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Reproductive Technology |
Institute of Reproduction and Development,2 Monash University, Clayton, 3168 Melbourne, Australia
National Stem Cell Centre,3 Melbourne 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, early development, fertilization, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Improvement in sperm freezing methods in recent years [2, 5, 6] provides more opportunities to establish archives of novel phenotypes. Nonetheless, sperm freezing remains difficult in some strains. The most successful sperm freezing protocols using cryoprotective solutions containing raffinose and skim milk were limited to hybrid F1 mouse strains [6] but were not efficient for cryoprotection of sperm of inbred strains such as BALB/c, C57BL/6J, and 129Sv/ImJ [2, 3]. Recently it was reported that removal of cell debris and separation of motile population of C57BL/6J sperm prior to freezing results in an increase in fertilization rates after thawing [7, 8].
The high rates of survival, fertilization, and development of mouse oocytes following intracytoplasmic sperm injection (ICSI) using Piezo micromanipulation system [9] has enabled research into fertilization with sperm of reduced quality. Immature [10], immotile [10, 11], and membrane-damaged mouse sperm [12, 13] have been shown to be capable of fertilizing oocytes at rates approaching normal sperm following ICSI.
As ICSI became a potential method to restore specific mouse strains and genetically altered mouse lines using sperm that fail to fertilize after conventional insemination in vitro, less complicated sperm-freezing methods not utilizing cryoprotectants such as freezing sperm unprotected [14] and freeze drying [15] have been explored. In both procedures sperm motility cannot be regained but fertilization can be obtained when a whole sperm or isolated sperm heads are injected directly into the oocyte cytoplasm [14–16]. However, implantation rates and live birth rates of embryos obtained from unprotected frozen sperm and freeze-dried sperm were lower than those obtained from fresh untreated sperm [12–16], even though the majority of oocytes fertilized with those sperm had a normal karyotype. The reduced development in vivo to live offspring, particularly from unprotected frozen sperm of inbred strains, was attributed primarily to chromosomal damage during sperm cryopreservation and storage [16].
The present study demonstrates that embryo development may also be reduced by the inability of sperm frozen without cryoprotectant to properly activate oocytes. The results show that artificial activation of oocytes with strontium after ICSI with frozen sperm enables embryo development to term in C57Bl/6J, 129Sv/ImJ, and F1 (C57BlxCBA) mouse strains.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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All media were prepared from analytical grade chemicals (BDH Chemicals Pty., Ltd., Melbourne, Australia; Sigma Chemicals Co., St. Louis, MO). Sperm were collected and capacitated in modified Tyrode medium [17] with no sodium pyruvate and sodium lactate. Oocytes were collected in Hepes-modified KSOMaa and cultured in modified KSOMaa [18]. Embryos were cultured in modified KSOMaa.
Ethics
Animal protocols used for this study were approved by Monash University Animal Ethics Committee. Experiments were conducted in accordance with the Australian National Health and Medical Research Council (NIH and MRC) code of practice for the care and use of animals for scientific purposes.
Animals
One F1 hybrid (C57BlxCBA) and two inbred (C57BL/6J and 129Sv/ImJ) strains of mice that are frequently used for N-ethyl-N-nitrosourea (ENU) mutagenesis and transgenesis studies were used for the study. Mice were kept under constant environmental conditions of 14L:14D and a temperature of 25°C.
Oocytes
Females were superovulated at 6 wk of age by an intraperitoneal injection of 10 IU eCG (Intervet, Sydney, Australia) in 0.2 ml saline followed by an intraperitoneal injection of 10 IU hCG (Intervet) in 0.2 ml saline 50 h later. At 13–14 h after hCG injection, oocyte-cumulus complexes were released from the oviducts into Hepes-KSOMaa containing 60 IU/ml hyaluronidase (type IV-S, Sigma) for 5 min. Oocytes were freed of adhering cumulus cells by gentle pipetting and washed and cultured in 20-µl drops of modified KSOMaa previously equilibrated under mineral oil (Sigma) in 5% CO2 in air at 37°C. Oocytes were incubated for 1–2 h at 37°C at 5% CO2 in air before they were used for IVF or ICSI.
Sperm
Sperm were collected from 12- to 16-wk-old males of the same strains as the oocyte donors. The cauda epididymides were dissected out and a small slit was made in the cauda before transfer to 2 ml of modified Tyrode medium [17] equilibrated in 5% CO2 in air at 37°C in a 5-ml plastic tube (Falcon, Becton Dickinson Labware, Lincoln Park, NJ). To increase the fertilization rate for the inbred strains, the cauda epididymides were transferred to 2 ml equilibrated modified Tyrode in 35-mm culture dish under oil. The tissue was removed from the tubes or dishes about 1 h later, and the sperm concentration was examined and adjusted to 3–5 x 106 sperm/ml. Sperm were capacitated for a total of 3 h before freezing and use for IVF or ICSI.
Freezing Sperm Without Cryoprotectants
About 0.2 ml of sperm suspension from the 5-ml plastic tubes was loaded into a 0.25-ml clear cryostraw (IMV Technologies, L'Aigle Cedex, France) with the aid of a plastic syringe. The open end of the straw was heat sealed and the straw was immediately plunged into a liquid nitrogen storage tank and stored there for 3–6 mo. We termed this procedure snap freezing and sperm frozen by that method snap-frozen sperm. For thawing, straws were removed from the storage tank and placed on a bench at room temperature. The thawed sperm solution was expelled from the straw and randomly selected sperm were used for ICSI.
In Vitro Fertilization
Twenty microliter drops of sperm suspension at a concentration of 3–5 x 106 sperm/ml were placed on a 35-mm culture dish (Falcon) after 1.5 h capacitation time and covered with mineral oil (Sigma). The drops were incubated for a further 1.5 h before 10–20 cumulus-free oocytes were transferred in a minimal volume of modified KSOMaa medium to each drop. Alternatively, 20–50 cumulus-free C57BL/6J and 129Sv/ImJ oocytes were placed in 2 ml capacitated sperm solution in a concentration of 3–5 x 106 sperm/ml under mineral oil.
Oocytes were removed from the sperm solution after 3 h, washed, and cultured at 37°C in modified KSOMaa under mineral oil under 5% CO2 in air. Six hours later, oocytes were examined for signs of normal fertilization. Oocytes with 2 pronuclei and a second polar body were regarded as fertilized. These oocytes were separated from unfertilized oocytes and cultured in modified KSOMaa at 37°C under 5% CO2 in air for either 24 h (two-cell stage) before being transferred to pseudopregnant females or 96 h for development to the blastocyst stage in vitro. The numbers of oocytes developing to two cells and blastocysts were recorded at 24 and 96 h of culture in vitro, respectively.
Intracytoplasmic Sperm Injection
Oocytes were micromanipulated on the lid of a sterile 150-mm plastic culture dish (Falcon) in Hepes-modified KSOMaa. Manipulation took place on the stage of an inverted microscope (Leica Microinstruments Wetzler GmbH, Leitz Wetzler, Germany) with the aid of a microinjection system (PiezoDrill, Burleigh Instrument Inc., Burleigh Park, NY) mounted on a Leica micromanipulator. Fresh untreated or thawed sperm were diluted (1:3) in 10% polyvinylpyrrolidone (ICN Biomedical Inc., Aurora, OH) solution (w/v in Hepes-modified KSOMaa). Sperm heads were separated from tails with a high Piezo pulse and aspirated into a glass micropipette with an internal diameter of 5 µm. Oocytes were held by a blunt glass pipette with an internal diameter of 20 µm. The zona pellucida of each oocyte was pierced with a lower Piezo pulse and the pipette was inserted under the zona pellucida pushing the oolemma toward the holding pipette. With the aid of a fine Piezo pulse, the oolemma was broken and a single sperm head was expelled into the cytoplasm of each oocyte while the pipette was gently withdrawn.
Manipulated oocytes were washed and cultured in modified KSOMaa. The number of oocytes that survived the injection and the number of pronuclear oocytes were recorded 6 h after injection. Normally fertilized oocytes were separated from the rest and cultured at 37°C under 5% CO2 in air for either 24 h (two-cell stage) before being transferred to pseudopregnant females or 96 h for development to the blastocyst stage in vitro. The number of oocytes cleaving to two cells and blastocysts 24 h and 96 h of culture in vitro, respectively, was recorded as for IVF.
Parthenogenetic Activation
Oocytes injected with snap-frozen sperm were cultured in modified KSOMaa containing 1.74 mM strontium (SrCl2, Sigma) as a replacement for 1.74 mM calcium (CaCl2, Sigma) for 6 h. Following activation, oocytes were washed and cultured in modified KSOMaa for 24 h before transfer at the two-cell stage to the oviducts of pseudopregnant females.
Chromosomal Analyses
Chromosomal analyses followed the method described by Kola et al [19]. Oocytes with two pronuclei at 6 h after sperm injection were placed in 100-µl culture drops containing vinblastine (ICN Biomedical Inc.) at a concentration of 6 ng/ml for 17–18 h. Vinblastine-treated oocytes were placed in hypotonic solution containing 15% fetal calf serum (Gibco, BRL, Life Technologies, Grand Island, NY) and 0.5% trisodium citrate (Sigma) for 1–2 min. Swollen oocytes were placed on ethanol-cleaned glass microscope slides, spread with drops of acetic acid/methanol (1:3, Sigma), dried, and stained with filtered Giemsa (BDH Chemicals) made in phosphate buffer (pH 6.8). The slides were examined with an Olympus light microscope using a 100x immersion oil lens. The number of chromosomes and gross chromosomal abnormalities in oocytes with clearly stained preparations was recorded.
Embryo Transfer
Two-cell embryos produced by IVF or ICSI with fresh or snap-frozen sperm were transferred to the oviducts of Day 1 pseudopregnant F1 females mated to vasectomized F1 males of proven sterility. Between 4 and 12 embryos were transferred to each female. Equal numbers of embryos were transferred to both oviducts. Females were allowed to give birth and the number of pups on the day of birth was recorded. Pups were left with their mothers until 4 wk of age before they were culled. The general well-being of the pups was examined to that age. This includes physical abnormalities, weight loss, excessive weight gain, and inactiveness.
Experimental Design
The aim of the study was to improve the viability of embryos produced from ICSI of snap-frozen sperm in inbred and hybrid mouse strains. The study contained two major experiments. Experiment 1 compared the effect of ICSI on embryo development in vitro and in vivo in relation to strain differences. Experiment 2 compared the outcome of ICSI using fresh and snap-frozen sperm and the effect of strontium activation on the viability of embryos produced from snap-frozen sperm in the three strains investigated.
Statistical Analyses
The results were analyzed by chi-square using Yates correction and analysis of variance tests as appropriate. Chi-square tests were used to compare the total number of oocytes fertilized, the number of two cells, blastocysts, and live pups between two groups or among several experimental groups. ANOVA was used to identify the differences in ovulation rates among the three mouse strains.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment 1: Effect of ICSI on In Vitro and In Vivo Development of 129Sv/ImJ, C57BL/6J, and F1 Embryos
The fertilization, in vitro development to 2 cells, and in vitro development to blastocysts rates of 129Sv/ImJ, C57BL/6J, and F1 oocytes following ICSI and IVF are presented in Tables 1 and 2. These rates were relatively high for the F1 strain following IVF with 93% (n = 242), 98% (n = 238) and 77% (n = 186) for fertilization and development to two-cell and blastocyst stages, respectively. Following ICSI, 75% of F1 oocytes survived and the fertilization and development to two cells were similar to IVF (Table 1). A significant drop (P < 0.001) in blastocyst rate was identified after ICSI when compared with IVF with only 55% of the fertilized F1 oocytes reaching this stage after ICSI.
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For the 129Sv/ImJ and C57BL/6J strains, fertilization rates using drops (Table 1) were significantly lower, compared with fertilization by ICSI (P < 0.001) from intact oocytes. Higher fertilization rates of oocytes from both inbred strains were obtained after using a large volume of sperm suspension for insemination (Table 2). Over 80% of the fertilized oocytes cleaved to two cells in both strains (Tables 1 and 2). The in vitro development to blastocysts of 129Sv/ImJ and C57BL/6J oocytes after modifying the insemination protocol was not examined. All embryos were transferred to recipient females for development in vivo. No differences in birth rates were identified following the transfer of embryos resulting from oocytes inseminated in drops or in a 2-ml sperm suspension (Table 3).
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The development of 129Sv/ImJ, C57BL/6J, and F1 embryos to term after ICSI was lower than that after IVF (Table 3). The differences were significant (P < 0.01) for the 129Sv/ImJ strain (Table 3) with only 31% of two-cell ICSI embryos developing to term, compared with 53% of IVF embryos. The lowest rate of development to term was observed for the C57BL/6J strain, regardless of the insemination procedure used. Significant differences in birth rates were found between C57BL/6J and F1 in IVF and ICSI (P < 0.01) and between C57BL/6J and 129Sv/ImJ after IVF (P < 0.01). Nonetheless, all pups reached 4 wk of age and had no physical abnormalities, excessive weight gain, or weight loss.
Experiment 2: Effect of Artificial Activation on Fertilization, Embryo Development In Vitro and to Term, Following ICSI with Snap-Frozen Sperm of 129Sv/ImJ, C57BL/6J, and F1 Mouse Strains
All snap frozen sperm were immotile after thawing at room temperature. The fertilization rates of oocytes injected with snap-frozen sperm were 84% (n = 39), 77% (n = 44) and 93% (n = 39) for 129Sv/ImJ, C57BL/6J, and F1, respectively. These were not different from fertilization rates by fresh sperm for all three strains (Table 4). However, the development to blastocysts in vitro was profoundly affected when snap-frozen sperm were used. Zygotes produced from snap-frozen sperm developed to blastocysts at rates of 41%, 16%, and 18% for 129SvImJ, C57BL/6J, and F1, respectively, compared with 73%, 41%, and 89% when fresh sperm were used. The differences were significant (P < 0.01) for the inbred strains and for the hybrid strain (P < 0.001). Transfer of two-cell embryos produced by ICSI of snap-frozen sperm resulted in pups in the F1 strain only. The proportion of pups born after ICSI with snap-frozen sperm in the F1 strain, though, was significantly lower (P < 0.001) than that born after ICSI with fresh sperm (Table 5).
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Treatment of oocytes injected with snap-frozen sperm with 1.7 mM Sr2+ resulted in the birth of pups in all three strains investigated (Table 5). For the F1 hybrid strain, the proportion of pups born from snap-frozen sperm was doubled. For the 129Sv/ImJ, the number of pups born following activation of the oocytes injected with snap-frozen sperm was similar to that obtained from oocytes injected with fresh sperm. Pups were also born from oocytes injected with C57BL/6J snap-frozen sperm following activation. However, the number of offspring was lower than that obtained from F1 and 129Sv/ImJ oocytes but not statistically different. Nonetheless, all pups reached 4 wk of age and had no physical abnormalities, excessive weight gain, or excessive weight loss.
Chromosome Analyses of 129Sv/ImJ Two Pronuclei Oocytes Following ICSI of Fresh and Snap-Frozen Sperm
The highest rate of development to blastocysts from oocytes injected with snap-frozen sperm was from the inbred strain 129Sv/ImJ. Nonetheless, no pups were born from the transfer of two-cell ICSI embryos produced with snap-frozen sperm. Therefore, we chose to examine the normality of chromosomes in zygotes produced by ICSI with snap-frozen sperm from this strain. Oocytes used for chromosome analyses were not treated for activation after injection with snap-frozen sperm.
Stained oocytes were divided into three groups based on chromosome appearance (Fig. 1): oocytes with a complete number and with normal structure of chromosomes in both pronuclei (Fig. 1a); oocytes with apparently reduced number of chromosomes mainly caused by chromosomes overlapping on each other (Fig. 1b); and oocytes with gross chromosomal breakages (Fig. 1c). When breakages and overlaps were identified in oocytes produced by snap-frozen sperm, they were usually from one of the two pronuclei (Fig. 1, b and c).
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From 21 oocytes injected with fresh sperm that were examined, 16 (76%) had normal chromosomes in both pronuclei, three (14%) had gross chromosomal breakages, and two (10%) had overlaps that prevented accurate analyses. From 24 oocytes injected with snap-frozen sperm, 10 (42%) had normal chromosomes in both pronuclei, six (25%) had gross breakages, and eight (33%) had overlapping chromosomes, which limited accurate analyses. The number of oocytes with normal chromosomes in both pronuclei was significantly lower following ICSI with snap-frozen sperm than with fresh sperm (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present experiments, sperm were frozen in modified Tyrode medium [17] containing 1.74 mM Ca2+. Sperm-fertilizing capacity after thawing using ICSI was normal but the pre- and postimplantation embryo development was reduced. Although freezing sperm without cryoprotectant affects sperm chromosome integrity [this study and 16], we identified that about 40% of 129Sv/ImJ oocytes injected with 129Sv/ImJ snap-frozen sperm had apparently normal chromosomes in both pronuclei. Similar proportions of the oocytes developed to blastocysts in vitro. Incubation of oocytes in Ca2+-free medium containing 1.74 mM Sr2+ for 6 h after ICSI enabled more embryos to develop to term in all three strains investigated. This indicates that the reduction in the ability of embryos to develop to blastocysts in vitro and term is not entirely related to sperm chromosome abnormalities resulting from the unprotected freezing method but also to the inability of sperm to effectively activate the oocytes.
Extrusion of a second polar body, formation of sperm and oocyte pronuclei, and early embryo development are not necessarily an indication of proper oocyte activation at fertilization. A complex cascade of events is initiated following the introduction of sperm heads into oocytes. This normally results in full oocyte activation, which includes the induction of oscillatory changes in intracellular calcium for resumption of meiosis and production of viable embryos [20, 21]. The process of mammalian oocyte activation during fertilization and more specifically after ICSI is not fully revealed and in mice may involve one or more activating factors [22–26]. Each can be differently affected by heat, cooling, freezing, and culture conditions, which may account for the reduced postimplantation embryo development following different sperm treatments [27–29].
Sperm gain full capacity to activate oocytes during maturation, which may be simply related to the quantity of the sperm-activating factor. Round spermatids of mice are unable to activate oocytes following injection into oocytes [10], but a single mouse elongating spermatid injected into mouse oocytes will induce sporadic intracellular Ca2+ increase [8]. Mouse elongating spermatids, however, cannot induce normal Ca2+ oscillations following injection into oocytes [30]. Injection of two elongating spermatids into oocytes can result in normal Ca2+ oscillations similar to that observed following the injection of fully matured sperm. Testicular sperm have the full capacity to activate mouse oocytes and support embryo development to blastocysts and to term [10].
It has been suggested that the sperm-activating factor is located at the equatorial segment of the sperm head [31] or in the perinuclear material [12]. An intact sperm nuclear matrix is necessary for the mouse sperm to participate in embryonic development. Exposure of sperm to dithiothreitol does not affect their ability to activate oocytes as evident by the extrusion of a second polar body and the formation of pronuclei but reduces the capacity of the embryos to develop to term [13]. Severe membrane damage following snap freezing and thawing may lead to partial loss or inactivation of the sperm-activating factor resulting in limited capacity of the sperm to activate oocytes. This limited activation as identified in the present study has also resulted in the extrusion of a second polar body, formation of pronuclei, and even early embryonic development. However, it was not sufficient to result in viable embryos and live offspring. The susceptibility of sperm-activating factor to cryodamage may also be strain specific. In the present study, a limited number of embryos produced from snap-frozen sperm of the hybrid F1 strain reached term without artificially activating the injected oocytes after ICSI. This was similar to the findings by Wakayama et al. [14] using another hybrid F1 (B6D2F1) mouse strain.
Activation by exposure to 5 mM Sr2+ in Ca2+-free medium for 45 min to 6 h enabled mouse embryo development to term following injection of oocytes with round spermatids [32], spermatocytes [33], heated sperm [29], and following nuclear transfer [34, 35]. Also, incubation of mouse oocytes in Ca2+-free medium containing 10 mM Sr2+ for longer than 2 h resulted in an increase in the number of inner cell mass cells and a limited postimplantation development of parthenogenetically activated oocytes [36]. Mouse oocytes activated with Sr2+ exhibit cytosolic Ca2+ oscillations, with a long-lasting increase of the first transient followed by subsequent oscillations, which resemble that found after sperm penetration [36, 37]. In addition, strontium does not induce oocyte chromosomal abnormalities [38].
In the present study, oocytes were activated by a 6-h exposure to 1.74 mM Sr2+ in Ca2+-free solution that was freshly prepared for each experiment. The 6-h exposure to 1.74 mM Sr2+ compensated for the deficiency of unprotected frozen sperm to activate oocytes. This concentration was lower than that used in other studies [29, 32–38] because the aim was to replace calcium concentration only.
The present study shows lower blastocyst and birth rates after ICSI for the hybrid and the inbred strains, compared with IVF. Nonetheless, ICSI is a powerful tool in the conservation of mouse strains when sperm are compromised following freezing and IVF outcome is limited or cannot be performed [5, 39]. A comparison of Ca2+ responses in human oocytes fertilized by subzonal insemination and ICSI showed a delayed, truncated Ca2+ response after ICSI [40]. However, Ca2+ oscillations are maintained in a typical way to normal fertilization. A more recent study reported that the time of initiation of Ca2+ release and oscillations is dependent on the method used to immobilize sperm before injection [41]. The reduced development observed after ICSI in the present study may be related to the invasiveness of the procedure. Taking into consideration the differences between strains in relation to modifications to optimal culture media and protocols used [8, 14, 16, 28, 42] and the need for activation of oocytes at fertilization, it is possible to generate sufficient progeny from fresh and frozen sperm for re-establishment of most genotypes used for transgenic and ENU mutagenesis purposes.
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FOOTNOTES |
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Received: 10 April 2003.
First decision: 29 April 2003.
Accepted: 17 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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