301 EVALUATION OF THE SPERM-MEDIATED GENE TRANSFER (SMGT) TECHNIQUE BY IN VITRO FERTILIZATION IN PIGS USING RecA PROTEIN

Sperm-mediated gene transfer (SMGT) is a transgenesis technique used in pigs mainly byAI (Lavitrano ML et al. 2002 Proc. Natl. Acad. Sci. USA 99, 14 230–14 235), and by intracytoplasmic spermi injection (ICSI) (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338), but up to now, it has not been reported by IVF (Bolling LC et al. 2003 Transgenics 4, 77–86). The aim of this study was to evaluate the efficiency of SMGT by IVF in pigs and the use of recombinase RecA to avoid possible exogenous DNA degradation by endonucleases. We designed a study with 3 experimental groups: (1) sperm incubated without exogenous DNA (control group), (2) sperm incubated with exogenous DNA (DNA group), and (3) sperm incubated with the complex RecA:DNA (RecA group). Ejaculates from mature boars were recovered and the seminal plasma was discarded to avoid detrimental effects on DNA binding. The spermatozoa were incubated with DNA or RecA-DNA and used as vectors for transferring linealized plasmid DNA [5.7 kb enhanced green fluorescent protein (EGFP)] into in vitro-matured porcine oocytes by IVF. Spermatozoa and oocytes were coincubated for 2 h in TALP medium; then, the fertilized oocytes were transferred into the culture drops with NCSU-23 medium. The binding of the DNA to the spermatozoa was confirmed by the use of enzymatic fluorescein-12-dUTP-labeled DNA and measured by flow cytometry. The total number of oocytes used was 584 (n = 59; n = 382; n = 143 for the 3 experimental groups, respectively). Embryos were examined for cleavage rate at 48 h after fertilization, and for embryo development at 144 h. Expression of EGFP in embryos was examined using a fluorescence inverted microscope. The results in our experiment showed that the coincubation of sperm with exogenous DNA induced a lower cleavage rate than when the RecA:DNA complex was used (DNA: 25.13 � 2.22 v. RecA: 41.26 � 4.13%, P < 0.05), and both no different from the control group (38.98 � 6.40). On the other hand, the production of blastocysts was similar in the 3 groups (Control: 21.74 � 8.79 v. DNA: 21.87 � 4.24 v. RecA: 15.25 � 4.72%) as well as the quality of the obtained embryos. The average number of cells per blastocyst was similar in the 3 groups (36.40 � 9.28 v. 37.26 � 3.32 v. 28.45 � 3.34, respectively). None of the produced embryos was detected for expressing protein EGFP. In conclusion, under our experimental conditions, IVF is not an effective technique for SMGT, whereas using ICSI-SMGT under the same conditions (DNA and DNA:RecA groups), we obtained a high percentage of transgenic embryos (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). Three main causes are hypothesized to be probably related to this conclusion: (i) the penetration of the oocytes is achieved only by the not DNA-bound viable spermatozoa in a competitive system, (ii) the DNA was only bound to altered membrane or dead spermatozoa, and (iii) the exogenous DNA is only present on sperm surface and in the process of fusion with oocyte membrane, the DNA is not internalized.

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Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Reproduction ◽

10.1530/rep-10-0129 ◽

2010 ◽

Vol 140 (2) ◽

pp. 259-272 ◽

Cited By ~ 33

Author(s):

Francisco A García-Vázquez ◽

Salvador Ruiz ◽

Carmen Matás ◽

M José Izquierdo-Rico ◽

Luis A Grullón ◽

...

Keyword(s):

Gene Transfer ◽

High Efficiency ◽

Fluorescent Protein ◽

Reactive Oxygen Species Generation ◽

Reca Protein ◽

Transgenic Animals ◽

Membrane Lipid ◽

Transgenic Pigs ◽

Exogenous Dna

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab286 ◽

2006 ◽

Vol 18 (2) ◽

pp. 250

Author(s):

M. G. Marques ◽

A. B. Nascimento ◽

V. P. Oliveira ◽

A. R. S. Coutinho ◽

M. E. O. A. Assumpção ◽

...

Keyword(s):

Culture Medium ◽

Cell Number ◽

Control Group ◽

Cleavage Rate ◽

Vitro Fertilization ◽

Electric Pulses ◽

Cell Numbers ◽

Blastocyst Rate ◽

And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

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335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab335 ◽

2011 ◽

Vol 23 (1) ◽

pp. 263

Author(s):

F. Pereyra-Bonnet ◽

A. Gibbons ◽

M. Cueto ◽

R. Bevacqua ◽

L. Escobar ◽

...

Keyword(s):

Fluorescent Protein ◽

Genetically Modified ◽

Fold Increase ◽

Egfp Expression ◽

Control Group ◽

Corpora Lutea ◽

Exogenous Dna ◽

Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

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404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab404 ◽

2007 ◽

Vol 19 (1) ◽

pp. 317

Author(s):

T. S. Kim ◽

Y. Cao ◽

H. T. Cheong ◽

B. K. Yang ◽

C. K. Park

Keyword(s):

Gene Transfer ◽

Transfection Efficiency ◽

The Other ◽

Control Group ◽

Dna Uptake ◽

Alkaline Lysis ◽

Exogenous Dna ◽

Other Hand ◽

Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

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68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab68 ◽

2008 ◽

Vol 20 (1) ◽

pp. 115

Author(s):

L. Attanasio ◽

A. De Rosa ◽

L. Boccia ◽

R. Di Palo ◽

G. Campanile ◽

...

Keyword(s):

Survival Rate ◽

In Vitro Fertilization ◽

Survival Rates ◽

Calf Serum ◽

Cumulus Cells ◽

Control Group ◽

Cleavage Rate ◽

Vitro Fertilization ◽

Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

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205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab205 ◽

2009 ◽

Vol 21 (1) ◽

pp. 200

Author(s):

S. Di Francesco ◽

E. Mariotti ◽

M. Rubessa ◽

G. Campanile ◽

R. Di Palo ◽

...

Keyword(s):

Embryo Development ◽

Cumulus Cells ◽

Bubalus Bubalis ◽

The Other ◽

Control Group ◽

Single Chain ◽

Cleavage Rate ◽

Chi Square ◽

Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

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Sperm-mediated gene transfer: effect on bovine in vitro embryo production

Zygote ◽

10.1017/s0967199412000147 ◽

2012 ◽

Vol 21 (4) ◽

pp. 325-329 ◽

Cited By ~ 3

Author(s):

Renata Simões ◽

Alessandra Corallo Nicacio ◽

Mario Binelli ◽

Fabiola Freitas de Paula-Lopes ◽

Marcella Pecora Milazzotto ◽

...

Keyword(s):

Gene Transfer ◽

Sperm Cell ◽

Dna Delivery ◽

Cell Interaction ◽

Control Group ◽

Exogenous Dna ◽

Incubation Periods ◽

Sperm Penetration ◽

And Function

SummaryThe technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

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402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab402 ◽

2007 ◽

Vol 19 (1) ◽

pp. 317

Author(s):

M. K. Gupta ◽

S. J. Uhm ◽

E. Y. Kim ◽

Y. H. Jung ◽

J. Y. Yu ◽

...

Keyword(s):

Gene Transfer ◽

Fluorescent Protein ◽

Standard Procedure ◽

Metaphase Plate ◽

Egfp Expression ◽

Cleavage Rate ◽

Cell Stage ◽

Transgenic Livestock ◽

Expression Rate

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5&percnt;) was similar to that of non-injected control oocytes (67.8 ± 0.4&percnt;). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1&percnt;. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1&percnt; when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1&percnt;). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

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Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model

Zygote ◽

10.1017/s0967199416000381 ◽

2017 ◽

Vol 25 (2) ◽

pp. 222-230 ◽

Cited By ~ 5

Author(s):

Daiane L. Bulgarelli ◽

Alessandra A. Vireque ◽

Caroline P. Pitangui-Molina ◽

Marcos F. Silva-de-Sá ◽

Ana Carolina J. de Sá Rosa-e-Silva

Keyword(s):

Embryo Development ◽

Germinal Vesicle ◽

Control Group ◽

Blastocyst Development ◽

Cleavage Rate ◽

Oocyte Vitrification ◽

Vitro Fertilization ◽

Parthenogenetic Activation ◽

Nuclear Maturation

SummaryThis study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus–oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

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Levels interleukine-4 and interleukin-17 (IL-4, IL-17) of blood in patients with habitual recurrent pregnancy loss, which occurred in a loop in vitro fertilization

HEALTH OF WOMAN ◽

10.15574/hw.2016.114.137 ◽

2016 ◽

pp. 137-139

Author(s):

K.P. Golovatyuk ◽

Keyword(s):

In Vitro Fertilization ◽

Conditioned Medium ◽

Mononuclear Cells ◽

Recurrent Miscarriage ◽

Interleukin 4 ◽

Control Group ◽

Interleukin 17 ◽

Vitro Fertilization ◽

Blood Mononuclear Cells

The objective: was to investigate the levels of cytokines IL-4 and IL-17 in serum and conditioned medium cultures of blood mononuclear cells (MNC) and evaluation association between their products and miscarriage, which occurred in IVF cycles. Patients and methods. We observed 240 patients with recurrent miscarriage, came in IVF cycles, and 100 apparently healthy fertile women in the control group. The concentrations of IL-4 and IL-17 in serum and conditioned medium of MNC cultures were determined. Results. The levels of IL-4 in the serum and conditioned medium in spontaneous and stimulated mitogen secretion was not significantly different from those in the control group, whereas IL-17 levels were higher than those in the control group serum, in conditioned media of stimulated and non-stimulated MNCs. Conclusion. Disregulation of activity of circulating blood mononuclear cells in women with recurrent miscarriage that followed IVF, is accompanied by increased secretion of IL-17 and almost constant production of IL-4 on the back of high stimulation index of production of these cytokines. Key words: in vitro fertilization, miscarriage, interleukin-4, interleukin-17, serum stimulated and non-stimulated mononuclear blood.

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