Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts
ABSTRACT
This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2–4-cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI.
INTRODUCTION
It has been reported that healthy mice offspring can be produced by intracytoplasmic sperm injection (ICSI) without activation of oocytes (Kimura & Yanagimachi 1995; Kimura et al. 1998; Perry et al. 1999). On the other hand, porcine oocytes require oocyte activation by procedures such as electrical pulse after ICSI (Lee & Yang 2004) or injection of CaCl2 solution during ICSI (Probst & Rath 2003). Lee and Yang (2004) demonstrated that an electrical strength of 2.2 kV/cm DC produced the best blastocyst rate following ICSI (34.4%). Furthermore, births of piglets derived from ICSI have been reported in several studies (Kolbe & Holtz 2000; Lai et al. 2001; Probst & Rath 2003), but a further improvement of ICSI is requested.
Fissore et al. (1998) reported that injection of sperm extracts (SE) from boar spermatozoa induced high rates of activation and cleavage in bovine and mouse oocytes. In our previous study (Matsuura & Maeda 2006; Matsuura et al. 2006), injection of SE from miniature pig spermatozoa induced comparatively high rates of porcine or bovine oocyte activation. If injection with SE were to support oocyte activation in ICSI, it might improve the rate of subsequent embryo development. However, there has been little research elucidating the effect of SE injection in porcine ICSI.
To investigate whether simultaneous injection with SE in ICSI is an effective activation method, we examined embryo development of porcine oocytes after injection with miniature pig sperm and SE.
MATERIALS AND METHODS
Care and treatment of all animals in this study were approved by the Institutional Animal Care and Use Committee of Hiroshima University.
Sperm preparation
Whole semen from Gettingen miniature pigs (2–3 years old) was collected weekly by a glove hand technique and filtered through cotton gauze to remove gel particles. The semen was then centrifuged at 400 g for 10 min to remove sediment. The supernatant was transferred into a new tube and centrifuged at 700 g for 5 min. The sperm pellets were suspended and washed twice in TALP-Hepes (TL-Hepes) medium (Bavister et al. 1983) at 700 g for 5 min. The washed spermatozoa were resuspended in a cell lysis buffer (75 mmol/L KCl, 20 mmol/L Hepes, 1 mmol/L EDTA, 10 mmol/L glycerophsphate, 1 mmol/L DTT, 200 µmol/L PMSF, 10 µg/mL pepstatin, 10 µg/mL leupeptin; pH 7.0), and then the sperm concentration was adjusted to 1 × 109/mL.
Extraction of SE and its adjustment
SE was extracted by Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL, USA) according to the manufacturer's instructions. In brief, the sperm suspension was centrifuged at 2500 g for 10 min. A sperm pellet was obtained by discarding the supernatant, and M-PER was used for each 100 mg of wet cell pellet. The lysate was shaken gently for 10 min by Mini Disk Rotor (BC-710; BIO CRAFT, Tokyo, Japan), and then it was centrifuged at 14 000 g for 15 min to remove cell debris. The supernatant was transferred to a new tube for further treatment, and then it was ultracentrifuged at 100 000 g (TL-100; Beckman Coulter Fullerton, CA, USA) for 1 h at 4°C. The clear supernatant was filtered through a 3000 molecular weight cut-off membrane (Microcon YM-3; Millipore Corporation, Bedford, MA, USA). The substances on the ultrafiltration membranes were washed twice with washing buffer solution containing of 75 mmol/L KCl and 20 mmol/L Hepes (pH 7.0), and then the protein concentration of the diluted substances was adjusted to 1.0 mg/mL with the same buffer (see the following procedure). The adjusted substances were stored at −80°C until use.
Porcine oocyte maturation
The ovaries were collected from prepubertal gilts (commercial crossbred) at a local slaughterhouse and transported within 2 h to the laboratory in 0.9% (w/v) saline solution containing 100 mg/mL kanamycin sulfate (Meiji Seika, Ltd, Tokyo, Japan) at 30°C. The follicular contents were recovered by cutting them from visible small antral follicles (about 2–5 mm in diameter) with a razor (make), and by scraping the inner surface of the follicle walls with a disposable surgical blade. Cumulus oocyte complexes (COCs) with uniform ooplasm and a compact cumulus cell mass were placed in phosphate-buffered saline (PBS) containing 0.01% (w/v) polyvinylpyrrolidone (PVP) (Sigma, St. Louis, MO, USA). They were washed twice with maturation medium (BSA-free NCSU37 Kikuchi et al. (2002)) supplemented with 0.6 mmol/L cysteine (Sigma), 10% FCS (Gibco BRL, Grand Island, NY, USA), 0.6 µg/mL porcine-FSH (Sigma), and 1.3 µg/mL equine-LH (Sigma). Twenty COCs were cultured for 48 h in a NUNC 48-well multidish (NUNC, Roskilde, Denmark) containing 300 µL of maturation medium.
Sperm collection for ICSI
Motile sperm for ICSI were collected by the swim-up procedure modified protocol of Lefebvre and Suarez (1996). In brief, sperm were suspended in TALP-Hepes (TL-Hepes) medium (Bavister et al. 1983). Then sperm suspension (1.0 mL) was transferred into a new tube (Falcon, Becton Dickinson Labware, Bedford, MA, USA). The tube was closely capped and placed in a 37°C incubator under 5% CO2 in humidified air at a 45° angle for 30 min. Subsequently, the top 500–700 µL containing actively motile sperm was removed with extreme care to avoid disturbing the interface of the two layers. The sperm concentration was adjusted to 1 × 105/mL, and then the sperm were used for ICSI.
Sperm and SE injection into oocytes
The injection procedure was based on the protocol by Kimura and Yanagimachi (1995). Five droplets (5 µL) were put on a 35 mm plastic dish (Falcon) in the row. These droplets were TCM-199 medium (Gibco BRL) supplemented with 10% PVP for pipette washing, TCM-199 medium supplemented with 10% PVP for immobilization of sperm, sperm suspension (1 × 105 sperm/mL), TCM-199 medium (Gibco BRL) containing 10% FCS (Gibco BRL) for ICSI, and SE adjusted to various concentrations (0.02, 0.04 and 0.08 mg/mL). Immobilized spermatozoa were aspirated into an injection pipette together with SE of 1.1 pL (the volume was calculated by the procedure described by Probst and Rath (2003)). ICSI was performed under an inverted microscope (Hoffmann Modulation-Contrast Optic). Two different micromanipulators were used. On the left hand side, a manually driven micromanipulator (Leitz, Germany) was used to fix oocytes. On the right hand side, a micromanipulator (Leitz, Germany) equipped with a Piezo inpact driving unit (PMAS-CT110; Primetech, Ibaraki, Japan) was used for sperm and SE injection. Both a spermatozoon and SE were then injected into an oocyte's cytoplasm using a Cell-Injector (Microinjector, CIJ-1; Shimadzu Corporation, Kyoto, Japan).
Evaluation of nuclear status and development
All injected oocytes were cultured as described by Kikuchi et al. (2002). The day ICSI was performed was defined as Day 0. The basic in vitro culture (IVC) medium was NCSU 37 medium containing 0.4% BSA (fraction V, Sigma). The oocytes were cultured for 48 h in an IVC medium supplemented with 0.17 mmol/L sodium pyruvate (Sigma) and 2.73 mmol/L sodium lactate (Sigma), then cultured in IVC medium with 5.55 mmol/L D-glucose (Sigma) from 48 to 168 h. After culturing for 48 h (Day 2), non-cleaved oocytes were evaluated for nuclear status. On the other hand, cleaved oocytes were cultured for more 120 h, and then the blastocyst rates were examined at 168 h (Day 7).
Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA). All percentage data were subjected to arc sine transformation before statistical analysis. Data were compared using the Tukey-Kramer honestly significant difference test. All experiments were repeated at least three times. Statistical comparisons were performed using JMP IN software (SAS Institute Inc., Cary, NC, USA). Differences were considered significant at P < 0.05.
RESULTS
Table 1 shows oocyte activation and embryo development of porcine oocytes after injection with miniature pig sperm and SE. There was no significant difference in rates of oocytes having 1 pronucleus and 1 polar body (1PN1PB) and 2 pronuclei and 1 polar body (2PN1PB) among all treatment groups. On the other hand, the rate of oocytes having 1 pronucleus and 2 polar bodies (1PN2PB) in SE (0.08 mg/mL) injection group (25.7%, parthenogenetic development) was significantly higher compared to other groups (P < 0.05). There was no oocyte with 2 pronuclei and 2 polar bodies (2PN2PB) in the SE (0.08 mg/mL) injection group, however oocytes with 2PN2PB were observed in the sperm injection group or sperm and SE injection groups. Furthermore, the rate of oocytes with 2PN2PB in sperm and SE (0.04 mg/mL) group (21.4%) was significantly higher compared to other groups (P < 0.05).
| Treatment | No. of treated oocytes | Number (mean% ± SEM) of oocytes | ||||||
|---|---|---|---|---|---|---|---|---|
| No. of activated or developed oocytes (%)(48 h after onset of culture) | No. of developed oocytes (%) (168 h after onset of culture) | |||||||
| Pronuclear stage | 2 to 4-cell stage | Blastocyst stage | Fragmentated | |||||
| 1PN1PB† | 1PN2PB‡ | 2PN1PB§ | 2PN2PB¶ | |||||
| SE (0.08 mg/mL) injection | 35 | 1 (2.9±3.3)a | 9 (25.7 ± 4.7)a | 1 (2.9 ± 3.3)a | 0 (0 ± 0.0)a | 8 (22.9 ± 2.3)a | 0 (0 ± 0.0)a | 3(8.6 ± 0.0)a |
| Sperm injection | 35 | 0 (0±0.0)a | 1 (2.9 ± 0.0)b | 0 (0 ± 0.0)a | 3 (8.6 ± 2.7)b | 8 (22.9 ± 1.5)a | 2 (5.7 ± 2.7)ab | 2(5.7 ± 2.7)a |
| Sperm and SE (0.02 mg/mL) injection | 54 | 0 (0±0.0)a | 1 (1.9 ± 2.7)b | 0 (0 ± 0.0)a | 7 (7.4 ± 2.2)b | 18 (33.3 ± 3.7)ab | 10 (18.5 ± 5.4)bc | 6(11.1 ± 2.3)a |
| Sperm and SE (0.04 mg/mL) injection | 42 | 0 (0±0.0)a | 2 (4.7 ± 2.3)b | 0 (0 ± 0.0)a | 9 (21.4 ± 1.7)c | 16 (38.1 ± 2.3)b | 9 (21.4 ± 3.0)c | 3(7.1 ± 0.3)a |
| Sperm and SE (0.08 mg/mL) injection | 38 | 0 (0±0.0)a | 1 (2.6 ± 3.3)b | 0 (0 ± 0.0)a | 4 (18.4 ± 1.0)b | 10 (26.3 ± 3.1)ab | 1 (2.6 ± 3.3)ab | 2(5.3 ± 2.9)a |
- † Oocytes with one pronucleus and without a second polar body.
- ‡ ‡Oocytes with one pronucleus and a second polar body.
- § §Oocytes with two pronuclei and without a second polar body.
- ¶ ¶Oocytes with two pronuclei and a second polar body.
- a–c Values within the same column with different letters differ significantly (P < 0.05).
In SE injection group (without sperm), developed oocytes at 2–4 stage were observed, but no blastocyst was obtained. On the other hand, blastocysts were obtained in all of sperm injection groups.
The rate of 2-4-cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%).
The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%).
The fragmentated oocytes were observed in all groups after 168 h culture period, however there were no significant differences among them.
DISCUSSION
It is well known that human oocytes do not require additional activation treatment after ICSI. This may be due to the fact that ICSI in human oocytes can generate enough calcium oscillations (Tesarik 1998). In contrast, porcine oocytes are not activated by just mechanical treatment with an injection pipette or the injected spermatozoon. In our previous studies, we showed that SE injection could elicit porcine oocyte activation effectively (Matsuura & Maeda 2006; Matsuura et al. 2006). Furthermore, SE injection decreased p34cdc2 kinase and MAP kinase activities, and oocyte activation as well as Ca2+ ionophore treatment (Matsuura & Maeda 2008). Therefore, the present study examined whether injection of SE extracted from miniature pig sperm is effective for embryonic development after ICSI in pigs.
In the sperm injection without SE, the rates of oocytes development to 2-4-cell and blastocyst stages were 22.9% and 5.7%, respectively. These results supported clearly the indication by Probst and Rath (2003) and Lee and Yang (2004) that porcine oocytes require oocyte activation after ICSI.
No blastocyst was obtained in SE injection group (without sperm), but blastocysts were obtained in all of sperm injection groups. Furthermore, the rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). From these results, it is clear that SE is effective for artificial activation of porcine oocytes and further development after ICSI, and that the optimal SE concentration is 0.04 mg/mL.
In sperm and SE (0.08 mg/mL) injection group, the rate of blastocyst stage (2.6%) was significantly lower than that of sperm and SE (0.04 mg/mL) injection group. We showed in our previous study that a higher concentration of SE was not effective for oocyte activation (Matsuura & Maeda 2006). Therefore, excessive activation factors (factor leased from sperm plus 0.08 mg/mL SE) might be harmful for oocyte activation and further development.
In SE injection group (without sperm), developed oocytes at 2–4 stage were observed, but no blastocyst was obtained. The reason might be due to haploid genome. Kure-bayashi et al. (1996) and Okada et al. (2004) showed that diploid embryos treated with cytochalasin B had a better chance to develop to the blastocyst stage than haploid embryos.
We have noted that SE injection induces oocyte activation, but the rate remains low compared to IVF, and that the ratio of decrease in both of p34cdc2 and MAP kinase activities in oocytes injected with SE is also low compared to IVF (Matsuura & Maeda 2008). Therefore, further study is required to improve the method of porcine oocyte artificial activation by SE injection.
ACKNOWLEDGMENTS
We wish to thank Dr Shaun Heaphy, University of Leicester for the critical reviewing of this manuscript.
