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The effect of okadaic acid on meiotic maturation of canine oocytes of different size
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Theriogenology 77 (2012) 46 –52 www.theriojournal.com
The effect of okadaic acid on meiotic maturation of canine oocytes of different size Federica Ariu*, Stefano Fois, Daniela Bebbere, Sergio Ledda, Irma Rosati, Maria Teresa Zedda, Salvatore Pau, Luisa Bogliolo Department of Pathology and Veterinary Clinic, University of Sassari, Via Vienna 2 07100 Sassari, Italy Received 12 January 2011; received in revised form 6 July 2011; accepted 6 July 2011
Abstract The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 M), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: ⬍110 m, 110 –120 m, ⬎120 m, and pre-incubated in OA 0.5 M for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 M for 1 h (30.8% P ⬍0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110 –120 m and ⬎120 m groups (P ⬍0.001) compared to control group, but a significantly higher proportion of the oocytes ⬎120 m pre-incubated with OA progressed to MII (51.3% P ⬍0.001). In contrast, smaller oocytes (⬍110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 M for 1 h, improves meiotic maturation. The culture of fully grown (⬎120 m) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation. © 2012 Elsevier Inc. All rights reserved. Keywords: Canine oocyte; Okadaic acid; Oocyte diameter
1. Introduction The lack of optimised conditions for in vitro maturation (IVM) of oocytes is the major obstacle for in vitro embryo production in canine species. The reproductive physiology of the dog is peculiar when compared with that of other species. Unlike other mammalian species, ovarian canine follicles luteinize prior to ovulation and the oocytes are ovulated at onset of the
* Corresponding author: Tel.: 0039079229568; fax: 0039079229414. E-mail address: [email protected] (Federica Ariu). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.07.013
first meiotic division (germinal vesicle). Preovulatory follicles release oocytes into the oviduct and nuclear maturation is completed 48 to 72 h post-ovulation in the presence of high circulating concentrations of progesterone [1,2,3] when the oocyte reaches the mid-portion of the oviduct [4]. From 1976 [5] to date, numerous studies have been conducted to improve success of in vitro maturation of bitch oocytes but an efficient protocol for IVM has not been established. Apart from the work recently published by Songsasen and Wildt [6], where 79.5% of oocytes recovered from follicles ⬎2 mm size reached the second metaphase (MII), usu-
F. Ariu et al. / Theriogenology 77 (2012) 46 –52
ally less than 20% of canine oocytes achieve nuclear maturation [7]. Moreover, only a single blastocyst has been produced after in vitro fertilization of in vitro matured oocytes [8]. The age of the donor bitch [9,10], the stage of the reproductive cycle [11,12], oocyte diameter [8,9], and the size of the ovarian follicle [6] are factors influencing meiotic competence of bitch oocytes cultured in vitro. In particular, oocyte diameter may be a useful selection parameter. It has been reported [8] that large oocytes (⬎120 m diameter) had a higher frequency of maturation than oocytes ⬍110 m diameter. The difficulty of finding an appropriate in vitro maturation system for canine oocytes clearly indicates the need to improve studies of the intrinsic mechanism leading to oocyte meiotic competence of canine oocytes and to identify specific signals inducing meiotic resumption and progression. In most mammals, oocytes resume meiosis in response to specific signals. It is well established that meiotic maturation is controlled by the cytoplasmic M-phase promoting factor (MPF), the key regulator of both mitotic and meiotic cell cycles, and the mitogen-activated protein kinases (MAPKs) which have been implicated in many signal transduction pathways. Among the substances activating M-phase kinases, Okadaic Acid (OA), a polyether fatty acid that specifically inhibits serine/treonine phosphatases 1 and 2A [13,14], induced meiotic resumption of oocytes of many species, including both competent and incompetent mouse oocytes [15]. It has been reported in pig and cattle that treatment of oocytes with OA results in acceleration of germinal vesicle breakdown (GVBD) and of H1 kinase activity [16]. Moreover, OA positively affects meiotic resumption of blue fox oocytes in vitro [17]. The objective of the present study was to determine the effect of OA on in vitro meiotic maturation of canine oocytes. In the first experiment the optimal concentration and exposure time of OA during in vitro maturation was investigated. The second experiment was performed to evaluate the effect of OA on in vitro meiotic resumption and progression until MII stage of oocyte with different diameter. 2. Materials and methods All chemicals in this study were purchased from Sigma Chemical Company (St. Louis. MO, USA) unless stated otherwise.
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2.1. Oocyte recovery Ovaries were collected from bitches at different oestrous phases during routine ovariohysterectomies at local veterinary clinics. The animals were of various breeds and aged 8 mo to 6 y. Reproductive tracts were placed immediately into PBS (Dulbecco’s Phosphate Buffered Saline), containing 1% penicillin (100 IU·mL–) and streptomycin (100 g · mL–) solution at 37 °C and transported to the laboratory. Within 2 h of collection the ovaries were washed free of blood in fresh PBS, placed in a TCM 199 medium buffered with 25 mM HEPES with penicillin–streptomycin (100 g · mL–) at 38.5 °C and then sliced repeatedly to release cumulus-oocyte complexes (COCs). Only COCs with two or more dense layers of cumulus cells, darkly granulated cytoplasm were selected for the experiment. 2.2. In vitro maturation The COCs were matured in TCM 199 supplemented with 20% estrous bitch serum (EBS) and 100 mM cysteamine. The bitch serum [18] for culture was heatinactivated for 30 min at 56 °C and stored at ⫺20 °C. After collection, oocytes were selected on the basis of the experimental design (see section 2.4.) and cultured in groups of 25–35 in 500 L of culture medium, under mineral oil, in a humidified atmosphere of 5% CO2, at 38.5 °C for 72 h. The Okadaic acid was added to the maturation medium and its influence was assessed in two different experiments as described in the experimental design. 2.3. Assessment of meiotic maturation At the end of the culture period (72 h) the meiotic stage of oocytes was evaluated. The oocytes were completely denuded via gentle pipetting with a fine bore glass pipette in TCM 199 buffered with 25 mM HEPES. Irrespective of the morphology of the oocytes, all oocytes were fixed and permeabilized for 3 min at room temperature in a small drop comprising PBS supplemented with 90% (v/v) glycerol and 0.1 mg/mL bis-benzimide (Hoechst 33342) on a glass slide. Subsequently, the oocytes were overlaid with a cover slip and incubated overnight at 4 °C. The nuclear configuration and chromatin morphology were evaluated under fluorescence microscopy (Olympus IX 70) with UV light to determine the stage of meiotic maturation. Oocytes in which chromatin was unidentifiable or not visible were considered as “unidentifiable oocytes.”
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F. Ariu et al. / Theriogenology 77 (2012) 46 –52
Table 1 Meiotic progression of bitch oocytes after incubation with Okadaic acid: effect of concentration and exposition time. Time CTR OA 2 M OA 0.5 M
1h 3h 20 h 1h 3h 20 h
Oocytes (n)
GV (%)
GVBD (%)
Stage of meiotic progression MI (%)
MII (%)
Unid. (%)
57 54 45 73 39 43 69
29 (50.9)a 3 (5.6)b 3 (6.7)b 3 (4.1)b 2 (5.1)b 2 (4.6)b 5 (7.2)b
14 (24.6) 46 (85.2) 36 (80.0) 60 (82.2) 20 (51.3) 34 (79.1) 58 (84.1)
0 0 0 0 5 (12.8) 1 (2.3) 0
4 (7.0)a 4 (7.4)a 4 (8.9)a 6 (8.2)a 12 (30.8)b 4 (9.3)a 6 (8.7)a
10 (17.5)a 1 (1.8)b 2 (4.4)b 4 (5.5)b 0b 2 (4.7)b 0b
GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; Unid, unidentifiable oocytes. Different superscripts within the same column indicate a significant difference a vs b P ⬍ 0.05.
2.4. Experimental design 2.4.1. Experiment 1 In this experiment, the optimal concentration and exposure time of OA during in vitro maturation of canine oocyte was investigated. Oocytes were cultured 1, 3, and 20 h in maturation medium supplemented with 0.5 or 2 M OA [19] and cultured up to 72 h in the same medium without OA at 38.5 °C, 5% CO2. At the end of in vitro maturation, assessment of nuclear maturation was performed according the method previously described. 2.4.2. Experiment 2 The goal of this experiment was to assess the effect of OA on meiotic maturation of canine oocyte in relation to their diameter. The diameter of the oocytes was measured with a video micrometer (Soft Imaging System GmbH, Analysis 2003) on a screen connected to a video camera recorder on an inverted microscope (Olympus IX 70). The video micrometer was calibrated with a stage micrometer, an X20 objective, and an X10 eyepiece. The diameter (excluding the zona pellucida) recovered was the mean of the two measurements made perpendicular to one another [12]. Oocytes were divided into three homogeneous groups according to their diameter: ⬍110 m, 110 –120 m, ⬎120 m. Based on the results of Experiment 1, we used the concentration of 0.5 M OA. The oocytes were preincubated for 1 h in TCM 199 ⫹ 20 % EBS, 100 mM cysteamine and 0.5 M OA. Then, oocytes were cultured up to 72 h in the same medium without OA at 38.5 °C, 5% CO2. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. 2.5. Statistical analysis Data were analysed using MINITAB Release 12.1 software package. Experiment 1 was repeated two
times and Experiment 2 five times. The effect of OA on the percentages of oocytes reaching various stages of nuclear maturation was evaluated by chi-squared test or by Fisher’s exact test, when appropriate. A probability level of P ⬍ 0.05 was considered as significantly different. 3. Results 3.1. Experiment 1 Results demonstrated (Table 1) that incubation with 2 M or 0.5 M OA for 1, 3, and 20 h showed a significant decrease (P ⬎0.001) in the proportion of oocytes that remained at the germinal vesicle stage compared to the control group. The percentage of oocytes reaching MII stage was significantly higher after incubation with 0.5 M OA for 1 h (30.8%, P ⬍0.001) compared to the other groups. Incubation with 2 M OA for 1, 3, and 20 h showed no significant differences in the percentage of oocytes reaching MII stage. 3.2. Experiment 2 Results shown in Table 2 indicated the effect of OA (0.5 M) in relation to oocytes of different size (Fig.1). The OA induced a significant decrease in the number of oocytes at the GV stage in ⬍110 m and 110 –120 m as compared to the control groups. The percentage of oocytes reaching MII in the 110 – 120 m OA group was significantly higher than the 110 –120 m control group. In contrast, the smaller oocytes (⬍110 m) did not develop to MII irrespective of OA exposure. The percentage of GV oocytes in the ⬎120 m OA group (17.9%) was similar to that in the ⬎120 m control group (16.7% P ⬍0.001), but a significantly higher proportion of the oocytes pre-incubated with OA
F. Ariu et al. / Theriogenology 77 (2012) 46 –52
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Table 2 Effect of okadaic acid on meiotic progression of canine oocytes with different diameter. Diameter oocytes (m)
OA
⬍110
— ⫹ — ⫹ — ⫹
110–120 ⬎120
Oocytes (n)
GV (%)
GVBD (%)
Stage of meiotic progression MI (%)
MII (%)
Unid. (%)
58 108 82 130 72 78
46 (95.8)a 78 (83)b 52 (68.4)c 38 (35.2)d 12 (17.6)e 14 (17.9)e
2 (4.2)a 16 (17)b 18 (23.7)c 52 (48.1)d 44 (64.7)e 24 (30.8)f
0 0 2 (2.6) 0 0 0
0a,b 0a 4 (5.3)b 18 (16.7)c 12 (17.7)c 40 (51.3)d
10 (17.3)a 14 (13.0)a 6 (7.3)a 22 (16.9) b 4 (5.5)c 0d
GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; Unid, unidentifiable oocytes. a-f Different superscripts within the same column indicate a significant difference a vs b P ⬍ 0.05.
progressed to MII than did the control oocytes (51.3% vs 16.7% P ⬍0.001). After treatment with OA, 38/40 oocytes ⬎120 m in MII showed a normal chromatin configuration in the MII plate (Fig. 2). The proportion of unidentifiable oocytes was significantly lower in oocytes 110 –120 m (7.3% vs 16.9%) and ⬎120 m (5.5% vs 0%) after incubation with OA compared to controls. 4. Discussion The results of this study demonstrate that OA increases meiotic resumption of canine oocytes greater than 110 m, with maturation rates to MII ranging from 16.7 to 51.3%. This is one of the highest rates of in vitro maturation obtained in canine oocytes. In a previous study [6], where 79.5% of oocytes reached MII, only approximately 20% of large oocytes (⬎120 m) completed nuclear maturation in vitro [8,12]. Our data revealed that the effect of OA is dose and time dependent and is significantly affected by the oocyte diameter. The maximum percentage of meiotic resumption and maturation rate to MII was observed when oocytes ⬎120 m diameter were cultured with 0.5 M OA for 1 h. In the first experiment, the optimal OA concentration and exposure time were investigated. Results demonstrated that incubation with OA showed a significant decrease in oocytes at germinal vesicle stage and that meiotic progression to MII was significantly improved after incubation with 0.5 M OA for 1 h. By varying the OA concentrations (2 M and 0.5 M) and exposure times (1 h, 3 h, 20 h) we identified the concentration-time combination more effective at inducing oocyte meiotic resumption. An increase was seen in oocytes incubated for 1 h with either 2 M and 0.5 M OA. This is similar to work of other authors who incubated mouse oocytes with OA [17]. In contrast, the
treatment with 2 M and 0.5 M for a longer time (20 h), increases the percentage of GVBD, compared to the control, but does not increase rates of MI and MII. Depending on the length of incubation and OA concentration, this molecule probably affects various pathways modulating the cell cycle. Previous work has shown that the exposure to OA results in different levels of expression depending on the experimental protocol: while microinjection of OA drives the oocytes into M-phase, with the formation of a metaphase spindle [19], continuous exposure to this agent results in an abortive M phase, without spindle formation [20,21]. Schwartz and Schultz [22], suggested that a long exposure to OA, in mouse oocytes, interferes with the normal progression of events controlled by protein phosphorylation and/or with changes in protein synthesis induced by meiotic maturation. In Xenopus Leavis [23], the microinjection of oocytes with OA was shown to induce GVBD during the formation of active MPF in a time between 30 – 60 min, obtaining percentages of meiotic maturation of 50%. Data from this study suggested that OA has an important role in activating various proteins used in meiotic maturation and convert pre-MPF into active MPF [23]. Moreover, in the mouse, Hampl et al [24] have showed that microinjection of oocytes with OA causes germinal vesicle breakdown and chromatin condensation with an increase of the percentages of meiotic maturation. In the canine species only one study has been performed on the effect of OA [17]. In this study the authors demonstrated that the addition of 2.5 M OA during culture of fox oocytes larger than 100 m induced 66% of oocytes to undergo GVBD-MI after 20 h of culture. However, the chromatin was abnormally condensed and the meiotic spindles were irregularly
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F. Ariu et al. / Theriogenology 77 (2012) 46 –52
Fig. 1. Canine oocytes with different diameter: (A) ⬍ 100 m, (B) 110 –120 m, (C) ⬎120 m.
assembled. The meiotic maturation to MII stage in oocytes incubated with OA was not investigated in this study. In the second experiment, we determined the effect of OA on the meiotic competence of canine oocytes in relation to oocyte diameter. Data obtained in other species demonstrated that OA induced meiotic resumption even in incompetently growing oocytes [15]. This is similar to our work where in the addition of 0.5 M OA during IVM induced a higher resumption of meiosis in all size groups of oocytes compared to the control groups; only the 110 – 120 m and ⬎120 m oocytes exposed to OA reached maturation to MII stage. The results of our work with OA canine oocytes offer direct correlation between oocyte size and meiotic competence. This is similar to work with different livestock species, which showed that meiotic competence and oocyte fertilization are related to follicular and oocyte diameter [25-29]. In this study, the diameter and meiotic competence of oocytes was not examined in relation to the status of the estrous cycle of the ovaries.We believe oocyte diameter is a useful selection parameter for canine oocyte in vitro maturation. According to the literature, oocyte diameter is an important factor when assessing the meiotic competence of canine oocytes collected from cortical follicles irrespective of the oestrous cycle ovary [8,12]. The relationships between stage of reproductive cycle, oocyte dimension, and meiotic competence are not completely clarified and results are still contradictory. Hewitt and England [9] reported that oocytes with the ⬎120 m size demonstrated a size-related ability to undergo meiotic maturation, irrespective of the oestrous cycle. Otoi et al. [8] showed a clear relationship between canine oocyte diameter and meiotic competence following in vitro culture. A significant decrease in oocytes greater than 120 m was found in anestrous period in comparison to estrous or dioestrus cycle [12]. It has been previously reported that the addition of OA could affect the spindle assembly and chromatin morphology in mouse oocytes [19,30]. We demonstrate that culture of canine oocytes OA at concentration as low as 0.5 M for short times (1h) maintains a normal morphology of MII oocyte chromatin. In conclusion, the addition of OA to the in vitro maturation system of canine oocytes increased meiotic resumption and maturation rates to MII. This is encouraging for the optimization of canine in vitro maturation. The biochemical-molecular mechanism of the action of OA in canine oocytes should be investigated in order to better understand which are the factors involved in canine oocyte meiotic resumption and progression to
F. Ariu et al. / Theriogenology 77 (2012) 46 –52
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Fig. 2. Canine oocytes ⬎120 m in vitro matured with OA 0.5 M,1h. (A) cumulus-oocyte complexes during IVM; (B) denuded MII oocyte; (C) MII oocyte stained with Hoechst 33342; (D) magnification of chromatin morphology in metaphase plate (MII, chromosomes of metaphase plate; PB, First polar body).
establish an in vitro system for the development of fully competent canine oocytes. Acknowledgments The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. The animal experiments were approved by the Animal Care and Use Committee of the University of Sassari. This work was supported by the Regione Autonoma della Sardegna fse Sardegna 2007–2013, Regional law August, 2007, n°7. References [1] Wildt DE, Chakraborty PK, Panko WB, Seager SW. Relationship of reproductive behavior serum luteinizing hormone and time of ovulation in the bitch. Biol Reprod 1978;18:561–70.
[2] Concannon PW, McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog. Reprod Fert 1989;39:3–25. [3] Reynaud K, Fontbonne A, Marseloo N, Thoumire S, Chebrout M, de Lesegno CV, Chastant-Maillard S. In vivo meiotic resumpion, fertilization and early embryonic development in the bitch. Reproduction 2005;130:193–201. [4] Tsutsui T. Gamete phisiology and timing of ovulation and fertilisation in dogs. Reprod Fert 1989;39:269 –75. [5] Mahi CA, Yanagimaci R. Maturation and sperm penetration of canine oocytes in vitro. Exp Zool 1976;16:189 –93. [6] Songsasen N, Wildt DE. Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes. Mol Reprod Dev 2005; 72:113–9. [7] Songsasen N, Wildt DE. Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci 2007;98:2–22. [8] Otoi T, Fujii M, Tanaka M, Ooka A, Suzuki T. Canine oocyte diameter in relation to meiotic competence and sperm penetration. Theriogenology 2000;54:535– 42.
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[9] Hewitt DA, England GCW. The effect of oocytes maturation and age upon oocyte nuclear maturation in vitro. Theriogenology 1998;49:957– 66. [10] Songsasen N, Yu I, Leibo SP. Nuclear maturation of canine oocytes cultured in protein-free media. Mol Reprod Dev 2002; 62:407–15. [11] Yamada S, Shimazu Y, Kawano Y, Nakazawa M, Naito K, Toyoda Y. In vitro maturation and fertilization of preovulatory dog oocytes. Reprod Fertil 1993;47:227–9. [12] Otoi T, Ooka A, Murakami M, Kurniani Karja NW, Suzuki T. Size distribution and meiotic competence of oocytes obtained from bitch ovaries at various stages of the oestrous cycle. Reprod Fertil Dev 2001;13:151–5. [13] Bialojan C, Takai A.Inhibitory effect of marine sponge toxin, okadaic acid, on protein phosphatases. Biochem 1988;256: 283–90. [14] Cohen P, Holmes CF, Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 1990 Mar;15:98 –102. [15] de Vantèry Arrighi C, Campana A, Schorderet-Slatkine S. Arole for the MEK-MAPK pathway in Okadaic Acid-induced meiotic resumption of incompetent growing mouse oocytes is controlled at both translational and posttranslational levels. Biol Reprod 2000;63:658 – 65. [16] Sun QY, Wu GM, Lai L. Regolation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro. Biol Reprod 2002;66:580 – 8. [17] Srsen V, Kalous J, Nagyova E, Sutovsky P, King WA, Motlik J. Effects of follicle-stimulating hormone, bovine somototrophin and okadaic acid on cumulus expansion and nuclear maturation of Blue fox (Alopex lagopus) oocytes in vitro. Zygote 1998;6:299 –309. [18] Otoi T, Fujii M, Tanaka M, Ooka A, Suzuki T. Effect of serum on the in vitro maturation of canine oocytes. Reprod Fertil Dev 1999b;11:387–90.
[19] Gavin AC, Vassalli JD, Cavadore JC, Schorderet-Slatkine S.Okadaic acid and p13 suc1 modulate the reinitiation of meiosis in mouse oocytes. Mol Repr Dev 1992;33:287–96. [20] Alexandre H, Van Cauwenberge A, Tsukitani Y, Mulnard J. Pleiotropic effect of okadaic acid on maturing mouse oocytes. Development 1991;112:971– 80. [21] Rime H, Huchon D, Jessus C, Goris J, Merlevede W, Ozon R. Characterization of MPF activation by okadaic acid in Xenopus oocyte. Cell Differ Dev 1990;29:47–58. [22] Schwartz DA, Schultz RM. Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and onecell embryos. Dev Biol 1991;145:119 –27. [23] Goris J, Hermann J, Hendrix P, Ozon R, Mervede W.Okadaic acid a specific protein phosphatase inhibitor, induced maturation and MPF formation in Xenopus laevis oocytes. EEBS 1989;245:91–94. [24] Hampl A., Eppig JJ. Translational regulation of the gradual increase in histone III kinase activity in maturing mouse oocytes. Mol Reprod Dev 1995;40:9 –15. [25] Lonergan P, Carolan C, Mermillod P. Development of bovine embryos in vitro following oocyte maturation under defined conditions. Reprod Nutr Dev 1994;34:329 –39. [26] Motlik J, Fulka J. Factors affecting meiotic competence in pig oocytes. Therigenology 1986;25:87–96. [27] Anguita B, Jimenez-Macedo AR, Izquierdo D, Mogas T, Paramio MT.Effect of oocyte diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity in prepubertal goat oocytes. Theriogenology 2007;67:526 –36. [28] Ledda S, Bogliolo L, Leoni G, Naitana S. Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep. Reprod Nutr Dev 1999;39:503– 8. [29] Otoi T, Yamamoto K, Koyama N, Tachikawa S, Suzuki T. Bovine oocyte diameter in relation to developmental competence. Theriogenology 1997;48:769 –74. [30] Sun QY, Breitbart H, Schatten H. Role of the MAPK cascade in mammalian germ cells. Reprod Fertil Dev 1999;11:443–50.
Theriogenology 77 (2012) 46 –52 www.theriojournal.com
The effect of okadaic acid on meiotic maturation of canine oocytes of different size Federica Ariu*, Stefano Fois, Daniela Bebbere, Sergio Ledda, Irma Rosati, Maria Teresa Zedda, Salvatore Pau, Luisa Bogliolo Department of Pathology and Veterinary Clinic, University of Sassari, Via Vienna 2 07100 Sassari, Italy Received 12 January 2011; received in revised form 6 July 2011; accepted 6 July 2011
Abstract The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 M), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: ⬍110 m, 110 –120 m, ⬎120 m, and pre-incubated in OA 0.5 M for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 M for 1 h (30.8% P ⬍0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110 –120 m and ⬎120 m groups (P ⬍0.001) compared to control group, but a significantly higher proportion of the oocytes ⬎120 m pre-incubated with OA progressed to MII (51.3% P ⬍0.001). In contrast, smaller oocytes (⬍110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 M for 1 h, improves meiotic maturation. The culture of fully grown (⬎120 m) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation. © 2012 Elsevier Inc. All rights reserved. Keywords: Canine oocyte; Okadaic acid; Oocyte diameter
1. Introduction The lack of optimised conditions for in vitro maturation (IVM) of oocytes is the major obstacle for in vitro embryo production in canine species. The reproductive physiology of the dog is peculiar when compared with that of other species. Unlike other mammalian species, ovarian canine follicles luteinize prior to ovulation and the oocytes are ovulated at onset of the
* Corresponding author: Tel.: 0039079229568; fax: 0039079229414. E-mail address: [email protected] (Federica Ariu). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.07.013
first meiotic division (germinal vesicle). Preovulatory follicles release oocytes into the oviduct and nuclear maturation is completed 48 to 72 h post-ovulation in the presence of high circulating concentrations of progesterone [1,2,3] when the oocyte reaches the mid-portion of the oviduct [4]. From 1976 [5] to date, numerous studies have been conducted to improve success of in vitro maturation of bitch oocytes but an efficient protocol for IVM has not been established. Apart from the work recently published by Songsasen and Wildt [6], where 79.5% of oocytes recovered from follicles ⬎2 mm size reached the second metaphase (MII), usu-
F. Ariu et al. / Theriogenology 77 (2012) 46 –52
ally less than 20% of canine oocytes achieve nuclear maturation [7]. Moreover, only a single blastocyst has been produced after in vitro fertilization of in vitro matured oocytes [8]. The age of the donor bitch [9,10], the stage of the reproductive cycle [11,12], oocyte diameter [8,9], and the size of the ovarian follicle [6] are factors influencing meiotic competence of bitch oocytes cultured in vitro. In particular, oocyte diameter may be a useful selection parameter. It has been reported [8] that large oocytes (⬎120 m diameter) had a higher frequency of maturation than oocytes ⬍110 m diameter. The difficulty of finding an appropriate in vitro maturation system for canine oocytes clearly indicates the need to improve studies of the intrinsic mechanism leading to oocyte meiotic competence of canine oocytes and to identify specific signals inducing meiotic resumption and progression. In most mammals, oocytes resume meiosis in response to specific signals. It is well established that meiotic maturation is controlled by the cytoplasmic M-phase promoting factor (MPF), the key regulator of both mitotic and meiotic cell cycles, and the mitogen-activated protein kinases (MAPKs) which have been implicated in many signal transduction pathways. Among the substances activating M-phase kinases, Okadaic Acid (OA), a polyether fatty acid that specifically inhibits serine/treonine phosphatases 1 and 2A [13,14], induced meiotic resumption of oocytes of many species, including both competent and incompetent mouse oocytes [15]. It has been reported in pig and cattle that treatment of oocytes with OA results in acceleration of germinal vesicle breakdown (GVBD) and of H1 kinase activity [16]. Moreover, OA positively affects meiotic resumption of blue fox oocytes in vitro [17]. The objective of the present study was to determine the effect of OA on in vitro meiotic maturation of canine oocytes. In the first experiment the optimal concentration and exposure time of OA during in vitro maturation was investigated. The second experiment was performed to evaluate the effect of OA on in vitro meiotic resumption and progression until MII stage of oocyte with different diameter. 2. Materials and methods All chemicals in this study were purchased from Sigma Chemical Company (St. Louis. MO, USA) unless stated otherwise.
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2.1. Oocyte recovery Ovaries were collected from bitches at different oestrous phases during routine ovariohysterectomies at local veterinary clinics. The animals were of various breeds and aged 8 mo to 6 y. Reproductive tracts were placed immediately into PBS (Dulbecco’s Phosphate Buffered Saline), containing 1% penicillin (100 IU·mL–) and streptomycin (100 g · mL–) solution at 37 °C and transported to the laboratory. Within 2 h of collection the ovaries were washed free of blood in fresh PBS, placed in a TCM 199 medium buffered with 25 mM HEPES with penicillin–streptomycin (100 g · mL–) at 38.5 °C and then sliced repeatedly to release cumulus-oocyte complexes (COCs). Only COCs with two or more dense layers of cumulus cells, darkly granulated cytoplasm were selected for the experiment. 2.2. In vitro maturation The COCs were matured in TCM 199 supplemented with 20% estrous bitch serum (EBS) and 100 mM cysteamine. The bitch serum [18] for culture was heatinactivated for 30 min at 56 °C and stored at ⫺20 °C. After collection, oocytes were selected on the basis of the experimental design (see section 2.4.) and cultured in groups of 25–35 in 500 L of culture medium, under mineral oil, in a humidified atmosphere of 5% CO2, at 38.5 °C for 72 h. The Okadaic acid was added to the maturation medium and its influence was assessed in two different experiments as described in the experimental design. 2.3. Assessment of meiotic maturation At the end of the culture period (72 h) the meiotic stage of oocytes was evaluated. The oocytes were completely denuded via gentle pipetting with a fine bore glass pipette in TCM 199 buffered with 25 mM HEPES. Irrespective of the morphology of the oocytes, all oocytes were fixed and permeabilized for 3 min at room temperature in a small drop comprising PBS supplemented with 90% (v/v) glycerol and 0.1 mg/mL bis-benzimide (Hoechst 33342) on a glass slide. Subsequently, the oocytes were overlaid with a cover slip and incubated overnight at 4 °C. The nuclear configuration and chromatin morphology were evaluated under fluorescence microscopy (Olympus IX 70) with UV light to determine the stage of meiotic maturation. Oocytes in which chromatin was unidentifiable or not visible were considered as “unidentifiable oocytes.”
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Table 1 Meiotic progression of bitch oocytes after incubation with Okadaic acid: effect of concentration and exposition time. Time CTR OA 2 M OA 0.5 M
1h 3h 20 h 1h 3h 20 h
Oocytes (n)
GV (%)
GVBD (%)
Stage of meiotic progression MI (%)
MII (%)
Unid. (%)
57 54 45 73 39 43 69
29 (50.9)a 3 (5.6)b 3 (6.7)b 3 (4.1)b 2 (5.1)b 2 (4.6)b 5 (7.2)b
14 (24.6) 46 (85.2) 36 (80.0) 60 (82.2) 20 (51.3) 34 (79.1) 58 (84.1)
0 0 0 0 5 (12.8) 1 (2.3) 0
4 (7.0)a 4 (7.4)a 4 (8.9)a 6 (8.2)a 12 (30.8)b 4 (9.3)a 6 (8.7)a
10 (17.5)a 1 (1.8)b 2 (4.4)b 4 (5.5)b 0b 2 (4.7)b 0b
GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; Unid, unidentifiable oocytes. Different superscripts within the same column indicate a significant difference a vs b P ⬍ 0.05.
2.4. Experimental design 2.4.1. Experiment 1 In this experiment, the optimal concentration and exposure time of OA during in vitro maturation of canine oocyte was investigated. Oocytes were cultured 1, 3, and 20 h in maturation medium supplemented with 0.5 or 2 M OA [19] and cultured up to 72 h in the same medium without OA at 38.5 °C, 5% CO2. At the end of in vitro maturation, assessment of nuclear maturation was performed according the method previously described. 2.4.2. Experiment 2 The goal of this experiment was to assess the effect of OA on meiotic maturation of canine oocyte in relation to their diameter. The diameter of the oocytes was measured with a video micrometer (Soft Imaging System GmbH, Analysis 2003) on a screen connected to a video camera recorder on an inverted microscope (Olympus IX 70). The video micrometer was calibrated with a stage micrometer, an X20 objective, and an X10 eyepiece. The diameter (excluding the zona pellucida) recovered was the mean of the two measurements made perpendicular to one another [12]. Oocytes were divided into three homogeneous groups according to their diameter: ⬍110 m, 110 –120 m, ⬎120 m. Based on the results of Experiment 1, we used the concentration of 0.5 M OA. The oocytes were preincubated for 1 h in TCM 199 ⫹ 20 % EBS, 100 mM cysteamine and 0.5 M OA. Then, oocytes were cultured up to 72 h in the same medium without OA at 38.5 °C, 5% CO2. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. 2.5. Statistical analysis Data were analysed using MINITAB Release 12.1 software package. Experiment 1 was repeated two
times and Experiment 2 five times. The effect of OA on the percentages of oocytes reaching various stages of nuclear maturation was evaluated by chi-squared test or by Fisher’s exact test, when appropriate. A probability level of P ⬍ 0.05 was considered as significantly different. 3. Results 3.1. Experiment 1 Results demonstrated (Table 1) that incubation with 2 M or 0.5 M OA for 1, 3, and 20 h showed a significant decrease (P ⬎0.001) in the proportion of oocytes that remained at the germinal vesicle stage compared to the control group. The percentage of oocytes reaching MII stage was significantly higher after incubation with 0.5 M OA for 1 h (30.8%, P ⬍0.001) compared to the other groups. Incubation with 2 M OA for 1, 3, and 20 h showed no significant differences in the percentage of oocytes reaching MII stage. 3.2. Experiment 2 Results shown in Table 2 indicated the effect of OA (0.5 M) in relation to oocytes of different size (Fig.1). The OA induced a significant decrease in the number of oocytes at the GV stage in ⬍110 m and 110 –120 m as compared to the control groups. The percentage of oocytes reaching MII in the 110 – 120 m OA group was significantly higher than the 110 –120 m control group. In contrast, the smaller oocytes (⬍110 m) did not develop to MII irrespective of OA exposure. The percentage of GV oocytes in the ⬎120 m OA group (17.9%) was similar to that in the ⬎120 m control group (16.7% P ⬍0.001), but a significantly higher proportion of the oocytes pre-incubated with OA
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Table 2 Effect of okadaic acid on meiotic progression of canine oocytes with different diameter. Diameter oocytes (m)
OA
⬍110
— ⫹ — ⫹ — ⫹
110–120 ⬎120
Oocytes (n)
GV (%)
GVBD (%)
Stage of meiotic progression MI (%)
MII (%)
Unid. (%)
58 108 82 130 72 78
46 (95.8)a 78 (83)b 52 (68.4)c 38 (35.2)d 12 (17.6)e 14 (17.9)e
2 (4.2)a 16 (17)b 18 (23.7)c 52 (48.1)d 44 (64.7)e 24 (30.8)f
0 0 2 (2.6) 0 0 0
0a,b 0a 4 (5.3)b 18 (16.7)c 12 (17.7)c 40 (51.3)d
10 (17.3)a 14 (13.0)a 6 (7.3)a 22 (16.9) b 4 (5.5)c 0d
GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; Unid, unidentifiable oocytes. a-f Different superscripts within the same column indicate a significant difference a vs b P ⬍ 0.05.
progressed to MII than did the control oocytes (51.3% vs 16.7% P ⬍0.001). After treatment with OA, 38/40 oocytes ⬎120 m in MII showed a normal chromatin configuration in the MII plate (Fig. 2). The proportion of unidentifiable oocytes was significantly lower in oocytes 110 –120 m (7.3% vs 16.9%) and ⬎120 m (5.5% vs 0%) after incubation with OA compared to controls. 4. Discussion The results of this study demonstrate that OA increases meiotic resumption of canine oocytes greater than 110 m, with maturation rates to MII ranging from 16.7 to 51.3%. This is one of the highest rates of in vitro maturation obtained in canine oocytes. In a previous study [6], where 79.5% of oocytes reached MII, only approximately 20% of large oocytes (⬎120 m) completed nuclear maturation in vitro [8,12]. Our data revealed that the effect of OA is dose and time dependent and is significantly affected by the oocyte diameter. The maximum percentage of meiotic resumption and maturation rate to MII was observed when oocytes ⬎120 m diameter were cultured with 0.5 M OA for 1 h. In the first experiment, the optimal OA concentration and exposure time were investigated. Results demonstrated that incubation with OA showed a significant decrease in oocytes at germinal vesicle stage and that meiotic progression to MII was significantly improved after incubation with 0.5 M OA for 1 h. By varying the OA concentrations (2 M and 0.5 M) and exposure times (1 h, 3 h, 20 h) we identified the concentration-time combination more effective at inducing oocyte meiotic resumption. An increase was seen in oocytes incubated for 1 h with either 2 M and 0.5 M OA. This is similar to work of other authors who incubated mouse oocytes with OA [17]. In contrast, the
treatment with 2 M and 0.5 M for a longer time (20 h), increases the percentage of GVBD, compared to the control, but does not increase rates of MI and MII. Depending on the length of incubation and OA concentration, this molecule probably affects various pathways modulating the cell cycle. Previous work has shown that the exposure to OA results in different levels of expression depending on the experimental protocol: while microinjection of OA drives the oocytes into M-phase, with the formation of a metaphase spindle [19], continuous exposure to this agent results in an abortive M phase, without spindle formation [20,21]. Schwartz and Schultz [22], suggested that a long exposure to OA, in mouse oocytes, interferes with the normal progression of events controlled by protein phosphorylation and/or with changes in protein synthesis induced by meiotic maturation. In Xenopus Leavis [23], the microinjection of oocytes with OA was shown to induce GVBD during the formation of active MPF in a time between 30 – 60 min, obtaining percentages of meiotic maturation of 50%. Data from this study suggested that OA has an important role in activating various proteins used in meiotic maturation and convert pre-MPF into active MPF [23]. Moreover, in the mouse, Hampl et al [24] have showed that microinjection of oocytes with OA causes germinal vesicle breakdown and chromatin condensation with an increase of the percentages of meiotic maturation. In the canine species only one study has been performed on the effect of OA [17]. In this study the authors demonstrated that the addition of 2.5 M OA during culture of fox oocytes larger than 100 m induced 66% of oocytes to undergo GVBD-MI after 20 h of culture. However, the chromatin was abnormally condensed and the meiotic spindles were irregularly
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Fig. 1. Canine oocytes with different diameter: (A) ⬍ 100 m, (B) 110 –120 m, (C) ⬎120 m.
assembled. The meiotic maturation to MII stage in oocytes incubated with OA was not investigated in this study. In the second experiment, we determined the effect of OA on the meiotic competence of canine oocytes in relation to oocyte diameter. Data obtained in other species demonstrated that OA induced meiotic resumption even in incompetently growing oocytes [15]. This is similar to our work where in the addition of 0.5 M OA during IVM induced a higher resumption of meiosis in all size groups of oocytes compared to the control groups; only the 110 – 120 m and ⬎120 m oocytes exposed to OA reached maturation to MII stage. The results of our work with OA canine oocytes offer direct correlation between oocyte size and meiotic competence. This is similar to work with different livestock species, which showed that meiotic competence and oocyte fertilization are related to follicular and oocyte diameter [25-29]. In this study, the diameter and meiotic competence of oocytes was not examined in relation to the status of the estrous cycle of the ovaries.We believe oocyte diameter is a useful selection parameter for canine oocyte in vitro maturation. According to the literature, oocyte diameter is an important factor when assessing the meiotic competence of canine oocytes collected from cortical follicles irrespective of the oestrous cycle ovary [8,12]. The relationships between stage of reproductive cycle, oocyte dimension, and meiotic competence are not completely clarified and results are still contradictory. Hewitt and England [9] reported that oocytes with the ⬎120 m size demonstrated a size-related ability to undergo meiotic maturation, irrespective of the oestrous cycle. Otoi et al. [8] showed a clear relationship between canine oocyte diameter and meiotic competence following in vitro culture. A significant decrease in oocytes greater than 120 m was found in anestrous period in comparison to estrous or dioestrus cycle [12]. It has been previously reported that the addition of OA could affect the spindle assembly and chromatin morphology in mouse oocytes [19,30]. We demonstrate that culture of canine oocytes OA at concentration as low as 0.5 M for short times (1h) maintains a normal morphology of MII oocyte chromatin. In conclusion, the addition of OA to the in vitro maturation system of canine oocytes increased meiotic resumption and maturation rates to MII. This is encouraging for the optimization of canine in vitro maturation. The biochemical-molecular mechanism of the action of OA in canine oocytes should be investigated in order to better understand which are the factors involved in canine oocyte meiotic resumption and progression to
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Fig. 2. Canine oocytes ⬎120 m in vitro matured with OA 0.5 M,1h. (A) cumulus-oocyte complexes during IVM; (B) denuded MII oocyte; (C) MII oocyte stained with Hoechst 33342; (D) magnification of chromatin morphology in metaphase plate (MII, chromosomes of metaphase plate; PB, First polar body).
establish an in vitro system for the development of fully competent canine oocytes. Acknowledgments The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. The animal experiments were approved by the Animal Care and Use Committee of the University of Sassari. This work was supported by the Regione Autonoma della Sardegna fse Sardegna 2007–2013, Regional law August, 2007, n°7. References [1] Wildt DE, Chakraborty PK, Panko WB, Seager SW. Relationship of reproductive behavior serum luteinizing hormone and time of ovulation in the bitch. Biol Reprod 1978;18:561–70.
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