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Reproductive Technology |
The Graduate School of Natural Science and Technology,3 Okayama University, Okayama 700-8530, Japan
Department of Animal Science and Technology,4 Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, early development, fertilization, gamete biology, ICSI, lyophilization, oocyte activation, pig, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of methods for the freeze-drying of spermatozoa is beneficial for the preservation of male genomes not only in laboratory rodents but also in farm animals. Because freeze-dried spermatozoa can be stored at room temperature or in ordinary refrigerators, the costs required for maintenance and shipping of spermatozoa would be reduced drastically. Therefore, preservation of spermatozoa in a freeze-dried condition will revolutionize animal husbandry as the development of the technique for cryopreservation did. Nevertheless, in farm animals, few reports have been published on the development of oocytes injected with freeze-dried spermatozoa, except in cattle [10].
The objective of the present study was to examine whether early fertilization of pig oocytes could be supported when they are microsurgically injected with freeze-dried spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Media
Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The medium used for maturation of oocytes was BSA-free NCSU 23 [11] supplemented with 0.1 mg/ml cysteine, 5 µg/ml insulin (I-6634), 10% (v/v) porcine follicular fluid (pFF), 10 IU/ml hCG (gonatropin; Teikoku-Zoki Co., Tokyo, Japan), 10 IU/ml eCG (Serotropin; Teikoku-Zoki), 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate. The pFF was withdrawn from superficial follicles (3–6 mm in diameter) on maturing pig ovaries using an 18-gauge needle fixed to a 10-ml disposable syringe. The drawn pFF was centrifuged at 1500 x g for 15 min at room temperature to remove any cells or debris, and the supernatant was stored at –20°C until used [12]. The medium used at ICSI was TL-Hepes-polyvinylalcohol (PVA; pH 7.4) composed of 114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO3, 0.34 mM NaH2PO4, 0.5 mM MgCl2, 2 mM CaCl2, 12 mM sorbitol, 10 mM Hepes, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 25 µg/ml gentamycin, 65 µg/ml potassium penicillin G, and 1 mg/ml PVA. The medium used for culture of oocytes after ICSI was NCSU 23 supplemented with 4 mg/ ml BSA (A-0281; fatty acid-free).
Preparation of Oocytes
Ovaries were obtained from maturing gilts at a local slaughterhouse and transported to the laboratory within 1–1.5 h in 0.9% (w/v) NaCl solution containing 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulfate at 37–39°C. Oocytes were aspirated from antral follicles (3– 6 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe and washed three times with maturation medium. Oocytes surrounded by compact and dense cumulus cell layers were selected. A group of 50 oocytes was introduced into a 500-µl drop of maturation medium covered with paraffin oil (no. 261-17; Nacalai Tesque, Kyoto, Japan), which had previously been equilibrated in an atmosphere of 5% CO2 in air at 39°C for at least 3 h in a four-well culture dish (no. 176740; Nunc, Roskilda, Denmark). The oocytes were then cultured for 44–48 h at 39°C under the same atmospheric conditions. After culture, oocytes were freed from cumulus cells by repeated passage through a fine pipette in maturation medium supplemented with 0.1% (w/v) hyaluronidase (H3506). After washing three times with TL-Hepes-PVA, the denuded oocytes were introduced into about 200 µl TL-Hepes-PVA in a 1.5-ml centrifuge tube and centrifuged at 7000 x g for 10 min to visualize the extruded first polar body. Ten to 15 oocytes with the first polar body were transferred into 5 µl of TL-Hepes-PVA covered with paraffin oil in a cover of a Petri dish (50 x 4 mm; Falcon no. 1006; Becton Dickinson Labware, Lincoln Park, NJ) and kept in an incubator (39°C) until used for ICSI.
Sperm Collection and Freeze Drying
Sperm-rich fractions collected from boars at a local livestock center were diluted four times with a solution composed of 23.5 mM sodium citrate, 11.9 mM NaHCO3, 6.3 mM EDTA-2Na, 46.7 mM Tris, 15.1 mM citric acid, 152.6 mM glucose, and 0.3 mg/ml gentamycin. This solution was essentially the same as Modena solution described by Weitze [13] as an extender for pig semen. After being transported to the laboratory within 2 h, the diluted semen (10 ml) was centrifuged at 600 x g for 10 min and the supernatant was removed. Then the pelleted spermatozoa were washed twice with 10 ml TL-Hepes-PVA by centrifugation at 600 x g for a period of 10 min each. Each of 150 µl of the final sperm suspension in 10 ml TL-Hepes-PVA was transferred into 1.5-ml centrifuge tubes, and the tubes were directly plunged into LN2 for 20 sec. The tubes were then placed in a precooled (–80°C) freeze-flask and the flask was attached to a freeze-drying system (Eyela; Tokyo-Rika-Kikai Co., Japan). The inside pressure was 39.9 x 10–3 mbar during lyophilization. After 4–24 h under lyophilization, the flask was removed from the system and the tubes were closed without removing oxygen and firmly sealed with parafilm and stored at 4°C or room temperature (25°C) for 1–6 mo. A portion of sperm suspension before freeze-drying was used for ICSI of fresh spermatozoa.
Rehydration of Freeze-Dried Spermatozoa
Freeze-dried sperm samples were rehydrated by adding 150 µl of milli-Q water into the tubes. The sperm suspension was then transferred into 15-ml centrifuge tubes and about 3 ml of TL-Hepes-PVA were added. A portion of the content of the centrifuge tubes was placed in ice water and the spermatozoa were sonicated for 3 sec (ten 0.3-sec bursts at 0.7-sec intervals) using 20% power output of a Branson Sonifier model 250 (Branson Ultrasonics, Danbury, CT). The separation into heads and tails was successful in more than 70% of the spermatozoa. Each 100 µl of the sperm suspension that was not sonicated was transferred into another centrifuge tube containing about 2 ml TL-Hepes-PVA. This sperm suspension (50 µl) was again diluted by adding 50 µl of TL-Hepes-PVA supplemented with 6% (w/v) polyvinylpyrrolidone (PVP-360). A 5-µl drop of the final sperm suspension was transferred onto a cover of a Petri dish adjacent to the drop containing oocytes that had been previously prepared.
ICSI
Microinjection of whole spermatozoa or isolated sperm heads into oocytes was performed at 25°C using a piezo-driven pipette that was prepared from borosilicate glass capillary tubes (Sutter Instrument Co., Novato, CA). The external and internal diameters of the tip of the injection pipette were 10–11 µm and 8–9 µm, respectively. A spermatozoon or a sperm head was aspirated into the injection pipette with a minimal amount of medium and the tip of the pipette was brought in contact with the zona pellucida of the oocyte, which was held by a holding pipette with an external diameter of 100–110 µm and an internal opening of 15–20 µm. The zona was drilled by applying two to three pulses (intensity 5, speed 3). The tip introduced into the perivitelline space was forced onto the oolemma and introduced into the cytoplasm. After a small amount of cytoplasm was once withdrawn to confirm that the tip was in the cytoplasm, a spermatozoon or a separated sperm head in a minimal amount of medium was expelled into the oocyte. Then the pipette was gently withdrawn from the oocyte. The injection of spermatozoa or sperm heads into oocytes was completed within 45 min after the preparation of oocytes. In one experiment, as the control, whole spermatozoa and isolated sperm heads before freeze-drying were used for ICSI. In that case, the final sperm suspension for ICSI was prepared with the same procedures as employed for obtaining freeze-dried samples without the process of freeze-drying.
Determination of Oocyte Activation after ICSI
All oocytes used for ICSI were washed three times with culture medium. After washing, 10–15 oocytes were transferred into 100 µl of the same medium covered with paraffin oil in a culture dish (35 x 10 mm; Falcon no. 1008) and cultured in a CO2 incubator (5% CO2 in air at 39°C). At 18–20 h after the start of culture, oocytes were fixed for about 72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% (w/ v) orcein in 45% (v/v) acetic acid in water, and examined for evidence of activation of oocytes and transformation of sperm nuclei using a phase-contrast microscope at a magnification of 200x or 400x. The oocytes in which intact sperm head, enlarged sperm head, or male pronucleus was observed were considered as successfully injected. The oocytes with one female pronucleus and a second polar body or with two female pronuclei without a second polar body were assessed as activated.
Examination of In Vitro Development of ICSI Oocytes
All oocytes used for ICSI were washed and cultured as described above. The oocytes were examined for their development after 48, 72, 144, and 168 h of culture under a dissecting microscope.
Examination of In Vitro Development of Oocytes Treated with Ca Ionophore after ICSI
The oocytes injected with sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4°C for 1 mo were treated with 5 or 10 µM Ca ionophore A23187 (CaA) in TL-Hepes-PVA for 2 min at 25°C. The treated oocytes were transferred into 100 µl of the same medium supplemented with 4 mg/ml BSA and kept at 25°C for 5 min. Then the oocytes were washed and cultured as mentioned above. The oocytes were examined for their development after 48, 72, 144, and 168 h of culture under a dissecting microscope.
Statistical Analysis
All proportional data were subjected to an arc-sine transformation, and the transformed values were analyzed using one-way or two-way ANOVA. When ANOVA revealed a significant effect, the treatments were compared by Fisher protected LSD least significant difference test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activation and Development of Oocytes Injected with Whole Spermatozoa or Isolated Sperm Heads Before and after Freeze Drying for 12–18 h and Stored at 25°C for 1 Mo
As shown in Table 1, significantly more (P < 0.05) oocytes were activated when they were injected with sperm heads than whole spermatozoa in both fresh and freeze-dried samples. However, the proportion of activated oocytes was significantly lower (P < 0.05) in freeze-dried than in fresh samples. In freeze-dried samples, but not in fresh samples, male pronuclear (MPN) formation was observed in significantly more (P < 0.05) oocytes activated after being injected with sperm heads than whole spermatozoa. There was no significant difference in the incidence of MPN formation in activated oocytes between fresh and freeze-dried sperm heads. As shown in Table 2, significantly more (P < 0.05) oocytes developed to the two- to four-cell stages when injected with sperm heads than whole spermatozoa, although these values were significantly lower (P < 0.05) than those obtained in fresh samples. Although, in fresh samples, injected oocytes developed to the morula and blastocyst stages, no development to the morula stage was observed in oocytes when freeze-dried samples were used.
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Activation and Development of Oocytes Injected with Sperm Heads Isolated from Spermatozoa Freeze-Dried for Various Times and Stored at 4 or 25°C for 1 Mo
As shown in Table 3, when injected with sperm heads, there were no significant differences in the proportion of activated oocytes and incidence of MPN formation (Fig. 2) among different times (4, 9, and 16 h) of freeze-drying and between different storage temperatures. However, these values were significantly decreased (P < 0.05) when spermatozoa were freeze-dried for 24 h. As shown in Table 4, the developmental ability of injected oocytes was gradually reduced as the time of freeze-drying was prolonged, particularly when freeze-dried spermatozoa were stored at 25°C. The development to the morula stage was observed only in oocytes injected with sperm heads isolated from spermatozoa freeze-dried for 4 h, but significantly fewer (P < 0.05) oocytes developed when freeze-dried spermatozoa were stored at 25°C than 4°C. The development to the blastocyst stage was observed only in oocytes injected with sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4°C (Fig. 3).
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Effect of Treatment of Oocytes with CaA after Being Injected with Sperm Heads Isolated from Spermatozoa Freeze-Dried for 4 h and Stored at 4°C for 1 Mo
As shown in Table 5, significantly more (P < 0.05) oocytes developed to the four-cell stage when injected oocytes were treated with 10 compared to 0–5 µM CaA. However, there were no significant differences in the proportions of oocytes developed to the two-cell, morula, and blastocyst stages among those treated with 0–10 µM CaA.
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Activation and Development of Oocytes Injected with Sperm Heads Isolated from Spermatozoa Freeze-Dried for 4 h and Stored at 4°C for Various Periods
As shown in Table 6, there were no significant differences in the proportion of activated oocytes and in the incidence of MPN formation in activated oocytes among oocytes injected with sperm heads isolated from spermatozoa stored 1, 3, and 6 mo. As shown in Table 7, however, the developmental ability of injected oocytes was significantly reduced (P < 0.05) with an increased period of storage; no development of injected oocytes to the blastocyst stage was observed when freeze-dried spermatozoa were stored for 3 and 6 mo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, activation and development of oocytes produced with freeze-dried pig spermatozoa were largely impaired compared with those produced with fresh spermatozoa. This difference in activation may indicate that substances responsible for oocyte activation such as sperm-borne oocyte activating factor [16] of sperm heads are damaged or inactivated during the freeze-drying process. Furthermore, it has been reported that the stability of the sperm nuclear matrix may be critical in the development of oocytes into live offspring [17]. In mice, at least 50% of spermatozoa had abnormal chromosomes after freeze-drying [8], and the incidence of abnormal karyotype in the zygote is directly correlated with embryo development [18]. Although we did not examine sperm plasma and chromosome integrity of pig spermatozoa after freeze-drying in the present study, chromosomal damage is a possible reason for impaired development of embryos produced with freeze-dried pig spermatozoa. It would be interesting to examine in further experiments whether calcium chelators like EGTA can prevent chromosomal damage of freeze-dried pig spermatozoa and thus the development of injected oocytes may be improved, as reported in mice [5].
In the present study, the proportion of activated oocytes, the incidence of MPN formation in activated oocytes, and the proportion of oocytes cleaved to the four-cell stage were significantly lower in the injection of whole spermatozoa than isolated sperm heads. During normal fertilization, the sperm plasma membrane does not enter the ooplasm [19]. In ICSI in mice, it has been reported that removal of sperm plasma membrane accelerates the activation of injected oocytes [20]. It has also been reported that membrane-damaged spermatozoa improves sperm head decondensation and production of blastocysts in ICSI oocytes in humans [21] and pigs [22]. Although the sperm plasma membrane is largely damaged or removed by the process of freeze-drying [4, 9], it may be possible that removal of the remaining sperm plasma and other membranes is accelerated by the additional treatment, the sonication, of rehydrated spermatozoa to separate into heads and tails. The further removal of the sperm membranes may make it easier for the sperm nucleus to mix with the ooplasm [23]. Thus, the injection of sperm heads isolated from spermatozoa rehydrated after freeze-drying may accelerate the activation of oocytes and MPN formation in activated oocytes. Nevertheless, the incidence of activation, MPN formation, and early development of oocytes was significantly lower when injected with freeze-dried than fresh, whole spermatozoa that have intact sperm membranes. As suggested in rabbits [9], the process of freeze-drying may induce chemical changes in the sperm membranes by which dissolution of the membranes becomes more difficult compared with fresh spermatozoa. However, further experiments are needed to clarify this point.
Although the freeze-dried systems used are different, spermatozoa have been freeze-dried for about 4 h in mice [8] and rabbits [9] or 12–18 h in mice [4] and cattle [10]. However, there have been no reports examining the effect of lyophilization time on the ability of spermatozoa to fertilize oocytes. The results of the present study demonstrate that the incidence of oocyte activation, MPN formation, and cleaved oocytes was significantly reduced when spermatozoa were freeze-dried for 24 h. Because about 50% of rehydrated spermatozoa had bent tails or were missing tails when freeze-dried for 24 h, a long time of freeze-drying may affect not only morphology or structure but also some substances responsible for oocyte activation, such as sperm-borne oocyte activating factor [16] of sperm heads. The most possible reason for such impairment may be that samples were overdried. In most experiments involving desiccation of proteins, bacteria, or plants, the final moisture reported for stable storage has been less than 5% [24]. When the moisture goes below a certain level, the molecular mobility decreased during desiccation begins to increase.
It has been reported that mouse oocytes injected with freeze-dried spermatozoa are readily activated without artificial stimulation [4–8]. However, in cattle, the developmental competence of oocytes after ICSI is improved by various stimuli for oocyte activation [25, 26]. Activation treatment has also enhanced the developmental competence of rabbit oocytes injected with freeze-dried spermatozoa [9]. Although about 90% of pig oocytes treated with 6.25 µM CaA can be activated [27], we did not observe any significant effects of CaA (5 and 10 µM) treatment of injected oocytes on the efficacy of blastocyst formation in the present study. It has been reported that about 4%–9% of pig oocytes matured in vitro developed to the blastocyst stage after being injected with fresh sperm heads and then stimulated with a direct current pulse [28]. However, about 13%–17% (in the present study) and 31%–45% (in other studies [14, 15]) of oocytes injected with fresh spermatozoa developed to the blastocyst stage without any stimulation for oocyte activation after ICSI. It may be possible that artificial stimulation of oocytes after ICSI is not necessary in pigs, like in mice [4–8], for their activation and development. This has been confirmed by Kolbe and Holtz [29], who observed that CaA treatment of pig oocytes following injection of frozen-thawed spermatozoa did not affect their further development. However, it has been found that a fixed field strength of a direct current pulse is beneficial to blastocyst formation of pig ICSI oocytes [22]. It would be interesting to examine if improved oocyte activation protocols will give higher blastocyst rates after ICSI with freeze-dried pig spermatozoa.
It has been demonstrated that long-term storage of freeze-dried mouse spermatozoa is not deleterious to their genetic integrity; as even after storage for up to 18 mo, spermatozoa can retain their ability to generate viable offspring [8]. Such long-term storage of freeze-dried spermatozoa is also possible in rabbits [9]. In the present study, however, although a similar incidence of activation and MPN formation was obtained in pig oocytes injected with freeze-dried sperm heads, the developmental ability of injected oocytes significantly decreased with increasing time of storage at 4°C. It has been known that DNA damage can occur when damaged cells are exposed to radical oxygen species [30]. Because, in the present study, freeze-dried samples were closed in the presence of air, it may be possible that DNA damage took place during storage. The protocols for freeze-drying, the storage conditions of dried spermatozoa, and procedures for rehydration need further modification to improve the fertilizing ability of freeze-dried pig spermatozoa stored for a long time.
In conclusion, we have demonstrated that pig spermatozoa can be freeze-dried and that these spermatozoa can subsequently induce activation and development to the blastocyst stage of oocytes after ICSI. Further experiments are needed, however, to examine whether pregnancies can be obtained following embryo transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 81 86 251 8388; kniwa{at}cc.okayama-u.ac.jp
Received: 23 April 2004.
First decision: 21 May 2004.
Accepted: 16 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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