Research Article
Development and quality of porcine embryos in different culture system and embryo-producing methods
- S-A. Ock, S-L. Lee, J-G. Kim, B-M. Kumar, S. Balasubramanian, S-Y. Choe, G-J. Rho
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- Published online by Cambridge University Press:
- 01 February 2007, pp. 1-8
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In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O2 or 5% O2 (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU−) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU −/+) on 20% O2 or 5% O2 (Group 4). IVF blastocysts did not differ significantly with O2 concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O2 concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 ± 16.1 vs. 24.0 ± 4.0 and 4.9 ± 9.0 vs. 22.8 ± 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O2 concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.
Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes
- H. Watanabe, H. Tateno, H. Kusakabe, T. Matsuoka, Y. Kamiguchi, Y. Fujise, H. Ishikawa, S. Ohsumi, Y. Fukui
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- 01 February 2007, pp. 9-14
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Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3–97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2–93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.
Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection
- M. Nakai, N. Kashiwazaki, A. Takizawa, N. Maedomari, M. Ozawa, J. Noguchi, H. Kaneko, M. Shino, K. Kikuchi
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- 01 February 2007, pp. 15-24
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Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.
The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.
Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey
- N. Chen, S-L. Liow, R. Bin Abdullah, WK. Khadijah Wan Embong, W-Y. Yip, L-G. Tan, G-Q. Tong, S-C. Ng
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- 01 February 2007, pp. 25-33
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Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 °C) without CO2 supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.
Ovine (Ovis aries) blastula from an in vitro production system and isolation of primary embryonic stem cells
- S-X. Zhu, Z. Sun, J-P. Zhang
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- 01 February 2007, pp. 35-41
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Livestock embryo production in in vitro systems has been highlighted due to the emergence of interest in embryo stem cells (ESC). ESC potency and their wide potential applications have been recognized in medicine, fundamental research fields and commercial markets due to ESC totipotency or pluripotency and self-renewal. Ovine ESC probably is a useful technical platform for transgenic livestock and animal cloning, but ESC lines have not yet been founded because of difficulties in ESC isolation and the lack of blastula materials. We have established an IVP (in vitro production) system in our laboratory, including in vitro maturation, in vitro fertilization and in vitro culture, to produce sheep blastula using fresh ovaries and testes collected from livestock production. This system can achieve rates of mature eggs and blastulas of 65 and 50% respectively, and can provide enough blastulas for ICM (inner cell mass) isolation. Furthermore, ESC-like clones were isolated from the ICM on ovine embryonic fibroblast (OEF) feeder cells and in ES-DMEM supplemented with the cell factors LIF and SCF, and these survived to the third passage, which was primarily identified by AKP staining and morphology. This work provides a basis for ovine ESC isolation and foundation of ESC lines.
Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen
- B. de Ávila Rodrigues, L. Carboneiro dos Santos, J.L. Rodrigues
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- 01 February 2007, pp. 43-53
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This study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus–oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 μg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6–8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6–8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.
Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts
- H-S. Kim, H-Y. Son, S. Kim, G-S. Lee, C-H. Park, S-K. Kang, B-C. Lee, W-S. Hwang, C-K. Lee
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- 01 February 2007, pp. 55-63
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The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.
Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions
- G. Sánchez Toranzo, J. Oterino, L. Zelarayán, F. Bonilla, M.I. Bühler
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- 01 February 2007, pp. 65-80
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It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm.
In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP3), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca2+. Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells.
The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes).
This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP3.
The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation.
Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell–cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP3 or Ca2+ through the gap junctions, which would increase the Ca2+ level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.
Is the sperm centrosome to blame for the complex polyploid chromosome patterns observed in cleavage stage embryos from an OAT patient?
- K. Chatzimeletiou, A. J. Rutherford, D. K. Griffin, A. H. Handyside
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- 01 February 2007, pp. 81-90
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Oligoasthenoteratozoospermia (OAT) is defined by a combined low count < 20 × 106 sperm/ml, poor motility < 50 % forward progression or < 25 % rapid linear progression and abnormal morphology (5–8 % normal using Kruger strict criteria) and has been associated with increased levels of sperm aneuploidy. Here we report on the cytogenetic findings from three ‘spare’ embryos from a couple that were referred for ICSI because of OAT. The embryos were processed for sequential FISH in three hybridization rounds using probes for chromosomes 3, 7, 9, 13, 17, 18, 21, X and Y. Molecular cytogenetic analysis of nine chromosomes revealed that all three embryos were female polyploid. One of them was uniformly tetraploid for all chromosomes tested, while the remaining two embryos showed evidence of abnormal postzygotic segregation of chromosomes, causing the derivative blastomeres to have uneven chromosomal constitution. In one of them in particular, the non-disjoining chromosomes showed preferential segregation to the same pole, rather than randomly moving towards either pole, suggesting an abnormal spindle and causing the derivative blastomeres to have significantly uneven chromosomal constitutions. The possible scenarios leading to polyploidy and chromosomal imbalance through cytokinetic failure and subsequent abnormal centrosomal distribution are outlined.
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- Published online by Cambridge University Press:
- 01 February 2007, p. 91
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