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* Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias, Segorbe, 12400 Castellón, Spain;
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Instituto de Medicina Reproductiva, 46009 Valencia, Spain; and
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Unidad de Reproducción Humana, H.U. La Fe de Valencia, 46009 Valencia, Spain
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: in vitro embryo production • oocyte maturation • porcine • recombinant human gonadotropin
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
Cytoplasmic maturation is assessed by the ability of oocytes to undergo early cleavage and embryo development after in vitro fertilization (IVF). Parthenogenetic embryo development, based on artificial activation (ATA) of oocytes, is used to study cytoplasmic maturation in pigs (Liu et al., 1997) because of high rates of polyspermy obtained with conventional IVF (Kren et al., 2003). Intracytoplasmic sperm injection (ICSI) is another option to avoid polyspermy (Wu et al., 2001); however, ICSI is not efficient in pigs due to low male pronuclear formation (Lee et al., 2003).
To increase the developmental ability of IVM oocytes, hormonal supplementation has been shown to enhance fertilization and embryo development of immature oocytes. Several IVM protocols for pig oocytes use PMSG and hCG as hormonal supplements. However, commercially available gonadotropins vary in purity, and contaminants in these products may influence the results within or among laboratories (Zuelke and Brackett, 1990; Choi et al., 2001). The use of recombinant hormones (rh) frees scientists and clinicians from the difficulties with the supply of hormones and reduces the potential of communicable diseases related to extraction and pooling of biological materials obtained from many donors (European Recombinant LH Study Group, 2001).
The aim of this work was to study the effect of rh-FSH and rh-LH on IVM of pig oocytes compared with a conventional hormonal supplementation with PMSG and hCG, as evaluated by the developmental ability of 3 types of pig embryos obtained by IVF, ICSI, or ATA.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culture Media
Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Química (Madrid, Spain). Media with bicarbonate were covered with mineral oil and were equilibrated overnight in a humidified atmosphere containing 5% CO2 in air at 38.5°C. Handling medium (HM199) was composed of Medium 199 supplemented with 25 mM HEPES, 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen, Barcelona, Spain), and antibiotics. Maturation medium (MM199) consisted of Medium 199 supplemented with 0.1% polyvinylalcohol, 0.57 mM Cys, 10 ng/mL of epidermal growth factor (EGF), and antibiotics, as described by Abeydeera et al. (2000), with hormonal modifications. Recombinant human FSH (Gonal-F 75, Serono, Madrid, Spain) and rh-LH (Luveris, Serono) and PMSG (Foligon, Intervet, Boxmeer, Holland) were solubilized in bidistilled water before freezing at –20°C at concentrations of 6.7, 6.7, and 500 IU/mL, respectively. Human chorionic gonadotropin (Sigma-Aldrich) was solubilized in MM199 before freezing at –20°C at a concentration of 20 IU/ mL and was used from both fresh and frozen stocks. In vitro fertilization was performed in Tyrode’s albumin lactate pyruvate medium (mTALP; Rath et al., 1999) consisting of 114.06 mM NaCl, 3.2 mM KCl, 8 mM Ca lactate 5H2O, 0.5 mM MgCl2·6H2O, 0.35 mM NaH2PO4, 25.07 mM NaHCO3, 10 mL/L of Na lactate, 1.1 mM Na pyruvate, 5 mM glucose, 2 mM caffeine, 3 mg/mL of BSA, 1 mg/mL of polyvinylalcohol, and antibiotics. Embryos were cultured in NCSU23 medium (Petters and Wells, 1993) supplemented with 4 mg/mL of BSA.
Preparation and Culture of Cumulus-Oocyte Complexes
Ovaries from prepubertal gilts were collected at a local abattoir and transported within 45 min to the laboratory in 0.9% (wt/vol) NaCl supplemented with antibiotics at 37°C. Cumulus-oocyte complexes (COC) were obtained by antral follicle (3 to 7 mm in diam.) puncture with an 18-gauge needle connected to a 10-mL syringe and prefilled with HM199.
In Vitro Maturation and Culture
Approximately 50 to 60 COC were cultured in 500 µL of medium in a Nunc 4-well multidish (Nunc, Roskilde, Denmark) for 44 to 46 h in a static culture system. During the first 22 to 23 h of culture, COC were incubated in MM199 supplemented with hormones (see below). Then, COC were washed twice and cultured in hormone-free MM199 for an additional 22 to 23 h. In vitro maturation and culture were performed at 38.5°C in an atmosphere of 5% CO2 and 100% relative humidity. After IVF, ICSI, or ATA, presumptive zygotes were washed twice in embryo culture medium and transferred to a Nunc 4-well multidish containing 500 µL of the same medium per well and incubated at 38.5°C in 5% CO2 in air.
In Vitro Fertilization, Intracytoplasmic Sperm Injection, and Chemical Activation
Sperm was supplied by a porcine AI center and stored at 17°C. The semen sample was submitted to gentle centrifugation (50 x g for 3 min) to eliminate foreign particles and dead cells. After the first wash, the cleanest fraction of semen was obtained and centrifuged again (1,250 x g for 4 min). The pellet was resuspended in mTALP, and the sperm concentration of the above mentioned suspension was calculated. In vitro fertilization consisted of coculture of approximately 50 IVM COC with 5 x 105 spermatozoa/mL in 500 µL of mTALP. After 6 h, the oocytes were gently washed to eliminate cumulus cells and sperm adhering to the zona pellucida. Finally, the embryos that were obtained were cultured in vitro under the conditions described.
For ICSI, matured oocytes were washed and transferred to HM199 drops. Intracytoplasmic sperm injection was conducted using an inverted microscope with attached micromanipulators, as described by Probst and Rath (2003) and García-Roselló et al. (2006), with minor modifications. In total, 10 to 15 microdrops were placed in each lid surrounding the central sperm drop containing 10% polyvinylpyrrolidone solution. The microdrops were covered with mineral oil. One single sperm was immobilized by crushing the midpiece with the tip of the injection pipette. The immobilized sperm was aspirated with the tail first. Thereafter, the injection pipette was moved into the drop containing the oocytes to be injected. A single oocyte was fixed by the holding pipette and with the polar body at the 6 or 12 o’clock position. The injection pipette was pushed through the zona pellucida and subsequently through the oolema into the cytoplasm at the 3 o’clock position. A small amount of ooplasm was aspirated into the injection pipette to ensure oocyte membrane penetration. The temperature was maintained at 38.5°C throughout the procedure using a heated microscope stage.
To obtain parthenogenetic embryos, metaphase II oocytes were activated by treatment with 5 µM ionomycin for 5 min. After ionomycin treatment, the oocytes were washed twice and incubated in 2 mM 6-dimethylaminopurine for 3.5 h (Boquest et al., 2002).
Experimental Design
In Exp. 1, COC were divided into 2 maturation groups. One group (A group) was supplemented with rh-FSH and rh-LH (0.1 IU/mL each), and the other group (B group) was supplemented with PMSG and hCG (10 IU/mL each). The effects of these 2 hormonal treatments on nuclear maturation and in vitro development of embryos obtained by IVF, ICSI, or ATA were studied. In Exp. 2, the effects of different hormonal concentrations of rh-FSH and rh-LH (0.5, 0.1, or 0.05 IU/mL) in maturation medium on nuclear maturation and in vitro development of embryos obtained by IVF were studied.
Assessment of Nuclear Maturation and Embryo Development
After in vitro maturation, the COC were briefly placed in HM199 supplemented with 1 mg/mL of bovine testes hyaluronidase and then gently pipetted through a small-bore pipette to strip the cumulus from the ova. The oocytes were evaluated under a stereomicroscope for the presence of the first polar body, which corresponded to nuclear matured oocytes. Regarding fertilization and embryo development, the rates of cleavage and blastocyst formation were evaluated at 48 and 168 h, respectively, after activation, IVF, or ICSI under a stereomicroscope. To observe the cell nuclei, the embryos developing to the blastocyst stage were fixed and stained in absolute ethanol with bisbenzimide, and the cell nuclei were evaluated under an epifluorescence microscope.
Statistical Analysis
At least 4 replicates were performed per treatment. Nuclear maturation, cleavage, or blastocyst formation rates were analyzed by a 
2 test. When a single degree of freedom was involved, the Yates’ correction for continuity was carried out. Results of the evaluation of cell nuclei were analyzed by the ANOVA test.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. No significant differences between hormonal treatments in cleavage or blastocyst rates of parthenogenetic, IVF, and ICSI embryos were detected. Total cell number of embryos was not significantly different in any experimental group (A: 31.3, 28.5, and 19.8 vs. B: 25.2, 25.5, and 20.6 to parthenogenetic, IVF, and ICSI embryos respectively; Table 1).
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. No effect of different hormonal concentrations on embryo development was observed (8.3, 13.0, and 5.7; P > 0.05). Total cell number of blastocysts, ranging from 17.1 to 29.6, was not significantly different between groups.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Li et al., 2003; Iwamoto et al., 2005; Koo et al., 2005; Yi and Park, 2005; García-Roselló et al., 2006) with a protocol based on rh-FSH and rh-LH on the developmental ability of 3 types of pig embryos obtained by IVF, ICSI, or ATA. Three different protocols were examined, because conclusions obtained in 1 IVM procedure for 1 type of embryo cannot be extrapolated to other types of embryo. Results obtained here allow us to substitute PMSG and hCG by rh-FSH and rh-LH without negative effects on these different types of pig embryos, but with the advantage of using more defined IVM conditions. The use of defined media would permit an appropriate quality control and repeatability among laboratories. Researchers have tested the use of recombinant gonadotropins or the effect of 1 hormone and its combination with others on in vitro embryo production (Anderiesz et al., 2000; Schoevers et al., 2003), but very few studies have compared differences between recombinant and pituitary gonadotropins, urinary gonadotropins, or both in embryo production in vitro (bovine: Ali and Sirard, 2002; sheep: Accardo et al., 2004). We did not find any papers studying differences between recombinant and nonrecombinant hormones on porcine in the published literature. Moreover, no reports comparing the effects of maturation media on embryos produced by 3 methods were found. Overall, controversial results were found in such studies. In the work of Ali and Sirard (2002) in bovine, oocytes matured with rh-FSH showed greater embryo development than those matured in pituitary gonadotrophin. When estradiol was added to IVM medium, greater differences in favor of rh were observed. Ali and Sirard (2002) indicated a possible differential effect of the recombinant product was caused either by a different glycosylation pattern or a contaminant in the pituitary FSH preparation. However, when evaluating sheep oocyte IVM, Accardo et al. (2004) showed that blastocyst rate was greatest in the PMSG-hCG group and was greater than in the rh-FSH-rh-LH group, although no significant differences between experimental groups were found in other reproductive variables, such as cleavage or lambing rates. In Accardo et al. (2004), the presence of contaminants, such as growth factors as indicated by Harper and Brackett (1993), could explain these results. In pigs, several studies have shown beneficial effects of EGF on oocyte maturation and its inclusion in the maturation medium (Abeydeera et al., 2000). Recently, it has been demonstrated that FSH and EGF have an additive effect on gilt oocytes (Ye et al., 2005). In our case, EGF was added to both experimental groups. So, the presence of EGF in both hormonal groups could have reduced embryo development differences between experimental groups.
In the present work, we also studied different hormonal concentrations to investigate optimal concentrations of rh-FSH and rh-LH in the media. In bovine, an increase in porcine FSH (from 50 to 500 ng/mL) provoked an increase in morula and blastocyst rates, but this increase was not significant when rh-FSH was used (Ali and Sirard, 2002). In the same work, an increase of LH (50 to 5,000 ng/mL) had no effect on subsequent embryo development. In other studies, no differences were found in cumulus expansion when rh-FSH was increased from 1 to 1,000 ng/mL, although the minor concentration was sufficient to expand approximately 90% of bovine COC (Calder et al., 2003). In pigs, Bing et al. (2001) observed that by increasing FSH concentration, an increase of metaphase II oocytes was obtained, but no effect was observed in male pronucleus formation. In this work, no differences in embryo development were found when hormonal concentration was increased from 0.05 to 0.5 ng/mL. It is possible that the range of variation in hormonal concentration in the present work is not large enough to detect significant differences and that the lowest concentration is sufficient to induce maturation of oocytes and further embryo development.
In conclusion, results obtained here permit us to substitute PMSG and hCG with rh-FSH and rh-LH and to produce, at the same level of efficiency, pig embryos obtained by IVF, ICSI, or ATA, but with more defined in vitro maturation conditions.
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Footnotes |
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2 Corresponding author: masilver{at}ivia.es
Received for publication September 20, 2006. Accepted for publication November 28, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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