37DEVELOPMENTAL POTENTIAL OF CLONE CELLS IN MURINE CLONE-FERTILIZED 
AGGREGATION CHIMERAS

In both murine and porcine preimplantation stage clones, mosaicism in gene expression has been observed, indicating variation in transcription of some genes between cells of the individual clone (Boiani M et al., 2002 Genes Dev. 16, 1209–1219; Park KW et al., 2002 Biol. Reprod. 66, 1001–1005). This observation raises the question as to whether all blastomeres within one early-stage clone are equivalent, or whether there are differences in developmental potential. To address this, we aggregated preimplantation-stage clone embryos with fertilized embryos and assessed contribution of Oct4-GFP expressing cells of clone origin in blastocysts and in vitro outgrowths. In normal embryos, the Oct4-GFP transgene is expressed during preimplantation stages and reflects expression of Oct4 protein. Mouse cumulus cell clones were produced from cells transgenic for Oct4-GFP (Szabó PE et al., 2002 Mech. Dev. 115, 157–160) as described (Boiani M et al., 2002 Genes Dev. 16, 1209–1219). Four-cell-stage clones and synchronous fertilized non-transgenic embryos were aggregated in micro-wells after removal of the zona pellucida using acid Tyrode’s solution. Aggregates were cultured to the blastocyst stage in -MEM supplemented with bovine serum albumin (0.4% w/v). All control chimeras produced from four-cell-stage fertilized non-transgenic and Oct4-GFP transgenic embryos formed blastocysts, and 15 of 20 had GFP-expressing cells. The majority of clone-wild-type aggregates developed to the blastocyst stage (35/40); however, contribution of GFP-expressing cells was observed in fewer blastocysts compared to controls (12/35; P<0.05). Contribution of GFP expressing clone cells to the ICM varied between 30% and 100% of cells as determined by subjective evaluation of GFP fluorescence overlaying bright-field images. During in vitro outgrowth formation of synchronous aggregation chimeras of clone and wild-type embryos, maintenance of clone contribution to the ICM mound was observed, but at a lower frequency (12% v. 34% at the blastocyst stage). The results suggest that aggregation with fertilized cells does not provide benefit to clone blastomeres during preimplantation stages. Possibly, clone blastomeres may not be competitive with wild-type blastomeres, or are developmentally asynchronous, which will be tested using asynchronous chimeras.

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P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Á Martíne. Moro ◽

I Lamas-Toranzo ◽

L González-Brusi ◽

A Pérez-Gómez ◽

P Bermejo-Álvarez

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Early Embryo ◽

Developmental Competence ◽

Success Rates ◽

Developmental Potential ◽

Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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Predictive value of bovine follicular components as markers of oocyte developmental potential

Reproduction Fertility and Development ◽

10.1071/rd13007 ◽

2014 ◽

Vol 26 (2) ◽

pp. 337 ◽

Cited By ~ 45

Author(s):

Satoko Matoba ◽

Katrin Bender ◽

Alan G. Fahey ◽

Solomon Mamo ◽

Lorraine Brennan ◽

...

Keyword(s):

Follicular Fluid ◽

Cumulus Cell ◽

Principal Component ◽

Transcript Abundance ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Developmental Potential ◽

Thecal Cells ◽

Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

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Nuclear transplantation using bovine primordial germ cells from male fetuses

Reproduction Fertility and Development ◽

10.1071/rd9951217 ◽

1995 ◽

Vol 7 (5) ◽

pp. 1217 ◽

Cited By ~ 11

Author(s):

F Delhaise ◽

FJ Ectors ◽

Roover R de ◽

F Ectors ◽

F Dessy

Keyword(s):

Nuclear Transfer ◽

Primordial Germ Cells ◽

Blastocyst Stage ◽

Cell Counting ◽

Nuclear Transplantation ◽

Cell Nuclei ◽

Cell Stage ◽

Developmental Potential ◽

Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

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Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

Zygote ◽

10.1017/s0967199407004492 ◽

2008 ◽

Vol 16 (2) ◽

pp. 93-110 ◽

Cited By ~ 7

Author(s):

Yong Tao ◽

Lizi Cheng ◽

Meiling Zhang ◽

Bin Li ◽

Jianping Ding ◽

...

Keyword(s):

Nuclear Envelope ◽

Developmental Change ◽

Early Stage ◽

Ultrastructural Changes ◽

Cell Stage ◽

Developmental Potential ◽

Reconstructed Embryos ◽

Low Efficiency

SummaryThe low efficiency of somatic cell nuclear transfer may be related to the ultrastructural deviations of reconstructed embryos. The present study investigated ultrastructural differences between in vivo-produced and cloned goat embryos, including intra- and interspecies embryos. Goat ear fibroblast cells were used as donors, while the enucleated bovine and goat oocytes matured in vitro as recipients. Goat–goat (GG), goat–cattle (GC) and goat in vivo-produced embryos at the 2-cell, 4-cell, 8-cell and 16-cell stages were compared using transmission electron microscopy. These results showed that the three types of embryos had a similar tendency for mitochondrial change. Nevertheless, changes in GG embryos were more similar to changes in in vivo-produced embryos than were GC embryos, which had more extreme mitochondrial deviation. The results indicate the effects of the cytoplast on mitochondria development. The zona pellucida (ZP) in all three types of embryos became thinner and ZP pores in both GC and GG embryos showed an increased rate of development, especially for GC embryos, while in vivo-produced embryos had smooth ZP. The Golgi apparatus (Gi) and rough endoplasmic reticulum (RER) of the two reconstructed embryos became apparent at the 8-cell stage, as was found for in vivo embryos. The results showed that the excretion of reconstructed embryos was activated on time. Lipid droplets (LD) of GC and GG embryos became bigger, and congregated. In in vivo-produced embryos LD changed little in volume and dispersed gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere illuminated that the nucleus function of reconstructed embryos was partly changed. In addition, at a later stage in GC embryos the nuclear envelope displayed infoldings and the chromatin was concentrated, implying that the blastomeres had an obvious trend towards apoptosis. The gap junctions of the three types of embryos changed differently and GG and GC embryos had bigger perivitelline and intercellular spaces than did in vivo-produced embryos. These results are indicative of normal intercellular communication at an early stage, but this became weaker in later stages in reconstructed embryos. In conclusion, inter- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe destruction. These ultrastructural deviations might contribute to the compromised developmental potential of reconstructed embryos.

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Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

Reproduction Fertility and Development ◽

10.1071/rd03062 ◽

2004 ◽

Vol 16 (6) ◽

pp. 617 ◽

Cited By ~ 3

Author(s):

Genevieve M. Magarey ◽

Karen E. Mate

Keyword(s):

Glucose Metabolism ◽

Intracytoplasmic Sperm Injection ◽

Tammar Wallaby ◽

Blastocyst Stage ◽

In Vitro Fertilisation ◽

Cell Stage ◽

Developmental Potential ◽

Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

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253. Calcium ionophore induction of marmoset oocyte activation

Reproduction Fertility and Development ◽

10.1071/srb05abs253 ◽

2005 ◽

Vol 17 (9) ◽

pp. 101

Author(s):

C. G. Grupen ◽

R. B. Gilchrist ◽

S. J. Schulz ◽

L. J. Ritter ◽

D. T. Armstrong

Keyword(s):

Assisted Reproductive Technologies ◽

Cumulus Cell ◽

Calcium Ionophore ◽

Reproductive Technologies ◽

Primate Species ◽

Oocyte Activation ◽

Cell Stage ◽

Developmental Potential ◽

Parthenogenetic Activation

The marmoset monkey (Callithrix jacchus) is a valuable model for developing assisted reproductive technologies in humans and endangered primate species. Calcium ionophore treatments have been used to induce parthenogenetic activation in a number of species, but the effectiveness of this reagent in initiating marmoset embryo development has not yet been reported. The aim of this study was to determine the developmental potential of in vitro matured (IVM) marmoset oocytes, following treatment with calcium ionophore. Immature oocytes from large (LA; >1.5 mm) and small (SA; 0.67–1.5 mm) antral follicles were isolated from the ovaries of FSH-primed animals and cultured in modified G2 medium for 26–30 h at 37.0°C in 6% CO2 in air. Meiotically mature oocytes were sequentially incubated with 5 μM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h and cultured in G1/G2 sequential medium at 37.0°C in 5% O2, 6% CO2, 89% N2 for 10 days. Cumulus cell expansion associated with LA oocytes (n=118) was greater than that of SA oocytes (n=212), as determined using well established classification criteria (2.7±0.1 v. 1.8±0.2; P<0.01). A greater proportion of LA oocytes completed meiosis to the metaphase-II stage compared with SA oocytes (85±7% v. 63±7%; P<0.05). Pronuclear formation was induced at similar rates in mature oocytes of both groups, but the rate of cleavage was higher for LA oocytes compared with SA oocytes (93±6% v. 66±5%; P<0.05). The number of cells per embryo was not different between the groups.This is the first study to demonstrate that calcium ionophore effectively induces parthenogenetic activation in IVM marmoset oocytes. However, the development of parthenotes was limited beyond the 8-cell stage. Further studies are needed to determine the cause of the developmental block.

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Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos

Zygote ◽

10.1017/s0967199411000566 ◽

2011 ◽

Vol 21 (1) ◽

pp. 31-51 ◽

Cited By ~ 72

Author(s):

W.S. Abd El Naby ◽

T.H. Hagos ◽

M.M. Hossain ◽

D. Salilew-Wondim ◽

A.Y. Gad ◽

...

Keyword(s):

Expression Analysis ◽

Follicular Development ◽

Cumulus Cells ◽

Cell Types ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Bovine Oocyte ◽

Cell Stage ◽

Preimplantation Stage

SummaryMicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

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2 THE IN VIVO DEVELOPMENTAL POTENTIAL OF PORCINE SKIN-DERIVED PROGENITORS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab2 ◽

2012 ◽

Vol 24 (1) ◽

pp. 112 ◽

Cited By ~ 1

Author(s):

M. T. Zhao ◽

X. Yang ◽

K. Lee ◽

J. Mao ◽

J. M. Teson ◽

...

Keyword(s):

Stem Cells ◽

Cell Lineage ◽

Pcr Amplification ◽

Cell Types ◽

Blastocyst Stage ◽

Cell Stage ◽

Germ Layers ◽

Developmental Potential

Skin-derived progenitors (SKP) are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes and chondrocytes, thus exhibiting characteristics similar to embryonic neural crest stem cells. SKP show distinct transcriptional profiles when compared with neurospheres/neural stem cells in the central nervous system (CNS) and skin-derived fibroblasts, indicating a novel type of multipotent stem cell derived from the dermis of the skin. However, it remains unclear whether SKP cells can produce ectoderm and mesoderm lineages or other germ layers in vivo, although oocyte-like structures can be induced from porcine SKP in vitro. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus the purpose of this study was to investigate the in vivo developmental potential of porcine SKP by chimera production. Porcine SKP cells and fibroblasts were isolated from the back skin of Day 35 to 50 GFP transgenic fetuses. Individual cells or clusters of male GFP transgenic SKP and skin-derived GFP-expressing fibroblasts were injected into pre-compact in vitro-fertilized (IVF) embryos, respectively and then transferred into corresponding surrogates 24 h post-injection. Additional injected embryos were cultured in PZM3 medium for another 2 days until the blastocyst stage and subsequently stained with Hoechst 33342. Interestingly, in some of the chimeras the injected SKP cells migrated and dispersed into different locations of the host blastocysts, whereas in others they remained as a cluster of cells within the chimeric blastocysts. In contrast, the fibroblast cells were not observed to spread around the host blastocysts. Two chimeric fetuses were recovered at the middle of gestation and a litter of viable piglets was born. Genomic DNA was extracted from various tissues of chimeric piglets and subjected to PCR amplification. Two chimeric fetuses and 2 out of 6 piglets carried the GFP transgene in SKP-derived chimeras, but GFP was not present in the fibroblast-derived chimeric fetuses (n = 6). Surprisingly, the GFP transgene was present in various tissues of two SKP-derived chimeric piglets, including lung, heart, liver, artery, kidney, brain, skin, muscle, gut, ovary, pancreas and stomach, thus representing the 3 germ layers (ectoderm, mesoderm and endoderm). In addition, SRY was detected in several tissues of the two GFP-positive female chimeric piglets, confirming the chimerism of these piglets. Therefore, it appears that porcine SKP can contribute to various cell types of the 3 germ layers and have a broader developmental potency than previously expected. Alternatively, pre-compact (4-cell and 8-cell stage) embryos may provide a unique environment for reprogramming skin-derived progenitors into a more primitive state by the process of embryonic compaction. This study was funded by NIH National Center for Research Resources (R01RR013438) and Food for the 21st Century at the University of Missouri.

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Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes

Zygote ◽

10.1017/s0967199401001393 ◽

2001 ◽

Vol 9 (4) ◽

pp. 339-346 ◽

Cited By ~ 65

Author(s):

Liangxue Lai ◽

Qingyuan Sun ◽

Guangming Wu ◽

Clifton N. Murphy ◽

Birgit Kühholzer ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Fluorescent Protein ◽

Blastocyst Stage ◽

Cleavage Rate ◽

Cell Stage ◽

Transgenic Embryos ◽

Green Fluorescent ◽

Pronuclear Formation

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8±17.3% vs 28.5±3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0±7.0% vs 63.3±12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0±11.6% vs 4.6±4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

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Production of cloned mice using oocytes derived from ICR-outbred strain

Reproduction ◽

10.1530/rep-17-0372 ◽

2017 ◽

Vol 154 (6) ◽

pp. 859-866 ◽

Cited By ~ 2

Author(s):

Yoshiaki Tanabe ◽

Hiroki Kuwayama ◽

Sayaka Wakayama ◽

Hiroaki Nagatomo ◽

Masatoshi Ooga ◽

...

Keyword(s):

Mouse Strain ◽

Cumulus Cell ◽

Inbred Strains ◽

Blastocyst Stage ◽

Biological Research ◽

Mouse Strains ◽

Cell Nuclei ◽

Cell Stage ◽

Developmental Potential ◽

Icr Mouse

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR-outbred mouse strain. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

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