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Regular Article |
a Transgenic and Embryonic Stem Cell Core, IMMAG, Medical College of Georgia, Augusta, Georgia 30912
b Department of Gynecology, Medical College of Georgia, Augusta, Georgia 30912
c Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, fertilization, gamete biology, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the first report of ICSI in mammals [8] this approach has been used in both research [9–12] and infertility treatments in humans [13, 14]. The ICSI procedure bypasses the effective biological mechanisms for selecting the fertilizing spermatozoon that are normally in place during the reproductive process. Therefore, apparently any randomly selected lyophilized spermatozoon can be used directly via ICSI for fertilizing mammalian oocytes [6, 7]. The efficiency of bovine ICSI is limited because of the necessity for additional oocyte activation before or after the ICSI procedure [12]. Various stimuli for oocyte activation, such as cyclohexamide [15, 16], cyclin-dependent kinase inhibitors [17, 18], calcium ionophores and electric current [19, 20], strontium chloride and phorbol esters [21, 22], and ethanol [23] have been suggested to initiate bovine embryonic development after ICSI. Studies with activation protocols demonstrated that the developmental competence of bovine embryos could be improved by treatment with ionomycin followed by 6-dimethylaminopurine (DMAP) after ICSI [15, 16, 24].
Recent progress achieved in mice with lyophilized spermatozoa [6, 21] holds promise for other species. The present report summarizes efforts to extend this approach to cattle in order to assess the effectiveness of lyophilized spermatozoa for production of bovine blastocysts in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design
The study consisted of 4 experiments. In experiment 1, lyophilized sperm injection and various stimuli to induce oocyte activation were combined to study effects on cleavage and subsequent development in vitro. For each replicate, 100–150 matured oocytes were split into 4 equal treatment groups, and experiments were repeated 4 times to reach statistical significance. Oocyte activation after ICSI were treated with 5 and 10 µM ionomycin alone or in combination with 1.9 mM DMAP (see details below).
In experiment 2, ICSI zygotes produced with frozen-thawed spermatozoa or with lyophilized spermatozoa were compared with each other, and were compared with sham-injected and activated oocytes to determine rates of pronuclear formation (16–18 h). Rates of cleavage and development to the blastocyst stage by 168 h were compared after ICSI with lyophilized or frozen-thawed sperm, or following sham injection, or chemical activation alone in experiment 3. Karyotyping by staining and sex determination through polymerase chain reaction (PCR) at the blastocyst stage were studied in experiment 4. Replicates included control and treated groups on any given day.
Media
The medium used for maturation of cumulus-oocyte complexes (COCs) was TCM-199 containing Earle salts (M-5019), 20% fetal bovine serum (FBS) (S11550, lot E0110; Atlanta Biologicals, Atlanta GA), 5 IU/ml of eCG (367222; Calbiochem Biosciences Inc., La Jolla, CA) and 5 IU/ml FSH (F-8174), 25 mM sodium bicarbonate (S-5761), 2.5 mM sodium pyruvate (P-4562), 0.1 mM cysteamine (C-8397), and 2.5 µg/ml gentamycin (P-4687). The pH was adjusted to 7.4 and the osmolality was adjusted to 280 mOsm/kg. For embryo culture, 50 ml of CR1aa medium was prepared by adding 112 mM NaCl (S-9888), 3 mM KCl (P-5405), 5 mM hemicalcium lactate (L-4388), 1 mM glutamine (G-5763), 0.4 mM sodium pyruvate (P-4562), 2.5 mg gentamycin (P-4687), 500 µl of modified Eagle medium amino acids (11130-051; Gibco BRL, Grand Island, NY), 1000 µl of Eagle basal medium amino acids (B-6766), and 150 mg of BSA (fatty acid-free, A-6003). Beginning on Day 4 of in vitro culture the medium was supplemented with 10% FBS (S11550, lot E0110; Atlanta Biologicals). For sperm swim-up and ICSI, Tyrode albumin lactate pyruvate medium [25] supplemented with 10 mM Hepes (Hepes-Tyrode albumin lactate pyruvate [TALP], H-7523) was used.
Oocyte Preparation
Groups of 15 COCs collected from ovaries harvested at a local slaughterhouse were matured in 100-µl droplets of maturation medium under mineral oil (M-8410) at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 22–24 h of in vitro maturation, the expanded cumulus cells were removed by vortexing for 3 min in Hepes-TALP medium containing 160 IU hyaluronidase (H-4272). Oocytes with an extruded first polar body were selected and transferred to 20 µl of fertilization-TALP [25] drops under mineral oil until ICSI was carried out. ICSI was performed in Hepes-TALP medium and embryos were transferred back to fertilization-TALP medium until activation was accomplished.
Sperm Preparation
A straw of frozen bull spermatozoa was thawed by immersion in a 37°C water bath for 15 sec. The thawed semen was layered over 1 ml of both 45% and 90% Enhance-S-plus (Conception Technologies Inc., San Diego, CA) in a 15-ml centrifuge tube. The tube was centrifuged at 1200 x g for 15 min to obtain the motile fraction of spermatozoa. The pellet containing the motile sperm fraction and 500 µl of 90% Enhance-S plus was carefully removed from the bottom of the tube by a 1-ml pipette and transferred to another 15-ml centrifuge tube. The spermatozoa were then resuspended in 2.0 ml of Hepes-TALP medium and centrifuged at 300 x g for 4 min. The supernatant was removed and 4 ml of modified Dulbecco modified Eagle medium (DMEM; 10315-026; Gibco) containing 10% FBS (S1550, lot E0110; Atlanta Biologicals), 1 M L-glutamine (25030-081; Gibco), 0.2 mM sodium pyruvate (1136-070; Gibco), 100x nonessential amino acids (11140-050; Gibco), 0.25 ml of l0x nucleosides (160 mg adenosine [A-4036], 170 mg guanosine [G-6264], 146 mg cytidine [C-4684], 146 mg uridine [U-3003], 48 mg thymidine [T-1895]), and 0.01 ml of penicillin-streptomycin (P-3539)/ml was added. Sperm concentration was adjusted to 0.5 x 105/ml; then 100 µl of sperm suspension was aliquoted into 1.0-ml Eppendorf tubes, and the tubes were plunged into a liquid nitrogen bath. The tubes were then placed in a precooled (-47°C) freeze-flask attached to a Flexi-Dry freeze dryer (FTS Systems Inc., Stone Ridge, NY). Inlet pressure was 190 x 103 mbar. About 12–18 h later, the flask was removed from the system and the tubes were closed and stored at 4°C for 1–3 mo before use. One hundred microliters of embryo-grade water (W-1503) was added to the dried spermatozoa for reconstitution. Then, 10 µl of spermatozoa were mixed with 90 µl of embryo-grade water containing 12% (wt/vol) polyvinylpyrrolidone (PVP; 102787; ICN Biochemicals Inc., Aurora, OH; average weight 360 kDa) for microinjection.
Sperm Microinjection
Oocytes were cultured in 5% CO2 in air at 37°C before and after microinjection until the activation treatment interval was completed. A small number of oocytes (15–20) were manipulated at any one time, whereas the remaining oocytes were maintained in culture. ICSI was performed at 400x magnification in 5-µl droplets of Hepes-TALP under mineral oil in a depression slide maintained at 22°C on the stage of a Nikon 200 inverted microscope. The injection pipette with an inner diameter at the tip of 7–9 µm was connected to a pair of Narishige micromanipulators, and the holding pipette was connected to an Airtram (Eppendorf, Westbury, NY). A selected spermatozoon was aspirated tail-first into the injection pipette after the tail was broken between the injection needle and the depression slide, and then moved to the drop containing the oocytes to be injected. Aspiration was used to break the oolemma and to draw a small volume of ooplasm into the pipette. The spermatozoon and the aspirated ooplasm were then expelled into the oocyte with a minimal volume of PVP solution. About 15 oocytes were placed in the micromanipulation drop and exposure to the Hepes-TALP medium lasted no longer than 20 min.
Activation and Further Development of the Oocytes
In the first experiment (see Experimental Design), two potential means of oocyte activation were compared. The following groups were included: ICSI with 5 or 10 µM ionomycin, or 5 or 10 µM ionomycin plus 1.9 mM DMAP slightly modified from that originally reported by Susko-Parrish et al. [25]. In brief, all oocytes were cultured for 15 min to 2 h before they were activated. The method consisted of exposing the oocytes to 5 or 10 µM ionomycin in Hepes-TALP containing 1 mg/ml BSA (fatty acid-free BSA, A-6003) for 5 min at 22°C after injection and then to Hepes-TALP containing 3 mg BSA/ml for 5 min to stop the activation process. Activated oocytes were washed in Hepes-TALP containing 3 mg BSA/ml, and cultured in the same medium for 15 min; then 15–20 oocytes were transferred to a 20-µl drop of 1.9 mM DMAP in CR1aa for 4 h. At the end of the 4-h incubation, activated oocytes were washed 3 times with Hepes-TALP containing 3 mg BSA/ml, and then transferred to 20 µl of CR1aa for further culture. On Day 4 of culture the medium was supplemented by adding FBS (10% vol/vol). This culture was maintained for a total of 7 days. The medium for embryo culture was refreshed by replacing 10 µl with fresh CR1aa each 48 h. Embryonic development was routinely assessed with an inverted microscope (200x) at 24-h intervals for up to 168 h after injection.
Cytological Procedures and Embryo Sexing
Sixteen to eighteen hours after ICSI, some oocytes were fixed overnight in methanol:acetic acid (3:1, vol/vol) and then stained with 1% aceto-lacmoid to reveal the presence of pronuclei. In some experiments presumptive zygotes and blastocysts were specially stained in order to visualize nuclear structures [26]. To determine cell numbers, blastocysts at 168 h after ICSI with lyophilized sperm, along with those from control treatments, were fixed in methanol:acetic acid (3:1, vol/vol) overnight and then stained with 4% Giemsa solution for 10 min. Nuclei were counted at 100x magnification.
Some blastocysts were examined for their cytogenetic composition as explained elsewhere [27, 28]. Briefly, these blastocysts were collected at 168 h after ICSI, and incubated with 0.04 µg/ml of colcemid (152-10-040; Gibco BRL) for 1 h and then transferred to a chilled hypotonic solution (a 1:3 mixture of 6.56 mM trisodium citrate and 75 mM KCl) for up to 2 h. Blastocysts were then treated in Carnoy fixative for 5 min and dropped onto a glass slide. Softening solutions containing methanol and glacial acetic acid were used to facilitate spreading, and blastocysts were viewed under a dissecting microscope. The slides were air-dried, then aged at 90°C for 30 min, stained with Giemsa, and examined (at 100x magnification) for chromosomal complements.
Embryo sex was determined using the polymerase chain reaction (PCR) technique of Zinovieva et al. [29]. Briefly, an expanded blastocyst was collected on Day 6 or 7 after ICSI, and transferred to a 0.6-ml PCR tube containing 5 µl of PBS and 1 mg/ml PVA after three consecutive washings with PBS + PVA (phosphate buffered saline plus polyvinyl alcohol). Embryos were then frozen individually at -20°C for further analysis. Five microliters of 2x PCR buffer containing 0.25 µg/µl proteinase K was added to the PCR tube containing a single blastocyst, and incubated for 1 h at 56°C, then for 10 min at 95°C for proteinase K inactivation. One microliter of the sample was added in 19 µl of standard PCR mix, and the first PCR was carried out (denaturing at 94°C for 5 min, annealing at 44°C for 30 sec, and polymerization at 72°C 30 sec for 4 cycles followed by 30 cycles at 94°C for 5 sec, annealing at 44°C for 5 sec, and polymerization at 72°C for 30 sec). One microliter of this reaction mixture was added to 19 µl of PCR mix, and the nested PCR (30 cycles) was performed (identical to the first PCR, except at 56°C for 5 sec and 72°C for 20 sec). Samples were analyzed by standard 1.5% agarose gel electrophoresis (1x TBE buffer at 90 V for 1 h).
Statistical Analysis
Each experiment was repeated at least 4 times. Data are reported as the number of oocytes that reached a particular developmental stage and expressed as a percentage of the original number of oocytes. SigmaStat (Jandel Scientific, San Rafael, CA) software package was used to analyze the data. One-way ANOVA after arc-sine transformation of the proportional data for cleavage and development was applied to assess statistical differences, and the Bonferroni t-test was used to determine differences among groups. A difference of P < 0.05 was taken to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment 1: Influences of Various Activation Protocols on Developmental Competence of Bovine Embryos after ICSI with Lyophilized Spermatozoa
As depicted in Table 1, cleavage and blastocyst development were inferior in the oocyte groups exposed to ionomycin (alone) compared with the groups in which ionomycin plus DMAP was employed. Significantly fewer blastocysts developed in the oocyte treatment groups that included ICSI with 5 µM and 10 µM ionomycin than in the groups treated with ionomycin plus DMAP (12.1% and 10.7% vs. 23.3% and 30.7%, respectively). The numbers of nuclei per blastocyst at 168 h after ICSI (all sperm-injected groups) ranged from 88 to 116 (n = 60; mean ± SEM, 102 ± 14) and were higher than those obtained in groups treated with ionomycin plus DMAP without ICSI, and which varied from 44 to 68 (n = 30; 271 mean ± SEM, 56 ± 12). Our observations suggested that 10 µM ionomycin for 5 min followed by 4 h of incubation with 1.9 mM DMAP was the preferred treatment. Although we tried to use the method suggested by Rho et al. [16] in which oocytes were cultured for 3 h after activation and then cultured in DMAP containing medium, we obtained inferior results (i.e., lower proportions of developmental stages), compared to the method suggested by Susko-Parrish et al. [25] (data not shown).
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Experiment 2: Cytological Observations 16–18 Hours after ICSI with Lyophilized, Frozen-Thawed Spermatozoa and Activation Control
Pronucleus formations of activated oocytes after ICSI were evaluated following aceto-lacmoid staining. Representative zygotes are shown in Figure 2. A summary of results (Table 2) after oocytes were injected with either frozen-thawed or lyophilized sperm cells and activated with 10 µM ionomycin alone shows lower proportions of oocytes with pronuclear formation (66.0% and 56.0%, respectively) than for comparable groups activated with 10 µM ionomycin plus 1.9 mM DMAP (82.0% and 74.0%, respectively). However, there were no other significant differences between the frozen-thawed and the lyophilized sperm groups (i.e., regarding intact sperm heads or presence of one pronucleus), which were activated either by ionomycin alone, or by ionomycin plus DMAP (Table 2). In sham-injected and activation groups (with no ICSI), pronuclear formation was observed in 3 oocytes out of 50 (6.0%) and 9 of 50 (18.0%) treated with ionomycin alone and in 9 of 50 (18.0%) and 13 of 50 (26.0%) treated with ionomycin plus DMAP. In spermatozoon-injected oocytes exhibiting one pronucleus the pronuclear structure involved a condensed sperm head.
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Experiment 3: Comparative Evaluation of Bovine Embryonic Development Obtained after ICSI with Frozen-Thawed or Lyophilized Spermatozoa, and after Sham Injection and Activation by Two Different Protocols
As in experiment 2, presumptive zygotes obtained by ICSI were activated either with 10 µM ionomycin alone, or 10 µM ionomycin plus 1.9 mM DMAP. Results of embryonic development from 4 different groups (frozen-thawed spermatozoa, lyophilized spermatozoa, sham injection, and activation) are shown in Table 3. Representative embryos resulting from ICSI are shown in Figure 3. No blastocysts developed from sham injection or activation when oocytes were activated with ionomycin alone; however, 19.3% (29 of 150) and 10.7% (16 of 150) of oocytes injected with frozen-thawed sperm or lyophilized sperm, respectively, developed to the blastocyst stage. On the other hand, when presumptive zygotes or oocytes were activated with ionomycin plus DMAP, blastocysts were obtained in 65 of 190 (34.2%) frozen-thawed, 59 of 199 (29.6%) lyophilized, 29 of 190 (15.3%) sham-injected, and 35 of 190 (18.4%) activated groups.
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Experiment 4: Sex Determination and Karyotyping Analysis of Bovine Blastocysts Obtained after Frozen-Thawed and Lyophilized Sperm Injection, Sham Injection, and Activation
After blastocysts were individually spread on slides for cytogenetic analysis, 5–10 spreads were counted for each. Then each blastocyst was classified as shown in Table 4 if more than 90% of spreads fell in one of the descriptive categories. A representative chromosomal spread for monoploid and diploid embryos (A and B, diploid; C and D, monoploid) is shown in Figure 4. Table 4 shows that most of the blastocysts that were subjected to evaluation were in the diploid category for frozen-thawed and lyophilized sperm injection groups. However, haploid and tetraploid embryos were also obtained in the sperm-injected groups. Some blastocysts obtained from sham injection and activation groups had diploid numbers of chromosomes. Male-determining chromosomes were seen only following ICSI with a spermatozoon.
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To corroborate our karyotyping results, we sexed bovine blastocysts obtained after injection of spermatozoa by PCR (Fig. 5). When chromosomes X and Y were documented for sexing, 29 of 50 XY and 21 of 50 XX karyotypes were detected in the frozen-thawed group, and 34 of 50 XY and 16 of 50 XX karyotpyes were detected for the lyophilized sperm group (Table 5). When we performed PCR on sham-injected and activated oocyte groups, inconclusive results were obtained (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Advancements in genetic engineering have resulted in the generation of thousands of genetically modified animals [34], and more transgenic lines are being added every day. Cryopreservation of these genetically modified spermatozoa and spermatozoa of some species is not possible with conventional techniques [35]. Lyophilization presents an excellent opportunity for potential application to such spermatozoa to allow their long-term storage with significant economic benefits. Lyophilization in combination with ICSI also allows a means for more economic usage of sperm cells because a few thousand spermatozoa cannot be lyophilized, stored for a long time, and used to obtain viable embryos. In this respect, lyophilization of sperm cells presents an excellent possibility for conservation of endangered animal species.
During the fertilization process, a large calcium oscillation is evoked in the oocyte by sperm entry [36]. This Ca2+ release in the oocyte triggers oocyte activation and early development in mammalian embryos [37]. Although the activation process has not been understood very well, it has been suggested that after sperm mingle with oocyte cytoplasm, a sperm cytosolic factor is released into the oocyte, which generates cytoplasmic calcium increase [38, 39]. Kimura et al. [39] suggested that a sperm-borne activating factor could be responsible for oocyte activation by joining to the nuclear matrix, and by calcium being released into the oocyte. Artificial stimulators such as ionomycin [10] or ethanol [23] are required for the induction of Ca2+ oscillations after ICSI [12] or nuclear transfer [40] in cattle. Activation protocols with ionomycin alone [7, 12, 15] or along with either an inhibitor of protein phosphorylation, DMAP [15, 16, 24], or cyclin-dependent kinase inhibitors [17, 18], have been successfully used for oocyte activation after ICSI. It has been proposed that oocyte activation requires either sperm entry to cytoplasm, which provides some activation factors, or some type of stimuli, which cause intracellular calcium increase [25]. Susko-Parrish et al. [25] demonstrated that when oocytes were treated consecutively with 5 µM ionomycin followed immediately by 1.9 mM DMAP they had a high rate of pronuclear formation (76%) and subsequent blastocyst formation (21%). Those researchers also demonstrated that sequential activation of bovine oocytes caused diploid embryos when they were matured in vitro for 18–20 h. The exact cascade of events is still unclear, but calcium oscillation evoked by ionomycin treatment could cause elevation of pH and changes in activity of cell cycle regulating proteins. Later, Rho et al. [16] slightly modified the protocol by including a 3-h culture period between ionomycin and DMAP treatments, and obtained 24% blastocysts. The rationale for a 3-h culture period was to allow oocytes to expel the second polar body before their exposure to the protein kinase inhibitor DMAP. Chung et al. [24] pretreated bovine spermatozoa with dithiothreitol to promote decondensation, and achieved 62% cleavage and 4.8% blastocyst development. Our results demonstrated that 52.1% two-cell and 10.7% blastocyst development were obtained when 10 µM ionomycin alone was employed; whereas 66.7% two-cell and 30.7% blastocyst development were observed when oocytes were activated with 10 µM ionomycin plus 1.9 mM DMAP after ICSI (Table 1). These results corroborate the findings of other laboratories [15, 25].
Higher rates of oocytes with two pronuclei were observed in the groups that were activated with ionomycin plus DMAP than in those that were activated by ionomycin alone (Table 2). However, in the absence of sperm injection some two-pronuclear zygotes were among the sham-injected and activated oocytes of either treatment regime (Table 2). It is not clear why the injection process or the spermatozoon itself is not enough to activate bovine oocytes after ICSI as in other mammalian species. It has been suggested that bovine spermatozoa have been packed more stably than other mammalian spermatozoa, including those of humans [41]; therefore, although ICSI bypasses all the biological mechanisms, a spermatozoon might not have enough time to complete the normal biological events such as sperm membrane destabilization to prepare for oocyte activation. Our results are somewhat contradictory with previous published results [24, 25]. At 16–18 h, two pronuclei were observed in 3 of 50 (6.0%) and in 9 of 50 (18.0%) sham-injected and activated groups of ionomycin alone treatment, respectively, whereas two pronuclei were observed in 9 of 50 (18.0%) and in 13 of 50 (26.0%) sham-injected and activated groups of ionomycin + DMAP, respectively (Table 2). Although there was a significant difference between ionomycin alone and ionomycin + DMAP treatments in our experiments, these are lower than those obtained in earlier experiments [25]. Significantly higher rates of cleavage, morula, and blastocyst stages were observed in sperm injected (frozen-thawed and lyophilized) than in the sham-injected and activated groups after ionomycin alone and ionomycin plus DMAP treatments (Table 3). The karyotyping analyses on those blastocysts obtained by either sham injection or activation revealed that when diploid, only X chromosomes were seen, whereas a certain distribution of X and Y chromosomes was found among sperm-injected blastocysts (Table 4). These results suggest that spermatozoa that were either frozen-thawed or lyophilized contributed to the activation process as well as to development to the blastocyst stage. Intracellular calcium oscillations may be caused by a sperm cytosolic factor, which was released by sperm interaction with receptors on the ooplasmic membrane and carried into the cytoplasm following sperm fusion [24, 37]. Studies carried out in mice imply that spermatozoa might carry a sperm-borne oocyte activating factor into the oocyte, which is released over time and binds to the nuclear matrix, thereby causing sperm decondensation and pronuclear formation [36, 39]. However, these processes are not well characterized in bovine species.
In addition, we obtained fewer activated oocytes (diploid) than has been reported in the literature [16, 24, 25]. Nevertheless, this is the first extensive ICSI study conducted in cattle to include karyotyping results at the blastocyst stage.
Embryo-sexing and preimplantation diagnosis of allele-specific variations are of significance for the embryo-transfer industry, and have been successfully employed in embryo transfer and in vitro fertilization programs in cows [29]. Our data verified that the sexing results obtained with karyotyping were comparable to those obtained with PCR shown in Table 5.
In conclusion, data presented here confirm that bovine spermatozoa can effectively be lyophilized for long-term storage, and reconstituted and injected into oocytes for initiation of embryo development. Encouragement in development of this system merits additional work to obtain pregnancies following embryo transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 A portion of this work was presented at the 27th Annual Conference of the International Embryo Transfer Society, Omaha, NE, in 2001. 
2 Correspondence: Levent Keskintepe, Sher Institute for Reproductive Medicine, 3121 South Maryland Avenue, Suite 300, Las Vegas, NV 89109. FAX: 702 892 9666; lkeskintepe{at}sherinstitute.com
Accepted: February 11, 2002.
Received: May 16, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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