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This Article Abstract Full Text (PDF) All Versions of this Article: 68/1/19    most recent biolreprod.102.007344v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bath, M. L. Search for Related Content PubMed PubMed Citation Articles by Bath, M. L. Agricola Articles by Bath, M. L.

Simple and Efficient In Vitro Fertilization with Cryopreserved C57BL/6J Mouse Sperm¹

^a The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%–12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%–88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%–63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.

assisted reproductive technology, fertilization, in vitro fertilization

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cryopreservation of mouse spermatozoa has become increasingly necessary for archiving purposes and for short-term removal of stock from overcrowded mouse rooms. Consequently, in recent years, a concerted effort has been made to establish a reliable method for the cryopreservation of mouse sperm, and now most laboratories use the raffinose/skim-milk method described by Nakagata [1]. This method is adequate for many hybrid strains and mice of mixed background, but it is far less effective for inbred strains, most notably the C57BL/6J mouse, which is commonly used in knock-out and transgenic experiments [2–4]. When aided by partial zona pellucida dissection (i.e., zona nicking) [2, 5] or intracytoplasmic sperm injection [6], frozen-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Why thawed C57BL/6J sperm have difficulty penetrating the intact zona pellucida is unknown, but it may be related to the low percentage that have progressive motility [2, 3] and to the presence of a large number of dead sperm during in vitro fertilization (IVF) [7]. The aim of the present study was to isolate the progressively motile spermatozoa from a thawed suspension of cryopreserved C57BL/6J sperm and to test their fertility by IVF and by retrieval of young.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals

Mice were obtained from the Walter and Eliza Hall Institute of Medical Research mouse breeding colony. They were maintained under routine husbandry procedures in accordance with the guidelines set out in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes [8].

Media

Tissue-culture grade or embryo-tested chemicals were obtained from Sigma Chemical Company (St. Louis, MO). Mineral oil was supplied by Cook IVF (Eight Mile Plains, QLD, Australia). Water (R 18 Ohm) was obtained in-house from a water-purification system (Millipore Australia, North Ryde, NSW, Australia). Media were prepared on the day before use and were filter-sterilized through 0.2-µm filters (Pall Gelman Laboratory, Ann Arbor, MI). The IVF and overnight (O/N) media were modifications of Quinn Basal X1 medium [9]. The sperm incubation (SI) medium contained no lactate or calcium. The lack of added calcium in the SI medium prevented acrosomal loss during sperm incubation [10]. The compositions of the SI, IVF, and O/N media are shown in Table 1.

Eggs were manipulated outside the incubator in IVF or O/N medium in which part of the bicarbonate was replaced with Hepes to give final concentrations of 4 and 21 mM, respectively. The pH was adjusted to 7.4 with 0.1 M NaOH.

Cumulus cells around eggs were removed by incubation of egg masses in Hepes-buffered IVF medium containing 0.05% (w/v) hyaluronidase.

Bicarbonate was added to all media from a 10x stock solution containing 5 µl/ml of phenol red solution. Hepes was added from a 0.25 M stock solution adjusted to pH 7.4 with 1 M NaOH. The Hepes solution contained 1 µl/ml of phenol red solution. The phenol red solution was prepared by dissolving 0.2 g of phenol red in 5 ml of 0.1 M NaOH.

The cryoprotectant was prepared by dissolving 1.93 g of raffinose pentahydrate in 8.8 ml of water. The osmolality was adjusted to 380 mOsm/kg H₂O by the addition of the appropriate volume of water [11]. The solution was filter-sterilized through a 0.22-µm membrane, stored overnight at 4°C, and warmed to room temperature before use.

Manipulation of Sperm

Sperm were transferred using standard pipettes with relatively wide-bore tips attached (volume, 200 µl [Greiner Labortechnik, Frickenhausen, Germany] and 10 µl [TF-400-L-R-S; Axygen Scientific, Union City, CA]).

Collection and Freezing of Sperm

Male C57BL/6J mice (age, 3–8 mo) were housed singly for at least 5 days before sperm collection. After killing a mouse by cervical dislocation, the caudae epididymides were removed and placed in 105 µl of raffinose solution in a flat-bottomed, 96-well tissue-culture plate. The tissue was cut into small pieces and the sperm allowed to disperse for 3 min. The epididymal tissue was removed and the plate gently shaken to distribute the sperm evenly before loading into six 0.25-ml freezing straws with 14.2 µl each of the sperm suspension. The straws were then sealed with straw-sealing powder (Genetics Australia, Bacchus Marsh, VIC, Australia) and placed in liquid nitrogen vapor for 10 min and then into liquid nitrogen, where they were stored.

Pre-Equilibration of Media

For holding eggs or egg masses before IVF, four 120-µl drops of IVF medium were placed in 60-mm, bacteriological-grade Petri dishes (Greiner Labortechnik) and overlaid with mineral oil. For IVF, four 200-µl drops of IVF medium were placed in 60-mm, tissue culture-grade Petri dishes (Nunc, Roskilde, Denmark). For culture of eggs overnight after IVF, four 120-µl drops of O/N medium were placed in 60-mm, tissue culture-grade Petri dishes. All dishes were transferred to an incubator and equilibrated overnight at 37°C in 5.5% CO₂ in air in a humidified atmosphere.

For incubation of sperm, 36 µl of SI medium were placed in the center of each 35-mm, bacteriological-grade Petri dish and overlaid with mineral oil. The dishes were left at room temperature overnight. Approximately 45 min before addition of sperm, the dishes were placed on a warming tray (37°C) and covered to exclude light and to retain heat.

Collection of Eggs

Female C57BL/6J mice (age, 26–28 days) were superovulated by i.p. injections of 10 IU of eCG (Folligon; Intervet, NSW, Australia) followed 48 h later by 10 IU of hCG (Chorulon; Intervet). At 12–13 h after the second injection, the mice were killed and their oviducts removed. The egg masses were isolated and treated with hyaluronidase to remove the cumulus cells. Eggs and egg masses were washed through several changes of Hepes-buffered IVF medium and then transferred to pre-equilibrated IVF medium. The use of bacteriological-grade dishes for holding egg masses discouraged the masses from sticking to the bottom of the dish.

Thawing of Sperm

Frozen sperm suspensions were thawed by removing a straw from the liquid nitrogen and, after 5 sec in air, were transferred to a 54°C water bath for 5 sec. The straw was wiped dry immediately after removal from the water bath.

Removal of Cell Debris

Cell debris present in the thawed sperm suspension included nonmotile sperm, epididymal tissue fragments, and small aggregations of motile sperm that have previously been referred to as asters [12]. Two methods were used to remove the cell debris.

Method 1 This was a two-step procedure. The first step enriched for progressively motile sperm, and the second step removed cell debris. A straw was cut just ahead of the column of thawed sperm suspension, and with the aid of a dissecting microscope, 12 µl of the suspension were transferred into a 36-µl drop of warm SI medium. The dish was placed on the warming tray, and the sperm were incubated in the dark for 30 min. During this time, many of the nonmotile sperm massed together. When a small volume (3 µl) of the massed sperm was removed by drawing the pipette tip from the periphery of the drop of medium toward the center, a wedge-shaped, sperm-free area was observed that immediately filled with single progressively motile sperm. These sperm were transferred in 3-µl volumes into the center of each of the four drops of pre-equilibrated IVF medium. Another 2–3 µl of massed sperm were removed, and again, 3 µl of motile sperm were transferred into the drops of IVF medium so that each drop received a total of 6 µl of motile sperm. Within 60 sec, many of the motile sperm had reached the periphery of the drop of IVF medium, allowing nonmotile sperm and asters to be removed from the center of the IVF drop in a volume of 20 µl or less.

Method 2 A small volume (1.5 µl) of thawed sperm suspension was transferred from the straw to the center of each drop of IVF medium. After the motile sperm had moved to the periphery, the cell debris was removed. Because this usually required removal of 20–40 µl of medium, this volume was replaced with fresh medium after the eggs were added.

In Vitro Fertilization

Usually, 24–27 eggs or one–two egg masses were placed around the periphery of each drop of IVF medium. The eggs and sperm were incubated together for 6 h at 37°C in 5.5% CO₂ in air in a humidified atmosphere. At the end of the 6-h incubation, the eggs were washed free of excess sperm in Hepes-buffered O/N medium and then transferred to the pre-equilibrated O/N medium. Twenty-four hours after the start of the IVF, the number of 2-cell embryos obtained was recorded. The fertility of the sperm (i.e., fertilization rate) was defined as the percentage of 2-cell embryos obtained.

Embryo Transfer

Two-cell embryos were transferred to the right oviduct of 0.5-day (CBA x C57BL/6J)F1 pseudopregnant mice. Each mouse received 7–10 embryos.

Statistics

In the present study, the main interest was to compare the fertilization rates of thawed sperm after three treatments: 1) direct transfer of sperm to the IVF medium; 2) direct transfer of sperm to the IVF medium, followed by removal of nonmotile cell debris and asters; and 3) isolation of the motile sperm by the two-step procedure. Comparison of the differences between the means for each treatment was done by the paired-sample Student t-test after arcsine transformation of the fertilization rates. Differences between the means were considered to be significant when P < 0.05 was achieved.

	RESULTS

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Fertilization after Removal of Cell Debris

Denuded eggs were inseminated with 1.5 µl of thawed sperm suspension (treatment 1), 1.5 µl of thawed sperm suspension with the cell debris removed (treatment 2), or 6 µl of motile sperm isolated by the two-step procedure (treatment 3; equivalent to 1.5 µl of thawed sperm suspension). The fertilization rates of sperm from 10 donor mice after each of these treatments are shown in Table 2. In Figure 1, the mean ± SEM is shown for each treatment group. Comparison of the mean fertilization rates for treatments 1 and 2 (5% and 35%, respectively) demonstrated unequivocally that the removal of cell debris during IVF substantially increased the rate of fertilization. The higher fertilization rate obtained after removal of dead sperm occurred even though the sperm concentration in the IVF drop would have been lower than that after treatment 1. A further increase in fertilization rate to 65% was achieved by using the two-step procedure. This possibly resulted from better removal of cell debris plus an increased concentration of progressively motile (i.e., functional) sperm.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Fertilization rates (mean ± SEM) obtained after treatment 1, 2, or 3 of the cryopreserved sperm from the 10 mice shown in Table 2. The difference between treatment groups was significant (P < 0.0001). (See Materials and Methods for definitions of treatments)

Reproducibility of the Fertilization Rate

Table 3 shows the fertilization rates obtained on separate days for 10 mice using the two-step procedure to isolate the progressively motile sperm. The mean fertilization rates of the sperm from the 10 mice were 65% and 66% on Day 1 and Day 2, respectively.

Fertilization of Denuded Eggs and Egg Masses

Table 4 shows that thawed sperm were able to fertilize egg masses and not just denuded eggs (P = 0.28). The average fertilization rates of the sperm from the six mice shown in Table 4 were 50% for denuded eggs and 45% for egg masses, indicating that the presence of cumulus cells had no influence on the ability of the sperm to fertilize the eggs.

Increased Eggs Increases the Number of 2-Cell Embryos

When the same volume of sperm, isolated by the two-step procedure, was used to inseminate 25–27 or 55–64 denuded eggs in each 200-µl drop of IVF medium, the higher number of eggs yielded more 2-cell embryos (530 from the sperm of four mice) than the lower number of eggs (289 from the sperm of the same four mice), although this was at the expense of a significantly reduced fertilization rate (mean, 70% vs. 55%; P = 0.0007). This result may have been associated with the reduced number of sperm per egg in the group with the higher number of eggs. In practical terms, increasing the number of eggs is a way of increasing the yield of 2-cell embryos and, hence, of pups born per aliquot of sperm thawed. The fertilization rates for the sperm of the four individual mice are shown in Table 5.

To determine if the development of 2-cell embryos was the result of fertilization and not parthenogenic activation of the eggs, an aliquot of sperm was thawed and refrozen by plunging the straw directly into liquid nitrogen. After a second thaw, the sperm were used in the two-step procedure. No motile sperm were observed in the IVF drop, and none of the denuded eggs developed into a 2-cell embryo. This result is shown in Table 5.

Newborn Mice from Frozen Sperm

In total, 1546 two-cell embryos were transferred to pseudopregnant recipient mice, and 785 (51%) young were born 19–20 days later. On any one day, 39–232 embryos were transferred, and 40%–63% of the transferred embryos produced live-born healthy pups.

	DISCUSSION

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In recent years, attempts at IVF using cryopreserved C57BL/6J mouse sperm have yielded few fertilized eggs [2–5, 13, 14], leading Sztein et al. [3] to conclude that IVF with these sperm would not be improved without the aid of special techniques, such as zona nicking or intracytoplasmic sperm injection. However, the results of the present study show that efficient fertilization rates with cryopreserved C57BL/6J mouse sperm can be achieved simply by removing nonmotile sperm, epididymal tissue fragments, and asters from the thawed sperm suspension. This explains previous findings demonstrating that an increased concentration of sperm in the IVF without the removal of cell debris does not improve the fertilization rate [3].

Few mouse sperm survive the freeze-thaw process, with less than 10% retaining progressive motility [11]. Consequently, a large number of dead and moribund sperm are inadvertently transferred with the sperm suspension to the IVF dish. In addition, epididymal tissue fragments and soluble substances are also transferred, and these may have detrimental effects on fertilization. Exactly how dead, moribund, or aggregated sperm or how epididymal tissue or soluble substances interfere with fertilization is unknown [7], but the explanation must take into account that they do not prevent cryopreserved C57BL/6J mouse sperm from fertilizing zona-nicked eggs [2, 5].

In the present study. it was assumed that the sperm capable of fertilizing eggs would be found among the progressively motile population. Usually, these sperm are isolated by the swim-up method [15]. Although swim-up has been tried with thawed mouse sperm, it has been unsuccessful, possibly because of the presence of an overwhelmingly large number of dead sperm [3, 7]. The advantages of the "swim-out" technique described in the present study are 1) that the sperm suspension is not centrifuged and pipetting is minimal, thereby avoiding mechanical damage [12]; 2) that the sperm can be visualized while isolating the motile sperm; and 3) that two steps are taken to remove the cell debris, with one selecting for the progressively motile sperm and the other aimed at removing most of the remaining nonmotile sperm and asters. The disadvantage is that a relatively large volume of thawed sperm suspension, containing a high concentration of sperm, is required, but this is greatly outweighed by the large number of 2-cell embryos obtained. These embryos are not compromised in any way. The production of live-born after transfer of the 2-cell embryos to pseudopregnant recipient mice is, in the author's experience, no different from that of 2-cell embryos transferred after IVF with fresh sperm or after in vivo fertilization.

In conclusion, a procedure has been devised for reproducibly efficient IVF and retrieval of young using cryopreserved sperm of C57BL/6J mice. The procedure requires no special technical skills or equipment. High fertilization rates are obtainable, and production of young is prolific.

Since submitting this manuscript, a paper has been published [16] showing that separating motile sperm before freezing increases the postthaw fertilization rate in C57BL/6J and BALB/c mice from 16% to 40% and from 14% to 51% respectively. An increase in fertilization rate was also found for FVB and DBA/2 mice [16]. These results suggest that removal of cell debris after thawing may also increase the fertilization rate of other strains in addition to C57BL/6J. This possibility is under investigation.

	ACKNOWLEDGMENTS

 
The author thanks D. Postma and A. Naughton for taking care of the mice, Drs. A. Egle and G. Smyth for help with the statistics, Drs. L. Robb and A. Strasser for critical reading of the manuscript, and E. Bonnici for editorial assistance.

	FOOTNOTES

 
¹ Supported by grants from the National Cancer Institute (R01 CA 43540) and the National Health and Medical Research Council, Canberra, Australia.

² Correspondence: Mary L. Bath, The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. FAX: 61 3 9347 0852; bath{at}wehi.edu.au

Received: 13 May 2002.

First decision: 8 June 2002.

Accepted: 8 July 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Nakagata N. Cryopreservation of mouse spermatozoa. Mamm Genome 2000 11:572-576[CrossRef][Medline]
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3. Sztein JM, Farley JS, Mobraaten LE. In vitro fertilization with cryopreserved inbred mouse sperm. Biol Reprod 2000 63:1774-1780[Abstract/Free Full Text]
4. Thornton CE, Brown SD, Glenister PH. Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses. Mamm Genome 1999 10:987-992[CrossRef][Medline]
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8. National Health and Medical Research Council. Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Canberra, ACT, Australia: Australian Government Publishing Service; 1997
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10. Fraser LR. Ca²⁺ is required for mouse sperm capacitation and fertilization in vitro. J Androl 1982 3:412-419[Abstract]
11. An TZ, Iwakiri M, Edashige K, Sakurai T, Kasai M. Factors affecting the survival of frozen-thawed mouse spermatozoa. Cryobiology 2000 40:237-249[CrossRef][Medline]
12. Katkov II, Mazur P. Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa. J Androl 1998 19:232-241[Abstract/Free Full Text]
13. Songsasen N, Leibo SP. Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 1997 35:255-269[CrossRef][Medline]
14. Sztein JM, Noble K, Farley JS, Mobraaten LE. Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation. Cryobiology 2001 42:28-39[CrossRef][Medline]
15. Hinting A, Lunardhi H. Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologia 2001 33:343-346[CrossRef][Medline]
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This Article Abstract Full Text (PDF) All Versions of this Article: 68/1/19    most recent biolreprod.102.007344v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bath, M. L. Search for Related Content PubMed PubMed Citation Articles by Bath, M. L. Agricola Articles by Bath, M. L.