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Reproductive Technology |
a Department of Animal Science/Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, fertilization, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Species differences in the response to ICSI have been found. In mice, humans, hamsters, and rabbits, ICSI alone is sufficient to activate the oocytes for further embryonic development [3, 11–15]. In cattle, however, additional parthenogenetic activation after ICSI is necessary to activate the oocytes [16]. In pigs, it is still unclear whether activation of the oocytes is beneficial to development of ICSI embryos. Intracytoplasmic injection of round spermatids combined with electrical activation (EA) significantly improved pronucleus formation in porcine oocytes [17] as was also found in the mouse [3] and the rabbit [18], for which ICSI of sperm requires no additional activation. Conflicting data have been reported for the effect of artificial activation after ICSI in pigs. With no activation treatments, comparable blastocyst rates have been obtained for ICSI and in vitro fertilization (IVF) [19], and live births have been obtained from in vivo-matured oocytes injected with fresh sperm with or without activation treatment [8, 9]. Improved fertilization rate and blastocyst development, however, were reported by Lai et al. [10], who subjected in vitro-matured oocytes to activation treatment after intracytoplasmic injection of frozen-thawed sperm.
In human ICSI studies, oocytes that have formed both the male and female pronuclei are considered fertilized [20, 21]. Although all oocytes received an injected sperm, <100% of ICSI oocytes manifested further development. Therefore, sperm entry is no longer a good criterion for successful fertilization. In humans, 70–80% of ICSI oocytes that failed to fertilize remained at the metapahse II (MII) stage and were therefore not activated (no reversal of MII arrest) [20, 21]. Additionally, many of the uncleaved human ICSI oocytes did not form the male pronucleus even after the oocytes had been activated [21, 22], suggesting that activation of human oocytes is not always associated with male pronucleus formation. It is unclear whether these effects also occur in pigs. Therefore, the current study was designed to analyze 1) the effects of additional activation stimulation of pig oocytes following intracytoplasmic injection of frozen-thawed sperm and 2) the association between male pronucleus formation and oocyte activation after ICSI treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries of prepubertal gilts were obtained from a local slaughterhouse and transported to the laboratory in PBS at 30°C within 1 h. Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3–7 mm in diameter) with an 18-ga needle into a disposable 10-ml syringe and washed three times with Tyrode lactate-Hepes (TL-Hepes) [23]. Twenty to 25 COCs were matured in 100-µl droplets of BSA-free NCSU23 [24] supplemented with 10% porcine follicular fluid, 1% modified Eagle medium (MEM) with nonessential amino acids (Sigma, St. Louis, MO), 0.2 mM pyruvate (Sigma), 0.1 mg/ml cysteine (Sigma), and 10 IU/ml porcine FSH (Vetrepharm, London, ON, Canada) at 39°C for 44 h without changing the medium.
Intracytoplasmic Injection of Spermatozoa
The oocytes were denuded of cumulus cells by repeated pipetting following 44 h of maturation. Because the cytoplasm of pig oocytes contains large numbers of dark lipid granules, oocytes were centrifuged for 10 min in an Eppendorf centrifuge at 12 000 x g in 200 µl of TL-Hepes to detect the formation of the first polar bodies. High-quality intact oocytes with a visible polar body were selected for further use. Frozen-thawed boar semen (Swine Genetics International, Cambridge, IA) was centrifuged in a percoll gradient (45%/90%) at 900 x g for 15 min. The pellet was then washed once with 5 ml of TL-Hepes and resuspended in 1 ml of TL-Hepes. Spermatozoa were centrifuged (400 x g for 5 min) and resuspended in injection medium (TL-Hepes:10% polyvinylpyrrolidone solution, 1:1). A microdrop (5 µl) of injection medium under light mineral oil was placed in the lid of a 60-mm sterile culture dish, which was positioned on an inverted microscope (Olympus, Tokyo, Japan) equipped with Leica micromanipulators. The steps of sperm injection are shown in Figure 1. A spermatozoon was aspirated into the injection pipette (Fig. 1A), and an oocyte was secured by a holding pipette (Fig. 1B). The sperm was expelled into the cytoplasm of the oocyte (Fig. 1C).
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Oocyte Activation
Following sperm injection, the injected oocytes were washed and preincubated for 2 min in pulse medium containing 0.25 M mannitol with 0.01% polyvinyl alcohol, 0.5 mM Hepes, 100 M CaCl2·H2O and 100 M MgCl2·6H2O (pH 7.2). Electrical stimulation to induce activation was delivered with a BTX Electro Cell Manipulator (Biotechnologies and Experimental Research, San Diego, CA) to a chamber with two parallel platinum wire electrodes (200 µm outside diameter) spaced 1 mm apart and covered with pulse medium. Oocytes were exposed to an electrical pulse (a 10-sec pulse at 0.05 kV/cm AC followed by a 30-µsec pulse at 1.7 kV/cm DC at room temperature) 0.5–1 h before or after ICSI. For chemical activation, calcium ionophore A23187 (Sigma) was dissolved in dimethylsulfoxide (DMSO; Sigma) at a concentration of 2 mM. A stock solution of A23187 in DMSO was stored at -20°C and diluted to 75 µM of A23187 in culture medium prior to treatment of oocytes. The oocytes were treated for 5 min at 25°C to induce chemical stimulation following ICSI. For experiment 2, a cycloheximide (CH) stock solution (1 mg/ml in culture medium stored at -20°C) was diluted to 10 µg/ml in culture medium. After EA or A23187 treatment, the oocytes were treated with CH in culture medium for 5 h. Oocytes were then thoroughly washed with culture medium prior to being transferred into fresh culture medium.
Embryo Culture and Examination
After ICSI with or without various activation treatments, all oocytes were transferred to 50-µl drops of culture medium supplemented with 1% MEM with nonessential amino acids (Sigma) and 0.4 mg/ml BSA at 39°C under 5% CO2. Cleavage and blastocyst formation (Fig. 1D) were examined at 48 h and 168 h (7 days) after ICSI.
Forty-eight hours after ICSI, oocytes that exhibited no evidence of development were fixed, and their DNA was stained with Hoechst 33342 to identify pronuclei, metaphase chromosomes, sperm heads, and polar bodies. Oocytes that had been cultured for 168 h were also examined to determine mean cell numbers. To further investigate the association between sperm head decondensation and activation, oocytes were stained with Hoechst dye 16–20 h following ICSI to identify pronuclei, metaphase chromosomes, sperm heads, and polar bodies. These oocytes were examined under a fluorescence microscope (Nikon, Tokyo, Japan) equipped with image analysis software. Images were prepared using Adobe Photoshop 5.0 software (Adobe Systems, Mountain View, CA) and archived digitally on an erasable magnetic optical disk.
The morphology of the injected sperm heads in the cytoplasm of oocytes was classified into three categories according to Ahmadi and Ng [25]: 1) intact sperm head, 2) decondensed or swelling sperm head, and 3) an enlarged nucleus.
Experimental Design
The study was comprised of three experiments. In the first experiment, we examined the effect of EA, before or after ICSI, on porcine oocyte fertilization and subsequent development. In experiment 2, we compared the effects of different stimuli (EA, A23187, and combined treatment with EA or A23187 and CH after ICSI) on rates of fertilization and subsequent development. Oocytes that had not cleaved 48 h after ICSI were stained with Hoechst 33342 to determine the reason for fertilization failure. Cytological studies were undertaken in experiment 3 to determine the causes of failed and abnormal fertilization 16–20 h after ICSI. The association between sperm head decondensation and oocyte activation was assessed (after Hoechst staining) by the extrusion of the second polar body and formation of pronuclei.
Statistical Analysis
Treatment effects on oocyte activation, fertilization, and development rates were analyzed with one-way ANOVA using the general liner model procedure of the Statistical Analysis System (Cary, NC). Three to five replicates were evaluated for each treatment in each experiment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 1, in which oocytes were subjected to treatments by ICSI alone or ICSI with EA before or after sperm injection, the majority of oocytes survived the mechanical injection (Table 1). Normal two-cell embryos (Fig. 2B) and blastocysts (Fig. 1D) were formed from all ICSI treatment groups, either with or without additional activation stimulus. Electrically stimulated ICSI oocytes, however, had significantly higher rates of cleavage and blastocyst development than did those treated with ICSI alone (P < 0.05). The timing of activation, either before or after ICSI, did not significantly affect blastocyst development.
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When the nuclear morphology of all ICSI oocytes/embryos was examined by DNA staining after 7 days of in vitro culture, nearly all oocytes that survived the injection were activated (released from MII arrest), even in the ICSI alone group. This activation rate was determined retrospectively by adding the number of oocytes with at least one pronucleus and the number of embryos that had developed to at least the two-cell stage (Table 1). Oocytes remaining at MII (failure of activation) were only present in the ICSI alone group and constituted a very small portion of oocytes that survived the injection (3/87). EA, however, significantly improved the fertilization rates, which were also determined retrospectively by adding the number of oocytes with two pronuclei and the number of embryos that had developed to at least the two-cell stage (Table 1).
Examination of the nuclear status of oocytes that failed to cleave 48 h after ICSI revealed asynchronous formation of male and female pronuclei. More than 70% of uncleaved oocytes were not fertilized because they failed to form the male pronucleus and had intact (Fig. 2, C–E), decondensed, or swelling sperm heads. These oocytes, however, were completely activated because they were released from MII arrest and had formed the female pronucleus (Fig. 2F and Table 2). Among oocytes that failed to cleave, additional activation treatment by electrical stimulus appeared to increase the percentage of fertilized oocytes (Table 2). These results suggest that activation treatment may improve male pronucleus formation.
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Examination of the nuclear status of embryos that cleaved but failed to form blastocysts after 168 h of in vitro culture revealed that the majority of these embryos also failed to form the male pronucleus (Table 3). They contained either an intact sperm head or a decondensed or swelling sperm head, even though they had undergone at least one round of cellular division, had the morphology of multicell embryos under normal light microscopy, and contained multiple nuclei under fluorescent microscopy (Fig. 2, E and F, and Table 3). Thus, these embryos had developed from only the female pronucleus and failed at various stages of development before reaching the blastocyst stage.
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The second experiment was designed to determine different combinations of electrical and chemical activation treatments on the development of ICSI embryos. Activation treatments by EA, EA + CH, A23187, or A23187 + CH did not significantly change cleavage or blastocyst rates (P > 0.05, Table 4) or improve the retrospectively determined fertilization rates (P > 0.05). Examination of the nuclear status of oocytes that failed to cleave 48 h after ICSI (Table 5) and that of embryos that did cleave but failed to form blastocysts (data not shown) revealed that, as in experiment 1, the majority of these oocytes/embryos failed to form male pronuclei and contained either intact or partially decondensed sperm heads. Male and female pronucleus development was not improved by the inhibition of protein synthesis using CH following ICSI.
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In experiments 1 and 2, activation and fertilization rates were determined retrospectively after the completion of in vitro embryo culture. To directly study the association between male pronucleus formation and oocyte activation, cytological observations of the nucleus were undertaken before the first mitosis (16–20 h after ICSI) to further determine the causes of failed and abnormal fertilization. Table 6 summarizes the nuclear morphology of porcine oocytes 16–20 h after ICSI followed by EA or A23187 treatments. The oocyte activation rates did not significantly differ between treatment with ICSI alone (97%) or ICSI with additional activation by EA (96.6%) or A23187 (100%). These results corroborate findings from experiment 1 in which ICSI alone was sufficient to release the MII-arrested oocytes and to induce formation of the female pronucleus. Among oocytes that were not fertilized, >50% of them had either intact or partially decondensed sperm heads (ICSI, 6/12; ICSI + EA, 4/8; ICSI + A23187, 6/10). Experiment 3 results together with those from experiments 1 and 2 demonstrated that the principal cause of fertilization failure following ICSI is not failure of oocyte activation but failure of male pronucleus formation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of fertilization comprises several postfusion events: release of oocyte MII arrest, extrusion of cortical granules and a second polar body, and the transformation of sperm nucleus and oocyte chromosomes into male and female pronuclei, respectively. These changes are thought to be initiated by Ca2+ oscillations in the oocytes after sperm-oocyte fusion. Three models or hypotheses have been proposed to explain the activation of oocytes by sperm, which trigger Ca2+ oscillations in the oocytes. Under the conduit model, the fusion of sperm with the oocyte's plasma membrane transfers the sperm's internal Ca2+ into the oocyte, triggering a cascade of Ca2+-induced Ca2+ release. Under the conduct model, the binding of sperm to the oocyte's surface receptors activates GTP-binding proteins (G proteins), which in turn stimulate the release of Ca2+. Under the content model, the action of a soluble oocyte-activating factor (sperm factor) released from the spermatozoon upon gamete fusion creates Ca2+ oscillations in the oocytes.
In the present study, virtually all oocytes that survived sperm injection were activated, regardless of artificial activation treatments. In experiments 1 and 2, oocyte activation rates were retrospectively determined, and in experiment 3 oocyte activation and fertilization were assessed by nuclear morphology before the first mitosis. The results of these experiments demonstrated that the presence of sperm alone was sufficient to activate the pig oocytes, and therefore either the conduit model or the content model may be operating, whereas binding of the sperm to the oocyte's cytoplasm membrane may not be required for pig oocyte activation. The use of frozen-thawed sperm for ICSI may also have improved the activation rates because membrane damage [10, 26] occurring during sperm cryopreservation may have facilitated the release of oocyte activation factor(s) from sperm into the oocytes.
We obtained nearly 100% oocyte activation after ICSI even without activation treatments, suggesting that ICSI alone is sufficient to release of porcine oocytes from MII arrest. However, only approximately 50% of all activated oocytes were successfully fertilized, as determined by formation of both the male and female pronuclei. The failure of male pronucleus formation was the major cause for the failure of fertilization in activated ICSI embryos. These observations differ from those in human oocytes, in which a lack of oocyte activation is responsible for 70–80% of fertilization failure [20, 21, 27, 28]. Our results in the pig imply that oocyte-activating factors in the sperm are still functional after freeze-thawing. However, these factors were not involved in the transformation of sperm head to male pronucleus. The failure of sperm head decondensation, therefore, may be attributed to the inadequacy of the oocytes. Experimental evidence in the fruit fly Drosophila melanogaster supports this hypothesis [29]. A specific gene mutation in the female flies can cause defects in oocytes that render them unable to transform normal sperm from wild-type males to male pronuclei during fertilization. Upon activation of these defective oocytes by normal sperm, only the female pronucleus formed. These oocytes can undergo cleavage division from the female pronuclei. As a result, haploid Drosophila embryos form from the female pronuclei, and these embryos die at early embryonic stages [29]. Because these observations are very similar to those in the present study where we found intact or partially decondensed sperm heads in unfertilized oocytes and in preblastocyst embryos, we surmise that pig oocytes subjected to ICSI treatments are deficient in their capability to induce sperm head transformation into male pronuclei. In the mouse, specific cytoplasmic factors responsible for decondensing the sperm head exist in oocytes. These factors have been found in matured oocytes and in the nuclei but not in the cytoplasm of germinal vesicle oocytes [30]. These sperm transformation activities, however, disappear within a few hours after oocyte activation [31]. Therefore, a subgroup of in vitro-matured pig oocytes may have reduced levels of sperm-decondensing factor(s) because of precocious activation of the oocytes prior to and during ICSI due to prolonged handling of oocytes and extended exposure to room temperature.
We evaluated the nuclear maturity of the oocytes after centrifugation, and only those that had extruded the first polar body were selected for injected. However, cytoplasmic maturity of these MII oocytes could not be analyzed without sacrificing the oocytes. Oocytes with complete nuclear maturity may not be completely matured in the cytoplasm. In vitro-matured oocytes (44 h maturation) may lack cytoplasmic factors such as glutathione [32] or ATP [33], both of which are required for the transformation of the sperm into the male pronucleus. The inadequacy of these factors may have also contributed to the failure of fertilization. However, these problems do not seem to affect IVF oocytes; we have never observed IVF oocytes with only female pronucleus formation and development (data not shown). Additionally, the beginning of sperm-induced Ca2+ oscillations is substantially delayed after ICSI [34], and this delay may also be related to improper male pronucleus formation. Furthermore, abnormalities in the chromatin of frozen-thawed sperm may also cause the failure of sperm head transformation. However, these abnormalities may not be a major cause of fertilization failure because DNA cross-linking created by oxidative stress in human sperm does not prevent male pronucleus formation after ICSI [35]. The failure of male pronucleus formation observed the porcine oocytes does not appear to be caused by incomplete oocyte activation because incomplete oocyte activation would result in the nuclear morphology of anapahse/telophase or metaphase III stages. None of these nuclear stages were observed in these oocytes, suggesting complete activation of all the porcine oocytes in our study.
Activation treatment after ICSI more than doubled blastocyst development compared with the ICSI alone group. Similar results were also obtained by Lai et al. [10]. It may be that the amount of oocyte-activating factor(s) released by the injected frozen-thawed sperm was only enough to partially initiate the physiological cascade of normal fertilization, i.e., to release the oocyte from MII arrest. Additional external stimulations such as EA and calcium ionophore treatment in combination with CH may have allowed the oocytes to continue the cascade of activation events.
In this study, we demonstrated that sperm nuclear swelling after ICSI is independent of oocyte activation and that an external activation protocol can enhance normal fertilization and preimplantation development of ICSI embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 860 486 0534; jyang{at}canr.uconn.edu
Received: 1 July 2002.
First decision: 21 July 2002.
Accepted: 28 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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