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Embryo |
Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl 26100 Cremona, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, implantation, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of androgenetic embryos instead has been studied mainly in mice [17–28]. In the literature are reported two methods of androgenetic embryo construction. The first is the replacement of the female pronucleus with a male pronucleus in the zygote and represents the traditional method used in mice and recently applied also in sheep [14]. The second method is the in vitro fertilization of enucleated oocytes [25]. It has been applied only in mice and has been very successful, given that, for the first time, it has been demonstrated that androgenetic embryos can develop not only up to the 6- to 8-somit stage as in the case of the pronuclear transfer [17] but also to the 25-somite stage [26]. The application of the first method to the bovine is limited because the size difference between the two pronuclei is not so obvious as in mice. Therefore, it is impossible to be certain of the origin of the pronuclei. In addition, bovine zygotes have an opaque cytoplasm that requires a centrifugation step to visualize the pronuclei. For this reason, the only possibility for correctly identifying the male pronucleus in bovine is to fertilize enucleated oocytes by intracytoplasmic sperm injection (ICSI) or by in vitro fertilization (IVF).
It is known that androgenetic mouse embryos have lower developmental ability already at early preimplantation stages compared with parthenogenetic and gynogenetic embryos [25]. In farm animals, the only attempt to investigate developmental potential of androgenetic embryos was done in sheep, but the data are limited [14]. In bovine, there is no published information in contrast with the considerable amount of data available on parthenogenetic pre- and postimplantation development [13, 15, 16]. So the question of the developmental capacity of bovine androgenetic embryos is still unanswered.
The aim of our work was to compare the developmental ability of bovine androgenetic embryos constructed by different methods versus parthenogenetic and biparental IVF embryos. In addition, we investigated the possibility of reprogramming the androgenetic nuclei by nuclear transfer into enucleated oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine ovaries were collected at the abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, ITS (insulin, transferrin, selenium), 100 µg/ml sodium pyruvate, 90 µg/ml L-cystein, 720 µg/ml glycine, 7 nl/ml ß-mercaptoethanol, gonadotropins (0.05 IU/ml FSH and 0.05 IU/ml LH; Pergovet 75, Serono). Oocytes were cultured at 38.5°C in 5% CO2 in humidified air.
Enucleation of Oocytes
At 15 h of maturation, the oocytes were denuded of granulosa cells by vortexing in the presence of hyaluronidase and then were returned to maturation medium and monitored every hour for the extrusion of the polar body. Starting from 16 h of maturation, oocytes with freshly extruded polar body were stained with Hoechst 33342 in the presence of cytochalasin B (5 µg/ml). Enucleation was performed by the aspiration of polar body and associated metaphase II plate in a minimal volume of cytoplasm by micropipette of 25- to 30-µm diameter. Completeness of enucleation was ascertained by the identification of metaphase chromosomes within the enucleation pipette under ultraviolet light. All manipulations were in medium synthetic oviduct fluid (SOF) [29] supplemented with 20 mM Hepes, 6 mg/ml BSA, and modified eagle medium amino acids (SOF-Hepes).
In Vitro Fertilization
Motile spermatozoa were obtained by centrifugation of frozen-thawed semen on a Percoll discontinuous density gradient for 40 min at 750 x g. Viable spermatozoa were washed in Tyrode albumin lactate pyruvate Ca2+-free and pelleted by centrifugation for 10 min at 400 x g.
After 22 h of maturation, the control oocytes, partially denuded of the cumulus cells, and enucleated oocytes were cultured in four-well plates containing 0.3 ml/well of fertilization medium SOFaa [29] without glucose supplemented with 1 µg/ml heparin, 20 µM D-penicillamine, 100 µM hypotaurine, 1 µM epinephrine, and sperm at 0.5–1 x 106/ml for 16–18 h in 5% CO2 and 5% O2 in humidified air at 38.5°C.
After incubation with sperm, presumptive zygotes were denuded of cumulus cells by vortexing and transferred to 20-µl culture drops of medium SOF amino acids (SOFaa) in 5% CO2 and 5% O2 in humidified air at 38.5°C under mineral oil.
Piezo-ICSI
For ICSI, the sperm suspension was prepared at a final concentration of 4 x 106/ml and diluted 1:1 with 12% polyvinylpyrrolidone. The injection of sperm in matured oocytes (control) and enucleated oocytes was performed after 22 h of maturation in SOF-Hepes with the help of Piezo impact Micro Manipulator PMM-150 (Prime Tech Ltd., Ibaraki, Japan).
Pronuclear Transfer
At 17 h of IVF in pronuclear transfer (PT) experiments, embryos were washed from sperm and centrifuged 3 min at 11 000 x g in 45% Percoll in SOF-Hepes to displace granules to one of the cell poles in order to visualize pronuclei by differential interference contrast optic (DIC). Pronucleus with small volume of cytoplasm was enucleated with a pipette 25–30 µm in diameter and transferred into the perivitelline space of another single pronuclear zygote. All manipulations were in SOF-Hepes in the presence of cytochalasin B (5 µg/ml). The cytoplast-karyoplast constructions were fused in 0.3 M mannitol solution, containing 50 µM CaCl2 and 100 µM MgCl2, by single direct-current-pulse of 1.2 Kv/cm applied for 30 µsec at 20–22.5 h after IVF.
Cloning of Androgenetic Embryos
Advanced D4 androgenetic embryos were treated with nocodazole (0.2 µg/ml) for 16–18 h. Zona pellucida was digested by pronase treatment (Sigma protease, P 8811; Sigma-Aldrich, St. Louis, MO) and blastomeres were separated by gentle pipetting in SOFaa with aphidicoline (0.1 µg/ml) to arrest them in G1/S-phase of the cell cycle. In the presence of aphidicoline, freshly cleaved blastomeres, selected for cloning, were transferred under the zona pellucida of enucleated oocytes, fused as described above and activated at 24 h of in vitro maturation (IVM) with 5 µM ionomycin in SOF-Hepes for 4 min followed by 4 h of culture in 10 µg/ml cycloheximide + 5 µg/ml cytochalasin B in modified SOFaa.
Parthenogenetic Activation
In order to obtain haploid parthenogenetic embryos, matured oocytes (with first polar body) were activated with 5 µM ionomycin for 4 min either at 22 h of IVM, cultured in IVM medium for 2 h and treated by 2 mM 6-dimethylaminopurine (DMAP) for 4 h, or at 28 h of IVM followed by 4 h of culture in 10 µg/ml cycloheximide. In order to obtain diploid parthenogenetic embryos, matured oocytes were activated with 5 µM ionomycin for 4 min either at 24 h of IVM followed by 2 mM 6-DMAP treatment for 4 h or at 28 h of IVM followed by 4 h of culture in 10 µg/ml cycloheximide + 5 µg/ml cytochalasin B.
In Vitro Culture
All embryos were cultured in medium SOFaa. During embryo culture, half of the medium was renewed on Day 3 with fresh SOFaa and on Day 6 with TCM 199 with 16 mg/ml BSA (Day 0 was the day of IVF, ICSI, and activation). Cleavage was assessed at 48 h, the rate of compacted morulae (CM Day 6) and the rate of blastocysts (BL Day 7) were recorded on Days 6 and 7, respectively. At 168 h of culture, some blastocysts were fixed in ethanol:acetic acid (3:1) overnight and stained with lacmoid to evaluate their cell numbers.
Sex Determination of ICSI Embryos by Polymerase Chain Reaction (PCR)
Day 7 blastocysts were subjected to sex determination as described elsewhere [30]. Briefly, embryos washed in BSA-free SOF-Hepes were transferred into lysis buffer with 150 µg/ml proteinase K for 30 min at 37°C. Proteinase K was subsequently inactivated at 99°C for 8 min. Afterward, primers, oligonucleotides, and DNA polymerase were added and the samples were denaturated at 95°C for 3 min, followed by 40 cycles of amplification at 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min with final extension of 72°C for 5 min using bovine DNA-specific and bovine Y-chromosome-specific primers [30]. The resulting PCR products were electrophoresed on a 2% agarose gel and visualized under ultraviolet light.
Embryo Transfer
Holstein-Friesian heifers were synchronized using standard protocols. Seven days after the onset of estrus, recipients with a palpable corpus luteum received one to two blastocysts (fresh or after thawing) transferred to the uterine horn ipsilateral to the corpus luteum. From Day 21, the animals were scanned (5-MHz linear probe, Sonovet 600; Medison, Seoul, Korea) weekly for pregnancy diagnosis. Animals were treated according to DLG 116/92 regarding animal experimentation in Italy.
Statistical Analysis
Differences between the experimental groups were verified using chi-square test and Student t-test; P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, the rate of fertilization of enucleated oocytes (n = 2627), estimated by visualization of pronuclei by DIC, after displacement of cytoplasmic lipids, was 56.6%. Most zygotes (94%) were haploid.
About 89% of haploid androgenetic embryos were able to cleave, but only a few of those cleaved were able to compact (2.7%) and to form blastocysts on Day 7 (1.8%; Table 1). The morula compaction and blastocyst formation appeared to be retarded and, in fact, another three blastocysts were formed on Days 8, 9, and 10.
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A minority (5.4%) of the zygotes were polyspermic and usually possessed only two pronuclei. These diploid IVF-androgenetic embryos cleaved at a high rate (93.5%). About a fifth of them were able to progress to the compacted morula stage (22.3% of cleaved). On Day 7, half of the compacted morulae (11.5% of cleaved) developed to blastocysts.
In order to increase the diploid androgenetic embryo production, we used the combination of IVF of enucleated oocytes with pronuclear transfer (IVF + PT). The cleavage of diploid (IVF + PT) embryos was not reduced after nuclear transfer manipulation in comparison with IVF diploid androgenotes. Moreover, the compaction and blastocyst formation of diploid (IVF + PT) embryos were significantly higher than in the IVF group (49.3% versus 22.3% and 31.3% versus 11.5%, respectively, P < 0.05; Table 1).
Development of Cloned Androgenetic Embryos
Day 4 androgenetic embryos, with an average cell number of 19 ± 4 (n = 13) were used for cloning. Only 43.6% of blastomeres were able to cleave during the first 3 h after removal of nocodazole (8 ± 3 blastomeres per embryo) and these synchronized blastomeres were fused with enucleated oocytes.
The cleavage rate of cloned embryos was significantly lower than in all other groups of androgenetic embryos (75%, P < 0.05). In contrast, the development to blastocyst of the cleaved embryos (28/72, 38.9%; Table 1) was similar to that of diploid (IVF + PT) androgenotes and control biparental embryos.
Development of Androgenetic Embryos Created by Piezo-ICSI
The development of androgenetic and biparental control embryos constructed by piezo-ICSI is summarized in Table 2. The rate of cleavage did not differ between groups of haploid androgenotes and control diploid biparental embryos (22–25%). In contrast, only 16% of diploid androgenotes cleaved (P < 0.05). About 21% of cleaved control ICSI embryos compacted and formed expanded blastocysts on Day 7, with an average cell number of 147 ± 26, but neither diploid nor haploid cleaved androgenetic embryos developed up to the morula compaction stage.
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The cytological analysis of uncleaved embryos showed that about a quarter of uncleaved haploid embryos (14/61, 23%) possessed sperm head with no or little decondensation, 59% of embryos were arrested at the pronuclear stage and 18% at the mitotic metaphase stage.
In the group of diploid androgenotes, except embryos with no or little signs of sperm head decondensation (26/142, 18.3%), 21.8% of embryos were with asynchronous sperm head decondensation, 38% were arrested at the two pronuclear stage and 21.8% at the mitotic metaphase stage.
Development of Parthenogenetic Embryos
The developmental capacity of parthenogenetic embryos is shown in Table 3. The cleavage rate of haploids was significantly lower than diploids. Within this latter group, the cleavage rate differed significantly in relation to the activation protocol, being higher in the 6-DMAP group.
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The development of cleaved haploids to the compacted morula stage tended to be lower compared with diploids. This difference in the developmental ability became significant at the blastocyst stage, when only 3% of the cleaved embryos in the case of cycloheximide activation and 14.5% in the case of 6-DMAP activation formed blastocysts, in comparison with 43–46% of diploid parthenogenetic embryos (Table 3).
Cell Number of Day 7 Blastocysts
The cell number of Day 7 androgenetic blastocysts is presented in Table 1. The cell number of the two haploid blastocysts was 38 and 71. The retarded Day 8, Day 9, and Day 10 blastocysts had 123, 67, and 48 cells, respectively.
The cell number of diploid androgenotes and cloned androgenetic embryos was in the range of 58–250, but the average number (123 ± 32) was significantly lower compared with control biparental blastocysts (185 ± 60, P < 0.05; Table 1).
The cell number of Day 7 parthenogenetic blastocysts is presented in Table 3. The cell number of haploid blastocysts was in the range of 62–103, with an average number of 90 ± 11.
The cell number of diploid parthenogenotes was in the range of 61–167, and the average value did not differ from the cell number of haploid parthenogenotes and tended to be lower compared with (IVF + PT) diploid androgenetic (99 ± 29 and 100 ± 23 versus 123 ± 32, P = 0.055).
Sex Ratio in Day 7 Diploid Androgenetic Blastocysts
A total of 21 diploid androgenetic BL Day 7 were sexed by PCR. Seven and 14 embryos were identified as female and male, respectively. The resulting ratio of XX and XY androgenotes was within the expected rate of 1:2.
Embryo Transfer
The transfer of five diploid Day 7 androgenetic blastocysts to three recipients resulted in one pregnancy (33.3%), which interrupted between Day 28 and Day 35.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown that the developmental capacity of diploid bovine androgenotes is influenced by the method of production. Although piezo-ICSI of enucleated oocytes for production of diploid androgenetic embryos gives rise to cleaved embryos at a rate comparable with ICSI controls, none of such embryos progressed to compaction and beyond. On the other hand, ICSI in bovine has been associated with several developmental anomalies such as insufficient sperm head decondensation [32–34], retardation [35], and asynchronous pronuclear formation. Therefore, all these abnormalities, associated with the ICSI technique itself, certainly contribute to developmental failure of ICSI-androgenotes.
In the group of IVF diploid androgenotes, we observed a decline in the postcleavage development compared with the IVF + PT group and IVF controls. This decline may be due to possible haploidization of some embryos during immediate cleavage or to asynchronous pronuclear formation or to inadequate remodeling of two sperm nuclei in a single oocyte. In contrast, no difference in development was observed between IVF + PT embryos and IVF controls. Most likely, the absence of the above-mentioned problems linked to ICSI and the correct ploidy allows the IVF + PT group to develop at the same rate as controls.
In this study, we compared the developmental ability of bovine haploid androgenotes and parthenogenotes. While most haploid androgenotes arrested their development at early stages of cleavage and only a few were able to progress through morula compaction, 24–37% of haploid parthenogenotes normally compacted. As in rabbits [36–38], the drop in development of bovine haploid parthenogenotes became more evident at the blastocyst stage. The decrease of developmental ability was also related to the activation protocol: the 6-DMAP treatment resulted in a 14.5% blastocyst rate (21.2% [31]) in comparison with a 3% rate after cycloheximide treatment (4% [39] to 7% [4]).
When we compared the developmental capacity of diploid bovine parthenogenetic and androgenetic embryos (IVF + PT), we found no differences in development up to the compacted morula stage. In contrast, at the blastocyst stage, we observed a higher development of diploid parthenogenetic embryos compared with diploid androgenetic embryos that, nevertheless, formed blastocysts at a similar rate as control biparental IVF embryos. This finding is in agreement with previous data reported in the mouse, where diploid parthenogenetic embryos develop to blastocysts very efficiently (84–94% [11]) while diploid androgenetic embryos show poor developmental ability already at early preimplantation stages (43% [25], 45–56% [26]).
Thus, in bovine, the haploid condition determines an arrest of embryo development at the early cleavage stages, presumably at the time of maternal-embryonic transition of gene transcription, while, on the contrary, the development of diploid parthenogenetic and androgenetic embryos (IVF + PT) is equal to control IVF biparental embryos. Similarly, in sheep, the development of diploid androgenotes to the morula-blastocyst stage is equal to diploid gynogenotes and control IVF embryos [14]. Therefore, the preimplantation development of bovine diploid androgenotes is very efficient in comparison with normal biparental IVF embryos if we take into account that 25% of the androgenotes (1 XX: 2 XY: 1 YY), that are expected to contain YY chromosomes, are destined to arrest after a few cleavage divisions [40].
We have used diploid androgenetic embryos at the morula stage for production of cloned embryos. The metaphase rate, 3 h after removal of embryos from nocodazole, was slightly lower (43.6% versus 51–54%) than in IVF control 16-cell stage embryos as reported by Tanaka [41] and Alberio et al. [42]. This difference may be due to the slightly more advanced stage of the embryos used in our study for nuclear transfer (19 ± 4 cells versus 16 cells) or to the androgenetic nature of the embryos. Although the development of constructed nuclear transfer (NT) androgenetic embryos was lower at the cleavage and blastocyst stages in comparison with NT embryos constructed with blastomeres of IVF morulae as reported by Tanaka [41], the blastocyst rate of the androgenetic cloned embryos was equal to control IVF embryos cultured under the same conditions.
Cell-count experiments revealed that bovine parthenogenetic and androgenetic embryos developed slowly and Day 7 blastocysts had fewer cells than biparental IVF controls. As for parthenogenetic murine [43, 44] and porcine embryos [45, 46], the cell number of bovine haploid parthenogenetic blastocysts tended to be lower compared with diploid parthenogenotes and (IVF + PT) androgenotes.
There was no difference in cell number among NT and control IVF blastocysts in our previous experiments on somatic cell nuclear transfer [47]. In this study, androgenetic cloned blastocysts had lower cell numbered than IVF controls, similar to their nuclear donors. This finding indicates that the cleavage kinetics have not been modified by nuclear transfer.
There are several reports on postimplantation development of parthenogenetic embryos in the mouse [9, 10, 12, 48, 49], sheep [5, 14], pig [13], and bovine [3, 15, 16]. In particular, in the mouse, parthenogenetic development can reach the forelimb stage [48, 49], and in bovine, parthenogenetic embryos can establish pregnancies that are maintained up to 27–32 days [3] or 48 days after transfer of single parthenogenote [15] and aggregated parthenogenetic bovine embryos can delay estrous up to 67 days [16]. A considerable amount of information is published also on the fate of mouse androgenetic embryos. In this species, XX and XY androgenotes develop not only to the blastocyst stage [50] but also to embryonic Day 9.5 [26] at theoretical rates (1:2). At that stage, some androgenotes have 20–25 somites, even though they have not rotated and have a still-open neural tube, suggesting that such imbalanced development is a result of the onset of the imprinting effect [26]. In addition, it has been demonstrated that random X chromosome inactivation occurs in murine androgenetic embryos [27, 51]. In contrast, in bovine, to our knowledge, there are no studies on the development of androgenetic embryos following transfer in recipients. Therefore, this is the first report to show that bovine androgenotes are able to implant and to survive up to Day 28.
We showed by PCR that the sex ratio of diploid androgenetic blastocyst is equal to the theoretically expected 1:2. So our results are in agreement with recent data in the mouse [27], indicating that XX bovine androgenetic embryos are able to develop at least to the blastocyst stage.
In normal diploid mouse zygotes, active demethylation of the paternal genome but not of the maternal genome takes place within 7–8 h of fertilization. Barton and colleagues [28] have reported that this selective epigenetic remodeling does not occur in uniparental diploid embryos. These authors have shown that the cellular machinery of the fertilized egg cannot remethylate the additional paternal genome in diploid androgenetic embryos or demethylate the additional maternal genome in diploid gynogenetic embryos. Therefore, major methylation reprogramming defects arise in uniparental diploid embryos. Most likely, the limited development of diploid androgenetic and parthenogenetic embryos is correlated to the inappropriate methylation status of the additional paternal or maternal genomes.
In conclusion, we have shown that bovine androgenetic embryos constructed by IVF undergo cleavage at high rates. However, only a few cleaved haploid androgenetic embryos progressed to the compacted morula and blastocyst stages. In contrast, diploid androgenotes constructed by IVF and pronuclear transfer, but not those constructed by IVF only, develop to blastocysts at the same rate as control IVF embryos, although cell number was lower. Following transfer of diploid androgenetic embryos in recipient heifers, one pregnancy was established and maintained up to Day 28. Finally, cloning of morula-stage diploid androgenotes did not increase the rate of cell division.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Cesare Galli, Laboratorio di Tecnologie della Riproduzione, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy. FAX: 39 0372 436133; cesare{at}galli2.191.it
Received: 6 August 2003.
First decision: 25 August 2003.
Accepted: 7 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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