1 Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, 2 School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, 3 Animal Cloning Institute, Dongshin University, Naju 520-714 and 4 Department of Veterinary Medicine, Kangwon National University, Chunchon 200-701, Korea
5 To whom correspondence should be addressed. E mail: firstlee{at}snu.ac.kr
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Abstract |
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Key words: blastocyst/ICSI/male pronucleus/open-ended tube/pig
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Introduction |
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Materials and methods |
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IVF
Frozenthawed boar semen was used for IVF. The media for sperm preparation and IVF were calcium- and magnesium-free phosphate-buffered saline (PBS) and modified Tris-buffered medium (mTBM), respectively. The PBS for sperm washing consisted of 136.9 mmol/l NaCl, 2.7 mmol/l KCl, 1.5 mmol/l KH2PO4, 8.1 mmol/l Na2HPO4, 0.5 mmol/l sodium pyruvate, 5.6 mmol/l glucose and 0.1% (w/v) bovine serum albumin (BSA). The mTBM consisted of 113.1 mmol/l NaCl, 3 mmol/l KCl, 7.5 mmol/l CaCl2, 20 mmol/l Tris (T-1410, Trizma base), 11 mmol/l glucose, 5 mmol/l sodium pyruvate and 0.8% (w/v) BSA (catalogue no. A-3311). Frozen semen was thawed at 39°C for 1 min in a water bath, diluted in 10 ml of PBS and centrifuged twice at 350 g for 3 min. The sperm pellet was resuspended in mTBM to give a concentration of 10 x 106 sperm/ml. At the end of the IVM culture, oocytes were freed from cumulus cells by repeated pipetting in the IVM medium containing 0.5 mg/ml hyaluronidase for 1 min and washed twice in mTBM. Twenty to 30 oocytes in 5 µl of mTBM were introduced into a 40 µl fertilization drop covered with mineral oil, and 5 µl of sperm suspension was added to each fertilization drop to give a final sperm concentration of 1 x 106 sperm/ml. Gametes were incubated for 8 h in a humidified atmosphere of 5% CO2 and 95% air (Yoon et al., 2000).
ICSI
The same lot of frozenthawed boar semen used for IVF was employed for ICSI. A semen straw was thawed at 39°C for 1 min. The semen (100 µl) was placed gently in the bottom of a 2.5 ml centrifuge tube containing 1 ml of Dulbeccos phosphate-buffered saline (DPBS) supplemented with 0.1% PVA and incubated in a humidified atmosphere of 5% CO2 and 95% air for a swim-up procedure with the cap covered. After 50 min, 0.5 ml of supernatant was collected from the top of the tube and centrifuged at 350 g for 3 min in a 2.5 ml centrifuge tube. The supernatant was removed and the sperm pellet was stored in a 5% CO2 incubator at 39°C prior to ICSI. In-vitro matured oocytes freed from the cumulus cells and exhibiting a first polar body and normal morphology were washed twice and transferred in a 7 µl drop of TLH containing 0.01% PVA. A small quantity of sperm pellet was placed in the centre of a 7 µl drop of DPBS containing 0.1% PVA overlaid with light white mineral oil. In conventional ICSI, injection pipettes are generally purchased from Humagen, Inc. (Charlottesville, VA; 10-MIC-S, 30° angled). Using an inverted microscope (Olympus IX50, Melville, NY) with micromanipulators (Narishige, Tokyo, Japan), individual spermatozoa were immobilized by scoring the tail, aspirated into an injection pipette and moved to the oocyte-containing drop. An oocyte was immobilized with its polar body at either the 6 or 12 oclock position by a holding pipette, and then the spermatozoon was injected and mixed with a small quantity of cytoplasm (Martin, 2000). In the modified ICSI, a thin-walled borosilicate microtube (G-1; Narishige) with an outer diameter (OD) of 1.0 mm and an inner diameter (ID) of 0.9 mm was pulled using a micropuller (PC-10; Narishige). The sharp tip of the pulled capillary tube was bevelled on a platinumiridium filament of a microforge (MF-900; Narishige) to achieve an angle of 30°. A small opening with many irregular short spikes was made at the tip of the injection pipette by inserting it into the open end of a holding pipette and bending the tip to break it just before ICSI (Figure 1). In this way, an injection pipette with an ID of 34 µm was regularly made. Under a microscope and using a manipulator without an injector (LABOVERT FS, Leitz, Germany), motile spermatozoa were aspirated into a 34 µm injection pipette connected with open-ended tubing by moving the pipette tip close to the sperm tail or head. The spermatozoa were aspirated tail or head first, and the equatorial region of the sperm head became stuck at the tip of the injection pipette (Figure 2A and B). Only motile spermatozoa captured around the boundary of a drop were moved to a drop containing oocytes, directly injected into the oocyte cytoplasm and mixed with cytoplasmic components thoroughly (Figure 3) by open tubing regulated by mouth.
In-vitro culture
At 8 h after insemination, oocytes were freed from cumulus cells and spermatozoa by repeated pipetting. Sperm-injected or in-vitro fertilized oocytes were washed three times in North Carolina State University (NCSU)-23 medium and transferred to 30 µl drops of NCSU-23 supplemented with 0.4% BSA. The ICSI and IVF oocytes were cultured for 18 and 10 h at 39°C under 5% CO2, 7% O2 and 88% N2, respectively, to identify MPN formation, and then cultured for another 168 h to count the total cell number of expanded blastocysts.
Assessment of male pronucleus formation and embryo development
At 18 h after ICSI or IVF, oocytes mounted on a glass slide were fixed for 10 min at 34°C in ethanol containing 25% (v/v) acetic acid, stained with 1% (w/v) orcein in 45% (v/v) acetic acid solution and examined for sperm decondensation or MPN formation under a phase-contrast microscope (Axiophot, Carl Zeiss, Germany) at x400 magnification. The oocytes were classified into three groups (Wei et al., 1999); (i) one MPN with the presence of one female pronucleus (FPN) and two polar bodies (PBs): (ii) one MPN with or without the presence of an FPN; and (iii) one MPN or one decondensed sperm head (DSH) with or without the presence of an FPN. The rate of survival and cleavage and total cell number of blastocysts after Hoechst staining were monitored at 48 and 168 h after ICSI or IVF, respectively.
Statistical analysis
The differences in MPN formation and embryo development among experimental groups were analysed using one-way ANOVA after arcsine transformation to maintain homogeneity of variance. Post hoc analyses to identify between-group differences were performed using the least significant difference (LSD) test. The non-parametric Wilcoxon test with Dunns method for multiple comparisons was used to determine the statistical significance in the total cell number of blastocysts among experimental groups. All data were presented as the arithmetic mean ± SEM. All analyses were performed using SAS (SAS Institute, version 8.1).
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Results |
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Discussion |
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In normal fertilization, the oolemma fuses with the post-acrosomal region of the sperm head plasma membrane, followed by sperm incorporation, DSH and formation of MPN with activation of oocytes (Bavister, 1989). Oocyte activation is thought to be initiated by Ca2+ oscillations (Parrington et al., 1996
) which occur by the release of sperm-borne oocyte-activating factor (SOAF) during the process of zona pellucida (ZP) penetration and fusion of the sperm and the oocyte plasma membranes (Kasai et al., 1999
). Therefore, for successful ICSI outcomes, it could be more desirable to damage the sperm head. However, the conventional ICSI procedure has focused only on manipulating the sperm tail to achieve sperm immobilization and disruption of the plasma membrane (Bourne et al., 1995
; Dozortsev et al., 1998
; Martin, 2000
; Wu et al., 2001
; Horiuchi et al., 2002
). In this study, we developed a modified sperm injection method by using an open-ended mouth tube and by using PVA instead of PVP which has been commonly used in conventional ICSI to decrease the movement of motile sperm and to control the pressure of oil in the injection pipette despite its detrimental effects on development of injected oocytes (Ashwood-Smith, 1971
; Tesarik et al., 1994
; Feichtinger et al., 1995
). The medium used for sperm drops containing PVA is known to keep the wall of the injection pipette less sticky over a longer period of time than BSA-containing medium, resulting in less adherence of mineral oil and debris to the injection pipette (Kuretake et al., 1996
). In the modified ICSI, a fast-moving spermatozoon swimming around the boundary of a drop was readily aspirated either tail or head first and stuck via the head equatorial region onto the sharp tip of the injection pipette. The negative pressure in the injection pipette connected to the open-ended tubing allowed easy sperm handling and conferred slight physical damage on the sperm head just before injection. The continuous negative pressure also prevented the spermatozoon from becoming detached from the pipette. The sperm tail was maintained parallel with the injection pipette. During the injection procedure, the sperm head membrane could be severely injured when sperm penetrate through the ZP and oolemma. Although the disulfide bonds of the sperm head formed during sperm passage through the epididymis make the nuclei of mature eutherian spermatozoa resistant to physical and chemical disruption (Yanagida et al., 1991
), the plasma or nuclear membrane of the sperm head might be damaged by mechanical penetration of the sperm head into the ooplasm in the modified ICSI. After sperm injection through the ZP, the head membrane-damaged spermatozoon was well mixed with the sticky ooplasm by repeated passage into and out of an injection pipette regulated by mouth using open-ended aspiration tubing. In this way, the success rate of sperm injection was close to 100% in the modified ICSI. The extensive damage to the sperm head plasma membrane during ICSI could be responsible for the high fertilization rate shown in the modified ICSI compared with that in conventional ICSI, perhaps due to more effective release of SOAF. Similar to our results, in human conventional ICSI, the fertilization rate was significantly higher when immature spermatozoa were immobilized in a more aggressive way (Palermo et al., 1996
). Overall, relatively low fertilization rates in the conventional (27.9%) and modified ICSI (46.7%) were observed in this study. It has been demonstrated that the outcomes of ICSI are known to be different according to the origin of sperm and oocytes, oocyte centrifugation before sperm injection, and activation treatment of oocytes, among others (Shoukir et al., 1998
; Griffiths et al., 2000
). Using in-vitro matured centrifuged oocytes and fresh semen, a 52% fertilization rate (MPN + FPN) was reported (Kim et al., 1998
). A 69% cleavage rate was reported using in-vivo matured centrifuged oocytes and fresh semen (Martin, 2000
), whereas Kolbe et al., (2000
) reported only a 26% cleavage rate under the same conditions. Thus, the relatively low fertilization rates shown in this study may be due to use of frozenthawed semen. In support of this idea, improved fertilization was achieved in conventional (45.6%) and modified ICSI (73.5%) using fresh boar semen (our unpublished results). In this study, the cleavage rates in the conventional (48.7%) and modified ICSI (60.6%) were not low compared with those of previous reports (Kolbe et al., 2000
; Martin, 2000
).
In conventional ICSI, the injection of excessive amounts of micromanipulation medium into oocytes could occur when the whole spermatozoon is expelled from the injection pipette, and this may have an adverse effect on subsequent embryonic development (Thadani, 1980). The large quantity of medium persisting around the injected sperm head may prevent intermingling of the ooplasm with sperm intracellular components, for instance, the nucleus and SOAF (Kimura et al., 1995
). The quantity of medium injected with the spermatozoon in conventional ICSI was shown to be proportional to the length of the sperm tail (Thadani, 1980
), suggesting that diminishing the length of the sperm tail by cutting it could help minimize the volume of injected medium. In the modified ICSI, without diminishing the length of the sperm tail, a smaller volume of medium was introduced into the cytoplasm during the head-first injection procedure than during tail-first sperm injection. The volume of medium injected during these two procedures was checked with phenol red-coloured solution in preliminary experiments (data not shown)
The injection pipettes routinely used in the conventional ICSI procedure must have an appropriate diameter to avoid difficulties in moving spermatozoa in and out of the pipettes (Payne, 1995). Generally, the diameter of the injection pipette in ICSI is determined in proportion to the size of the sperm head. Severe disruption of the oocyte cytoplasm during injection has been reported when an injection pipette was wider than 8 µm (Payne, 1995
). The higher levels of fertilization achieved in human ICSI than in domestic animals (Hsu et al., 1999
) was explained as partially due to the smaller size of the injection pipette, the lack of a need to centrifuge oocytes before ICSI and the relatively short length of a human sperm tail (Van Steirteghem et al., 1993
; Tesarik, 1996
). As in human conventional ICSI, in the modified ICSI, we achieved higher rates of normal fertilization by using a small injection pipette and by injection of head membrane-damaged sperm, which allowed the injection procedure without centrifuging oocytes and diminishing the length of the sperm tail. Additionally, the injection pipette used in the modified ICSI enabled us to eliminate the need to aspirate cytoplasmic material deep into the injection pipette in order to identify the rupture of ooplasmic membrane and to help mix tail-damaged sperm with cytoplasmic materials, which is currently carried out in conventional ICSI.
In conclusion, using an animal model, we believe the present study provides sufficient technical advances to replace conventional ICSI with the modified ICSI, which is more effective and also avoids unnecessary procedures involved in conventional ICSI.
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Acknowledgements |
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References |
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Submitted on April 4, 2003; resubmitted on June 28, 2003; accepted on July 10, 2003.