scholarly journals 156 ENHANCED BOVINE EMBRYONIC DEVELOPMENT AFTER MICROFLUIDIC CUMULUS CELL REMOVAL POST-FERTILIZATION

2005 ◽  
Vol 17 (2) ◽  
pp. 228
Author(s):  
A. Reeder ◽  
R. Monson ◽  
D. Beebe ◽  
B. Lindsey ◽  
J. Rutledge
Keyword(s):  
Laminar Flow ◽  
Embryonic Development ◽  
Cell Biology ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Fertilization Rates ◽  
Flow Profiles ◽  
Kinetics Of ◽  
Bovine Blastocyst

Microfluidic technologies are increasingly being used in cell biology and embryology research. In order to manipulate an embryonic environment microfluidics take advantage of miniscule media amounts. With the use of pressure heads and laminar flow profiles, a presumptive zygote can be gently manipulated in a microfluidic device for removal of the supporting cumulus cells post-fertilization. Presumptive embryos were assigned at random to three cumulus removal treatments at 48 h post-fertilization: vortexing (3 min), handstripping (with 135-μm-ID stripping pipette), and microfluidics. Blastocyst rates were determined through Day 8 post-fertilization. Rates were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA), accounting for replicates and treatment. Kinetics of development were also impacted as larger proportions of embryos in the microfluidic group reached the blastocyst stage before embryos of the vortex or hand-stripping treatments. These data suggest that cumulus cell removal in a gentle fashion is associated with enhanced embryonic development in the bovine. Table 1. Comparison of cumulus cell removal techniques on bovine blastocyst rates

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

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

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 &gt; 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

scholarly journals Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 421-437 ◽  
Author(s):  
Pouneh Maraghechi ◽  
László Hiripi ◽  
Gábor Tóth ◽  
Babett Bontovics ◽  
Zsuzsanna Bősze ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Expression Level ◽  
Preimplantation Embryos ◽  
Regulatory Function ◽  
Low Level ◽  
Non Coding Rnas

MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3′UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

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

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
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.

195 SUPPRESSION OF SETDB1 DURING BOVINE EMBRYONIC DEVELOPMENT RESULTS IN PRE-IMPLANTATION MORTALITY AND DECREASED TRANSCRIPTION OF TRANSPOSABLE ELEMENTS

2015 ◽  
Vol 27 (1) ◽  
pp. 188
Author(s):  
M. D. Snyder ◽  
J. H. Pryor ◽  
K. J. Veazey ◽  
M. D. Peoples ◽  
G. L. Williamson ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Transposable Elements ◽  
Fluorescent Microscopy ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Treatment Groups ◽  
Commercial Supplier ◽  
Morula Stage ◽  
Student’S T

Organization of chromatin structure by the combinatorial patterns of DNA methylation and post-translational histone modification is essential for the establishment and maintenance of proper transcriptional programs that result in the coordination of embryonic development. We previously observed that suppression of transcripts encoding SET domain, bifurcated 1 (SETDB1) using small interfering RNAs (siRNA) is embryonic lethal, with SETDB1-suppressed embryos (n = 361) arresting immediately before the blastocyst stage (blastocyst rate: Control 44.9 ± 4.9% and NULL injected 25.7 ± 6.0%). Studies in rodents indicate SETDB1 is a crucial regulator of transposable elements and that the precise epigenetic regulation of these elements is a key aspect of transcriptional programs controlling pluripotency and placentation. To better characterise the molecular basis of the observed mortality, we analysed expression of the bovine Long Interspersed Nuclear Element 1 family (LINE1) of transposable elements via quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). Mature bovine oocytes were obtained from a commercial supplier (De Soto Biosciences, Seymour, TN, USA) and IVF performed by standard laboratory protocol. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes divided into 3 different treatment groups: non-injected control (CNTL), non-targeting siRNA injected control (siNULL), and zygotes injected with siRNAs targeting SETDB1 (siSETDB1). Each embryo was injected with ~100 pL of siRNAs (10 µM) in fluorescent dextran solution. All zygotes were verified as injected by fluorescent microscopy and then cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) medium supplemented with 4 mg mL of BSA (Probumin, EMD Millipore, Darmstadt, Germany). Groups of embryos (15–20) from each treatment were lysed at the 4-cell, 8-cell, and morula stages, RNA extracted, and analysed by RT-qPCR using GAPDH and YWHAZ as reference genes. A two-way ANOVA and a Student's t-test were used to analyse the results from the RT-qPCR. As expected, siSETDB1-injected morulae displayed dramatic reduction in the level of Setdb1 transcripts as compared to siNULL control (96%; P < 0.05). Preliminary analysis of LINE1 transcripts at the morula stage indicated siSETDB1-injected embryos displayed a 75% reduction compared to the siNULL. Whether alteration in LINE1 regulation contributes to the developmental arrest and embryonic mortality of siSETDB1-injected embryos is under investigation.

scholarly journals Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 425-435 ◽  
Author(s):  
Radek Procházka ◽  
Michal Petlach ◽  
Eva Nagyová ◽  
Lucie Němcová
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Human Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Epidermal Growth

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus–oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competencein vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression ofAREGandEREGreached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2,TNFAIP6, andHAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression ofCYP11A1in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
F. Forell ◽  
C. Feltrin ◽  
L. C. Santos ◽  
A. D. Vieira ◽  
U. M. Costa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Technological Development ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
National Council ◽  
Immature Oocytes ◽  
Oocyte Recovery

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

scholarly journals MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

2015 ◽  
Vol 308 (6) ◽  
pp. E525-E534 ◽  
Author(s):  
Bo Pan ◽  
Derek Toms ◽  
Wei Shen ◽  
Julang Li
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Specific Effect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
And Function

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion ( HAS2, PTGS2) and oocyte maturation ( CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), zona pellucida 3 ( ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

scholarly journals Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Jason R Herrick ◽  
Amber M Brad ◽  
Rebecca L Krisher
Keyword(s):  
Embryonic Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Pentose Phosphate ◽  
The Other ◽  
Nuclear Maturation ◽  
Maturation In Vitro ◽  
Intracellular Content ◽  
Cytoplasmic Maturation

The objectives of this study were to manipulate metabolism of glucose through glycolysis and the pentose phosphate pathway (PPP) in porcine oocytes during in vitro maturation, and determine the effects of this manipulation on meiotic progression, intracellular glutathione (GSX) concentrations and embryonic development. Cumulus-oocyte complexes isolated from abattoir ovaries were matured (40–44 h) in Purdue Porcine Medium for maturation alone (control) or supplemented with pyrroline-5 carboxylate (PC, 0.1 μM; PPP stimulator), diphenyleneiodonium (DPI, 0.1 μM; PPP inhibitor), dinitrophenol (DNP, 10 μM; glycolytic stimulator), hexametaphosphate (HMP, 100 μM; glycolytic inhibitor), PC + HMP or DNP + DPI. At the conclusion of in vitro maturation, cumulus cells were removed and oocytes were randomly allocated for analysis of GSX, metabolism and nuclear maturation, or in vitro fertilization and embryo culture. Both DPI and DNP + DPI decreased (P ≤ 0.05) the activity of glycolysis and the PPP, increased (P ≤ 0.05) the percentage of immature oocytes, and decreased (P ≤ 0.05) the proportion of mature oocytes compared with control oocytes and oocytes from the other treatments. Embryonic development (cleavage and blastocyst stage) and the intracellular content of GSX were also decreased (P ≤ 0.05) following exposure to DPI or DNP + DPI compared with control oocytes and oocytes from the other treatments. Oocyte metabolism, nuclear maturation, GSX content and embryonic development were unaffected (P > 0.05) following exposure to PC, DNP, HMP or PC + HMP. Our results suggest that metabolism of glucose through the PPP and/or glycolysis plays a key role in the control of nuclear and cytoplasmic maturation of porcine oocytes in vitro.

254 REGULATORY microRNA IN THE BIDIRECTIONAL COMMUNICATION OF BOVINE OOCYTES AND THE SURROUNDING CUMULUS CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 284
Author(s):  
D. Tesfaye ◽  
W. S. Abd El Naby ◽  
M. D. Hossain ◽  
A. Gad ◽  
D. Salilew-Wondim ◽  
...  
Keyword(s):  
Small Rna ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Cell Group ◽  
Cell Stage ◽  
Bidirectional Communication ◽  
Major Genome ◽  
System Data

MicroRNA (miRNA) are small molecules (˜22 nucleotide in length) that influence the expression of hundreds of genes for numerous biological processes including development. In this study we aimed to investigate the presence and role of miRNA in the bidirectional communication of oocyte and cumulus cells. For this, triplicate pools each containing 1600 immature and mature oocytes and their corresponding cumulus cells were used for miRNA isolation using miRNeasy® Mini Kit (Qiagen, Hilden, Germany). From each oocyte and cumulus cell group, 50 ng of small RNA was used for reverse transcription using RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD, USA). The resulting small RNA cDNA was used as a template to profile 88 human miRNA related to cell development and differentiation using SYBR green-based real-time PCR system. Data analysis was preformed using the comparative Ct method after normalization using endogenous control RNA (SNORD44, SNORD47, SNORD48, and U6). In addition, miR-205 and miR-210 were used for localization in pre-implantation stages of embryo using 3′digoxigenin labeled, LNA- modified in situ oligonucleotide probes (Exiqon, Vedbaek, Denmark). The result of the PCR array revealed a total of 34 and 49 miRNA to be greatly abundant in immature and mature oocyte, respectively, compared with the corresponding cumulus cells, whereas only 5 and 4 miRNA were enriched in cumulus cells compared with immature and matured oocytes, respectively. Based on expression intensity, 6 oocyte enriched (miR-205, miR-150, miR-96, miR-122, miR-146a, and miR-146b-5p) and 2 cumulus-cell enriched (miR-452 and miR-210) were selected for expression analysis in pre-implantation-stage embryos and in oocyte and cumulus cells matured with or without cumulus and oocyte factors, respectively. All oocyte-specific miRNA were found to be greatly abundant in early stages of embryo development and drop after 4-cells until the blastocyst stage, following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in pre-implantation-stage embryos, in which signals were greater until the 4-cell stage and reduced thereafter. However, miR-210 and miR-452 showed no defined profile. miR-205, miR-150, miR-122, miR-146a, miR-146b-5p, and miR-452 were found to be abundant at a greater level (P < 0.05) in oocytes matured without cumulus cells compared with those matured in the presence of cumulus cells. The expression of miR-205, miR-150, and miR-122 in cumulus cells was greater in the presence of oocyte cytoplasm during maturation, whereas 16-fold increases in relative abundance of miR-210 were observed in oocyte- optimized cumulus cells. These results evidenced that oocyte and cumulus cells have a distinct set of miRNA, which is dependent on the bidirectional communication of the oocyte and the surrounding cumulus cells. Moreover, maternal miRNA were found to persist until the major genome activation in bovine.

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