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a Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gamete biology, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, a modified method for separating motile from nonmotile spermatozoa with Sephadex beads [6] was used to increase the viable sperm population for freezing and in vitro fertilization (IVF). Spermatozoa were cryopreserved with 18% raffinose and 3% skim milk [7, 8]. Comparisons of IVF rates were made between non-separated and separated fresh and cryopreserved spermatozoa from a hybrid (B6D2F1) and various inbred strains (DBA/2, C57BL/6J, FVB, and BALB/c) of mice. Preimplantation and postimplantation development of embryos generated by IVF were also compared. A preliminary report of these data appeared elsewhere [9].
We used these 4 inbred strains because they are important for mutagenesis and transgenesis studies, and because they are difficult to cryopreserve. C57BL/6J is one of the most widely used substrains of all the inbred mouse strains, and one of the parental strains of commonly used hybrids. The FVB/N strain is used extensively to produce transgenic mice because of its prominent pronuclei, which facilitates gene injection, and because of its ability to expedite a high level of germ line transmission in chimeras produced from FVB host blastocysts injected with embryonic stem cells. The BALB/c strain is used widely for monoclonal antibody production and in mutagenesis studies. DBA/2 is a parental strain for some hybrids and an important strain in mutagenesis studies as a recommended host for several tumors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
Animals
Mice were obtained at 6 wk of age. B6D2F1 (C57BL/6 ;ts DBA/2), DBA/2, and CD-1 mice were obtained from the National Cancer Institute (Raleigh, NC); C57BL/6J and BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME); and FVB mice were obtained from Charles River Laboratories (Wilmington, MA). Mice had ad libitum access to a standard diet and were maintained in a temperature- and light-controlled room (22°C, 14L:10D) in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996).
Media
All culture media and Dulbecco PBS were prepared from the individual components. PBS was used to separate motile spermatozoa on a Sephadex column, T6 medium [10] was used for IVF, Hepes-buffered CZB medium (Hepes-CZB, [11] was used for washing oocytes after IVF, and mKSOMAA medium [12] was used for culturing to the blastocyst stage. The mKSOMAA medium contained 5.56 mmol/L glucose, 4 mg/ml BSA, and amino acids. The essential and nonessential amino acids in the latter medium were obtained from Gibco BRL (Grand Island, NY). T6 and mKSOMAA media were maintained in an atmosphere of 5% CO2 in air, and Hepes-CZB and PBS were maintained in air alone.
Sperm Collection and Separation of Motile Spermatozoa
Epididymal spermatozoa were obtained from males at 8–16 wk of age. One cauda epididymis from each male was used as the source of nonseparated spermatozoa; it was placed in 50 µl of cryoprotectant solution in an organ tissue culture dish (3513037; Falcon, Bedford, MA). The other cauda epididymis was placed in 250 µl of PBS in order to separate motile spermatozoa on a Sephadex column (see below). The epididymal contents were expressed from the cauda epididymides with needles and the tissue was discarded. In both preparations the spermatozoa were allowed to disperse for 2–5 min at room temperature.
Separation of motile populations of spermatozoa was performed on the basis of a method described elsewhere [6] and was carried out at room temperature (25°C). A column of Sephadex G-25 (G-25-150, Sigma; dry bead diameter 50–150 µm) suspended in PBS was prepared in a 1-ml sterile syringe, and its outlet was plugged with glass wool. A Sephadex suspension was prepared in a 5-ml tube and transferred with a Pasteur pipette into the syringe to make a column up to about the 500-µl mark after the suspension had settled.
The columns were washed 2–3 times by passing approximately 0.5 ml of PBS through the column each time. The total sperm suspension from a single cauda epididymis (250 µl), after dispersion in PBS, was placed on the top of the Sephadex column bed. A further 250 µl of PBS was added to the top of the column as soon as the sperm suspension had completely entered the column and no further liquid was being eluted. The eluent was collected into a clean 1.5-ml Eppendorf tube. The motile-rich fraction of spermatozoa was recovered in about 250 µl of eluent. A sample of the eluent was taken to assess sperm concentration and motility, and the remainder was centrifuged at 720 ;ts g for 10 min. The supernatant was removed and the spermatozoa were resuspended in 20–50 µl of cryoprotectant solution to give a final concentration of 20–40 ;ts 106 spermatozoa/ml.
Sperm Evaluation
The following assessments of sperm samples were made before and after separation with Sephadex.
Sperm concentration and motility Samples of sperm suspension (1–10 µl) were taken to evaluate concentration and motility using a hemocytometer. Before separation they were diluted to 60;ts with PBS, but after Sephadex separation the samples were not diluted because of their lower density. The numbers of motile and nonmotile spermatozoa were assessed using the method outlined by the World Health Organization [13]. Concentration was expressed as the number of spermatozoa per milliliter, and motility as the proportion of motile spermatozoa in the sample.
Membrane integrity Membrane integrity was assessed by a modification of the method described by Harrison and Vickers [14], which relies on the different characteristics of fluorescent dyes, 6-carboxyfluorescein diacetate and propidium iodide. Spermatozoa were stained in the solution consisting of PBS, 20 µmol/L 6-carboxyfluorescein diacetate, and 7.3 µmol/L propidium iodide. Slides were examined immediately on a Nikon epifluorescent microscope using standard fluorescein and rhodamine filter sets. Membrane-intact spermatozoa fluoresced green when intracellular esterases convert the membrane-permeable 6-carboxyfluorescein diacetate dye to the impermeant carboxyfluorescein. Spermatozoa with damaged membranes do not retain esterase activity and fluoresce red with the normally impermeable DNA binding fluorescent dye, propidium iodide. At least 500 spermatozoa were counted per sample.
Sperm Freezing
Cryoprotectant solution Spermatozoa were frozen by modifying a method described by Nakagata [2]. Briefly, the cryoprotectant solution containing 18% D(+)-raffinose pentahydrate (w/v) and 3% skim milk (w/v) was prepared by dissolving 3.6 g of D(+)-raffinose pentahydrate and 0.6 g of skim milk in 20 ml of glass-distilled water at 60°C. The solution was centrifuged at 10 000 ;ts g for 20 min at room temperature. The supernatant was removed, filtered through a sterile 0.45-µm Millipore (Bedford, MA) filter, and stored at -20°C in 0.5-ml aliquots in sterile 1.5-ml polystyrene Eppendorf tubes. Immediately before use, an aliquot was thawed and warmed to 37°C.
Freezing Epididymal spermatozoa that had been directly expressed into the cryoprotectant (i.e., nonseparated sperm) were loaded into 250-µl straws (Edwards Innovations, Spring Valley, VA) immediately after dispersion (10–15 µl per straw). The straws were sealed with Critoseal (Oxford Labware, St. Louis, MO) and placed in a plastic holder, which floated on the surface of liquid nitrogen in a liquid nitrogen storage container for 15 min before immersion in the liquid phase. The separated motile populations of epididymal spermatozoa were similarly loaded into straws immediately after dispersion in the cryoprotectant, then sealed, frozen, and stored. Samples were stored for up to 1 mo.
Thawing Straws were removed from the storage container and immersed into a 37°C water bath for 10–15 min. The contents of a straw were expressed into a Petri dish, and an appropriate amount was added immediately to the prepared drop of T6 media for subsequent capacitation and IVF.
Oocyte Collection and IVF
Oocytes were obtained from 8- to 12-wk-old mice after superovulation with i.p. injections of 5 IU eCG (Calbiochem, La Jolla, CA) and 5 IU hCG (Calbiochem) given 48 h apart. BALB/c mice responded poorly to gonadotropin treatment, and low and variable numbers of viable oocytes were obtained for IVF in all replicates. Oviducts were removed 14–15 h after the hCG injection and placed in PBS in a Petri dish. Oviducts were transferred singly beneath the mineral oil (Squibb & Sons, Princeton, NJ) in close proximity to the fertilization drop. The cumulus-oocyte complexes were released from the ampulla into the oil by rupturing the oviduct with the aid of a 25-gauge needle, and then moved into the fertilization drop.
In all instances, gametes used for IVF were from the same strain. The method for sperm capacitation and IVF with T6 medium has been described elsewhere [10]. Essentially, 200-µl drops of T6 medium (subsequently referred to as fertilization drops) were overlaid with mineral oil in a plastic culture dish (60-mm diameter) and equilibrated overnight at 37°C in an humidified atmosphere of 5% CO2 in air. Each dish contained 200-µl drops to which nonseparated and separated spermatozoa were added. The volume of spermatozoa added to the fertilization drop depended on the concentration of spermatozoa in the samples after dispersion in the cryoprotectant solution. Generally, 2.5–10.0 µl of spermatozoa were added to each fertilization drop to give final concentrations of about 1–2 ;ts 106/ml spermatozoa for separated and nonseparated samples of spermatozoa, respectively. Separated samples exhibited higher motility and reduced agglutination, and preliminary experiments showed that lower concentrations of about 1 ;ts 106/ml of separated spermatozoa were suitable for IVF.
Nonseparated and separated spermatozoa were incubated in T6 medium for about 60 and 30 min, respectively, before oocytes were added. The shorter incubation time for separated samples was based on the reduction in capacitation time required for mouse spermatozoa after centrifugation [6]. The contents of 4 oviducts were released into each fertilization drop. After incubation for 4 h, the oocytes were washed through several changes of Hepes-CZB medium followed by at least 1 wash in mKSOMAA medium. Only morphologically normal oocytes were selected for culture.
Preimplantation Culture
For embryo culture, 50-µl drops of mKSOMAA medium were set up in a plastic culture dish (351007; Falcon), overlaid with mineral oil, and equilibrated overnight at 37°C in a humidified atmosphere of 5% CO2 in air. After washing the oocytes free of spermatozoa, groups of 10 oocytes were placed in each culture drop and incubated for up to 96 h. Oocytes were scored for pronucleus formation (activation) at 6 h after the commencement of culture, and the number of 2-cell embryos (fertilization) were scored after 24 h in culture. Embryos were either left to progress through preimplantation development to the blastocyst stage, and scored after 48, 72, and 96 h in culture, or they were transferred to pseudopregnant recipients at the 2-cell stage.
Embryo Transfer
Two-cell embryos were transferred to the oviducts (5–10 per oviduct) of CD-1 females mated with vasectomized CD-1 males on Day 1 of pseudopregnancy. Embryos generated with nonseparated spermatozoa were transferred to 1 oviduct, and embryos generated with separated spermatozoa were transferred to the contralateral oviduct of the same pseudopregnant female. The number of implantation sites and fetuses were recorded at Day 15 of gestation.
Experimental Design
Experiments were designed to compare IVF of oocytes with nonseparated samples of epididymal spermatozoa containing varying proportions of nonmotile and motile cells with samples of highly motile spermatozoa separated on a Sephadex column. Fertilization and development rates were compared between fresh and frozen spermatozoa. In order to reduce possible variation in the fertility of spermatozoa between males within a strain, a single male was the source of spermatozoa for both fresh and frozen, and nonseparated and separated spermatozoa in each experimental replicate. The experiment was replicated three times for each strain. In experiments with each inbred strain, the B6D2F1 hybrid gametes were included as controls for IVF and culture.
The rate of fertilization was calculated from the proportion of 2-cell embryos developing from the number of morphologically normal oocytes inseminated, and the overall success rate in development to the blastocyst stage from the proportion of blastocysts developing from the same population of normal oocytes inseminated.
Statistical Analysis
Chi-square, likelihood ratio, and the Fisher exact probability tests were used for analyzing all responses. Computations were performed using KyPlot version 2.0-beta 13 software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatozoa from the different strains varied in concentration from 20 to 40 ;ts 106/ml, and motility varied from 50% to 70%. In preliminary experiments we established that sperm samples separated with Sephadex have a significantly higher proportion of motile spermatozoa than nonseparated samples. Motility in B6D2F1 hybrid males increased from 73.6% ± 2.4% to 95.8% ± 1.0% (P 0.001), whereas the sperm concentration decreased 6-fold, from 32.5 ± 1.5 ;ts 106/ml to 5.84 ± 0.9 ;ts 106/ml (P < 0.001). The proportion of viable spermatozoa also increased after separation (from 53.7% ± 3.2% before separation to 80.6% ± 3.2% after separation; P < 0.01).
In Vitro Fertilization
Our initial comparison of Tyrode medium (T6) and Toyoda medium (TYH) [15] for sperm capacitation and IVF revealed no difference between the proportions of B6D2F1 oocytes fertilized with B6D2F1 spermatozoa (both >97%) and little difference in embryo development to the blastocyst stage (92% vs. 89%). Therefore, we chose T6 medium for capacitation and IVF, and mKSOMAA medium for embryo culture in all subsequent experiments. The rate of fertilization and blastocyst formation (90% and 86%, respectively; 4 replicates, n = 298) was not affected by spermatozoa that had been previously exposed to cryoprotective solutions (i.e., raffinose and skim milk).
Fertility Comparison of Separatedand Nonseparated Spermatozoa
Data for fertilization with fresh and frozen nonseparated and separated epididymal spermatozoa are presented in Table 1 and Figure 1. Levels of statistical significance for comparisons within strains are shown in Figure 1. Responses between replicates were similar within a strain, and the data have been combined. Overall fertilization with fresh spermatozoa was optimal for B6D2F1, FVB, and DBA/2 strains, but was significantly lower in C57BL/6J and BALB/c strains. In all strains the rate of fertilization with nonseparated spermatozoa was significantly lower after freezing (P < 0.01 for BALB/c; P < 0.001 for other strains). Fertility improved considerably with the use of frozen spermatozoa that had been separated before freezing; this difference was greater in some strains than others (B6D2F1, C57BL/6J, and BALB/c, P < 0.001; FVB and DBA/2, P < 0.05). Significant differences occurred between nonseparated and separated fresh spermatozoa of B6D2F1 and C57BL/6J strains (P < 0.001), and to a lesser extent for the BALB/c strain (P < 0.05), but there was no difference with FVB and DBA/2 strains. Fertilization was lower after sperm separation only in the B6D2F1 strain, and this was probably due to an increase in polyspermy. A further reduction in sperm density might obviate this problem. There were no significant differences between fertilization with fresh or frozen separated spermatozoa from B6D2F1, FVB, and DBA/2 strains, but fertilization was significantly lower with C57BL/6J and BALB/c strains (P < 0.001 and P < 0.01, respectively). However, in the latter strains, fertilization was 2–3 times higher when separated spermatozoa were used than when nonseparated frozen sperm were used. Overall, spermatozoa that were frozen after Sephadex separation were more fertile than nonseparated frozen samples in all strains examined. A dramatic improvement in fertility occurred with C57BL/6J and BALB/c strains, the 2 strains in which IVF outcomes are poor when freshly collected spermatozoa are used.
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Preimplantation Development
Preimplantation development after 72 and 96 h of culture is summarized in Table 1. Although high proportions of two-cell B6D2F1, FVB, C57BL/6J, and DBA/2 embryos developed to the morula and blastocyst stages, significantly fewer C57BL/6J and DBA/2 embryos formed to the blastocyst stage. Suboptimal conditions for culture (e.g., in the gas phase, various levels of medium, oxygen, or both) may account for the reduction in development to the blastocyst stage. Significantly more blastocysts developed from embryos that were generated from separated fresh and frozen BALB/c spermatozoa than from nonseparated spermatozoa (78% and 83% vs. 50% and 45%, respectively). Because the number of observations is relatively small, the variations in development may reflect the quality of the oocytes obtained after superovulation (see above) or earlier penetration by separated spermatozoa that had been centrifuged [6]. The overall efficiency of blastocyst formation, as calculated from the number of normal oocytes inseminated, confirms the beneficial effect of separating motile populations of spermatozoa and freezing them (Table 1).
Postimplantation Development
In an initial study of postimplantation development of 2-cell embryos derived from IVF with nonseparated and Sephadex-separated, fresh epididymal spermatozoa, no differences were found in the proportion of implants and fetuses at Day 14.5 of gestation. Therefore, postimplantation data for embryos derived from nonseparated and Sephadex-separated spermatozoa have been combined for both fresh and frozen spermatozoa, respectively, in all 5 strains (Table 2). The proportion of embryos that implanted was similar for FVB, DBA/2, and BALB/c strains, regardless of sperm treatment before IVF, but it differed significantly for B6D2F1 (P < 0.05) and C57BL/6J (P < 0.001) strains.
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Fetal development was similar between embryos derived from fresh and frozen spermatozoa in all strains except BALB/c (P < 0.001), although the number of embryos transferred in this strain was low. Comparing postimplantation development in the different strains, fetal development was similar in B6D2F1, FVB, and C57BL/6J strains, but significantly lower in DBA/2 (P < 0.0l) and BALB/c strains (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fertility of cryopreserved spermatozoa as assayed by IVF was higher in all strains when sperm samples were separated on Sephadex before freezing. The greatest improvement was obtained with separated and cryopreserved samples from C57BL/6J (40%) and BALB/c (51%) strains, but this was still significantly lower than for the other strains (77%–89%). This may reflect a greater sensitivity to freezing, thawing, or both, and it would be worthwhile to examine the extent of membrane and cellular injury occurring in the different strains. Mazur et al. [1] have listed an entire series of different factors that may contribute to injury, including cell volume changes induced by osmotic imbalance, mechanical stress such as centrifugation, and O2 free radical damage. Lower fertilization rates may also be due to the proportion and concentration of normal motile spermatozoa in the thawed samples falling below the threshold necessary to achieve high rates of sperm penetration.
Separation of motile populations of spermatozoa before freezing allows a more realistic assessment of cryoinjury to be made after thawing. In addition, it is important to have viable and motile populations of spermatozoa for unconventional cryostorage techniques such as freeze-drying [17] and rapid freezing without cryoprotection [18], because viability cannot be assessed after storage when membrane integrity and sperm motility have been compromised. A disadvantage of Sephadex separation is the relatively low number of spermatozoa recovered in the column eluent (about 15% of the total number of spermatozoa added to the column). The sperm concentration is further reduced after centrifugation. Nevertheless, the number of spermatozoa recovered was adequate in the present experiments, but it would be necessary to modify the protocol in strains in which significantly fewer spermatozoa can be obtained from the epididymis. In such instances, intracytoplasmic injection (ICSI) of spermatozoa into oocytes may be a more appropriate means of generating embryos because the technique requires relatively few sperm cells (unpublished observations). ICSI has been shown effective in producing progeny from embryos derived from freeze-dried spermatozoa and from spermatozoa frozen without cryoprotection [17, 18].
The greatest improvement was accomplished with both fresh and frozen separated, motile populations of spermatozoa in strains that normally have low success rates with IVF using fresh gametes. Previously, Nakagata et al. [19] achieved higher rates of fertilization with C57BL/6J spermatozoa (>70%) when the zonae pellucida of C57BL/6J oocytes were partially dissected before insemination. However, partial dissection carries the danger of polyspermy and requires that zygotes be cultured in vitro to the morula or blastocyst stage before embryo transfer because blastomeres have a tendency to escape through the slit in the zona pellucida during early stages of embryonic development. Sephadex separation of motile spermatozoa has an advantage over partial dissection in that the zona pellucida of the oocyte remains intact and embryo transfer can be performed at the two-cell stage. Also, because the zona pellucida stays intact, the risk of transmitting viruses or bacteria to the potential embryo is lower.
A high proportion of 2-cell embryos from B6D2F1 and FVB strains developed to the blastocyst stage in vitro (>90%). In contrast, development of 2-cell embryos from C57BL/6J and DBA/2 strains was lower and more variable (56%–72%, and 56%–81% for the 2 strains, respectively), indicating some of the difficulties inherent in the culture of certain inbred strains through preimplantation development [20]. Results were similar for BALB/c strains, although development to the blastocyst stage by embryos generated with fresh and frozen separated spermatozoa (78% and 83%) appeared to be greater than it was with nonseparated spermatozoa (50% and 45%). However, the number of BALB/c embryos cultured was much lower because of the difficulties in obtaining viable oocytes from superovulated females. Further study is necessary to establish whether fertilization with Sephadex-separated spermatozoa has any real effect on subsequent embryonic development.
In conclusion, separation of motile populations of epididymal spermatozoa for IVF has a positive effect on the rate of fertilization in certain mouse strains. After freezing of the separated motile samples of spermatozoa, the rate of fertilization was increased in all the strains examined. The technique is recommended for improving the success of IVF in strains that have relatively low success rates and when the ICSI technique may be unavailable.
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FOOTNOTES |
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1 This material is based on work that was performed as part of the National Cooperative Program on Mouse Sperm Cryopreservation, which is funded by the National Institute of Child Health and Human Development and the National Center for Research Resources, National Institutes of Health grant U0 1HD38205. 
2 Correspondence: Monika A. Szczygiel, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; szczygie{at}hawaii.edu
3 Current address: Laboratory of Cell Toxicology, Food and Drug Safety Center, Hatano Research Institute, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan
Accepted: February 4, 2002.
Received: December 26, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 comparisons are indicated below the figure