130 ANTI-APOPTOTIC pTEGT SHOWS A DIFFERENTIAL mRNA TRANSCRIPT LEVEL DURING IN VITRO MATURATION AND EARLY DEVELOPMENT OF PORCINE IVP EMBRYO

2006 ◽  
Vol 18 (2) ◽  
pp. 173
Author(s):  
J.-Y. Kim ◽  
J.-H. Kim ◽  
H.-Y. Choi ◽  
B.-W. Kim ◽  
S. J. Uhm ◽  
...  
Keyword(s):  
Early Development ◽  
In Vitro Maturation ◽  
Transcript Level ◽  
Early Embryo ◽  
Mrna Transcript ◽  
Rt Pcr ◽  
Mrna Transcript Level ◽  
Embryonic Genome ◽  
Apoptotic Effect

It is well known that very early development of the mammalian pre-implantation embryo is regulated by gene transcripts and proteins stored in the oocyte and that the embryonic genome gains control of development following 1 to 3 cleavage divisions. An active transcription and translation is required for chromatin condensation and germinal vesicle breakdown in pig oocyte. The transition from maternal to embryonic control of development is a gradual event, and following this transition, the maternally derived transcripts and proteins are gradually degraded. Successful embryonic development is dependent on the temporal and stage-specific expression of proper genes, but information on specific gene expression during early stages before zygotic gene activation (ZGA) is limited. Before activation of the embryonic genome, mRNA and proteins synthesized during oocyte growth and maturation contribute to early development. In this study, we compared the mRNA transcripts level among porcine immature, in vitro-matured and cleaved 2- to 4-cell stage embryos after in vitro fertilization to identify genes that show differential mRNA transcript levels during maturation and very early embryonic development. For the first strand cDNA synthesis, oligo (dT) primers were added to the total RNA isolated from each sample. Using annealing control primer (ACP)-based GeneFishing PCR, we detected tens of different genes showing differential mRNA transcript level (DRTL) and nine DRTL genes were identified to be KCRF, CAMSAP1, SMP1, FLJ20647, LOC132321, NADH1, NADH6, HERC3, and TEGT. Of 9 DRTL genes, TEGT showed higher mRNA transcript level at the immaturation stage, and mRNA transcript levels of the other 8 genes were increased after in vitro maturation. Therefore, we focused on TEGT (testis enhanced gene transcript), which is highly expressed in testis and also in oocytes before in vitro maturation. Differential mRNA transcripts pattern of CAMSAP1 and TEGT were confirmed using RT-PCR and real-time RT-PCR. Porcine TEGT (pTEGT) was cloned and sequenced to have an ORF of 714 bp nucleotides and to encode an integral membrane protein. When overexpressed in HEK293 cells, pTEGT suppressed apoptosis induced by etoposide. We found that pTEGT, but not TEGT-C (C-terminal deletion mutant), inhibited etoposide- and staurosporine-induced cell death. Next, we found that introduction of TEGT siRNA suppressed the anti-apoptotic effect of TEGT. Interestingly, expression of TEGT suppressed etoposide-induced ERK activation, suggesting that ERK phosphorylation is involved in the anti-apoptotic function of the gene. Several reports showed that apoptosis and MAP kinase signaling pathways play important roles in oocyte maturation and early embryo development. Therefore, the anti-apoptotic effect of TEGT was suggested to play a key role in the normal ooctye maturation and early embryo development. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2358
Author(s):  
Katarzyna Suwik ◽  
Emilia Sinderewicz ◽  
Dorota Boruszewska ◽  
Ilona Kowalczyk-Zięba ◽  
Joanna Staszkiewicz-Chodor ◽  
...  
Keyword(s):  
Early Development ◽  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Peroxisome Proliferator ◽  
Early Embryo Development ◽  
Quality Markers

Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1—early, 2—developing, 3—expanded, 4—hatched); quality stages: A—high quality, B—moderate quality, C—low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

Progesterone receptor-dependent regulation of genes in the oviducts of female mice

2014 ◽  
Vol 46 (16) ◽  
pp. 583-592 ◽  
Author(s):  
Lisa K. Akison ◽  
Michael J. Boden ◽  
David J. Kennaway ◽  
Darryl L. Russell ◽  
Rebecca L. Robker
Keyword(s):  
Progesterone Receptor ◽  
Critical Role ◽  
Early Embryo ◽  
Rt Pcr ◽  
Morphological Defects ◽  
Embryo Transport ◽  
Polymerase Chain ◽  
Comprehensive Study

Oviducts play a critical role in gamete and embryo transport, as well as supporting early embryo development. Progesterone receptor (PGR) is a transcription factor highly expressed in oviductal cells, while its activating ligand, progesterone, surges to peak levels as ovulation approaches. Progesterone is known to regulate oviduct cilia beating and muscular contractions in vitro, but how PGR may mediate this in vivo is poorly understood. We used PGR null mice to identify genes potentially regulated by PGR in the oviducts during the periovulatory period. Histologically, oviducts from PGR null mice showed no gross structural or morphological defects compared with normal littermates. However, microarray analysis of oviducts at 8 h posthuman chorionic gonadotropin revealed >1,000 PGR-dependent genes. Using reverse-transcription polymerase chain reaction (RT-PCR) we selected 10 genes for validation based on their potential roles in oocyte/embryo transport and support. Eight genes were confirmed to be downregulated ( Adamts1, Itga8, Edn3, Prlr, Ptgfr, Des, Myocd, and Actg2) and one upregulated ( Agtr2) in PGR null oviducts. Expression of these genes was also assessed in oviducts of naturally cycling mice during ovulation and day 1 and day 4 of pregnancy. Adamts1, Itga8, Edn3, Prlr, and Ptgfr were significantly upregulated in oviducts at ovulation/mating. However, most genes showed basal levels of expression at other times. The exceptions were Prlr and Ptgfr, which showed pulsatile increases on day 1 and/or day 4 of pregnancy. This is the first, comprehensive study to elucidate putative PGR-regulated genes in the oviduct and reveals key downstream targets potentially mediating oocyte and embryo transport.

Cytoplasmic and nuclear determinants of the maternal-to-embryonic transition

2008 ◽  
Vol 20 (1) ◽  
pp. 45 ◽  
Author(s):  
Anilkumar Bettegowda ◽  
Kyung-Bon Lee ◽  
George W. Smith
Keyword(s):  
Subsequent Development ◽  
Oocyte Quality ◽  
Early Embryo ◽  
Success Rates ◽  
Embryo Production ◽  
Maternal Mrna ◽  
Time Period ◽  
Embryonic Genome ◽  
Production Success

Although improvements in culture systems have greatly enhanced in vitro embryo production, success rates under the best conditions are still far from ideal. The reasons for developmental arrest of the majority of in vitro produced embryos are unclear, but likely attributable, in part, to intrinsic and extrinsic influences on the cytoplasmic and/or nuclear environment of an oocyte and/or early embryo that impede normal progression through the maternal-to-embryonic transition. The maternal-to-embryonic transition is the time period during embryonic development spanning from fertilisation until when control of early embryogenesis changes from regulation by oocyte-derived factors to regulation by products of the embryonic genome. The products of numerous maternal effect genes transcribed and stored during oogenesis mediate this transition. Marked epigenetic changes to chromatin during this window of development significantly modulate embryonic gene expression. Depletion of maternal mRNA pools is also an obligatory event during the maternal-to-embryonic transition critical to subsequent development. An increased knowledge of the fundamental mechanisms and mediators of the maternal-to-embryonic transition is foundational to understanding the regulation of oocyte quality and future breakthroughs relevant to embryo production.

scholarly journals Using RT-PCR and glutathione level to study the effect of follicular fluid on in vitro maturation and gene expression of sheep oocytes

2019 ◽  
Vol 26 (6) ◽  
pp. 1216-1222 ◽  
Author(s):  
M. Al-Mutary ◽  
M. Al-Ghadi ◽  
A. Al-himaidi ◽  
D. Iwamoto ◽  
Y. Al-anazi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Glutathione Level ◽  
Rt Pcr
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