A modified method for ICSI in the pig: injection of head membrane-damaged sperm using a 3–4 µm diameter injection pipette

Hwan Yul Yong1,2, Byoung Sik Pyo3, Ji Young Hong4, Sung Keun Kang1, Byeong Chun Lee1,5, Eun Song Lee4 and Woo Suk Hwang1,2

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Conventional ICSI to date was focused only on tail membrane damage to achieve sperm immobilization and disruption of the plasma membrane, even though liberation of soluble sperm factors is achieved by disruption of the sperm head membrane. METHODS: A modified method for ICSI was developed: head membrane-damaged spermatozoa aspirated tail or head first were injected into the ooplasm using a 3–4 µm diameter injection pipette connected to an open-ended aspiration tube regulated by mouth. The efficiency of this modified ICSI was compared with that of conventional ICSI and IVF. RESULTS: When spermatozoa aspirated tail first were injected, a decondensed sperm head was more frequently observed in the oocyte cytoplasm with the modified ICSI (80.0%) than with conventional ICSI (55.7%) or IVF (63.5%) (P < 0.001). The rates of male pronucleus (MPN) formation in the modified ICSI or IVF were significantly higher (50.7 and 39.7%, respectively) than in conventional ICSI (27.9%) (P < 0.001). The rates of survival, cleavage and embryo development to blastocyst were significantly higher in the modified ICSI (71.7, 60.6 and 17.5%) than in conventional ICSI (48.1, 48.7 and 10.5%) (P < 0.001). No significant differences in MPN formation and embryo development to blastocyst were observed between the tail- and head-first sperm aspiration. CONCLUSION: Our results demonstrated that, in the pig, the procedures of pursuing, capturing and immobilizing a spermatozoon and producing deliberate damage to the tail membrane in conventional ICSI were not required in the modified ICSI. We believe that the present study provides sufficient technical advancement to replace conventional ICSI with the modified ICSI, which is more effective and also avoids unnecessary procedures involved in conventional ICSI.

Key words: blastocyst/ICSI/male pronucleus/open-ended tube/pig


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ICSI has been employed extensively to overcome male factor infertility in humans and to elucidate fundamental mechanisms of fertilization in animals. The main procedures of ICSI (conventional ICSI) to date include immobilization of a spermatozoon by damaging the tail, complete aspiration into an injection pipette and mixing an injected spermatozoon with ooplasm by aspirating cytoplasmic material together with the sperm cell inside the injection pipette. In the conventional ICSI procedure, sperm immobilization and disruption of the plasma membrane before injection are considered critical for successful fertilization, because they are beneficial for sperm handling and for liberation of soluble sperm factors that are believed to induce oocyte activation (Svalander et al., 1995Go; Vanderzwalmen et al., 1996Go; Dozortsev et al., 1997Go). In conventional ICSI, a motile spermatozoon is mechanically immobilized by manipulating the sperm tail in various ways, including scoring (Horiuchi et al., 2002Go; Martin, 2000Go), crushing (Bourne et al., 1995Go), vigorous swiping (Wu et al., 2001Go), several piezo pulses (Dozortsev et al., 1998Go) or repeated aspiration in and out of the injection pipette (Pope et al., 1998Go). A chemical agent, polyvinylpyrrolidone (PVP), is also used in many laboratories to slow down sperm movement and to facilitate the breaking of the sperm tail despite its detrimental effects on survival and development of injected oocytes (Tesarik et al., 1994Go; Feichtinger et al., 1995Go). Overall, the conventional ICSI procedure has focused only on manipulating the sperm tail to achieve sperm immobilization and disruption of the plasma membrane, even though liberation of soluble sperm factors is achieved by disruption of sperm head membrane. Currently, Triton X-100 and dithiothreitol (DTT) are the only chemical reagents known to damage the sperm head membrane, but these reagents were shown to have detrimental effects on development of oocytes after injection (Szczygiel et al., 2002Go). Until now, there has been no mechanical way to damage the sperm head membrane in conventional ICSI. Accordingly, the present study was designed to modify the conventional ICSI technique to improve its outcome in the pig. A 3–4 µm injection pipette connected to an open-ended aspiration tube regulated by mouth was used to pick up a spermatozoon without using an injector and PVP treatment. Spermatozoa were aspirated either head or tail first into the injection pipette. The usefulness and efficiency of the modified ICSI technique were evaluated by monitoring sperm head decondensation, male pronucleus (MPN) formation and embryo development to blastocyst.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro maturation
Ovaries were obtained from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% (w/v) NaCl solution at 30 to 35°C within 2 h of slaughter. After washing ovaries three times in 0.9% (w/v) NaCl solution, immature oocytes were aspirated with an 18-gauge hypodermic needle attached to a 5 ml disposable syringe. After pooling follicular contents in a 15 ml conical tube, the supernatant was discarded. Cumulus–oocytes complexes (COCs) in the sediment were rinsed three times in Tyrode’s lactate–HEPES (TLH) medium supplemented with 0.01% polyvinyl alcohol (PVA). Unless indicated otherwise, reagents were obtained from Sigma-Aldrich Co. (St Louis, MO). The COCs enclosed by more than three layers of compact cumulus cells and containing oocytes with an evenly granulated ooplasm were selected for in-vitro maturation (IVM). The IVM medium was tissue culture medium (TCM)-199 consisting of Earle’s salts, L-glutamine (Life Technologies Inc., Rockville, MD), 26.2 mmol/l NaHCO3, 3.05 mmol/l glucose, 0.91 mmol/l sodium pyruvate, 0.57 mmol/l L-cysteine, 75 µg/ml kanamycin, 20 ng/ml epidermal growth factor, 5 IU/ml pregnant mare’s serum gonadotrophin (PMSG)/HCG (Intervet, Boxmeer, The Netherlands) and 10% (v/v) porcine follicular fluid (pFF). The pFF was aspirated from antral follicles 3–7 mm in diameter from prepubertal gilt ovaries. After centrifugation at 1600 g for 30 min, supernatants were collected and filtered sequentially through 1.2 and 0.45 µm syringe filters (Gelman Sciences, Ann Arbor, MI). Prepared pFF was then stored at –20°C until use. A group of 30–50 COCs was cultured in 500 µl of IVM medium at 39°C in a humidified atmosphere of 5% CO2 and 95% air. After culturing for 22 h, COCs were transferred to PMSG- and HCG-free IVM medium and cultured for another 20 h.

IVF
Frozen–thawed 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., 2000Go).

ICSI
The same lot of frozen–thawed 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 Dulbecco’s 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 o’clock 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 platinum–iridium 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 3–4 µm was regularly made. Under a microscope and using a manipulator without an injector (LABOVERT FS, Leitz, Germany), motile spermatozoa were aspirated into a 3–4 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., 1999Go); (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 Dunn’s 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of fertilization methods on MPN formation and DSH
The rates of pronucleus formation in IVF, conventional and modified ICSI with tail-first aspirated sperm are summarized in Table I. Polyspermic fertilization was found in 22 of 63 oocytes after IVF, which showed more than two MPNs or DSHs. The rates of normal fertilization and total MPN formation in the modified ICSI were significantly higher (P < 0.001) than in conventional ICSI (46.7 and 50.7% versus 21.3 and 27.9%, respectively). With the exception of polyspermic fertilization, the formation rates of DSH and MPN were significantly higher (P < 0.001) in the modified ICSI than in conventional ICSI and IVF (80.0% versus 55.7 and 63.5%, respectively).


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Table I. Comparison of male pronucleus formation following IVF, conventional and modified ICSI with tail-first aspirated sperm injection
 
Effect of fertilization methods on embryo development to blastocyst
As shown in Table II, the survival rate of oocytes in the modified ICSI with tail-first aspirated sperm was significantly higher (P < 0.001) than in conventional ICSI (71.7% versus 48.1%, respectively). The cleavage rate was significantly higher (P < 0.001) in the modified ICSI than in conventional ICSI or IVF (60.6% versus 48.7 or 53.2%, respectively). The rate of development to expanded blastocyst was significantly higher in the IVF group (P < 0.001) than in the conventional or modified ICSI (19.4% versus 10.5 or 17.5%, respectively). No significant difference was found in the total cell number of blastocysts among treatment groups.


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Table II. Comparison of survival rate and embryo development following IVF, conventional and modified ICSI with tail-first aspirated sperm injection
 
Comparison of MPN formation and embryo development to blastocyst in the modified ICSI with tail- or head-first aspirated sperm
The rates of MPN formation, survival, cleavage, blastocyst formation and total cell number of expanded blastocysts between the two aspiration methods were not significantly different (Tables III and IV).


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Table III. Comparison of male pronucleus development following modified ICSI with tail- or head-first aspirated sperm
 

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Table IV. Comparison of embryo development following modified ICSI with tail-or head-first aspirated spermatozoa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we demonstrated that the modified ICSI with tail- or head-first aspirated spermatozoa achieved higher rates of MPN formation and embryo development to blastocyst than conventional porcine ICSI or IVF.

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, 1989Go). Oocyte activation is thought to be initiated by Ca2+ oscillations (Parrington et al., 1996Go) 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., 1999Go). 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., 1995Go; Dozortsev et al., 1998Go; Martin, 2000Go; Wu et al., 2001Go; Horiuchi et al., 2002Go). 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, 1971Go; Tesarik et al., 1994Go; Feichtinger et al., 1995Go). 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., 1996Go). 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., 1991Go), 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., 1996Go). 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., 1998Go; Griffiths et al., 2000Go). Using in-vitro matured centrifuged oocytes and fresh semen, a 52% fertilization rate (MPN + FPN) was reported (Kim et al., 1998Go). A 69% cleavage rate was reported using in-vivo matured centrifuged oocytes and fresh semen (Martin, 2000Go), whereas Kolbe et al., (2000Go) 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 frozen–thawed 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., 2000Go; Martin, 2000Go).

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, 1980Go). 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., 1995Go). The quantity of medium injected with the spermatozoon in conventional ICSI was shown to be proportional to the length of the sperm tail (Thadani, 1980Go), 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, 1995Go). 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, 1995Go). The higher levels of fertilization achieved in human ICSI than in domestic animals (Hsu et al., 1999Go) 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., 1993Go; Tesarik, 1996Go). 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.


    Acknowledgements
 
This study was supported by a grant from the Advanced Backbone IT Technology Development (IMT2000-C1-1). The authors are grateful for a graduate fellowship provided by the Ministry of Education, through the BK21 program. We thank Dr Barry D.Bavister for his valuable editing of the manuscript.



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Figure 1. The preparation of an injection pipette. (A) The pulled capillary tube was inserted into the end of a holding pipette. (B) The bending point of the inserted tip was determined empirically and (C) the tip was bent gradually until (D) the tip of the pipette was broken. All of these procedures were performed in the drop of oocytes just before sperm injection. The broken piece located in the holding pipette was expelled before holding the oocytes.

 


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Figure 2. A diagram showing an immobilized spermatozoon at the tip of the injection pipette, captured tail (A) or head first (B). The sperm tail before mixing with cytoplasm is straightened towards the opposite end of the tip of the injection pipette by negative pressure (A).

 


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Figure 3. A diagram showing a spermatozoon injected into the ooplasm. Note the mechanical damage to the membrane of the sperm head equatorial region when it penetrates the zona pellucida (ZP) and ooplasm membrane. Repeatedly aspirating the head membrane-damaged sperm in and out of the injection pipette facilitates mixing of the whole spermatozoon with sticky cytoplasmic components.

 

    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 4, 2003; resubmitted on June 28, 2003; accepted on July 10, 2003.