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RIKEN Kobe Institute,3 Center for Developmental Biology, Laboratory for Genomic Reprogramming, Chuo-ku, Kobe City, Hyogo 650-0047, Japan
Department of Life Science,4 Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, embryonic development, fertilization, gamete biology, ICSI, osmolarity, sperm, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The generally accepted method for sperm preservation is freezing in liquid nitrogen at –196°C; however, liquid nitrogen is not readily available to many small-scale farmers, especially in developing countries. Although preservation of sperm without freezing has been reported (e.g., mouse spermatozoa could fertilize oocytes after being stored for 7 day at 4–6°C in medium [15] or in mineral oil [16] for 8 days), these spermatozoa completely lost their in vitro fertilization capacity. Using the intracytoplasmic sperm injection (ICSI) technique, Kishikawa and colleagues obtained normal live fetuses using immotile spermatozoa retrieved 20 days after death, which were maintained in a refrigerator at 4°C [17].
To our knowledge, a system for preservation of mammalian spermatozoa in a simple medium at room temperature or low temperature, without freezing, has not been reported. Recently, Wakayama et al. [18] reported that mouse spermatozoa can be freeze dried without losing their genetic and reproductive potential and that reconstituted lyophilized sperm can support normal development when injected directly into mature oocytes. Simplified assisted reproductive technology of this type could potentially improve sperm preservation on small farms in remote areas of developing countries.
The collection and preservation of sperm sometimes takes place under field conditions that are far from ideal, for example, when reproductive material is harvested from severely injured or sick wildlife or from animals killed by hunters. In this context, the development of a simple medium for preserving sperm without freezing is needed, especially to facilitate transportation of sperm from the farm to the laboratory or between countries.
Since the successful development of the ICSI technique in humans [19], this approach has been applied in cows [20], mice [21], and pigs [22]. Using the ICSI technique surmounts several difficulties related to the quality of sperm, such as low motility, low viability [17, 18, 23], and polyspermy [22], as well as problems arising in laboratory studies when mutant lines of interest exhibit defects in male reproductive function. In a recent report, Szcygiel et al. [24] showed that ICSI is more efficient than in vitro fertilization (IVF) for generating mouse embryos from cryopreserved material.
To develop a system for sperm preservation in a simple medium without freezing, the effects of BSA supplementation, storage temperature, and the osmolarity of the medium on the viability and competence of sperm is examined. Additionally, to circumvent the difficulties involved in transporting spermatozoa in liquid nitrogen, we developed a two-step preservation system in which the sperm was first held in a simple medium, with high osmolarity, for 1 wk (the maximum duration for sperm transportation) at room temperature, and then stored at –20°C. We then examined the viability of the preserved spermatozoa and their ability to support normal embryonic development and live births after ICSI and embryo transfer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B6D2F1 mice (C57BL/6 x DBA/2) were used to collect oocytes and spermatozoa. All animals were maintained in accordance with the Animal Experimental Hand Book at the Center for Developmental Biology, RIKEN.
Oocyte Collection
Female B6D2F1 mice (8–10 wk old) were superovulated by administration of 5 IU equine chorionic gonadotropin (eCG) followed 48 h later by 5 IU human chorionic gonadotropin (hCG). Oocytes were collected from the oviducts about 16 h after injection of hCG. After collection, cumulus cells were dispersed with 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO) in droplets of HEPES-buffered CZB medium (HEPES-CZB) [25]. After several minutes, the oocytes were transferred to fresh droplets of HEPES-CZB and were denuded of almost all cumulus cells by gentle pipetting. Denuded oocytes that had a homogeneous ooplasm were selected and resuspended in new droplets of KSOMaa medium (potassium simplex optimized medium with amino acids) (Specialty Media, Phillipsburg, NJ) containing 1% BSA (Sigma), which had been previously covered with paraffin oil (Nacalai Tesque, Kyoto, Japan). The oocytes were then cultured at 37°C, in a 5% CO2 atmosphere until use.
Sperm Collection and Preservation
Sperm were collected from two epididymides of a mature B6D2F1 male for each experiment and incubated for 30 min at 37°C in 5% CO2 in air before preservation.
In Experiment 1, the sperm were collected in KSOMaa containing 0, 1, or 4 mg/ml BSA and then stored at room temperature (RT, 27°C).
In Experiment 2, based on the results of Experiment 1, the sperm were collected in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and stored at 4, 27, or 37°C (5% CO2 incubator).
In Experiment 3, the sperm were stored at 4°C in KSOM-BSA medium with an osmolarity of 271.43 (the usual osmolarity of KSOM), and 300, 400, 500, 600, 700, 800, 1000, 1500, or 2000 mOsmol; the osmolarity was adjusted by addition of 0, 0.84, 3.78, 6.72, 9.67, 12.61, 15.55, 21.46, 36.15, or 50.86 mg/ml NaCl to the KSOM-BSA medium, respectively [26].
In Experiment 4, the sperm were first stored for 1 wk in KSOM-BSA with an osmolarity of 271 or 800 mOsmol and then stored at –20°C for 1, 2, or 3 mo. Spermatozoa were stored in plastic 1.5-ml sampling tubes (Assist Co. Ltd., Tokyo, Japan) at a concentration of 5–6 x 106 spermatozoa/ml.
Sperm Head Microinjection
All preserved spermatozoa were washed three times with PBS at the end of the preservation period, and 1-µl aliquots of the sperm solution were placed in droplets (
10 µl) of HEPES-CZB containing 12% polyvinylpyrolidone (Mr 360 kDa; Wako, Japan) in a micromanipulation chamber. In all experiments, fresh sperm were included as controls. Intracytoplasmic sperm injection was performed as described previously [21]. Briefly, after washing, the sperm heads were separated from the tail by subjecting the head-tail junction to a few piezo pulses. ICSI was performed at RT. After 20 min of recovery at RT, the oocytes were cultured in KSOM for preimplantation development.
Observation of Sperm Motility and Immunofluorescent Labeling of Sperm
Immediately after the incubation or after being transferred into a series of 1.5-ml sampling tubes of different osmolarities (271–2000 mOsmol) of preservative medium, the sperm were assessed under a phase-contrast microscope by using a hemocytometer to determine the proportion of motile sperm. A motile sperm was defined as a cell having a progressive or nonprogressive motion, with nonprogressive sperm showing clear flagellar movement but no change in position. Immotile sperm included all nonmoving cells without flagellar motion and sperm heads without a flagellum. Sperm motion was analyzed by calculating the average number of immotile sperm, counted by hemocytometer.
For immunofluorescent examination, the fresh or stored sperm (which had been stored at different osmolarities for 2 mo) were pelleted by centrifugation and then fixed with 3.5% paraformaldehyde in Dulbecco PBS containing 0.1% (w/v) polyvinyl alcohol (PBS-PVA) for 30 min at room temperature. After being washed twice in 1% (w/v) BSA in PBS (PBS-BSA), the sperm pellets were incubated with 20 µg/ml fluorescein isothiocyanate-conjugated peanut agglutinin (Sigma) in PBS-BSA for 30 min. After two rinses with PBS-BSA, sperm DNA was stained with 400 µg/ ml propidium iodide (Sigma) for 20 min. After two additional rinses, the sperm pellets were mounted in a drop of antifade medium (Vector Laboratories, Inc., Burlingame, CA) on a microscopic slide and observed under an epifluorescence microscope.
Culture for Development and Embryo Transfer
KSOMaa medium containing 1 mg/ml BSA was used for embryo development. The injected oocytes in each experimental group were cultured in droplets (1 µl per oocyte) of media under paraffin oil in a plastic dish in a CO2 incubator. The stages of embryonic development were evaluated under an inverted microscope at 24-h intervals. Embryo transfer was conducted 72 h after ICSI; 8–10 morula or blastocysts were transferred to the uterus of each surrogate mother (ICR mice) on Day 3 of pseudopregnancy following mating with vasectomized ICR males.
The developmental competence of sperm was measured using the percentage of sperm-injected oocytes that reached the morula or blastocyst stage after 72 h in vitro. The full-term competence of sperm was defined as the percentage of sperm-injected oocytes that resulted in live offspring.
Statistical Analysis
Each experiment was repeated five to eight times to obtain 150–200 oocytes per treatment. The data were subjected to arcsine transformation in each replication. The transformed values were analyzed using one-way ANOVA. Values of P less than 0.05 were considered to indicate statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The developmental competence of sperm stored in KSOMaa medium without BSA was significantly lower than that of sperm stored with 1 or 4 mg/ml BSA (Fig. 1). Without BSA supplementation, sperm stored for 7 days supported development to the morula or blastocyst stage in 14% of the oocytes; sperm stored longer than 7 days were no longer developmentally competent. For ICSI conducted after 4 days of storage, the developmental competence of sperm stored in medium without BSA was lower (43%, P < 0.05) than that of sperm stored in 1 or 4 mg/ml BSA (61% and 68%, respectively). After 9 days of storage, 16% of sperm stored in medium supplemented with 1 mg/ml BSA remained developmentally competent. Supplementation with 4 mg/ml BSA extended the period of sperm competence to 14 days, with 3% of the sperm-injected oocytes developing to the morula and blastocyst stage.
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The frequency of activated oocytes after injection of stored sperm was always higher at any given time compared with the rate of oocytes that developed to the morula and blastocyst stage. Increasing the concentration of BSA from 0 to 4 mg/ml resulted in a prolongation of the competence of sperm activation from 11 to 18 days after storage at RT (Fig. 1).
Effects of Storage Temperature on Spermatozoa Viability and In Vitro Embryonic Development after Injection into Mature Mouse Oocytes
Figure 2 summarizes the effects of the storage temperature on the developmental competence of stored sperm. Sperm stored at 37°C for 5 days supported embryonic development to the morula/blastocyst stage in 15% of the sperm-injected oocytes. Sperm held at RT (27°C) remained developmentally competent for 2 wk; however, sperm held at 4°C remained competent after 24 days of storage, at which time 13% of the sperm-injected oocytes reached the morula/blastocyst stage.
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Effects of Osmolarity of Preservation Medium on Spermatozoa Viability, Embryonic Development, and Live Births after Injection into Mature Mouse Oocytes
Table 1 summarizes the effects of exposure to preservation KSOMaa media, with different osmolarities, on sperm motility. Increasing the osmolarity from 271 mOsmol to 2000 mOsmol significantly decreased sperm motility. Immediately after placement in 800 mOsmol media, all sperm motion ceased. At the time of collection, all sperm had intact acrosomes (Fig. 3A), but after 60 days of storage at 4°C, the acrosomes were lost (Fig. 3B).
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The developmental competence of sperm after storage in preservation medium with different osmolarities at 4°C is shown in Figure 4. Intracytoplasmic sperm injection was conducted using sperm stored in preservation medium with osmolarities of 271, 300, 400, 500, 600, 700, 800, 1000, 1500, or 2000 mOsmol. After 10 days of storage, there were no significant differences in the developmental competence of the stored sperm. However, after 20 days of storage, the developmental competence was significantly higher for spermatozoa that were stored in the 500- to 2000-mOsmol media (64–74%) than for those stored in the 271- to 400-mOsmol media (35–41%; P < 0.05). After storage for 30 days, the developmental competence remained significantly higher for sperm that were stored in 500- to 2000-mOsmol media (46–69%) than for those stored in 271- to 400-mOsmol media (9–29%; P < 0.05); the highest rates of sperm competence were obtained with 700-, 800-, and 1000-mOsmol media (64%, 69%, and 61%, respectively).
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The competence of sperm to support development to full term after storage in media with different osmolarities was determined by transferring morula/blastocyst-stage embryos into surrogate mothers (Fig. 5). After storage for 60 days, the full-term competence was significantly higher for sperm that were stored in 800-mOsmol medium (39%) than for sperm stored in 700-, 1000-, 1500-, or 2000-mOsmol media (18%, 20%, 17%, and 2%, respectively, P < 0.05, Fig. 5). In contrast, when ICSI was performed using sperm stored for 60 days in low-osmolarity media (271–500 mOsmol), all the oocytes were arrested before the compaction stage (Fig. 4, 60 days; and Fig. 6A). Finally, after 70 days of storage, only sperm stored in 800- or 1000-mOsmol media could support development to full term after injection into mature oocytes and subsequent embryo transfer (14%, Fig. 6B, a); the offspring that resulted from this procedure had normal reproductive ability (Fig. 6B, b).
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Effects of Two-Step Preservation on Spermatozoal Viability, Embryonic Development, and Live Births after Injection into Mature Mouse Oocytes
Increasing the osmolarity of the preservation medium from 271 to 800 mOsmol for the first week of storage (first step) resulted in increased sperm competence after storage at –20°C for 1–3 mo (second step) (Fig. 7). Only spermatozoa that were preserved in the 800-mOsmol medium could support the full-term development of embryos after 2 or 3 mo of storage (Fig. 7). The successful rate of full-term development of oocytes injected with sperm stored for 1 mo in this two-step preservation process with 800 mOsmol (31%) is similar to the previous work of Wakayama et al., in which sperm were stored at –20°C immediately after sperm collection [23].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm stored in media containing BSA have higher viability, an increased ability to activate oocytes after ICSI, and retain their developmental competence longer than sperm stored in BSA-free medium. Serum albumin is normally required to support sperm capacitation and its inclusion in media used for IVF is preferred. Recently, Bavister et al. [27] demonstrated that sperm capacitation and fertilizing competence in vitro is dependent on the presence of the native albumin molecule. Our data demonstrate that BSA not only preserves sperm activation capacity but also lengthens the period of time that spermatozoa can be stored at detrimental temperatures (RT) while retaining viability, developmental competence, and full-term competence after injection into oocytes. Inclusion of BSA in the storage medium enables sperm to be preserved for at least 20 days and still be competent to activate oocytes.
Sperm stored for 5 days at 37°C retain developmental competence (Fig. 2) and full-term competence (4%) when used in ICSI. Decreasing the storage temperature from 37 to 4°C results in increased sperm viability, developmental competence (Fig. 2), and full-term competence (data not shown). Sato et al. [28] indicate that spermatozoa kept for up to 3 days at 22°C can still fertilize oocytes, although with a relatively low efficiency. Red deer spermatozoa stored within the epididymides at 5°C and retrieved as long as 4 days after the death of the animal retain some viability [29]. Moreover, boar spermatozoa stored at 18°C for 72 h show a rapid decline in enzymatic activities and concomitant acrosomal reactions [30]. In this study, we used the ICSI technique for fertilization, thereby permitting use of sperm stored for up to 5 days at 37°C, 14 days at 27°C, and 24 days at 4°C, while retaining useful levels of developmental competence (Fig. 2), in spite of essentially no spermatozoal motility. These findings clearly indicate that, although sperm viability, developmental competence, and full-term competence in IVF applications are strongly dependent on the storage temperature, the use of ICSI for fertilization overcomes reduced sperm motility and prolongs the period of usability of stored sperm. Recent studies indicate that motility of sperm is not essential for fertilization because, when nuclei of nonviable spermatozoa are injected into oocytes, live offspring are produced [17, 23].
In our study, we observed that increasing the holding temperature results in an increase in motion of the spermatozoa immediately after collection; however, these conditions lead to a rapid decrease in motility after 1 day compared with sperm held at lower temperatures (4°C). It is possible that the high motility of sperm at higher temperatures increases the metabolism of the spermatozoa, leading to a rapid consumption of stored energy reserves and a consequent decline in sperm viability in comparison with sperm held at low temperatures.
An important finding in our study was the relationship between the osmolarity of the sperm preservation medium and the viability and developmental competence of stored sperm. Interestingly, we found that increasing the osmolarity of the preservation medium resulted in decreased sperm motility, but sperm stored in high osmolarity media retained a greater ability to activate oocytes and higher levels of developmental competence. Guthrie et al. [31] found that bovine sperm motility decreases abruptly when the cells are incubated in hypo- or hyperosmotic solutions relative to the normal level of motility at 290 mOsmol. When human sperm from fertile subjects are suspended in media with osmolarities ranging from 300 to 600 mOsmol, the proportion of motile sperm and the kinetic characteristics progressively reduce with increasing osmolarity, and sperm motility is nearly abolished in a 600-mOsmol medium [32]. Thus, our study confirms that increasing the osmolarity of the preservation medium results in a decreased motility in mouse sperm and that all motility ceases in an 800 mOsmol medium. However, immobile sperm are not synonymous with dead sperm; sperm stored in a high osmolarity medium activated oocytes and supported full-term development after injection into mature oocytes. Moreover, although all the spermatozoa lost the acrosome after 2 mo of storage in a high-osmolarity medium, the stored sperm retained developmental and full-term competence. These findings suggest that the immediate cessation of sperm motion before storage may help conserve energy and decrease the process of catabolism and might increase the viability of the sperm after long-term preservation. Similarly, most media used for sperm cryopreservation are supplemented with glycerol, which decreases the motion of sperm before freezing, but increases postthaw motility and viability [33, 34].
A previous study showed that the ability of spermatozoa to activate oocytes spontaneously is not destroyed by lyophilization or freezing without cryoprotection in a simple Tris-HCl buffer containing 50 mM EGTA and 50 mM NaCl [35]. Wakayama et al. [23] have determined that freeze-dried mouse spermatozoa are all immobile and dead in the conventional sense. When injected into oocytes, however, their nuclei can support normal embryonic development even after 3 mo of preservation in a dried state. Furthermore, plasma membrane integrity of sperm is not essential for fertilization and full-term development if sperm nuclei are injected directly into oocytes [23]. Taken together with our results, these findings indicate that dead sperm are not lifeless.
Thus, we have developed a new method for the preservation of sperm using only a simple medium and salt, which permits retention of the developmental and full-term competence of sperm after 70 days of storage without freezing. These results cause us to speculate that high osmolarity is causing sperm death and no more motility but may be inhibiting degradation of DNA (full-term competence of sperm is an exact evidence in this study).
The results of Experiment 4 again show that high osmolarity of preservation medium in the first step prolongs the period of viability to up to 3 mo for sperm stored at –20°C. Thus, immediate cessation of sperm motility before preservation may be inhibited enzymatic activity and maintain a high degree of normal DNA integrity, results in live births after ICSI. It has been clearly demonstrated that sperm preserved at –20°C without cryoprotection immediately after collection can support full-term development after 4 wk of storage [23]. Thus, the application of high technology in the laboratory (ICSI) allows us to transport spermatozoa from the field under low-technology conditions. By carrying the sperm in a pocket, sealed in a tube containing a simple medium with a suitable osmolarity (adjusted by addition of salt), we can transport sperm from country to country, knowing that the sperm will retain reproductive competence for up to 3 mo when later stored at –20°C in the laboratory. In this study, we used KSOM as a simple medium for preservation because this medium is commonly used for culture of mouse embryo in our laboratory. However, our goal is to apply this to field work; therefore, using just water and salt for sperm preservation is required for the further study.
In conclusion, we have developed, for the first time, a simple method for sperm preservation using a hyperosmotic medium without freezing as the initial approach. Mouse spermatozoa are able to activate oocytes, support development to the blastocyst stage, and produce live offspring after 70 days of storage at 4°C in a simple medium with the osmolarity adjusted to 800 mOsmol by the addition of NaCl. Our results also suggest that using a two-step process of preservation—first holding in 800-mOsmol medium at RT for up to 1 wk (the transport time) and then storage at –20°C—permits retention of full-term competence for up to 3 mo. The combination of laboratory modernity (ICSI) and a simple method of sperm preservation might allow the transportation of spermatozoa from farm to laboratory or between countries without cryopreservation.
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FOOTNOTES |
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2 Correspondence: Nguyen Van Thuan, RIKEN Kobe Institute, Center for Developmental Biology, Laboratory for Genomic Reprogramming, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe City, Hyogo 650-0047, Japan. FAX: 81 78 306 3095; nvthuan{at}cdb.riken.jp
Received: 22 July 2004.
First decision: 30 August 2004.
Accepted: 29 September 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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