Induction and inhibition of oocyte maturation by EDCs in zebrafish
© Tokumoto et al; licensee BioMed Central Ltd. 2005
Received: 05 November 2005
Accepted: 09 December 2005
Published: 09 December 2005
Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen.
In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation.
Among agents tested, tamoxifen (TAM) and its metabolite 4-hydroxytamoxifen (4-OHT) showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B) induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP) had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent.
These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.
Fish oocytes provide an appropriate experimental system with which to investigate the molecular mechanisms controlling meiosis and the embryonic cell cycle. Several factors responsible for the regulation of meiotic maturation in fish oocytes have been identified. These include the isolation and characterization of a fish maturation-inducing hormone (MIH) and the components of maturation-promoting factor (MPF) (cdc2, the catalytic subunit, and cyclin B, the regulatory subunit) 
Oocyte maturation in fish is triggered by MIH, which acts on receptors located on the oocyte membrane and induces the activation of MPF in the oocyte cytoplasm . During the course of maturation, oocytes undergo drastic morphological changes associated with progression of the meiotic cell cycle, among which breakdown of the oocyte nuclear envelope (germinal vesicle breakdown, GVBD) occurring at the prophase/metaphase transition is usually regarded as a hallmark of the progress of oocyte maturation. In a number of teleost species, C21 steroids have been shown to be potent initiators of GVBD in vitro and to be present at high levels in plasma of fish undergoing final oocyte maturation. Among C21 steroids, however, only two were identified as naturally occurring MIH in fish: 17α, 20β-dihydroxy-4-pregnen-3-one (17, 20β-DHP) in amago salmon  and 17α, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S), in the Atlantic croaker and spotted sea trout . Testosterone, as well as other C19 steroids, induces GVBD only at high concentrations. Estradiol-17β and other C18 steroids are generally not effective inducers of oocyte maturation in fish . Recently, a strong candidate for the MIH receptor, membrane progestin receptor (mPR), was identified [6, 7]. In zebrafish, two types of mPRs, α and β, were identified . 17α, 20β-DHP has been shown to induce oocyte maturation by stimulating the de novo synthesis of cyclin B, a regulatory subunit of MPF .
Several endocrine-disrupting chemicals or EDCs, Kepon and o,p-DDD, have been reported to antagonize MIH-induced meiotic maturation of fish oocytes in vitro . EDCs such as methoxychlor and ethynyl estradiol also antagonize frog oocyte maturation. One of EDCs, diethylstilbestrol (DES), is a nonsteroidal substance which was prescribed during the late 1940s to early 1970s to pregnant women to prevent abortion, preeclampsia, and other complications of pregnancy. Male and female offspring exposed in utero to DES may develop multiple dysplastic and neoplastic lesions of the reproductive tract, along with other changes, during development . In a previous study, we found that treatment of oocytes with DES alone induces maturation in goldfish and zebrafish . The results suggested that DES might interact with mPR to induce maturation. In the present study, we examined stimulatory and inhibitory effects of other EDCs on zebrafish oocyte maturation and discussed possible groups of DES interacting with the MIH receptor.
Zebrafish were purchased from aquatic dealer and were maintained at 28.5°C on a 14 h light/10 h dark cycle . 17, 20β-DHP, DES, TAM, 4-OHT and 17β-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO). 17α-Estradiol, ethynylestradiol, butyl benzyl phthalate, di (2-ethylhexyl) phthalate and pentachlorophenol were obtained from Wako Pure Chemical Industries (Osaka, Japan). Other chemicals were purchased as follows: resveratorol, Calbiochem (Darmstadt, Germany); DDTs, AccuStandard (New Haven, CT); bisphenol A, Nacalai Tesque (Kyoto, Japan); p-nonylphenol, Kanto Chemical Co. (Tokyo, Japan); 4-octylphenol, Aldrich Chemical Co. (Milwaukee, WI).
Oocyte preparation and in vitro culture
Gravid female zebrafish which possesses full-grown immature oocytes were selected from a group of mixture of 10–50 male and female that were keep in 20 cm × 25 cm square and 25 cm high acryl case with continuous out-flow water. Ovaries of zebrafish were isolated from sacrificed females and placed in fresh zebrafish Ringer's solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.2) and washed with the same solution. Ovaries were dissected into ovarian fragments (each containing 2–10 oocytes) manually by using fine forceps. Fully-grown immature oocytes were exposed in vitro by incubating ovarian fragments in 4 ml of zebrafish Ringer's solution containing each agent (from a 1000-fold stock in ethanol) at 25.0°C or room temperature with gentle agitation (40 rpm). To assess the maturation processes, germinal vesicles (GVs) were examined under a binocular microscope (SMZ645, Nikon, Tokyo, Japan) after placing the oocytes in clearing solution  or GVBD was assessed by scoring the oocytes that became transparent . %GVBD was determined in more than twenty oocytes.
Preparation of oocyte and egg extracts
Intact follicles were carefully isolated by using fine forceps. Groups of twenty intact follicles were transferred to a 1.5-ml Eppendorf micro centrifuge tube and crushed with 5 strokes of a plastic pestle in 200 μl of sample buffer for SDS-PAGE. The samples were centrifuged at 5,000 rpm for 5 min at 4°C in a fixed-angle rotor (MX-300 micro centrifuge, TOMY, Tokyo, Japan). The supernatant (100 μl) was collected for electrophoresis and immunoblotting.
SDS-PAGE and immunoblotting
Antiserums, which recognize the protein band of zebrafish cyclin B1 and mPRα produced in previous study were used . Proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE with 10% gel) by the method of Laemmli , and transferred to Immobilon membrane (Millipore, Billerica, MA). Membranes were blocked in 5% non-fat powdered milk, and incubated with primary antibodies for 1 hr at room temperature. Immunocomplexes were visualized using the ECL detection kit (Amersham Biosciences, Uppsala, Sweden).
Relative potency of various substances in inducing and preventing fish oocyte maturation
The relative effectiveness of 17,20β-DHP and sixteen other agents including EDCs and several steroid hormones in inducing and preventing GVBD was investigated using zebrafish oocytes (Fig. 1). As described previously, DES exhibited strong activity to induce GVBD like 17,20β-DHP. As predicted given their structural similarity to DES, TAM and 4-OHT were also effective in inducing GVBD. TAM and 4-OHT induced 10–20% GVBD at 10 μM. They were relatively less potent than DES and induced 60–70% GVBD even at the highest concentration. As shown in the goldfish oocytes , two estrogens did not induce GVBD at the concentration tested. Nine kinds of EDCs: DDTs, phthalates and phenols, did not induce GVBD as in goldfish. The inhibitory effects of EDCs on 17,20β-DHP-induced oocyte maturation were also examined. Of the fourteen agents tested, only PCP was effective in inhibiting GVBD without pre-incubation (Fig. 1B). When oocytes were treated with 10 μM PCP in addition to 17,20β-DHP, maturation was completely prevented. Methoxychlor also inhibited GVBD when oocytes were pre-incubated with this agent.
EDCs induce natural oocyte maturation
Inhibition of oocyte maturation by PCP
Effect of natural steroids on inducing oocyte maturation
In this study, the effects of EDCs on the maturation of zebrafish oocytes were examined. We found that treatment of oocytes with nonsteroidal substances; TAM and 4-OHT, alone induced maturation as did DES. The morphology and an intracellular molecular event induced by EDCs and 17, 20β-DHP were indistinguishable and suggest that, at least qualitatively, EDCs and 17, 20β-DHP induced the same type of maturation.
TAM and its metabolite 4-OHT share structural similarity with DES and have been shown to have estrogenic effects the same as DES . TAM is a non-steroidal anti-estrogen commonly used in the treatment of advanced breast cancer and as adjuvant therapy following surgery in early stage disease . Its use has also been approved for reducing the incidence of breast cancer among high-risk women . TAM acts as an estrogen antagonist in mammary tissue, but mimics the agnostic effects of estrogen in bone and in the cardiovascular system . In the uterus, TAM acts as a partial estrogen agonist . Previously, we predicted that TAM and 4-OHT might have maturation-inducing activity given their structural similarity to DES. As predicted, these compounds did possess maturation-inducing activity. In general, the potency and biological properties of a compound can be predicted from its structure. It is, therefore, possible to clarify the contribution of a particular structure to the biological response of a target. DES and tamoxifens both have an ethylated stilbene. Studies using DES analogues and tamoxifens indicated that an ethylated stilbene is essential to act as a ligand for mPR to induce oocyte maturation. To consider the possible site of interaction between DES and the MIH receptor, we compared the ability of progestines to induce the maturation of zebrafish in this study.
All of the progestins examined except pregnenolone induced a dose-related stimulation of oocyte maturation similar to that induced by 17,20β-DHP, although potencies were significantly different: 17,20β-DHP > 17α-Prog > 17α-Preg> Prog > Preg (Fig. 5). Conversion of the 20-keto group to a 20β-hydroxy group was the single stimulatory alteration to form a natural MIH, 17,20β-DHP. The results show that addition of a single hydroxyl group to position 17α of progesterone also increased the ability to induce GVBD. Conversion of the 3-hydroxy group to a 3-keto group was also had a stimulatory effect because the potency differed between progesterone and pregnenolone and between 17α-Prog and 17α-Preg. This analytical approach to the structure-activity relationships of steroids revealed that 20β-OH, 17α-OH and 3 = O are critical groups that contribute to the interaction with the MIH receptor. The oxygen atom is thought to make a hydrogen bond with residues in the receptor protein. In the case of 17,20β-DHP, it is thought that three oxygen atoms contribute to a hydrogen bond. DES may mimic the hydrogen bond generated by 20β-OH and 3 = O. Loss of a third hydrogen bond may explain the weak activity of DES to induce oocyte maturation.
Effects of endocrine-disrupting chemicals on oocyte maturation
Effect on oocyte maturation
Pickford and Morris 
inhibition, spotted sea trout
Das and Thomas 
Fort et al. 
stimulation, goldfish zebrafish
Tokumoto et al. 
Although, we have no data to account for the molecular mechanism for preventing maturation by PCP at present, it is hypothesized that PCP acts as an antagonist for 17,20β-DHP, based on the result that no pre-incubation was required for inhibition of 17,20β-DHP-induced oocyte maturation. Previously, we have shown induction of oocyte maturation in fish by an endocrine disrupting chemical (EDC), DES . Recently we have cloned and identified membrane progestin receptor α (mPRα), a strong candidate for MIH receptor . By using the antibody for mPRα, it is suggested that DES induced maturation via interactions with mPRα . Interactions between the MIH receptor and EDCs have been demonstrated previously in fish. Among EDCs, Kepone and o,p'-DDD, have been reported to antagonize binding of 20β-S to the MIH receptor in spotted sea trout, further suggesting that EDCs may interact with mPRs . Direct evidence of the interaction of EDCs with the MIH receptor remains to be obtained.
Based on our findings that TAM and 4-OHT possess the inducing activity for oocyte maturation as well as DES, ethylated stillbene could be a structure interacting with MIH receptor. These agents induce physiological oocyte maturation. PCP, a widely used biocide, completely blocked oocyte maturation. These results demonstrated that EDCs also disrupt progesterone-dependent system as well as estrogen-dependent one.
The physiological significance of effects of EDCs will be confirmed by binding assay which show direct evidence of the interaction of EDCs with the MIH receptor.
This work was supported by the CREST Research Project of the Japan Science and Technology Corporation to Y.N. and Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17570175 to T.T.). Part of this study was performed as the National Institute for Basic Biology Cooperative Research Program (4–110 and 5–108 to T.T). M.T. is a Research Fellow of the Japan Science and Technology Corporation.
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