253. Calcium ionophore induction of marmoset oocyte activation

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.

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

2004 ◽  
Vol 16 (2) ◽  
pp. 141
Author(s):  
S. Eckardt ◽  
N.A. Leu ◽  
K.J. McLaughlin
Keyword(s):  
Early Stage ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Wild Type ◽  
Cell Stage ◽  
Developmental Potential ◽  
Preimplantation Stage ◽  
Transgenic Embryos ◽  
Clone Cells

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&lt;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.

scholarly journals Efficient marmoset genome engineering by autologous embryo transfer and CRISPR/Cas9 technology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yukiko Abe ◽  
Harumi Nakao ◽  
Motoki Goto ◽  
Moe Tamano ◽  
Michinori Koebis ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Genome Engineering ◽  
Assisted Reproductive Technologies ◽  
Small Body ◽  
Common Marmoset ◽  
Reproductive Technologies ◽  
Reproductive Capacity ◽  
Primate Species ◽  
Novel Method

AbstractGenetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for “Reduction” in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.

1 Assessing the energy status of porcine embryos by means of biodynamic imaging

2020 ◽  
Vol 32 (2) ◽  
pp. 125
Author(s):  
I. Lorenzo ◽  
Z. Li ◽  
M. Torres ◽  
Z. Machaty ◽  
D. Nolte
Keyword(s):  
Sodium Azide ◽  
Assisted Reproductive Technologies ◽  
Imaging System ◽  
Reproductive Technologies ◽  
Optical Measurements ◽  
Energy Status ◽  
Embryo Viability ◽  
Cell Stage ◽  
Developmental Potential ◽  
Bioluminescence Assay

Assisted reproductive technologies are powerful tools for enhancing production in livestock or treating infertility in humans. Unfortunately, the success rate of the technologies is rather low. A major reason for the poor efficiency is the lack of methods to reliably assess the developmental potential of the embryos before transfer into recipients. Therefore, a noninvasive method to ensure the selection of only the best embryos for transfer would be highly desirable. Biodynamic imaging is a compelling new microscopy that uses intracellular Doppler spectroscopy to perform label-free, noninvasive optical measurements of cellular fitness. The aim of this study was to investigate whether biodynamic imaging can be used to assess the energy status of the embryos, which may be indicative of their viability. Porcine oocytes matured invitro were parthenogenetically activated by an electrical pulse and cultured for 2 days. The parthenotes were then divided into two groups, and approximately half of them were incubated for an additional 2 days in the presence of 20mM sodium azide. Sodium azide is an inhibitor of oxidative phosphorylation and is known to block ATP production. The rest of the embryos were cultured without sodium azide and used as a control to indicate normal ATP levels. At the end of the culture period embryos that reached the 8- to 16-cell stage were evaluated by our biodynamic imaging system to assess their energy status, after which they were lysed and their ATP contents were determined by means of a bioluminescence assay. A total of 68 embryos (32 treated with the inhibitor and 36 control) were evaluated. The ATP content analysis showed that the control embryos had significantly more ATP than those treated with sodium azide as determined by Student's t-test (5.04±1.07 vs. 1.31±0.57; P&lt;0.05). A correlative study was then completed where biodynamic biomarkers were used to classify embryos to estimate the ability of biodynamic imaging to identify embryos with high or low energy status. A set of 13 biomarkers representing each embryo as a feature vector was used to train a classifier. We found that the cross-validated classifier had a sensitivity and specificity of ~80%. In addition, a receiver-operator curve constructed by varying the ATP threshold of the independent bioluminescence assay had an area-under-the-curve of 0.81. These results indicate that biodynamic imaging is able to determine the energy status of the embryos noninvasively and has great potential in the assessment of embryo viability.

scholarly journals The role of nitric oxide in parthenogenetic activation of pig oocytes: A review

2008 ◽  
Vol 52 (No. 11) ◽  
pp. 363-377 ◽  
Author(s):  
J. Petr ◽  
E. Chmelíková ◽  
L. Tůmová ◽  
M. Ješeta
Keyword(s):  
Nitric Oxide ◽  
Calcium Ionophore ◽  
Signalling Pathways ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Complex Processes ◽  
Parthenogenetic Development ◽  
Adequate Stimulus ◽  
Signalling Cascade

Parthenogenetic activation of mammalian oocytes with artificial stimuli is commonly applied in various reproductive biotechniques, e.g. cloning using nuclear transfer. For this reason, many studies focus on oocyte activation <I>in vitro</I>. Recently we have described the activation of pig oocytes using nitric oxide. This activating stimulus is very specific in many aspects. However, it does not provide an adequate stimulus for parthenogenetic development. It was shown that nitric oxide stimulated some signalling pathways which are inactive in conventional treatments for parthenogenetic activation, e.g. the cGMP-dependent signalling cascade. On the other hand, nitric oxide does not stimulate certain signalling pathways involved in oocyte activation after calcium ionophore, e.g. the PKC signalling cascade. The aim of this review is to characterize the complex processes induced in oocytes after treatment with nitric oxide. Perspectives for further research and the application of nitric oxide for parthenogenetic activation of oocytes are outlined.

scholarly journals Follicle-stimulating hormone mediates the consumption of serum-derived glycogen by bovine cumulus-oocyte complexes during in vitro maturation

2021 ◽  
pp. 2512-2517
Author(s):  
Ludymila F. Cantanhêde ◽  
Cristiane T. Santos-Silva ◽  
Marcelo T. Moura ◽  
José C. Ferreira-Silva ◽  
Júnior M. B. Oliveira ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Follicle Stimulating Hormone ◽  
Cumulus Cell ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Independent Manner ◽  
Oocyte Developmental Competence ◽  
Stimulating Hormone

Background and Aim: Oocyte in vitro maturation (IVM) is an appealing approach for several assisted reproductive technologies and dissecting oocyte maturation. Nonetheless, IVM leads to lower developmental competence and usually relies on undefined, serum-containing media. Therefore, biochemical profiling aimed to explore fluctuations in IVM media content during the acquisition of oocyte developmental competence. Materials and Methods: Bovine cumulus-oocyte complexes (COCs) underwent IVM in TCM199 medium with Earle's salts, supplemented with 2.0 mM L-glutamine, 10% fetal bovine serum, antibiotics, and 0.05 IU/mL porcine follicle-stimulating hormone (FSH+) or vehicle control (CTL) medium for 22 h. Results: FSH withdrawal (CTL) diminished several processes associated with the acquisition of oocyte developmental competence, such as reduced cumulus cell expansion, diminished estradiol synthesis (FSH+: 116.0±0.0 pg/mL vs. CTL: 97.6±18.0 pg/mL), and lower oocyte nuclear maturation rate (FSH+: 96.47% vs. CTL: 88.76%). Fresh media formulations (i.e., TCM199 with FSH or vehicle) were indistinguishable under biochemical profiling threshold conditions. Biochemical profiling showed similar total protein and lipid concentrations between groups. Further, total sugar concentrations diminished from fresh media to their post-IVM counterparts, albeit in an FSH-independent manner. Glycogen concentrations remained unaltered after IVM within CTL media, albeit were substantially lower after IVM under FSH+ conditions. Conclusion: FSH mediates the consumption of serum-derived glycogen by bovine COCs during IVM and implies that serum-free media should contain increased glucose concentrations to facilitate the acquisition of oocyte developmental competence.

36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 126
Author(s):  
V. Hall ◽  
D. Compton ◽  
P. Stojkovic ◽  
M. Nesbitt ◽  
M. Herbert ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Developmental Competence ◽  
Host Cells ◽  
Cell Stage ◽  
Double Labeling ◽  
Immunocytochemical Analysis ◽  
Parthenogenetic Activation

The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.

123 LONG-TERM PRODUCTION OF IN VITRO-MATURED PORCINE OOCYTES AND EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 161
Author(s):  
A. M. Paprocki ◽  
C. M. Syverson ◽  
R. W. Koppang ◽  
J. R. Dobrinsky
Keyword(s):  
Assisted Reproductive Technologies ◽  
Genetic Material ◽  
Reproductive Technologies ◽  
Scientific Data ◽  
Developmental Competence ◽  
Embryo Production ◽  
Pump System ◽  
Developmental Potential ◽  
Parthenogenetic Embryos

Although in vivo matured, ovulated, or both, oocytes provide the finest genetic material for use in assisted reproductive technologies (ART), their en masse production requires livestock production facilities, staff and associated overhead, is expensive and labor intensive, their harvest involves surgical or laparoscopic expertise, and large yields needed for en masse daily embryo production are cumbersome and very costly. In vitro-matured (IVM) oocytes have long been a practical gamete source for ART, including in vitro fertilization, ICSI and cloning. Rather than using conventional IVF to produce embryos, we employ in vitro oocyte activation for the production of diploidized parthenogenetic embryos, removing problems associated with variable embryo production due to polyspermic inseminations. In this way, we can produce a repeatable and consistent supply of mature oocytes, advanced embryos, or both, used in product testing, quality control, transgenic or cloned (or both) embryo production, in vitro development controls, as well as in-house culture control embryos for customer scientific data sharing. In this study, we observe mature oocyte and parthenogenetic embryo production over a complete year as control information for our laboratory. Additionally, colleagues may use these data for comparison in their own scientific mission. At least 3 times a month for 12 consecutive months, ovaries were collected from mature females at an abattoir and transported to our laboratory. Cumulus–oocyte complexes were aspirated from 4–6 mm follicles with an 18-gauge needle fixed to a vacuum pump system. Only COC surrounded by two or more layers of compact cumulus investment and containing oocytes of equal size were placed into a commercial TCM-199-based IVM system (Minitube of America Inc., Verona, WI, USA). After 42 h IVM, mature oocytes were isolated from their expanded cumulus and subjected to chemical (ionomycin/DMAP) parthenogenetic activation based on US Patent 5,496,720. Embryos were cultured 120 h in NCSU-23, then cultured for an additional 48 h in NCSU-23 (no BSA) supplemented with 10% FBS. A minimum of 1504 premium and 4604 standard oocytes (Minitube of America Inc.) were placed into IVM. Both premium (1364, 90.7%) and standard (4061, 88.2%; P > 0.05) oocytes are used to produce mature oocytes (MO). Of 781 premium MOs made into diploidized parthenogenetic embryos, 459 (58.8%) developed into blastocysts (61.3 cells/embryo). Of 2068 standard MO made into diploidized parthenogenetic embryos, 914 (44.2%; P < 0.05) developed into blastocysts (64.7 cells/embryo). En masse in vitro maturation of oocytes can supply a repeatable and consistent supply of mature oocytes for use in assisted reproductive technologies. These MO have the developmental potential to form blastocysts in vitro and enable scientists to infer developmental competence of in vitro-produced embryos for research and commercial use.

127 L-Carnitine Supplementation During In Vitro Maturation Improves Developmental Competence of Canine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 203 ◽  
Author(s):  
A. Salama ◽  
M. Fathi ◽  
M. R. Badr ◽  
A. R. Moawad
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Mammalian Species ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Cell Stage ◽  
Nuclear Maturation

In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL−1) or without LC supplements (0 mg mL−1; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL−1 LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL−1 LC supplemented group than the 0.3 mg mL−1 supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL−1 LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL−1 supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.

scholarly journals Transcriptional profiling by RNA-Seq of peri-attachment porcine embryos generated by a variety of assisted reproductive technologies

2013 ◽  
Vol 45 (14) ◽  
pp. 577-589 ◽  
Author(s):  
S. Clay Isom ◽  
John R. Stevens ◽  
Rongfeng Li ◽  
William G. Spollen ◽  
Lindsay Cox ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Transforming Growth Factor ◽  
Assisted Reproductive Technologies ◽  
Expression Profiles ◽  
Reproductive Technologies ◽  
Fertilization In Vitro ◽  
Oocyte Activation ◽  
Attachment Development

Substantial mortality of in vitro manipulated porcine embryos is observed during peri-attachment development. Herein we describe our efforts to characterize the transcriptomes of embryonic disc (ED) and trophectoderm (TE) cells from porcine embryos derived from in vivo fertilization, in vitro fertilization (IVF), parthenogenetic oocyte activation (PA), and somatic cell nuclear transfer (SCNT) on days 10, 12, and 14 of gestation. The IVF, PA, and SCNT embryos were generated with in vitro matured oocytes and were cultured overnight in vitro before being transferred to recipient females. Sequencing of cDNA from the resulting embryonic samples was accomplished with the Genome Analyzer IIx platform from Illumina. Reads were aligned to a custom-built swine transcriptome. A generalized linear model was fit for ED and TE samples separately, accounting for embryo type, gestation day, and their interaction. Those genes with significant differences between embryo types were characterized in terms of gene ontologies and KEGG pathways. Transforming growth factor-β signaling was downregulated in the EDs of IVF embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signaling were aberrantly regulated. Expression of genes involved in chromatin modification, gene silencing by RNA, and apoptosis was significantly disrupted in ED cells from SCNT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various embryo types during peri-attachment development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from assisted reproductive technologies.

scholarly journals Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

2017 ◽  
Vol 114 (29) ◽  
pp. E5796-E5804 ◽  
Author(s):  
Ye Yuan ◽  
Lee D. Spate ◽  
Bethany K. Redel ◽  
Yuchen Tian ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Fourfold Increase

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called “FLI medium,” improves nuclear maturation of oocytes in cumulus–oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus–oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

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