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From the * University of Murcia, Department of
Veterinary Physiology, Faculty of Veterinary Science, Murcia, Spain; and the
Department of Animal Reproduction, Nacional
Institute of Agricultural Research and Technology (INIA), Madrid, Spain.
| Correspondence to: Pilar Coy, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain (e-mail: pcoy{at}um.es). |
| Received for publication June 17, 2005; accepted for publication September 26, 2005. |
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Abstract |
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Key words: Capacitation, sperm cryopreservation, acrosome reaction, early development
According to Probst and Rath (2003), the current main problem arising from porcine ICSI is the low proportion of blastocysts obtained, and it has been suggested (Kren et al, 2003) that this low proportion is a consequence of the fact that porcine ICSI fertilized oocytes are unable to induce sperm head decondensation yielding functional male pronuclei (Lee et al, 2003). In addition to the contradictory results regarding the convenience of employing whole spermatozoa or isolated heads (Kwon et al, 2004; Lee and Yang, 2004), 3 considerations (related to sperm factor) should be evaluated for male pronuclear formation ability and subsequent normal fertilization post-ICSI. The first consideration is that cryopreservation procedures could induce DNA fragmentation of the sperm or damage to the sperm centriole, leading to failed fertilization and embryo cleavage (Billard, 1983; Kim et al, 2002; Baumber et al, 2003; Martin et al, 2004). Few data have been reported demonstrating differences after injection of fresh or frozen ejaculated (the most available source of sperm cells) spermatozoa into in vitro matured (the most available source of oocytes) pig oocytes. Second, the use of purified samples of spermatozoa to ensure that only membrane-intact sperm cells are used could be an important point when an increase in the ICSI yield is the ultimate aim. In this case, the advantage of using a short and simple sperm treatment, compared to a complex procedure, to ensure membrane integrity should be pondered. Third, the impact of injecting the intact acrosome vesicle into the oocyte has been reported to be a factor causing delayed male chromatin decondensation and male pronuclear formation (Katayama et al, 2002a), but more categorical results are necessary before we can include the induction of the acrosome reaction in the porcine ICSI protocols.
The aim of this study was to evaluate the impact on porcine ICSI yield of 1) sperm cryopreservation, 2) sperm washing and selection procedure, and 3) the induction of artificial acrosome reaction with a calcium ionophore before the injection. Our experimental outcomes, in terms of oocyte activation, pronuclear formation, and embryo development postfertilization by ICSI, are presented here and discussed.
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Materials and Methods |
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Media and Chemicals
Unless otherwise indicated, all chemicals and reagents were purchased from
Sigma-Aldrich Química S.A. (Madrid, Spain). The oocyte maturation
medium was NCSU-37 (Petters and Wells,
1993) supplemented with 0.57 mM cysteine, 1 mM dibutyryl cAMP, 5
µg/mL insulin, 50 µM ß-mercaptoethanol, 10 IU/mL eCG (Folligon;
Intervet International B.V., Boxmeer, Holland), 10 IU/mL hCG (Chorulon;
Intervet International B.V.), and 10% porcine follicular fluid (vol/vol).
The medium used for embryo micromanipulation was Dulbecco phosphate-buffered saline (DPBS) supplemented with 10% fetal calf serum (FCS). After microinjection, oocytes recovered in TALP medium (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 Na lactate, 1.1 mM Na-pyruvate, 5 mM glucose, 2 mM caffeine, 3 mg/mL bovine serum albumin (BSA; A-9647), 1 mg/mL polyvinylpyrrolidone (PVA), and 0.17 mM kanamycin sulfate.
The embryo culture medium was NCSU-23 containing 0.4% BSA (A-8022), 75 µg/mL potassium penicillin G, and 50 µg/mL streptomycin sulfate (Macháty et al, 1998).
Oocyte Collection and In Vitro Maturation
Within 30 minutes of slaughter, ovaries from prepubertal gilts were
transported to the laboratory in saline (0.9% wt/vol NaCl) containing 100
µg/mL kanamycin sulfate at 37°C and were then washed once in 0.04%
(wt/vol) cetrimide solution and twice in saline. Oocyte-cumulus cell complexes
were collected from non-atretic follicles (3–6 mm in diameter), washed
twice in 35-mm plastic Petri dishes containing DPBS supplemented with 4 mg/mL
PVA, and then washed twice more in maturation medium previously equilibrated
for at least 3 hours at 38.5°C under an atmosphere of 5% CO2
and 100% humidity. Only oocytes harvested within 2 hours of slaughter
(Matás et al, 1996) with a complete dense cumulus oophorus were matured. Groups of 50 oocytes were
cultured in 500 µL maturation medium for 20–22 hours at 38.5°C
under an atmosphere of 5% CO2 and 100% humidity. Once cultured,
oocytes were washed twice, transferred to fresh maturation medium without
hormonal supplements or dibutyryl-cAMP, and cultured for an additional
20–22 hours (Funahashi and Day,
1993).
Sperm Collection and Treatments
Fresh semen was collected from stud boars of known fertility by the
gloved-hand method. After collection, the sperm-rich fraction was immediately
transported to the laboratory and diluted in Beltsville thawing solution (BTS)
at 15°C. Fresh diluted spermatozoa were used on the same day of
collection.
Semen samples were also cryopreserved using the straw freezing procedure described by Westendorf et al (1975), with minor modifications as indicated below. Diluted semen was held at 15°C for 2 hours and were later centrifuged at 800 x g for 10 minutes. The supernatant was discarded and the semen pellet was resuspended with lactose-egg yolk extender (LEY; 80 mL of 11% lactose and 20 mL egg yolk) to provide 1.5 x 109 spermatozoa/mL. After further cooling to 5°C over a 120-minute period, 2 parts of LEY-extender semen was mixed with a LEY-extender solution containing 1.5% Orvus Es Paste (Equex-Paste; Minitüb, Tiefenbach, Germany) and 9% glycerol. The final semen concentration to be frozen was 1 x 109 spermatozoa/mL in 3% glycerol. The diluted and cooled semen was loaded into 0.5-mL straws (Minitüb), sealed, transferred to a programmable freezer (Icecube 1800; Minitüb), and frozen horizontally in racks. The freezing rate was 1°C/min from 5°C to -4.5°C, holding for 1 minute at -4.5°C, and then 30°C/min from -4.5°C to -180°C. Frozen straws were stored in liquid nitrogen until use.
Thawing of cryopreserved spermatozoa was performed by straw immersion in a 52°C water bath for 11 seconds. Thawed semen was resuspended in BTS at 37°C and centrifuged at 100 x g for 10 minutes to remove the residual additive employed for cryopreservation.
The Percoll (Pharmacia, Uppsala, Sweden) sperm cell pretreatment used in all experiments of this study involved layering a 0.5-mL aliquot of semen on a discontinuous 45% and 90% (vol/vol) Percoll gradient (Parrish et al, 1995) and centrifuging at 700 x g for 30 minutes. Peletted cells collected from the bottom of the 90% fraction were washed in TALP medium by centrifugation at 100 x g for 10 minutes. This second sperm pellet was resuspended in DPBS supplemented with 10% FCS (Biological Industries, Haemek, Israel) to give a final concentration of 5 x 105 spermatozoa/mL.
The DPBS sperm cell pretreatment used in the second experiment of this study involved the centrifugation of 10 mL of fresh semen at 1200 x g for 3 minutes, after which the pellet was resuspended in DPBS supplemented with 10% FCS to reach the final concentration of 5 x 105 spermatozoa/mL.
In the last experiment of this study, the acrosome reaction was induced artificially in sperm samples with a 15-minute incubation period in TALP medium containing 1 µM or 5 µM of calcium ionophore (A23187) at 38.5°C under an atmosphere of 5% CO2 and 100% humidity. Aliquots of 100 µL from each sperm cell treatment were supplemented with 5 µL fluorescein isothiocyanate–labeled peanut agglutinin (FITC-PNA; 200 µg/mL) and 5 µL propidium iodide (PI; 200 µg/mL) and were maintained at 38°C for 5 minutes and finally fixed with 10 µL paraformaldehyde (1% vol/vol in saline solution). Spermatozoa were then examined under an epifluorescence microscope and divided into 4 categories according to their FITC-PNA/PI staining pattern, as follows: a) live acrosome intact spermatozoa, sperm cells with no FITC-PNA or PI staining; b) live acrosome-reacted spermatozoa, sperm cells with FITC-PNA acrosome staining; c) dead acrosome intact spermatozoa, sperm cells with PI nuclear staining; and d) dead acrosome-reacted spermatozoa, sperm cells with PI nuclear staining and FITC-PNA acrosome staining.
ICSI
Oocytes cultured for 44 hours in maturation medium were mechanically
denuded by gentle aspiration with a pipette. Denuded oocytes were washed twice
in supplemented DPBS medium and transferred to ICSI drops. ICSI was conducted
on a heated microscope at 200x magnification using a Nikon Diaphot 300
inverted microscope with attached micromanipulators. Only fully matured MII
oocytes were microinjected. The ICSI medium used was DPBS supplemented with
10% FCS. Prior to ICSI, oocytes were loaded on 4-µL microinjection drops
placed onto the lid of a Petri dish (1 oocyte/drop). In total, 10 to 15
microdrops were placed in each lid surrounding central sperm drops, which
resulted from a mixture of 4 µL of DPBS-FCS and 1 µL of the sperm
suspension. Microdrops were covered with mineral oil (Sigma-Aldrich, M-8410).
ICSI was performed as described by Probst and Rath
(2003). Briefly, 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, positioning 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 in order to ensure oocyte membrane penetration.
Subsequently, the immobilized spermatozoon was released into the cytoplasm.
The temperature was maintained at 38.5°C throughout the procedure using a
heated microscopic stage. Injected oocytes were placed in TALP medium.
Zygotes were fixed with acetic alcohol 18–20 hours after microinjection, stained with 1% (wt/vol) lacmoid, and examined at 400x magnification under a phase contrast microscope. Oocytes with 1 pronucleus and 1 sperm inside, either decondensed or not, were classified as "1PN & 1Sperm." Oocytes with 2 pronuclei were classified as "2PN" (Figure, a). Oocytes with only 1 pronucleus without sperm inside or with 3 pronuclei were designated as "other" (Figure, b).
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Statistical Analysis
In this study, data are presented as means plus or minus standard error of
the mean (SEM) after being fitted to the binomial variable model. In our first
and last experiments, data were analyzed by 1-way analysis of variance
(ANOVA). In our second experiment, the 2-way ANOVA was used.
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Results |
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In our second experiment, the impact of the sperm treatment (DPBS vs Percoll) and sperm cell donor (2 boars were used) on ICSI performance was evaluated. As shown in Table 3, when the ICSI performance was assessed by the in vitro embryo development post-ICSI, a sperm treatment effect, as well as a boar effect, was not detected. However, as shown in Table 4, the DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P = .0467). Relative to the observed boar effect, significantly higher blastocyst formation proportions were obtained when ICSI was performed with spermatozoa collected from boar A (P = .0003). Independently of the treatment and sperm cell donor used, significant differences in blastocyst cell numbers were not detected.
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In order to determine whether the injection of acrosome-reacted spermatozoa would benefit our ICSI results, in a third experiment, fresh sperm samples collected from a single donor and treated by the Percoll gradient method were submitted to different concentrations (1 and 5 µM) of A23187, a calcium ionophore capable of artificially inducing acrosome reaction. In a first assay, the proportions of live and dead spermatozoa that retained or did not retain an intact acrosome after a 15-minute incubation with either concentration were determined and compared with unexposed control samples. A total of 2400 sperm cells were analyzed per treatment in 4 replicates. As we can see on Table 5, while the smallest of the concentrations of calcium ionophore enriched a population of live acrosome-reacted sperm cells (1445 out of 2400 treated cells, 60%), the highest concentration enriched a population of dead acrosome-reacted sperm cells (1385 out of 2400 treated cells, 58%). As expected, the majority of control spermatozoa (75%) retained their acrosome vesicle and were alive at the end of the incubation period.
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In a subsequent assay we evaluated and compared the impact of both sperm cell treatments on oocyte activation and fertilization after ICSI. In Table 6, the outcomes are presented. In no case were the differences among treatments observed in the proportion of oocyte activation and fertilization significant. Despite these results, an evaluation was conducted of a possible later impact of both sperm cell calcium ionophore treatments on embryo development after ICSI. Again, significant differences were not detected (Table 7), and as in previous experiments, the proportion of blastocyst formation and number of cells per blastocyst were very poor.
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Discussion |
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In our first experiment, the impact on the ICSI efficiency of fresh and cryopreserved sperm was evaluated and compared. Both types of spermatozoa were washed in a Percoll gradient prior to the fertilization process in order to ensure that only membrane-intact sperm cells were used in the ICSI procedure. We imposed these experimental conditions because our hypothesis was that possible outcome differences should be due to nuclear or cytoplasmic alterations (White, 1993) rather than membrane destabilization processes similar to capacitation (Maxwell and Johnson, 1997), both described during freezing/thawing procedures. Our ICSI results, which show a proportion of oocytes developing at least 1 pronucleus with fresh spermatozoa that was significantly higher than the proportion obtained with frozen-thawed spermatozoa, indicate that sperm cryopreservation in porcine induces cytoplasmic/nuclear alterations that compromise oocyte activation. By interpreting these results, taking into account previous reports in porcine describing a cytosolic sperm factor as the main factor responsible for oocyte activation (Macháty et al, 2000), it seems plausible that only spermatozoa without alterations in this factor could induce oocyte activation correctly. Cryopreservation and temperature shock may indeed denature this crucial factor for meiosis resumption. The lower cleavage proportion observed after ICSI with frozen-thawed spermatozoa indicates that sperm cryopreservation may also affect other nuclear/cytoplasmic factors required for transition through first mitosis. These factors could include 1) the sperm chromatin, leading to DNA fragmentation and failed fertilization and embryo cleavage, and 2) the sperm centriole, also leading to failed fertilization and embryo cleavage. With regard to sperm DNA fragmentation, several studies have reported that cryopreservation could result in increased sperm DNA fragmentation levels (Baumber et al, 2003; Martin et al, 2004). With regard to the sperm centriole, previous reports have suggested potential damage of the centriole after sperm cryopreservation (Billard, 1983; Kim et al, 2002).
These results confirm previous reports (Kolbe and Holtz, 1999) in which the injection of fresh ejaculated and frozen-thawed epididymal spermatozoa was compared and in which higher cleavage proportions were observed in the first case. In practical terms, our porcine ICSI results indicate that fresh spermatozoa provide better results than frozen spermatozoa. The use of fresh sperm cells will, however, not replace the use of frozen-thawed spermatozoa from gamete banks in laboratories that depend on sperm availability. Our porcine ICSI results also highlight important species differences, since they are in complete contrast with the results observed in rodent species, in which oocyte activation and progression through first mitosis are not compromised by the sperm freezing procedures frequently used before ICSI.
Sperm preparations for ICSI differ among laboratories. In our second experiment, we compared 2 sperm-washing and selection procedures frequently used. The first procedure comprised sperm wash and resuspension in DPBS medium supplemented with FCS, and the second comprised centrifugation through a Percoll gradient. The results obtained showed no significant differences in oocyte activation and fertilization proportions post-ICSI between treatments, and these proportions were similar to those previously reported under different conditions (Martin, 2000; Wu et al, 2001; Katayama et al, 2002b). However, when embryo in vitro development after ICSI was evaluated, a sperm treatment effect on the cleavage proportion and a strong boar effect on the proportion of blastocyst formation were detected. The reduced progression through first mitosis after ICSI with spermatozoa submitted to a Percoll treatment seems to indicate that in porcine, fewer embryo toxicity problems develop in response to ICSI when it is performed with DPBS-washed and selected sperm. In addition, the DPBS sperm-washing procedure requires less labor and is less expensive than the Percoll sperm treatment. The much higher proportion of blastocyst formation obtained with the sperm cells collected from boar A highlights the importance of careful selection of the sperm cell donor before ICSI in porcine. We considered it important to perform this evaluation, since we could not find any ICSI reference in pigs assessing this possible boar effect known in conventional IVF (Gadea and Matás, 2000). These results confirm previous evidence that regardless of the fertilizing ability of a particular sperm sample, the genetic background of the sperm cell donor modulates, to a great extent, the efficiency of ART.
In our third experiment, we tried to evaluate the impact on ICSI efficiency of the use of acrosome-reacted and non–acrosome reacted spermatozoa. The acrosome vesicle of a sperm cell has different enzymes, which can damage the oocyte when introduced in the ooplasm during ICSI (Tesarik and Mendoza, 1999). This does not happen in IVF, because only the acrosome-reacted sperm cells are able to fertilize. Some authors have observed that oocytes injected with acrosome-intact spermatozoa delay the onset of male chromatin decondensation and male pronucleus formation (Katayama et al, 2002a). We hypothesized that sperm pretreatment with calcium ionophore, an acrosome reaction inductor, could have a positive effect on ICSI outcomes. Two calcium ionophore concentrations were tested, enriching two significantly different sperm cell populations. The smallest of the concentrations enriched a population of live acrosome-reacted sperm cells and the highest concentration, a population of dead acrosome-reacted spermatozoa. However, neither of these 2 sperm cell populations were able to confirm our hypothesis. Differences in oocyte activation, pronuclear formation, and embryo development were not found between them or in relation to untreated control sperm samples after fertilization by ICSI. These results are in agreement with previous attempts in the porcine species, which used sperm heads from epididymal frozen-thawed spermatozoa (Nakai et al, 2003). However, in bovine (Goto et al, 1990), mouse (Lachan-Kaplan and Trounson, 1995), and sheep (Gómez et al, 1997), it was observed that the acrosome reaction or membrane alterations previous to ICSI were necessary to facilitate sperm head decondensation and fertilization. It is possible that porcine oocytes have a specific tolerance to acrosomal contents of the same species, as has been proposed (Sathananthan et al, 1997; Kimura et al, 1998). Considering all reported information and our experimental results, we conclude that the induction of the acrosome reaction before ICSI is beneficial depending on the species, but is probably not important in porcine. Moreover, from this experiment we also could observe that neither the live nor dead status of the spermatozoa affects the efficiency of ICSI in porcine. In particular, porcine behave like rodent species, in which it was shown that dead frozen and dehydrated sperm can be used successfully to generate offspring by ICSI (Wakayama et al, 1998).
In conclusion, the results of this study show that standardization and simplification of the ICSI procedure in pigs can be achieved by using fresh spermatozoa pretreated and selected with a single washing procedure in DPBS supplemented with FCS. They also indicate that sperm cell donor selection should be carefully performed for improved results. In addition, the results of this study also indicate that the acrosome reaction and live-dead status of the sperm cells used are parameters that do not affect the efficiency of the ICSI procedure in pigs. However, the relatively low percentage of blastocyst formation observed, although fertilization and cleavage proportions were always higher than 40%, underlines the problem that porcine ICSI zygotes still have limited ability to develop within current in vitro culture systems.
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Acknowledgments |
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Footnotes |
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