Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.     

Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is a popular method used in assisted conception, and live offspring have been born from a variety of species, including humans. In ICSI, sperm chromatin is introduced into the oocyte together with the acrosome, a structure that does not enter the oocyte during normal fertilization. We compared sperm chromatin remodeling, the potential of embryos to develop in vitro, and DNA synthesis in mouse embryos obtained from in vitro fertilization (IVF) and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development. Sperm chromatin was examined on air-dried, Giemsa-stained preparations at 30-min intervals for up to 4.5 h postfertilization. In all experimental groups, the oocytes underwent activation and formed pronuclei with similar rates. However, the dynamics of sperm chromatin remodeling in ICSI and IVF embryos varied. In ICSI, chromatin remodeling was more asynchronous than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome prior to injection with calcium ionophore A23187 but not with detergent Triton X-100 allowed more synchronous chromatin remodeling, timely DNA synthesis, and good embryo development. Our data have significance for the refinement of the molecular and biologic mechanisms associated with ICSI for current and future applications.     

In vitro developmental competence of ICSI-derived activated ovine embryos

The present study was conducted to determine the necessity for activation after intracytoplasmic sperm injection (ICSI) in sheep. The effect of chemical stimulation with either 5 μM ionomycin (I) for 5 min or ionomycin + 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h on the efficiency of ICSI, was compared in six experimental groups: (1) ICSI, (2) ICSI + I, (3) ICSI + I + 6DMAP, (4) Sham, (5) Sham + I, and (6) parthenogenetics (Sham and parthenogenetic groups were used as controls). In the present study, ovine oocytes needed additional chemical stimulation, after conventional ICSI, to activate (female pronucleous formation) and to form zygotes with male and female pronuclei (2PN). The percentage of cleaved embryos obtained and developed to blastocyst stage was higher (P < 0.001) for ICSI-derived zygotes, followed by activation (I and I + 6DMAP; 18.2 and 22.5%, respectively) than ICSI and Sham injection without activation (3.0 and 0.0%, respectively). There was, however, no significant difference between activation protocols I or I + 6DMAP. Furthermore, there was no significant difference among chemically activated, ICSI-derived zygotes in term of hatchability rate; however, the percentage was significantly higher in parthenogenetic and IVF groups than ICSI and Sham injection. In conclusion, neither sperm alone nor mechanical activation was sufficient for ovine oocyte activation and pronuclei formation. Therefore, in our study conditions for in vitro embryo development, chemical activation of oocytes must be considered an essential part of the ICSI procedure in sheep.     

Microtubules and parental genome organisation during abnormal fertilisation in humans

We analysed the distribution of beta-tubulins, acetylated alpha-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, beta-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.     

Sperm DNA adducts impair fertilization during ICSI but not during IVF

Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization) and 118 ICSI (intracytoplasmic sperm injection) first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999). DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI). The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002) and motility (p=0.0037) but not by DNA adducts concentration (p=0.30528), whereas during ICSI was positively influenced by sperm motility (p=0.04669) and negatively by DNA adducts concentration (p=0.00796). DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.     

Developmental potential of bovine androgenetic and parthenogenetic embryos: a comparative study

In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.     

Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.     

In vitro development of domestic cat embryos following intra-cytoplasmic sperm injection with testicular spermatozoa

The objective was to assess the ability of testicular spermatozoa to fertilize in vitro matured domestic cat oocytes and support blastocyst formation in vitro following intra-cytoplasmic sperm injection (ICSI). After IVM, oocytes were randomly and equally allocated among treatment groups (ICSI with testicular spermatozoa, ICSI with ejaculated spermatozoa, sham ICSI, and control IVF). At 18 h after either injection or insemination, the percentage of fertilized oocytes (per total metaphase II oocytes) was approximately 65% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which was less (P < 0.05) than control IVF (approximately 90%). On Day 7, the percentage of cleaved embryos (per total metaphase II oocytes) was approximately 60% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which also was less (P < 0.05) than control IVF (approximately 85%). After ICSI with testicular spermatozoa, the percentage of blastocysts (per total cleaved embryos) was approximately 11.0%, which was less (P < 0.05) than ICSI with ejaculated spermatozoa (approximately 21.0%); the latter was less (P < 0.05) than control IVF (approximately 43.0%). No blastocyst formation was observed after sham ICSI. For the first time in the domestic cat, this study demonstrated the fertilizing ability and developmental potential of intra-testicular spermatozoa delivered directly into intra-ovarian oocytes matured in vitro.     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.     

Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating

The procedures of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are routinely used in modern medicine to overcome infertility and, in animal husbandry, to propagate lines with compromised fertility. However, there remains concern that manual selection and injection of whole sperm into oocytes could contribute to pre- and postnatal developmental defects. To address this, we have used gene expression profiling and immunophenotyping to characterize offspring generated by these procedures. We used gametes from glutathione peroxidase 1 knockout (Gpx1-/-) mice as a sensitized screen responsive to oxidative stress from artificial reproduction technologies (ART). There were no differences between IVF and ICSI derived offspring in gene expression patterns, and minor differences in hematopoietic parameters. Furthermore there were only minor differences between these IVF and ICSI pups and those derived from natural mating. These data demonstrate for the first time in that there is no significant phenotypic affects of ICSI when compared to IVF and we identified a relatively minor influence of the artificial fertilization methods on phenotype of offspring compared with natural mating. These observations would support the use of ICSI for derivation of mutant mouse lines and may be of some importance for the use of this technique in human ART.     

Oocyte activation: lessons from human infertility

During fertilization, the spermatozoon penetrates through the cumulus cells and the zona pellucida that surrounds the oocyte, before it binds and fuses with the oocyte plasma membrane to induce activation. In vitro fertilization (IVF) studies performed in non-human mammals have contributed extensive knowledge regarding the mechanisms by which the spermatozoon activates the meiotic-arrested oocyte to resume meiosis, cleave and develop into an embryo. Although IVF has been used extensively for treating subfertile couples, not all of them were able to benefit from this procedure. In intracytoplasmic sperm injection (ICSI), one viable spermatozoon only is sufficient for successful fertilization of a single oocyte. Moreover, the injected fertilizing spermatozoon bypasses several physiological barriers, compared with IVF, which together could explain the high success rate for this procedure. ICSI has also allowed the identification of sperm components that are required for successful fertilization.