The Influence of Some Amino Acids in a Chemical Defined Media on In Vitro Maturation and ICSI Fertilization of Swine Oocytes

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1 Bulletin UASVM Animal Science and Biotechnologies, 67(1-2)/2010 Print ISSN ; Electronic ISSN X The Influence of Some Amino Acids in a Chemical Defined Media on In Vitro Maturation and ICSI Fertilization of Swine Oocytes Iulian ROMAN, Vasile MICLEA, Andrea HETTIG, Marius ZĂHAN, Ileana MICLEA, Florin VARO-GHIURU, Alexandru RUSU University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, 3- Manastur Street, Cluj-Napoca, Romania; antonius.roman@gmail.com Abstract. Supplementation of a chemical defined in vitro maturation (IVM) media with some amino acids was examined in the terms of oocyte maturation and fecundation rate following intracytoplasmic sperm injection (ICSI). Addition of glutamine resulted in a higher (p<0.0) in vitro maturation rate, compared to the total absence of amino acids in media, but in terms of fecundation rate, none of the amino acids used increased the fecundation rate in our research. Keywords: ICSI, IVM, swine oocytes, amino acids INTRODUCTION Amino acids play important roles as osmolytes (Anbari and Schultz, 1993), (Palacin et al., 1998), intracellular buffers (Edwards et al., 1998) and energy substrates (Rose-Hellekant et al., 1998), and they improve in vitro embryonic development and implantation after transfer to surrogate mice (Van Winkle and Campione, 1996). Amino acids enhance embryonic development and blastocyst formation in mice (Monis and Bavister, 1990), hamsters (Gardner and Lane, 1993) and rats (Miyoshi et al., 199). Many studies have investigated the influence of essential or nonessential amino acids in a group on oocyte maturation or embryo development. Amino acids had stimulatory or inhibitory effects on hamster embryo development depending on individual amino acids (Bavister and Arlotto, 1990), but few studies have examined the effect of individual amino acids in a chemically defined medium on pig oocyte maturation and subsequent development after fertilization (Hong et al., 2004; Kobayashi et al., 2006). Nevertheless, in these studies the in vitro fecundation (IVF) was carried out by the classic technique, a technique were the incidence of polyspermy is high. The polyspermy problem is solved using the ICSI technique, since only one sperm is placed inside the oocyte. However, the already low blastocyst proportion obtained through traditional IVF after embryo culture (Machaty et al., 1998), drops drastically after ICSI (Probst and Rath, 2003). The reasons behind this failure that could be related to the oocyte, to the sperm or to the zygote development, need to be investigated. MATERIALS AND METHODS The boar semen was collected by gloved hand method from two commercial crossbreed boars. The volume, concentration and mobility were examined on the fresh semen, and the good quality semen was further diluted with Beltsville Thawing Solution (BTS) and stored at 17ºC. Ovaries were collected from slaughtered gilts from a local abattoir and transported within hours from scarification in a sterile saline solution. Oocyte collection was carried out 424

2 by aspiration with a 21G needle attached to a 10 ml syringe then the oocytes are introduced in harvest media (M199, Sigma-Aldrich, Germany) supplemented with 2.2g/l NaHCO3 and antibiotics (Penicillin sodium salt and Streptomycin sulfate). Ten oocytes were placed in micro-drops (3 µl) of NCSU 37 medium, supplemented with various concentrations of amino acids, depending of treatment, and hormones (10 UI/ml FHS; 10 UI/ml hcg). After hours of culture in %CO2 atmosphere, high humidity and 37ºC, the oocytes were mechanically denuded using a 100µl micropipette and examined under a reverse contrast microscope for the maturation status. We have analyzed the morphological shape of the oocyte, the presence or absence of the first polar body, the ovoplasm granulation and the perivitelin space. The mature oocytes were injected with a single spermatozoon, using a Narishige micromanipulation station, equipped with Eppendorf holding pipettes and injection needles, under a inverted contrast heated stage Olympus XI-1 microscope. The mature oocytes were placed with the first polar body at 6 or 12 o clock, and the injection was made at 3 o clock. Diluted semen was three times centrifuged at 800 x g, for 6 minutes, and the pellet was re-suspended in TALP medium. Sperm immobilization was carried out in a 0µl drop of 10% Polyvinylpyrrolidone (PVP) in TALP medium. Injected oocytes were placed in 30µl drops of NCSU 23 medium (Long et. al., 1999) supplemented with 0µM β-mercaptoethanol, 0µM Cysteine, and 0.4% bovine serum albumin, and covered with sterile paraffin oil. The fecundation rate was assessed 48 hours after injection in terms of embryo formation. The treatments for maturation media were according to the intrafollicular amino acid concentration, assessed by Hong and Lee (2007). Therefore, the treatments were: Control (without amino acid addition), +GLN 0.18 mm Glutamine, +CYS 0.7 mm Cysteine, + ANS mm Asparagine and + LYS mm Lysine. RESULTS AND DISCUSSION After hours of incubation, the oocytes were examined, and the results are showed in Tab. 1. Influence of amino acid addition over the in vitro maturation of porcine oocytes Tab. 1 No. of Maturation % (Mean ± Treatment N repetitions S.D) Control ± a + GLN ± b + CYS ± ab + ASN ± a + LYS ± 8.24 ab NOTE: Different letters between treatments denote significant differences (p<0.0, One-way ANOVA, Tukey-Kramer Multiple Comparisons Test). The results show significant differences between +GLN treatment, control and +ASP treatments. No differences were fond between +ASN and control, asparagine being the single amino acid determined in the follicular fluid that increases it s concentration as the follicle grows from < 3mm to > 8 mm in diameter. The glutamine treatment is the only who gave similar results as using consecrated IVM media (Roman et al., 2010; Hong and Lee, 2007), with addition of serum components, follicular fluid, or complexes of amino acids. High variability was found between repetitions in the +CYS treatment, illustrated by the high standard deviation, witch is probably the reason why this treatment does not offer significant 42

3 benefits in our research, because it is well known the important role of this amino-acid for the cytoplasmic maturation (Kishida et al., 2004). Addition of glutamine to the defined chemical media gave the best results in our study, as expected, because GLN is present in almost every media used for porcine oocyte manipulation, maturation or fecundation, and together with glutamic acid, alanine and glycine are the most abundant amino acids present in the intrafollicular fluid (Hong and Lee, 2007). Forty-eighth hours after ICSI, the injected oocytes were examined for embryo formation, and the results are showed in Tab. 2. Tab. 2 Influence of amino acid addition in IVM media on the fecundation rate Treatment N (injected oocytes) No. of repetitions Embryo formation % (mean ± S.D) Control + GLN + CYS + ASN + LYS ,86 ± 42.33a 62,66 ± 34,19a ± 31.83a ± 21.67a 3.43 ± 24.44a No significant differences were found between treatments, probably due to the high standard deviation resulted, because the average percent between treatments (e.g. +GLN vs. + LYS) seem to be very different. Also, in the case of fecundation rate, the best results were when 0.18mM Glutamine are added to the IVM medium, similar to the fecundation rate obtained normally when IVM medium is supplemented with serum components and follicular fluid (Roman et al., 2010). No doubt that to reduce the high variation among treatments it is needed to increase the number of injected oocytes and the number of repetitions, in order to eliminate as much as possible the oocyte degeneration caused by the microinjection technique. Looking strictly to the average values obtained, we can assume that ASP and GLN have a benefic effect over the fecundation rate, with similar results. Also we have observed slight differences in terms of oocyte quality, especially between +GLN and the other treatments. As presented in figure 1 the perivittelin space has a uniform distribution and the cytoplasm is well shaped and the oolema without irregularities. In the case of control and +ASN or + LYS treatments (Fig. 2), we can observe a slightly larger perivittelin space and a overall irregular shape. We assume that is a consequence of rapid consumption of medium compounds (amino acids and energetic substances), and by consequence the decrease of IVM medium osmotic pressure. Fig. 1. In vitro matured porcine oocyte with + GLN treatment, prepared for injection Fig. 2. In vitro matured porcine oocyte with + ASN treatment, prepared for injection 426

4 Hong et al. (2004) suggests that amino acid addition to porcine IVM media is better expressed in the presence of gonadotrophines and EGF (Epidermal Growth Factor), due to the fact that the exogenous hormones induces changes in the oocyte metabolism and the amino acid impact is higher. Some studies have quantified the effect of amino acid addition to the IVM media (Hong and Lee, 2007; Hong et al., 2004) in terms of decrease of polyspermie following IVF, but in our case this aspect was bypassed by the use of ICSI technique. The addition of Cysteine to IVM medium did not improve the fecundation rate in our study, despite the fact that is one o the most important amino acids for porcine IFV and further embryo development, used in the production of glutathione, needed for sperm chromatin decondensation and male pronuclear formation (Kobayashi et al., 2006). CONCLUSIONS - Addition of glutamine to a chemical defined in vitro maturation medium improves the maturation rate of porcine oocytes collected from slaughtered gilts. - Addition of individual amino acids such as asparagine and lysine in concentration found in the follicular fluid do not improve the IVM rate or the fecundation rate, in the absence of other follicular or serum components. - Despite the fact that in our study the fecundation rate is not sustained by statistical differences, we assume that glutamine and asparagine improve the cleavage and embryo formation, when used in a chemical defined IVM medium. Acknowledgements. This research was financially supported by CNMP project no /2007 and by CNCSIS project no. 1081/2009. REFFERENCES 1. Anbari, K. and R.M. Schultz (1993). Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryo in vitro. Mol Reprod Dev, 3: Bavister, B.D. and T. Arlotto (2009). Influence of single amino acids on the development of hamster one-cell embryos in vitro. Mol Reprod Dev; 2: Edwards, L.J., D.A. Williams, D.K. Gardner (1998). Intracellular ph of the mouse preimplantation embryo: amino acids act as buffer of intracellular ph. Hum Reprod,13: Empar, Garcıa-Rosello, Pilar Coy, Francisco Alberto Garcıa Vazquez, Salvador Ruiz, Carmen Matas (2006). Analysis of different factors influencing the intracytoplasmic sperm injection (ICSI) yield in pigs. Theriogenology, 66: Gardner, DK. and M. Lane (1993). Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod, 48: Hong, J. and E. Lee (2007). Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development. Theriogenology, 68: Hong, J., H.Y. Yong, B.C. Lee, W.S. Hwang, J.M. Lim, E.S. Lee (2004). Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media. Theriogenology, 62: Kobayashi, M., E.S. Lee, Y. Fukui (2006). Cysteamine or b-mercaptoethanol added into a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection. Theriogenology, 6: Machaty, Z., B.N. Day, R.S. Prather (1998). Development of early porcine embryos in vitro and in vivo. Biol Reprod, 9:

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