japanese eel, Anguilla japonica. Gen. Comp. Endocrinol. 125, 9-16.Pant, N., Pant, A.B.,

PT

Chaturvedi, P.K., Shukla, M., Mathur, N., Gupta, Y.K., Saxena, D.K., 2013. Semen quality of environmentally exposed human population: The toxicological

CC E

consequence.

Environ.

Sci.

Pollut.

Res.

20,

8274-8281.

https://doi.org/10.1007/s11356-013-1813-8

A

Picard, A., Capony, J.P., Brautigan, D.L., Doree, M., 1989. Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish. J. Cell Biol. 109, 3347-3354. https://doi.org/10.1083/jcb.109.6.3347

Preece, E.P., Hardy, F.J., Moore, B.C., Bryan, M., 2017. A review of microcystin detections in Estuarine and Marine waters: Environmental implications and human

health

risk.

Harmful

https://doi.org/10.1016/j.hal.2016.11.006

Algae

61,

31-45.

Puddick, J., Prinsep, M.R., Wood, S.A., Kaufononga, S.A.F., Cary, S.C., Hamilton, D.P., 2014. High levels of structural diversity observed in microcystins from microcystis CAWBG11 and characterization of six new microcystin congeners. Mar. Drugs 12, 5372-5395. https://doi.org/10.3390/md12115372 Qiao, Q., Liu, W., Wu, K., Song, T., Hu, J., Huang, X., Wen, J., Chen, L., Zhang, X., 2013. Female zebrafish (Danio rerio) are more vulnerable than males to

142-143, 272-282. https://doi.org/10.1016/j.aquatox.2013.07.002

IP T

microcystin-LR exposure, without exhibiting estrogenic effects. Aquat. Toxicol.

SC R

Rogers, E.D., Henry, T.B., Twiner, M.J., Gouffon, J.S., Mcpherson, J.T., Boyer, G.L.,

Sayler, G.S., Wilhelm, S.W., 2011. Global gene expression profiling in larval zebrafish exposed to microcystin-LR and microcystis reveals endocrine

U

disrupting effects of cyanobacteria. Environ. Sci. Technol. 45, 1962-1969.Sagata,

A

https://doi.org/10.1002/bies.950190105

N

N., 1997. What does Mos do in oocytes and somatic cells? BioEssays 19, 13-21.

Tokumoto, T., Yamaguchi, T., Ii, S., Tokumoto, M., 2011. In vivo induction of oocyte and

ovulation

in

M

maturation

zebrafish.

PLoS

One

6.

ED

https://doi.org/10.1371/journal.pone.0025206 Wang, L., Wang, X., Geng, Z., Zhou, Y., Chen, Y., Wu, J., Han, X., 2013. Distribution of microcystin-LR to testis of male Sprague-Dawley rats.

PT

Ecotoxicology 22, 1555-1563. https://doi.org/10.1007/s10646-013-1141-2 WHO, 2004. Guidelines for drinking water quality, in: World Health Organization. p.

CC E

516. https://doi.org/10.1525/jer.2014.9.2.1

WHO. 2003. Guidelines for safe recreational water environments. Coastal and fresh waters, volume 1.

A

Zampolla, T., Zhang, T., Rawson, D.M., 2008. Evaluation of zebrafish (Danio rerio) ovarian Follicle viability by simultaneous staining with Fluorescein Diacetate and Propidium Iodide. Cryoletters 29, 463-475. Zhang,

M.,

Ouyang,

H.,

Xia,

G.,

2009.

The

signal

pathway

of

gonadotrophins-induced mammalian oocyte meiotic resumption. Mol. Hum. Reprod. 15, 399. https://doi.org/10.1093/molehr/gap031

Zhang, J., Chen, J., and Xia, Z., 2013. Microcystin-LR exhibits immunomodulatory role in mouse primary hepatocytes through activation of the NF-κB and MAPK signaling pathways. Toxicol. Sci. 136, 86-96. Zhao, Y., Xie, L., Yan, Y., 2015. Microcystin-LR impairs zebrafish reproduction by affecting oogenesis and endocrine system. Chemosphere 120, 115-122. https://doi.org/10.1016/j.chemosphere.2014.06.028

cytoskeletal

disruption

in

animal

cells.

Toxicon

101,

92-100.

SC R

https://doi.org/10.1016/j.toxicon.2015.05.005

IP T

Zhou, M., Tu, W.W., Xu, J., 2015. Mechanisms of microcystin-LR-induced

Zhu, Y., Rice, C.D., Pang, Y., Pace, M., Thomas, P., 2003. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an

U

intermediary in meiotic maturation of fish oocytes. Proc. Natl. Acad. Sci. U.S.A.

N

100, 2231-2236. https://doi.org/10.1073/pnas.0336132100

A

Zhu, Y., Zhong, X., Zheng, S., Ge, Z., Du, Q., Zhang, S., 2005. Transformation of immortalized colorectal crypt cells by microcystin involving constitutive

M

activation of Akt and MAPK cascade. Carcinogenesis 26, 1207-1214.

c o n tr o l 1 μ g /L

*

1 .5

**

1 0 μ g /L 1 0 0 μ g /L

1 .0

*

*

0 .5

h 4

h

0 .0

2

A

CC E

P P 2 A (m m o l/g * m in )

2 .0

PT

ED

https://doi.org/10.1093/carcin/bgi069

Figure 1. PP2A activity in zebrafish oocytes exposed to MC-LR for 2 and 4 h (mean ± SEM, n =

A

CC E

PT

ED

M

A

N

U

SC R

IP T

6). Asterisk (*) and (**) indicate significant differences at P < 0.05 and P <0.01 between MC-LR treated groups and control group, respectively.

IP T SC R U N A M ED PT CC E A Figure 2. Effects of MC-LR on ERK, p38 and JNK phosphorylation in zebrafish oocytes. The Western blot results of each target protein (A) and the phosphorylated protein/total protein fold change (B) in oocytes exposed to MC-LR for 2 h, the Western blot results of each target protein (C)

SC R

IP T

and the phosphorylated protein/total protein fold change (D) in oocytes exposed to MC-LR for 4 h (mean ± SEM, n = 3). Asterisk (**) indicates significant differences at P <0.01 between MC-LR treated groups and control group.

*

N

U

1 .0

/L g

/L

μ

g

0 1

1

ED

0

μ

g μ 1

c

o

n

tr

o

/L

l

0 .0

M

A

0 .5

0

M P F ( U /g )

1 .5

PT

4 h

A

CC E

Figure 3. MPF activity in zebrafish oocytes exposed to MC-LR for 4 h (mean ± SEM, n = 3). Asterisk (*) indicates significant differences at P < 0.05 between MC-LR treated groups and control group.

IP T SC R U N A M ED

A

CC E

PT

Figure 4. The expression and location of Cyclin B in oocytes treated with MC-LR for 4 h. Control group (A), 1 μg/L group (B), 10 μg/L group (C) and 100 μg/L group (D).

IP T SC R U N A M ED PT CC E A

Figure 5. Morphologic observation of GV and GVBD oocytes.

100

* G V B D (% )

80

60

40

IP T

20

/L g

/L

μ

g

SC R

0 0

0

1

1

c

1

o

n

μ

μ

g

tr

o

/L

l

0

4 h

M

A

N

U

Figure 6. Effects of MC-LR on the GVBD rates of oocytes (mean ± SEM, n = 6). Asterisk (*) indicates significant differences at P < 0.05 between MC-LR treated groups and control group.

ED

*

*

PT

( μ g /g p r o t e in )

100

50

CC E

VTG

150

/L g

/L

μ 0 1

0

0 1

1

μ

μ

g

g

o tr n

o

c

A

/L

l

0

4 h

Figure 7. VTG content in zebrafish oocytes exposed to MC-LR for 4 h (mean ± SEM, n = 6). Asterisk (*) indicates significant differences at P < 0.05 between MC-LR treated groups and control group.

A ED

PT

CC E

IP T

SC R

U

N

A

M

D 2 .0

M C -L R M C -L R + U 0 1 2 6

a

a

a

a

1 .0

a

a b

b

SC R

p - E R K /E R K

b 1 .5

0 .5

g μ 0 0 1

1

1

0

μ

μ

g

g

/L

/L

/L

0 .0

N

U

4 h

E

b

a

g

μ 0 0 1

1

0

μ 1

4 h

CC E

/L

ED /L

PT

g

/L

0 .0

b

g

b

a

a

a

1 .0

0 .5

A

M

a

a

μ

p -p 3 8 /p 3 8

2 .0

A

2 .5

1 .5

IP T

c o n tr o l

c o n tr o l M C -L R M C -L R + S B 2 0 3 5 8 0

F 2 .0

c o n tr o l

b

M C -L R

a

p - J N K /J N K

1 .5

M C -L R + S P 6 0 0 1 2 5

a a

a

a

1 .0

a b

b

0 .5

/L g

/L

1

1

0

0

0

μ

μ

g

/L g μ 1

IP T

0 .0

SC R

4 h

N

U

Figure 8. Effects of MC-LR and MAPK inhibitor on ERK, p38 and JNK phosphorylation in oocytes. The Western blot results of each target protein (A-C) and the phosphorylated protein/total protein fold change (D-F) in oocytes exposed to MC-LR and ERK, p38, JNK inhibitor for 4 h (mean ± SEM, n = 3). Different letters indicate statistical differences (P < 0.05). M C -L R + M A P K in h ib ito r

A

100

*

M

*

60

S +

+

/L

/L

g

g

μ 0 1

0 1

0

0

/L g 1

0

0

μ

B

P S

2 1 0 U +

0

0

1

CC E

c

o

n

μ

tr

g

o

/L

l

0

PT

20

μ

40

6

ED

G V B D (% )

80

4 h

A

Figure 9. Effects of MAPK inhibitors on GVBD rates of oocytes treated with 100 μg/L MC-LR (mean ± SEM, n = 6). Asterisk (*) indicates significant differences at P <0.05 between treated groups and control group.

A ED

PT

CC E

IP T

SC R

U

N

A

M