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Reproductive Technology |
Department of Animal Science/Center for Regenerative Biology,5 University of Connecticut, Storrs, Connecticut 06269
Institute for Biogenesis Research,6 University of Hawaii Medical School, Honolulu, Hawaii 96822
Faculty of Agriculture and Life Sciences,7 Hirosaki University, Hirosaki 036-8561, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, in vitro fertilization, oocyte development, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Natural fertilization (i.e., the union of a mature oocyte with a sperm) triggers oocyte activation and embryo development [6, 7]. In most mammals, including humans and rabbits, the centrosome serves as a microtubule-organizing center in the zygotes [8–13]. Brought into the oocyte by the spermatozoon, the centrosome plays a critical role in assembly of the microtubule network that brings the paternal and maternal pronuclei to the center of the newly formed zygote [14]. The mouse and, perhaps, all common laboratory rodents are exceptional among mammals in that they do not require sperm centrosomes for fertilization and embryonic development [14, 15]. To date, the mouse remains the only species in which freeze-dried sperm have been shown to support term development. However, because mouse spermatozoa do not contribute centrosomes for the zygote, the possibility exists that freeze-dried spermatozoa of species that contribute centrosomes may not be able to achieve fertilization or support full-term development.
The objective of the present study was to test whether freeze-dried sperm could support full-term development in rabbits, a species for which the centrosome from sperm is required for successful fertilization and development. Here, we report that the freeze-drying process does little damage to rabbit sperm chromosomes, and high blastocyst development and a full-term pup could be generated from intracytoplasmic sperm injection (ICSI) of freeze-dried rabbit sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experimental procedures were approved by the Institutional Animal Care and Use Committees of the University of Connecticut and the University of Hawaii. Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Sperm Collection and Freeze-Drying
Semen was obtained from a Dutch-belted rabbit using an artificial vagina [16]. Semen was collected (one ejaculation per replicate) for fresh sperm experiments. Rabbit freeze-dried sperm was prepared by a modification of the technique described by Wakayama and Yanagimachi [2]. Briefly, a 100-µl aliquot of freshly collected semen was placed at the bottom of a 5-ml plastic centrifuge tube containing 1–2 ml of EGTA buffer (50 mM EGTA, 50 mM NaCl, and 10 mM Tris-HCl; buffer pH adjusted to 8.0–8.2) [3]. After a 10-min incubation at 37°C to allow spermatozoa to swim into the buffer, the upper 1 ml of the medium was collected. A 100-µl aliquot was placed at the bottom of a 2-ml glass ampule and immediately plunged into liquid nitrogen for 20 sec. The ampule was connected to a freeze-drying machine (Labconco, Kansas City, MO) and lyophilized for 4 h before flame-sealing [3]. The inside pressure of the vial was 23–40 x 10–3 mbar during sealing. Ampules were kept at 4°C for 12–24 mo before shipping at ambient temperature by express mail from Honolulu, Hawaii, to Storrs, Connecticut. They were kept at 4°C for another 12–18 mo before use.
Sperm Live/Dead Staining
The spermatozoa were simultaneously incubated with 100 nM SYBR 14 and with 12 µM propidium iodide, according to the standard protocol (Live/Dead Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR). Membrane-intact ("live") sperm are stained by SYBR 14 (green), whereas membrane-broken ("dead") sperm are stained by propidium iodide (red).
Examination of Rabbit Sperm Chromosomes
Rabbit freeze-dried sperm chromosomes were examined by injecting a single rabbit spermatozoon into a mouse oocyte using a micromanipulator with a piezo-electric actuator (PMM Controller, model PMAS-CT150; Prima Tech, Tsukuba, Japan). Approximately 5 h after ICSI of mouse oocytes, the oocytes with two distinct pronuclei and second polar bodies were placed in CZB medium [17] containing 6 ng/ml of vinblastin to arrest them at metaphase of the first cleavage. The eggs were then freed from the zona pellucida with 0.5% pronase and processed for the preparation of chromosome spreads onto clean glass slides [18]. Haploid sets of mouse and rabbit chromosomes were easily recognizable from each other because of species-specific differences in the structure and number of chromosomes.
Scanning Electronic Microscopy of Rabbit Spermatozoa
After rinsing with 0.1 M Hepes buffer and 0.05 M NaCl (250–260 mOs), spermatozoa were fixed for 1 h in 1.5% glutaraldehyde and 1.5% paraformaldehyde in the same buffer. They were then washed by three changes of Hepes buffer and placed on small glass cover-slips (12 x 12 mm) coated with 0.1% poly-L-lysine solution. Postfixation was done with 1% osmium tetroxide in Hepes-NaCl buffer for 1 h. Samples were then dehydrated in a series of increasing concentrations of ethanol, critical point-dried, sputter-coated with gold, and examined with a Zeiss DSM- 982 scanning electron microscope (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 2 kV.
Oocyte Recovery
Mature Dutch-belted female rabbits were induced to superovulate with two 3-mg and then four 4-mg s.c. injections of FSH (Folltropin-V; Vetrepharm Canada, Inc., London, ON, Canada) given 12 h apart, followed by an i.v. injection of 200 IU of hCG given 12 h after the final administration of FSH. At 16 h after administration of hCG, the animals were laparotomized, and their oviducts were flushed with Dulbecco modified PBS (DPBS; Gibco, Grand Island, NY) containing 3 mg/ml of BSA for collection of cumulus-oocyte complexes. The cumulus cells were removed by exposure to hyaluronidase (300 IU/ml in DPBS) for 1 min, followed by vortexing. Cumulus cell-free oocytes were washed and kept in KSOM medium [19] at 39°C in 5% CO2 in a humidified atmosphere for less than 30 min before sperm injection. A total of 27 does were used for oocyte recovery.
Intracytoplasmic Sperm Injection
One hundred microliters of sterilized, distilled water were added to a newly opened glass ampule to rehydrate freeze-dried spermatozoa. Approximately 5 µl of the sperm suspension were added into one 20-µl drop of M2 medium containing 12% (w/v) polyvinylpyrrolidone (molecular weight, 360 000; Sigma). The drop containing the spermatozoa was surrounded by eight micromanipulation drops (20 µl) of DPBS plus 20% fetal bovine serum. All drops were under mineral oil in a 60-mm Petri dish. A group of 5–10 oocytes was transferred into a manipulation drop. A single sperm was drawn, tail first, into an injection pipette and then washed in a drop of medium to remove EGTA, which might have adverse effects on oocyte activation and embryo development. A spermatozoon was then transferred into a second drop for further rinsing before injection into the cytoplasm of an oocyte, according to the technique described by Li et al. [20]. A single spermatozoon with tail, intact or fragmented, was injected into each oocyte. Injection was performed at room temperature and completed within 1 h after rehydration of freeze-dried spermatozoa. Oocytes that survived ICSI (oocyte appearing to be morphologically normal after 30 min) were used for the next steps.
Oocyte Activation
Some ICSI oocytes were incubated in KSOM medium with 0.1% BSA without any activation treatment. Others were subjected to one or two consecutive activation treatments [21]. Each consisted of a 5-min treatment with 10 µg/ml of calcium-ionophore A23187, followed by 1-h incubation in KSOM with 0.1% BSA containing 5 µg/ml of cycloheximide (CHX) plus 2 mM 6-dimethylaminopurine (6-DMAP). Oocytes without sperm injection but with activation treatments served as parthenogenetically activated controls.
In Vitro Culture of ICSI Oocytes and Embryo Transfer
Some ICSI oocytes were cultured in KSOM with 0.1% (w/v) BSA for the first 2 days, then transferred to KSOM with 1% (w/v) BSA for further culture for 4 days in a humidified atmosphere of 5% O2:5% CO2:90% N2 at 39°C. These oocytes were used for examination of in vitro blastocyst development.
For embryo transfer, activated ICSI oocytes were transferred into the oviducts of recipient does. The ovulation of the recipient does had been synchronized with that of the oocyte donors by i.m. injection of 1.2 µg of GnRH analogue (Cystorelin; Fort Dodge Laboratory, Fort Dodge, IA) 16 h before embryo transfer [22].
Immunocytochemistry and Laser-Scanning Confocal Microscopy
Following ICSI, oocytes were fixed and immunostained by antibodies following the routine protocol of our laboratory as described previously [23]. Briefly, the oocytes were fixed in Microtubule Stabilization Buffer Extraction Fixative (MTSB XF) [24] at 39°C for at least 60 min, then stored at 4°C overnight. The MTSB XF consisted of 0.1 M PIPES, 5 mM MgCl2, 2.5 mM EGTA, 50% (v/v) deuterium oxide, 0.01% (v/v) aprotinin, 1 mM dithiothreitol, 1 µM Taxol, and 0.5% (v/v) Triton-X 100. It contained 2% (w/v) formaldehyde to stabilize the oocyte's cytoskeleton. Fixed oocytes were washed in washing buffer (PBS containing 3 mM NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% normal goat serum, 0.1 M glycine, and 2% BSA) three times, for 15 min each time, and then left in washing buffer overnight at 4°C for blocking and permeabilization. Oocytes or embryos were then double-stained to visualize microtubules and DNA. Samples were incubated in mouse anti-tubulin (1:200; T-6199; Sigma) for 4 h at 37°C or overnight at 4°C. After three washes in washing buffer, the oocytes or embryos were incubated in fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (1:200, Fluka-10766; Sigma) for 4 h at room temperature. Some samples were incubated with rhodamin-phalloidin (1:1000; P-1951; Sigma) at 37°C for 30 min to stain actin filaments. Finally, the samples were washed, stained for DNA with 7.5 µM propidium iodide, mounted on glass slides, and examined with a laser-scanning confocal microscope (model TCS SP2; Leica, Mannheim, Germany).
Statistical Analysis
All data were analyzed by chi-square test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unlike live spermatozoa, freeze-dried spermatozoa were dead in the conventional sense. After rehydration, all sperm were motionless and stained as dead (Fig. 1). Approximately 16% of the freeze-dried spermatozoa had broken or no tails, whereas 100% of fresh spermatozoa had intact tails (Fig. 2). After freeze-drying, the connection between the sperm head and tail became so fragile that pipetting could easily cause head-tail separation. For many rehydrated sperm, the acrosome was detached from the head (not shown). In all freeze-dried sperm examined, damage to the plasma membranes of the head and tail were observed (Fig. 2). Despite such drastic physical alternations in sperm structures, the nuclei of freeze-dried spermatozoa seemed to be cytogenetically intact, because 92% (56/61) of oocytes injected with freeze-dried spermatozoa had normal chromosome constituents when examined before the first cleavage (Fig. 3).
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Development of ICSI Oocytes
Figure 4 shows the behavior of freeze-dried sperm heads (nuclei) after injection into oocytes. At the end of the first round of oocyte activation treatment (A23187 for 5 min and CHX + 6-DMAP for 1 h), oocytes were still not activated and had condensed sperm nuclei (Fig. 4a). When examined after a 1-h culture in KSOM, a small sperm aster was seen around a compact sperm nucleus (Fig. 4b). When oocytes were subjected to two consecutive activation treatments, they were activated and had decondensing or decondensed sperm nuclei with microtubular asters (Fig. 4c). Well-developed pronuclei of sperm and oocyte origin were discernible 3 h after activation treatment (Fig. 4d).
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Preimplantation development of oocytes injected with fresh or freeze-dried spermatozoa is summarized in Table 1. Parthenogenetic activation without sperm injection generated 43% blastocyst development. Without the two rounds of oocyte activation treatments, only 6% of the oocytes injected with fresh spermatozoa developed into blastocysts, whereas as much as 30% of oocytes developed to blastocysts after ICSI of fresh sperm and parthenogenetic activation, suggesting that the physical injection and the subsequent release of the sperm content in the oocytes are not sufficient to activate the oocytes for early embryo development. Additional stimulation is required to replace the activation effect of the sperm-oocyte fusion process during natural fertilization. Interestingly, ICSI of freeze-dried sperm followed by activation generated a rate of blastocyst development (24%) similar to that of ICSI with fresh sperm. This observation further demonstrates the minimal damaging effect of the freeze-drying process to the sperm's nuclear DNA.
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When a total of 230 embryos at the one-cell stage were transferred into eight pseudopregnant female rabbits (Table 2), one female became pregnant and delivered a full-term kit (weight at birth, 67.4 g) on the 33rd day after ICSI. The kit appeared to be normal but was dead because of dystocia, a common problem for a single birth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, poor oocyte development after ICSI using freeze- dried rabbit spermatozoa without activation (Table 1) could result from deterioration of sperm-born oocyte-activating factor (SOAF) [7] caused by freeze-drying. This is in contrast to mouse freeze-dried sperm ICSI, in which oocytes are readily activated without artificial stimulation [2–5]. Deterioration of SOAF because of freeze-drying may be minimized by improving the activation procedures. A recent study showed that more than 80% of rabbit oocytes developed to the blastocyst stage after repeated parthenogenetic stimulation combining chemical activation with electric current [28]. Activation of nuclear-transferred rabbit oocytes with inositol 1,4,5-trisphosphate, inducing repeated rises in intracellular Ca2+, led to improved postimplantation development of embryos [29].
Second, the plasma membranes of rabbit spermatozoa may persist around sperm nuclei for some time after sperm deposition inside the oocyte, which would retard the interactions between SOAF and the oocyte cytoplasm. Previous removal of sperm plasma membranes is known to accelerate the onset of oocyte activation after ICSI [30]. Despite the observation of membrane damage in all freeze-dried sperm examined, the lyophilization process may introduce chemical changes to the plasma and other sperm membranes, rendering them more difficult to be dissolved than those of fresh, intact sperm. Thus, we may be able to increase the efficiency of rabbit ICSI by proper removal of the sperm's plasma membranes. After all, the sperm plasma membrane never enters the ooplasm during normal fertilization [6].
The fact that we were able to generate blastocysts from ICSI with freeze-dried sperm at a rate similar to that from ICSI with fresh sperm suggests that the centrosomes in the freeze-dried sperm were perhaps not severely damaged. In mammals other than common laboratory rodents such as the mouse, the distal sperm centrosome is believed to play a critical role in the assembly of the microtubular (spindle) network, which brings the male and female pronuclei to the center of the zygote [8, 9, 14]. In the rabbit, zygotic centrosomes in naturally fertilized eggs are of paternal and maternal origin [31]. Because we selectively injected freeze-dried spermatozoa that had tails (either long or short), the proximal centrosome in the head-tail junction must have been injected into all oocytes during ICSI. Alternatively, it is possible that sperm centrosomes were damaged by freeze-drying, but they are not required for embryo development. Recent studies in amphibians and Drosophila sp. show that sperm centrosomes are not essential for spindle formation or normal embryonic development [32–35]. Injection of sperm heads (without tails) into oocytes led to births of viable offspring in cattle [36] and pigs [37]. Thus, it is possible that the blastocysts and the term kit we produced were developed from oocytes fertilized by the spermatozoa that had no functional centrosome. Nonetheless, the birth of a full-term rabbit kit reported here indicates that freeze-drying the spermatozoa of nonrodent species is possible. A day may come when spermatozoa of all mammalian species are kept freeze-dried at ambient temperature indefinitely.
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FOOTNOTES |
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2 Correspondence. FAX: 860 486 0534; jyang{at}canr.uconn.edu
3 Current address: Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210
4 Current address: Department of Biological Sciences, Asahikawa Medical College, Asahikawa, Hokkaido, Japan
Received: 26 November 2003.
First decision: 12 December 2003.
Accepted: 2 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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