Single in vitro bovine embryo production: Coculture with autologous cumulus cells, developmental competence, embryo quality and gene expression profiles

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30–40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60–70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

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Comparative Gene Expression Profiling in Human Cumulus Cells according to Ovarian Gonadotropin Treatments

BioMed Research International ◽

10.1155/2013/354582 ◽

2013 ◽

Vol 2013 ◽

pp. 1-13 ◽

Cited By ~ 21

Author(s):

Said Assou ◽

Delphine Haouzi ◽

Hervé Dechaud ◽

Anna Gala ◽

Alice Ferrières ◽

...

Keyword(s):

Gene Expression ◽

Embryo Quality ◽

Gnrh Antagonist ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cumulus Cells ◽

Periovulatory Follicles ◽

Compare Gene Expression ◽

Day 3 Embryo

Inin vitrofertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship within vitroembryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1andCOL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1RandGM2Afor HP-hMG;GREM1andOSBPL6for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2andPTX3) were related toin vitroembryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers ofin vitroembryo quality.

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Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes

Reproduction Fertility and Development ◽

10.1071/rd14086 ◽

2016 ◽

Vol 28 (3) ◽

pp. 278 ◽

Cited By ~ 6

Author(s):

Su-Jin Cho ◽

Kyeong-Lim Lee ◽

Yu-Gon Kim ◽

Dong-Hoon Kim ◽

Jae-Gyu Yoo ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cumulus Cells ◽

Nuclear Maturation ◽

Differential Gene ◽

Monitoring Gene Expression ◽

Follicle Aspiration

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus–oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus–oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P < 0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

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Gene expression profiles in mouse cumulus cells derived from in vitro matured oocytes with and without blastocyst formation

Gene Expression Patterns ◽

10.1016/j.gep.2017.05.002 ◽

2017 ◽

Vol 25-26 ◽

pp. 46-58 ◽

Cited By ~ 4

Author(s):

Yixin Xu ◽

Tuanping Zhou ◽

Li Shao ◽

Bei Zhang ◽

Kailu Liu ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cumulus Cells ◽

Blastocyst Formation

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Expression profiles of select genes in cumulus–oocyte complexes from young and aged mares

Reproduction Fertility and Development ◽

10.1071/rd14446 ◽

2015 ◽

Vol 27 (6) ◽

pp. 914 ◽

Cited By ~ 6

Author(s):

Lindsay Cox ◽

Dirk K. Vanderwall ◽

Kate C. Parkinson ◽

Alexis Sweat ◽

S. Clay Isom

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cumulus Cells ◽

Developmental Competence ◽

Differentially Expressed ◽

Initiation Factor ◽

Translational Initiation ◽

Human Oocytes ◽

Age Related

There is compelling evidence that oocytes from mares >18 years of age have a high incidence of inherent defects that result in early embryonic loss. In women, an age-related decrease in oocyte quality is associated with an increased incidence of aneuploidy and it has recently been determined that the gene expression profile of human oocytes is altered with advancing age. We hypothesised that similar age-related aberrations in gene expression occur in equine oocytes. Therefore, the aim of the present study was to compare gene expression profiles of individual oocytes and cumulus cells from young and aged mares, specifically evaluating genes that have been identified as being differentially expressed with advancing maternal age and/or aneuploidy in human oocytes. Expression of 48 genes was compared between 14 cumulus–oocyte complexes (COCs) from mares aged 3–12 years and 10 COCs from mares ≥18 years of age. Three genes (mitochondrial translational initiation factor 3 (IF3), heat shock transcription factor 5 (HSF5) and Y box binding protein 2 (YBX2)) were differentially expressed in oocytes, with all being more abundant in oocytes from young mares. Three genes (ADP-ribosylation factor-like 6 interacting protein 6 (ARL6IP6), BCL2-associated X protein (BAX) and hypoxia upregulated 1 (HYOU1)) were differentially expressed in cumulus cells, with all being more abundant in aged mares. The results of the present study confirm there are age-related differences in gene expression in equine COCs, which may be associated with the lower quality and decreased developmental competence of oocytes from aged mares.

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Oral propylene glycol modifies follicular fluid and gene expression profiles in cumulus–oocyte complexes and embryos in feed-restricted heifers

Reproduction Fertility and Development ◽

10.1071/rd17037 ◽

2018 ◽

Vol 30 (3) ◽

pp. 417 ◽

Cited By ~ 2

Author(s):

G. Gamarra ◽

C. Ponsart ◽

S. Lacaze ◽

F. Nuttinck ◽

A. Cordova ◽

...

Keyword(s):

Gene Expression ◽

Propylene Glycol ◽

Follicular Fluid ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Control Group ◽

In Vitro Production

Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400 mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.

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Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality

Zygote ◽

10.1017/s0967199413000658 ◽

2014 ◽

Vol 23 (3) ◽

pp. 367-377 ◽

Cited By ~ 14

Author(s):

Sandra Milena Bernal ◽

Julia Heinzmann ◽

Doris Herrmann ◽

Bernd Timmermann ◽

Ulrich Baulain ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Expression Profiles ◽

Expression Patterns ◽

Developmental Competence ◽

Bovine Embryo ◽

Global Methylation ◽

Oocyte Developmental Competence

SummaryCyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

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163 EFFECTS OF DIFFERENT IN VITRO MATURATION SYSTEMS ON EMBRYO DEVELOPMENT IN BOVINE PREPUBERTAL AND ADULT DONORS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab163 ◽

2014 ◽

Vol 26 (1) ◽

pp. 195

Author(s):

S. M. Bernal ◽

J. Heinzmann ◽

D. Herrmann ◽

U. Baulain ◽

A. Lucas-Hahn ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

In Vitro Maturation ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Developmental Competence ◽

In Vitro Production ◽

Lactating Cows

Prepubertal bovine females have been suggested as a source of oocytes in order to accelerate genetic gain and decrease the generation interval. However, prepubertal oocytes have a lower developmental competence than their adult counterparts. In vitro maturation (IVM) systems using cyclic AMP (cAMP) regulators and 30-h culture have been suggested to improve blastocyst in vitro production rates from bovine oocytes (Albuz et al., 2010). The present study evaluated the effects of an addition of the cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX), and cilostamide during extended IVM on blastocyst yields and gene expression in prepubertal and adult bovine females. Holstein-Friesian donors were submitted to ovum pick-up twice per week. Oocytes from groups of 12 animals, including lactating cows (>2 lactations) and prepubertal donors (6–10 months old) were used in the following treatment groups: TCM24 (24-h IVM, routine protocol/control), cAMP30 (2-h pre-IVM culture using forskolin-IBMX and 30-h IVM adding cilostamide), DMSO30 [2-h pre-IVM culture and 30-h IVM with dimethyl sulfoxide (DMSO)/vehicle control]. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. In vivo blastocysts were produced from superovulated cows and used for gene expression analysis. Cleavage rates, blastocyst formation, and mRNA abundance of selected genes were evaluated. The Glimmix procedure from SAS/STAT (SAS Institute Inc., Cary, NC, USA) was performed to compare blastocyst and cleavage rates. One-way ANOVA was implemented to evaluate gene expression. A total of 793 oocytes from the different sources were submitted to the IVM treatments. Cleavage rates (prepubertal donors: 64.6 ± 4%, 59.1 ± 6.4%, 53 ± 4.4%, cows: 55.1 ± 4.3%, 59 ± 6.5%, 50.8 ± 4.4%, for TCM24, cAMP30, and DMSO30, respectively; P > 0.05) and blastocyst/zygotes rates (prepubertal donors: 27 ± 6%; 21.8 ± 3.5%; 17.6 ± 2.4%; cows: 28 ± 3.3%; 27.7 ± 2.9%; 22.7 ± 3.2% for TCM24, cAMP30, and DMSO30, respectively; P > 0.05) did not differ among in vitro treatments. The mRNA relative abundance of the EGR1 gene was down-regulated 6-fold in all in vitro-produced blastocysts compared with their in vivo counterparts (P < 0.05). Gene expression profiles for SLC2A8, DNMT3B, BCL-XL, and PRDX1 were similar in in vitro and in vivo blastocysts. These results show similar embryo production patterns in prepubertal and adult donors. Furthermore, DMSO did not show effects on embryo developmental rates when used during IVM. The gene expression levels of EGR1 confirm our recent findings in blastocysts obtained from oocytes from slaughterhouse ovaries (data not presented), showing its usefulness as an embryo quality marker. These preliminary results indicate that oocyte developmental capacity in prepubertal donors can be similar to that of the adult donors without addition of cAMP modulators.

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Cumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes

Reproduction Fertility and Development ◽

10.1071/rd13061 ◽

2014 ◽

Vol 26 (6) ◽

pp. 855 ◽

Cited By ~ 25

Author(s):

A. Bunel ◽

A. L. Nivet ◽

P. Blondin ◽

C. Vigneault ◽

F. J. Richard ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Cumulus Cell ◽

Early Period ◽

Gene Expression Profiles ◽

Oocyte Quality ◽

Developmental Competence ◽

Qrt Pcr ◽

Blastocyst Rate

The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus–oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n = 3 cows, 12 oocyte collections) and qRT-PCR (n = 3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process.

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227 CONSTRUCTION OF STAGE-SPECIFIC cDNA MICROARRAY, AND ANALYSIS OF IN VITRO PRODUCED PRE-IMPLANATION STAGE BOVINE EMBRYOS FOR DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab227 ◽

2005 ◽

Vol 17 (2) ◽

pp. 264

Author(s):

S. Mamo ◽

C.A. Sargent ◽

N.A. Affara ◽

K. Wimmers ◽

S. Ponsuksili ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Expression Profiles ◽

Developmental Competence ◽

Cdna Clones ◽

Bovine Embryo ◽

Single Experiment

Microarray technology currently has wide acceptance as a research tool in the study of gene expression profiling, mainly as a result of its use for monitoring the expression profiles of thousands of genes in a single experiment. However, its use in analyzing gene expression in the pre-implantation stage of bovine embryo development has been limited for reasons such as the large amount of RNA required and the lack of bovine specific cDNA clone collections (Smith L and Greenfield A 2003 Hum. Mol. Genet. 12, 1–8). In this study, with the objectives of producing pre-implantation-stage-specific bovine cDNA clones and examining the developmental competence, eighty-two selected target clones of pre-implantation-stage-specific genes were prepared and spotted on the glass slide. Embryos were produced in vitro and mRNAs were isolated from contrasting probes of good quality matured oocytes and blastocyst-stage embryos using a Dynabead mRNA isolation kit by following the manufacturer's instructions. First-strand cDNA syntheses were primed with T7 Oligo d(T)21 primer, followed by random primed second-strand syntheses using a DOP master kit (Roche Diagnostics, Mannheim, Germany) and global amplification using the same primers used for the first- and second-strand syntheses. In vitro transcription was performed to amplify the RNA by using the AmpliScribe T7 transcription kit (EPICENTRE Technologies, Oldendorf, Germany), and the amplified RNA (aRNA) was purified using a RNeasy Mini kit (Qiagen, Hilden, Germany). Finally, the results of different RNA amplifications (aRNA) were tested by hybridization on microarrays and also using real-time PCR techniques. With these analyses, the sufficiency of the yield and linearity of amplification procedures were confirmed. Three micrograms each of aRNA were labelled with Cy3 and Cy5 dyes and hybridized to the array. After overnight incubation at 42°C, the slides were sequentially washed and scanned using an ArrayWorx biochip reader (Applied Precision, Marlborough, UK), and quantifications as well as all analyses were carried out using different TIGR software modules (Saeed AI et al. 2003 Biotechniques 34(2), 374–378). Analyses of the results of repeated hybridizations showed that 35 genes (43%), which belong to different functional groups, were differentially expressed between the two stages. Further independent analyses using real-time PCR confirmed the results of 25 genes. Hence, it is possible to conclude that the established methods can be used for large scale gene expression analysis, and the identified genes can be potential candidates for characterizing developmental competence.

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