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Arq. Bras. Med. Vet. Zootec. vol.55 no.3 Belo Horizonte June 2003
Activation of bovine oocytes by strontium combined or not with an electric pulse
Ativação de oócitos bovinos com estrôncio combinado ou não com pulso elétrico
C.L.V. LealI, II; S.C. MéoII; J.M. GarciaII
IDepartamento de Ciências Básicas ZAB/FZEA/USP Av. Duque de Caxias Norte, 225 13635-900 Pirassununga, SP
IIDepartamento de Medicina Veterinária Preventiva e Reprodução Animal - FCAV/UNESP
Keywords: cattle, activation, parthenogenesis, oocyte, strontium, electric pulse
O objetivo deste trabalho foi avaliar as taxas de ativação e de clivagem de oócitos bovinos tratados com estrôncio (10mm de SrCl2), após maturação in vitro por 27-28 horas. No experimento 1, os tratamentos foram: S4 ativação pelo estrôncio por 4 horas; S12 ativação pelo estrôncio por 12 horas; S30 ativação pelo estrôncio por 30 horas; e P ativação por pulso elétrico (3 pulsos de 1,0kv/cm). No experimento 2 os tratamentos foram: PS4 ativação combinada pelo pulso elétrico e pelo estrôncio por 4 horas; S4P ativação pelo estrôncio por 4 horas e pelo pulso elétrico; e PS30 ativação pelo pulso elétrico e pelo estrôncio por 30 horas. No experimento 1, todos os tratamentos apresentaram taxas similares de ativação (83-90%; P>0,05). Para clivagem, P foi melhor (53%; P<0,05) do que todos os tratamentos com estrôncio (6 a 28%). No experimento 2, o tratamento S4P apresentou melhor taxa de ativação (88%; P<0,05) do que PS4 e PS30 (60 e 68%, respectivamente). Para clivagem, observou-se o mesmo padrão, S4P (65%; P<0,05) e PS4 e PS30 (37% e 44%, respectivamente). Conclui-se que o estrôncio é capaz de ativar oócitos bovinos e sua combinação com pulso elétrico não melhora a ativação. Este é o primeiro relato demonstrando que o estrôncio ativa oócitos bovinos.
Palavras-chave: bovino, ativação, partenogênese, oócito, estrôncio, pulso elétrico
During fertilization, the spermatozoa, besides contributing with its DNA, is also responsible for activating the oocyte so it may complete meiosis and initiate embryonic development. With the growing interest in cloning by nuclear transfer (NT) (Macháty et al., 1999) and intracytoplasmic sperm injection (ICSI) (Suttner et al., 2000) activation has been more actively studied, since the transferred nucleus or injected sperm cell are not able to induce activation. Several artificial methods such as electric pulse, ethanol and others, are able to promote activation of the oocyte (Macháty et al., 1999). These techniques aim to mimic the sperm cell, which induces oscillations in intracellular calcium concentrations (Fissore et al., 1992; Sun et al., 1994), leading to the inactivation of MPF (Liu et al., 1998) and allowing the oocyte to resume meiosis and start embryo development. However, most of these treatments, although able to induce activation, do not induce the calcium oscillations (Macháty, Prather, 1998) and may be contributing for the lower efficiency of embryonic development after NT (Fissore et al., 1999). Strontium has shown to be efficient for activating mouse oocytes (Wakayama et al., 1998) and to produce intracellular calcium oscillations similar to that induced by the spermatozoa (Bos-Mikich et al., 1997). However, no data are available regarding the use of strontium to activate bovine oocytes.
The aim of the present study was to assess the activating ability of bovine oocytes cultured in the presence of strontium for different periods of time and if the association with an electric pulse would improve activation rates.
Oocytes were obtained by the aspiration of 3-7mm antral follicles of ovaries collected in slaughterhouse and those recovered presenting homogeneous cytoplasm and at least four layers of cumulus cells were selected for in vitro maturation (IVM). IVM was in TCM 199 + 10% FCS + FSH (0.5µg/ml) + LH (5µg/ml) + estradiol (1µg/ml) + 0.25mM sodium pyruvate + 75µg/ml kanamycin for 27-28 hours. Cumulus cells were removed by treatment with 0.2% hyaluronidase and pipetting. Denuded oocytes with an extruded first polar body were randomly assigned to the following treatments: Experiment 1 - 10mM SrCl2 in Ca2+-free TALP for 4, 12 or 30h (treatments S4, S12 and S30, respectively); electric pulse (treatment P) 3 pulses of 1.0 kV/cm for 100µs, at 5min intervals in 0.28M inositol + 100µM CaCl2 + 100µM MgSO4; Experiment 2 - the combinations PS4, S4P and PS30. After each treatment, the oocytes were cultured in TCM 199 + 5mg/ml BSA for 24 or 48h to assess activation (anaphase-telophase II and pronuclear stages) and cleavage rates, respectively. Oocytes were stained (10µg/ml Hoechst 33342 for 10min) to observe the chromatin by epifluorescence microscopy. All cultures were at 38.5oC under 5% CO2 in air.
Activation rates were high (above 83%, P>0.05) and similar for all treatments (Table 1). For cleavage rates (Table 1), S12 resulted in the lowest cleavage rate (6%, P<0.05) and P was the best treatment (53%, P<0.05). S4 and S30 resulted in intermediate cleavage rates (24 and 28%, respectively).
These results demonstrate that treatment of IVM oocytes with 10 mM SrCl2 activates bovine oocytes at high rates which are comparable to more traditional systems such as electric pulse. However, the cleavage rates were relatively low, indicating that the stimulus triggers initial stages of activation (release from MII arrest up to pronuclear formation) but is not enough to stimulate the next step of the first cell cycle (cleavage). Although SrCl2 is known to be quite effective for activating mouse oocytes (up to 100% activation; Kishikawa et al., 1999), evoking intracellular calcium oscillations similar to spermatozoa (Bos-Mikich et al., 1997), and resulting in high developmental rates (up to 46% blastocyst development; Otaegui et al., 1999), it seems less effective for bovine oocytes. This may be caused by different sensitivity of bovine oocytes to SrCl2 or inability to induce the proper calcium spiking pattern in this species. There is no available information on this regard for bovine oocytes. Also, inadequate concentration of strontium for bovine oocytes could be another reason, since the concentration used in this experiment was the same recommended for mouse oocytes (Bos-Mikich et al., 1997).
In order to try to improve the activation rates, combined treatments of strontium and electric pulse were tested and the results are shown in Tab. 2.
S4P presented the best activation and cleavage rates (88% and 65%, respectively) than those obtained for PS4 (60% and 37%, respectively) and PS30 (68% and 44%, respectively) (P<0.05). Although activation and cleavage rates were reasonable, there was no improvement after combining treatments. In fact, PS4 and PS30 treatments seemed to lower activation rates. It could be speculated that the electric pulse induced one large calcium wave (Robl et al., 1992 apud Macháty, Prather, 1998) and was possibly followed by a sequence of calcium waves (Bos-Mikich et al., 1997). Although this pattern would be more similar to that observed during fertilization (Nakada, Mizuno, 1998), it is also possible that calcium release was excessive under the conditions used in this study. Excess intracellular calcium can be toxic to oocytes (Loi et al., 1998), and this could have reduced viability of treated oocytes. On the other hand, the best combined treatment (S4P) may have caused first, a sequence of calcium waves, followed 4h later by a large wave induced by an electric pulse. The initial waves may have been enough to trigger MPF inactivation, but not to maintain it inactivated. Possibly, after a decrease in these calcium releases, the electric pulse may have given an additional stimulus to avoid MPF reactivation (Collas et al., 1993) and may have activated oocytes which had eventually not been activated by the previous stimulus. Besides, by the time the electric stimulus was given the oocyte would be aged, which may have facilitated activation (Barnes et al., 1993) due to lower levels of MPF (Tian et al., 2002). However, to better explain the observations made in this work, it would be necessary to determine if strontium is able to induce calcium oscillations and MPF inactivation in bovine oocytes. Indirectly, it can be inferred from our results that strontium is able to do so, since oocytes did activate and cleave, but to what extent this inactivation may have occurred remains to be determined. Adjustments to the protocol might provide better results.
According to the results obtained in this work, it can be concluded that: 1) strontium is capable of activating bovine oocytes; 2) the combination of strontium and electric pulse does not improve activation. This is the first report demonstrating that strontium can activate bovine oocytes.
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Suporte financeiro: FAPESP, Brasil, processo 98/12085-1
Recebido para publicação em 20 de agosto de 2002