Cryopreservation of equine oocytes: looking into the crystal ball

2020 ◽  
Vol 32 (5) ◽  
pp. 453 ◽  
Author(s):  
Tine De Coster ◽  
Daniel Angel Velez ◽  
Ann Van Soom ◽  
Henri Woelders ◽  
Katrien Smits
Keyword(s):  
Cumulus Cells ◽  
Short Exposure ◽  
Biological Factors ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Crystal Ball ◽  
Immature Oocytes ◽  
Cryoprotective Agents ◽  
The Past ◽  
Chromosome Alignment

Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

123 BOVINE OOCYTE VITRIFICATION USING THE CRYOTOP METHOD: EFFECT OF CUMULUS CELLS AND VITRIFICATION PROTOCOL ON SURVIVAL AND SUBSEQUENT DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 220
Author(s):  
X. L. Zhou ◽  
A. Al Naib ◽  
D. W. Sun ◽  
P. Lonergan
Keyword(s):  
Subsequent Development ◽  
Cumulus Cells ◽  
Theoretical Modelling ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Permeability Characteristics ◽  
Survival And Development ◽  
Blastocyst Rate ◽  
Improve Rate ◽  
Bovine Oocytes

The ability to successfully cryopreserve mammalian oocytes has numerous practical and economic ethical benefits that may positively affect animal breeding programs and assisted conception in humans. However, oocyte survival and development following cryopreservation remain poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocol, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. Bovine oocytes were collected from the ovaries of slaughtered cross-bred beef heifers. In Experiment 1, cumulus-enclosed and partially denuded GV and MII oocytes were vitrified in 15% EG + 15% DMSO + 0.5 M sucrose in 2 steps. In Experiment 2, GV oocytes were vitrified as above or using theoretical modelling based on permeability and osmotic tolerance characteristics (Wang et al. Reprod. Fertil. Dev. 21 141) in 30% EG +11.4% trehalose in 3 steps or 40% EG + 11.4% trehalose in 4 steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA, USA) 1% X-1000, 1% Z-1000, or both in 3 steps. The survival, cleavage, and blastocyst rate of cumulus-enclosed oocytes was significantly higher (ANOVA) than those of partially denuded oocytes when vitrified at GV (93.8% v. 81.3%, 65.8% v. 47.3%, 11.3% v. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% v. 91.8%, 35.2% v. 36.8%, 5.0% v. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher development competence than those vitrified at MII (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage, and blastocyst rates among 3 protocols (86.0% v. 92.8% v. 91.2%, 44.8% v. 54.4% v. 45.6%, 5.0% v. 5.4% v. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P < 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols.

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
G Palermo
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Time Lapse Imaging

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P &lt; 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P &lt; 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P &lt; 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P &lt; 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P &lt; 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

scholarly journals 276 THE EFFECT OF CUMULUS CELLS DURING MATURATION ON THE RISE IN THE CONCENTRATION OF INTRACELLULAR Ca2+ ([Ca2+]i) OF PORCINE OOCYTES INDUCEDBY INOSITOL 1,4,5-TRISPHOSPHATE

2005 ◽  
Vol 17 (2) ◽  
pp. 288
Author(s):  
T. Amano ◽  
T. Mori ◽  
K. Matsumoto ◽  
T. Watanabe ◽  
A. Iritani
Keyword(s):  
Cumulus Cells ◽  
Immature Oocytes ◽  
R Ratio ◽  
Transient Rise ◽  
Activation Rate ◽  
Sperm Penetration ◽  
Cytoplasmic Maturation ◽  
Student’S T ◽  
Rate Of Activation

Increase of inositol 1,4,5-triphosphate (IP3) in the cytoplasm of mammalian oocytes is said to be responsible for [Ca2+]i oscillation observed in the oocytes immediately after sperm penetration, and the [Ca2+]i oscillation is known to be essential for the development of embryos. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of oocytes and affecting the embryonic development. To obtain more information about the role of cumulus cells in cytoplasmic maturation, the effects of cumulus cells during maturation on the rise in [Ca2+]i and on the rate of activation of porcine mature oocytes induced by IP3 injection were investigated. The immature porcine oocytes were divided into three groups: COCs (intact cumulus-oocyte complexes), DOs (oocytes denuded of their cumulus cells), Co-culture (DOs attached to separated cumulus cells). These groups of immature oocytes were cultured in NCSU23 46 h for maturation. To examine the function of cumulus cells, two groups of immature oocytes were also prepared: DOs + pyruvate (DOs put into NCSU23 with pyruvate) and COCs-glucose free (COCs put into NCSU23 without glucose). The mature oocytes from each group were loaded with Ca2+ indicator fluorescent dye Fura2-AM, and then were irradiated by 340 nm and 360 nm of ultraviolet immediately after the injection of IP3. The intensities of emission light caused by the irradiation of 340 nm and 360 nm ultraviolet were recorded as E340 and E360. Since coupling of Ca2+ and the dye intensifies E340, but does not change E360, the level of [Ca2+]i was shown as R (ratio = E340/E360) in this study. Activation rate was calculated by counting the number of the oocytes that formed pronuclei by injection of IP3. ANOVA and Student's t-test were used in this study. Transient rise in [Ca2+]i was observed in the mature oocytes from every group. The peak R of the rise in [Ca2+]i of the mature oocytes derived from COCs, Dos, and Co-culture and induced by IP3 were 7.2, 4.0, and 6.9, respectively. The R of DOs was significantly lower than those of the others (P < 0.05). Also, the activation rate of the mature oocytes from DOs was significantly lower than those from COCs and Co-culture (31, 66, and 66%). The mature oocytes from DOs + pyruvate showed the same level of peak R compared with those from COCs (7.4 and 6.3), but COCs-glucose free showed a slight but significantly lower peak R compared with the mature oocytes from COCs (6.0 and 7.4, P < 0.05). In conclusion, cumulus cells appeared to support the rise in [Ca2+]i of porcine oocytes induced by IP3 during maturation and the following activation. Moreover, a function of cumulus cells supposedly produces pyruvate by metabolizing glucose and provides it to oocytes during maturation for promoting the cytoplasmic maturation. A part of this study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

scholarly journals Treating Complex Nervous System Vascular Disorders through a “Needle Stick”: Origins, Evolution, and Future of Neuroendovascular Therapy

Neurosurgery ◽  
2001 ◽  
Vol 48 (3) ◽  
pp. 463-475 ◽  
Author(s):  
L. Nelson Hopkins ◽  
Giuseppe Lanzino ◽  
Lee R. Guterman
Keyword(s):  
Gene Therapy ◽  
Central Nervous System ◽  
Nervous System ◽  
Molecular Basis ◽  
Nervous System Diseases ◽  
Central Nervous System Diseases ◽  
Biological Factors ◽  
Device Development ◽  
The Past ◽  
Established Treatment

Abstract IN THE PAST few decades, dramatic improvements have occurred in the field of neuroendovascular surgery. Endovascular therapy today is a well-established treatment modality for a variety of cerebrovascular and nonvascular central nervous system diseases. The foundation of this spectacular evolution was laid by the efforts of pioneering visionaries who often worked alone and under difficult, almost impossible, conditions. Ongoing device development and refinement have revolutionized the field at a dizzying, exhilarating pace. With a better understanding of the molecular basis of diseases and further advancements in gene therapy, neuroendovascular techniques have an enormous potential for application to the entire spectrum of central nervous system diseases as a minimally invasive vehicle for the delivery of biological factors.

Personality research—challenges for the future

1990 ◽  
Vol 4 (1) ◽  
pp. 1-17 ◽  
Author(s):  
David Magnusson
Keyword(s):  
Individual Development ◽  
Psychological Processes ◽  
Biological Factors ◽  
Methodological Issues ◽  
Personality Psychology ◽  
Psychological Phenomena ◽  
The Past ◽  
Personality Research ◽  
Individual Functioning ◽  
Emotional System

This article is about the lessons that can be learned from the mistakes of the past. After a critical, constructive analysis of current theorizing and research, important directions of future personality psychology are described against the background of a general theoretical framework. It is argued that individual functioning cannot be understood or explained if the environmental factors that are operating in the individual's interactions with the environment and the biological factors that are constantly interacting with the cognitive‐emotional system are not considered. Finally, the article focuses on conceptual and methodological issues that are of major importance for further progress in personality psychology, viz. (a) the match between level of psychological processes and type of data, (b) the nature of psychological phenomena studied in terms of variables, (c) the use of chronological age as the marker of individual development, and (d) the comparison between a variable and a person approach.

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 195 ◽  
Author(s):  
R. R. Payton ◽  
L. A. Rispoli ◽  
J. L. Edwards
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Heat Stress ◽  
Empirical Bayes ◽  
Rna Extraction ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Extracellular Region

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

IVM of mouse fully grown germinal vesicle oocytes upon a feeder layer of selected cumulus cells enhances their developmental competence

2019 ◽  
Vol 31 (6) ◽  
pp. 1068
Author(s):  
Federica Cavalera ◽  
Milena Simovic ◽  
Mario Zanoni ◽  
Valeria Merico ◽  
Silvia Garagna ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Development Rate ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Developmental Competence ◽  
Mouse Oocytes ◽  
Feeder Layers ◽  
Morula Stage ◽  
Bidirectional Exchange

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus–oocyte–complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.

scholarly journals Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 221-228 ◽  
Author(s):  
G-P Li ◽  
K L White ◽  
K I Aston ◽  
L N Meerdo ◽  
T D Bunch
Keyword(s):  
Conditioned Medium ◽  
Bovine Serum ◽  
Chromosomal Abnormalities ◽  
Cumulus Cell ◽  
Chromosome Complement ◽  
Cumulus Cells ◽  
Conditioned Media ◽  
Polyploid Cell ◽  
Chromosomal Anomalies ◽  
Blastocyst Development

The effects of bovine cumulus cell-conditioned medium on cloned bovine embryonic development and subsequent chromosome complement were examined using an air-dry procedure. Conditioned media were prepared using CR1aa supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). Nuclear-transferred embryos were reconstructed with nuclei from cumulus cells. Similar cleavage, morula, and blastocyst development was observed in conditioned media groups compared with the co-culture group. No differences (P > 0.05) were observed in the composition of blastocyst chromosomes after co-culture in different media, either with or without starvation of donor cells. The overall diploid blastocyst rate ranged from 75% to 84%. Chromosomal complement of blastocysts, however, was very different between conditioned medium and co-culture treatments. Overall incidence of chromosomal anomalies was 40% in conditioned medium, which was significantly higher (P < 0.001) than the co-culture group (20%). Moreover, a higher incidence (P < 0.05) of chromosomally abnormal blastocysts (41.5%) was observed after culture with FBS-containing conditioned medium than those cultured in BSA-containing conditioned medium (31.4%). No diploid improvement was observed after exchange of the culture system from conditioned medium to co-culture, or from co-culture to conditioned medium after the first 72 h of culture. The results of this study also indicated that the overall cell number was much lower (P < 0.01) in blastocysts with chromosomal abnormalities than those with a normal diploid state. We have concluded that medium conditioned with bovine cumulus cells increases the incidence of chromosomal anomalies in nuclear reconstructed embryos.

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