198 KARYOPHERIN ALPHA EXPRESSION IN PORCINE OOCYTES AND EMBRYOS PRODUCED BY IN VITRO FERTILIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 197
Author(s):  
X. Wang ◽  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Internal Control ◽  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Significance Level ◽  
Nuclear Localization Signals ◽  
Importin Α

Partitioning intracellular proteins between nuclear and cytoplasmic compartments is critically important for coordinating major cellular events involved in transcription and differentiation. Import of cytoplasmic proteins bearing classical nuclear localization signals (NLSs) into the nucleus is mediated by the importin α/β heterodimer. Importin α, also called karyopherin α (KPNA), serves to recognize the NLS-bearing cytoplasmic cargo. Six KPNA molecules have been characterized in human (KPNA1-6). Select KPNA molecules are known to be differently expressed in specific tissues; individual KPNA molecules also have specificity for unique NLS-bearing cargos. We hypothesized that transcripts for individual porcine KPNA molecules would be present at differing levels at specific stages of oocyte maturation and cleavage development, thereby reflecting the changing requirements for particular import pathways during this window of development. To test this hypothesis, we first identified the porcine orthologs of KPNA1-6. We also identified the open reading frame of a potentially novel KPNA, KPNA7. KPNA7 was highly represented in the porcine EST database from expressed sequence tags derived from oocytes and ovarian tissue. Transcript abundance of KPNA1-7 was determined in germinal vesicle (GV) and MII-stage (MII) porcine oocytes and 4-cell (4C) and blastocyst-stage (BL) porcine embryos using quantitative real-time PCR. mRNA was isolated from pools (50 200) of GV and MII oocytes and 4C and BL embryos produced by IVF. Transcripts for KPNA1-7 and YWHAG (internal control for transcript normalization) were amplified in duplicate across 3 to 5 replicates. Relative transcript abundance of these genes was measured using the comparative CT method; GV was taken as the calibrator stage. Data were analyzed using GLM procedures in SAS (SAS Institute Inc., Cary, NC, USA) with the significance level at 0.05, and differences were compared by Tukey’s post test. Our results showed that KPNA1 had a significant decrease in MII oocytes (4-fold, GV v. MII). Transcript abundance of KPNA2 was significantly higher in GV oocytes and 4C embryos than in MII oocytes (2-fold GV v. MII; 3-fold 4C v. MII); KPNA2 transcripts were not detectable in BL embryos. KPNA3 transcripts were reduced in BL embryos compared to GV oocytes (8-fold, BL v. GV). KPNA4 transcripts were increased at the 4-cell stage (18-fold, GV v. 4C). The transcripts of KPNA5 were detectable only in GV and MII oocytes. No significant changes in the amount of KPNA6 transcripts were detectable at the stages analyzed. Transcript levels of KPNA7 were reduced in BL embryos as compared to the GV oocytes (1165-fold, BL v. GV). Throughout all these stages examined, KPNA5 had the lowest transcript abundance and was not detectable in 4C and BL stages. Transcripts levels of KPNA7 were in higher abundance than KPNA1-6 in GV and MII oocytes. Results suggest that KPNA7 is a new member of the KPNA family. Our results also suggest that porcine oocytes and embryos, at discrete stages of development, have differing requirements for individual KPNA molecules.

scholarly journals Effects of melatonin and human follicular fluid supplementation of in vitro maturation medium on mouse vitrified germinal vesicle oocytes: A laboratory study

2021 ◽  
pp. 889-898
Author(s):  
Razieh Doroudi ◽  
Zohre Changizi ◽  
Seyed Noureddin Nematollahi-Mahani
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cell Stage ◽  
Human Follicular Fluid ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Ivf Outcomes

Background: Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved. Objective: As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate. Materials and Methods: Four hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups. Results: Development to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes. Conclusion: Employing an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation. Key words: Vitrification, Germinal vesicle, In vitro oocyte maturation, Melatonin, Follicular fluid.

144 EPIGENETIC ASYMMETRY IN HISTONE H3 LYSINE 9 DIMETHYLATION STATUS IN PRONUCLEAR STAGE PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 180
Author(s):  
M. Sega ◽  
R. Cabot
Keyword(s):  
In Vitro Fertilization ◽  
Histone H3 ◽  
Germinal Vesicle ◽  
Immunocytochemical Staining ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
H3k9 Dimethylation ◽  
Chi Square Analysis

Covalent modification of specific residues on the tail regions of core histone proteins has been shown to play a key role in regulation of the genome. Methylation of lysine 9 on histone H3 (H3K9) is associated with repression of transcription and formation of heterochromatin domains. The maternal- and paternal-derived pronuclei from pronuclear stage murine embryos have an asymmetric distribution of H3K9 dimethylation between the 2 pronuclei; maternal pronuclei have the H3K9 dimethylation, whereas the paternal pronuclei lack this modification. The aim of this study was to characterize the H3K9 dimethylation pattern in cleavage stage porcine embryos. Indirect immunocytochemical staining was performed using a commercially available antibody from Upstate (Charlottesville, VA) that recognizes the dimethylated form of lysine 9 on histone H3 and fluorescein isothiocyanate (FITC)-conjugated secondary antibody on germinal vesicle (GV) stage porcine oocytes and cleavage stage porcine embryos. Germinal vesicle stage oocytes were matured in vitro for 44 h in a chemically defined maturation medium (TCM 199 supplemented with 0.1% PVA, 0.069 mg/mL cysteine, 10 ng/mL EGF 0.5 IU/mL LH, and 0.5 IU/mL FSH) at 39�C in 5% CO2. Following in vitro fertilization, presumptive zygotes were cultured in NCSU23 medium containing 4 mg/mL BSA at 39�C in 5% CO2, 5% O2. Embryos were fixed in 3.7% paraformaldehide for 2 h and washed in PBS and 0.1% Tween20. H3K9 dimethylation is present in the nuclei of GV-stage oocytes (n = 24) and pronuclear (n = 13), 2-cell (n = 4), 4-cell (n = 9), blastocyst (n = 8) stage embryos. The analysis revealed 2 interesting findings. First, examination of thin optical sections through the nuclei of processed embryos on a confocal microscope revealed that in GV-stage oocytes and pronuclear, 2-cell and 4-cell stage embryos the dimethylated form of H3K9 was distributed throughout the nuclei at these developmental time points, whereas in blastocyst stage embryos, the dimethylated H3K9 was restricted to the nuclear periphery. Second, not all pronuclei within pronuclear stage embryos were positive for the dimethylated form of H3K9. To determine if differential H3K9 methylation pattern observed among pronuclei in 1-cell stage embryos was due to parental origin, we produced parthenogenic porcine embryos by electrically activating porcine oocytes in calcium containing activation medium and processed them as indicated above for fertilized oocytes (n = 13). All pronuclei found in pronuclear stage parthenogenic porcine embryos were positive for dimethylation of H3K9. Chi square analysis revealed this pattern to be different from that observed in pronuclear embryos produced by fertilization (P < 0.05). In summary we conclude that the pronuclei in 1-celled embryos produced by in vitro fertilization are differentially dimethylated at H3K9 and that the localization of dimethylated H3K9 changes during cleavage development.

183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

2014 ◽  
Vol 26 (1) ◽  
pp. 206 ◽  
Author(s):  
S. Chastant-Maillard ◽  
K. Reynaud ◽  
S. Thoumire ◽  
M. Chebrout
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fetal Calf Serum ◽  
Selection Process ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Sperm Head ◽  
Cell Stage ◽  
Vitro Fertilization

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).

scholarly journals Examining the quality and fertilization competence of bovine ejaculate with low progressive motility – should we give it a chance?

2021 ◽  
Author(s):  
Y. Li ◽  
D. Kalo ◽  
A. Komsky-Elbaz ◽  
Y. Zeron ◽  
Z. Roth
Keyword(s):  
Artificial Insemination ◽  
Calcium Ionophore ◽  
Transcript Abundance ◽  
Control Group ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Lower Volume

AbstractSpermatozoa progressive motility is positively correlated with fertilization competence. Bulls’ ejaculates with progressive motility lower than 50% are routinely rejected through the process of straw preparation, designated for artificial insemination of dairy cows. We examined the quality and fertility competence of ejaculates with relative low progressive motility (55–60%, n = 5; control) with those of very low progressive motility i.e. below the lower threshold, (20-45%, n = 5; rejected). Analysis revealed a lower volume for the control vs. rejected samples. Dip-Quick staining revealed a higher proportion of spermatozoa with abnormal morphology in the rejected group, in particular those with detached heads. Activation of spermatozoa with calcium ionophore, resulted by a lower proportion of activated spermatozoa in the rejected group. In addition, a higher proportion of spermatozoa with DNA damage were recorded in the rejected vs. the control samples. Following in-vitro fertilization, the proportion of oocytes that developed to the 2- and 4-cell stage embryos did not differ between groups. However, the proportion of embryos that further developed to blastocysts, was higher in the control group. Transcript abundance of selected genes in the blastocysts and the apoptotic index did not differ between groups, suggesting that the forming blastocysts were of the same quality. It is suggested that in specific cases, for example genetically superior bulls, ejaculates with very low progressive motility can be used for in vitro production of embryo. Further in vivo examinations, i.e. artificial insemination or transferring of embryos derived from these inferior ejaculates, might clarified this point.

178 TRANSCRIPT ABUNDANCE OF METHYLTRANSFERASES SPECIFIC FOR H3K9 DIFFER AT DISCRETE STAGES OF PORCINE OOCYTE AND CLEAVAGE STAGE EMBRYO DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 188
Author(s):  
M. N. Biancardi ◽  
L. Magnani ◽  
C. M. Johnson ◽  
R. A. Cabot
Keyword(s):  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Covalent Modification ◽  
Cleavage Stage ◽  
Transcript Levels ◽  
Vitro Fertilization ◽  
Multiple Comparison Test ◽  
Porcine Oocyte ◽  
Stage Embryo

Covalent modification of histone proteins plays a key role in transcriptional regulation. Methylation of the lysine residue 9 of histone protein 3 (H3K9) is globally remodeled during cleavage development. The aim of this study was to determine the relative transcript abundance of the 5 histone methyltransferases (HMTases) that have been shown to methylate H3K9, in porcine oocytes and cleavage stage embryos. We hypothesized that transcript levels for each of these HMTases (Suv39h1, Suv39h2, ESET, GLP, and G9a) would differ in their respective abundance at each developmental stage and that some would undergo dramatic changes in transcript abundance during development. To test this hypothesis, quantitative RT-PCR was performed on mRNA isolated from pools of 78–457 germinal vesicle (GV) and metaphase II (MII)-stage oocytes and 4-cell (4C) and blastocyst (BL)-stage embryos produced by either parthenogenetic activation (PA) or in vitro fertilization (IVF); each pool constituted an experimental replicate, and a minimum of 3 replicates were performed for each stage. Along with the 5 HMTases, transcripts for YWHAG were assayed and used as a normalizer. PCR data were quantified using Δ CT and 2ΔΔ CT methods; in the latter case, Δ CT values from the GV stage were used as the calibrator. All data were analyzed by ANOVA and Tukey’s multiple-comparison test. In GV oocytes, Suv39h2 transcripts were in the highest abundance, while ESET transcripts were in the lowest abundance, ESET transcripts being 1450-fold less abundant than Suv39h2 (P < 0.05). In MII oocytes, Suv39h2, GLP, and G9a transcripts were present in highest abundance and were not significantly different from each other; Suv39h1 transcripts were 18-fold less abundant, and ESET transcripts were 6650-fold less abundant than GLP transcripts (P < 0.05). ESET transcripts were present in the least abundance in 4C-PA embryos, 84-fold less than GLP (P < 0.05); no difference in transcript levels for Suv39h1, Suv39h2, GLP, and G9a were detected. ESET transcripts were not detectable 4C-IVF embryos. ESET transcripts were present in the least abundance in BL-PA embryos, 39-fold less abundant than GLP; there was no difference in transcript abundance between Suv39h1, Suv39h2, and G9a. In BL-IVF-stage embryos, ESET transcripts were in the lowest abundance, 97-fold less abundant than GLP (P < 0.05). Analysis of how transcripts for each individual HMTase change from the GV oocyte to the BL embryo revealed no significant change in G9a transcript abundance. GLP transcripts decrease 7-fold at the 4C stage (GV v. 4C, P < 0.05). No difference in relative abundance of ESET transcripts was detectable at GV, MII, or BL-IVF stages; ESET was not detectable in 4C-IVF embryos. SuvH1 transcripts decreased 9-fold in BL-IVF (GV v. BL, P < 0.05); SuvH2 transcripts decreased 21-fold at the 4C-IVF and BL-IVF stages (GV v. 4C, BL, P < 0.05). The differences in transcript abundance of these genes indicate that their expression may change in order to regulate the transcription of genes important in early development.

292. Influence of the in vitro environment on rat gametes and pre-implantation embryos

2005 ◽  
Vol 17 (9) ◽  
pp. 123
Author(s):  
N. R. Borg ◽  
M. K. Holland
Keyword(s):  
Protein Profile ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
In Vitro Systems ◽  
Interaction Research

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.

Levels of MMP2, TIMP1 and TIMP2 in Follicular Fluids in Women Undergoing in Vitro Fertilization and Their Relationship to Oocyte Quality

2019 ◽  
Vol 12 (2) ◽  
pp. 100-107
Author(s):  
Nina P. Ayvazova ◽  
Lyubomira O. Ilieva ◽  
Emiliana I. Konova ◽  
Milena A. Atanasova
Keyword(s):  
Matrix Metalloproteinases ◽  
Ovarian Tissue ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Oocyte Maturity ◽  
Vitro Fertilization ◽  
Follicular Fluids

Summary Recently, the important role of matrix metalloproteinases (MMPs) has been identified in follicular development and subsequent ovulation. Although the role of MMP in ovarian tissue remodeling during folliculogenesis has been well studied, the relationship between matrix protease activity and their inhibitors - Tisue inhibitors of matrix metalloproteinases (TIMP) and aging of the oocytes is still unclear. The present study aimed to establish the probable relationship between the expression levels of MMP-2 and TMP-1 and TIMP-2 in follicular fluid with the degree of oocyte maturity and quality. Follicular fluids from 20 women collected on the day of follicular puncture were tested for the presence of MMP-2, TIMP-1, and TIMP-2 using enzyme-linked immunosorbent assay (ELISA). The oocytes obtained were described in terms of maturity, morphology, and fertilization, as well as the embryo’s quality and rate of development. MMP-2 was significantly higher in follicular aspirates in the first prophase of meiosis - germinal vesicle (GV), compared to aspirates with first metaphase (MI) (p=0.011) and second metaphase (MII) of mature oocytes (p=0.010). The MMP-2/TIMP-1 ratio was significantly higher for GV compared to M1 (p=0.011), M2 (p=0.006) and atretic oocytes (p=0.032); (F(3, 71)=2.909, p=0.040). Based on our results, we can conclude that MMP-2 concentration in follicular fluids during the IVF / ICSI procedure had a significant relationship to oocyte maturation levels. It was significantly higher in the case of immature oocytes. On the other hand, oocytes with normal morphology were associated with a significantly higher MMP-2 concentration in follicular fluids.

scholarly journals Autologous Platelet-Rich Plasma Treatment Enables Pregnancy for a Woman in Premature Menopause

2018 ◽  
Vol 8 (1) ◽  
pp. 1 ◽  
Author(s):  
Konstantinos Sfakianoudis ◽  
Mara Simopoulou ◽  
Nikolaos Nitsos ◽  
Anna Rapani ◽  
Athanasios Pappas ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Plasma Treatment ◽  
Platelet Rich Plasma ◽  
Ovarian Tissue ◽  
Medical Knowledge ◽  
Premature Menopause ◽  
Vitro Fertilization ◽  
Autologous Platelet Rich Plasma ◽  
Autologous Platelet

This report presents the case of a woman aged 40 who has experienced premature menopause from the age of 35. Having rejected oocyte donation, she opted for intraovarian injection of autologous platelet-rich plasma with the aim to rejuvenate the ovarian tissue and enable the employment of her own gametes through in-vitro fertilization. Six weeks following the autologous platelet-rich plasma treatment, a significant reduction in the patient’s follicle-stimulating hormone (FSH) levels were noted. A natural in-vitro fertilization cycle led to a biochemical pregnancy, resulting in a spontaneous abortion at the 5th week of pregnancy. This is the first report of a successful autologous platelet-rich plasma application leading to pregnancy in menopause. This report uniquely contributes to the medical knowledge and challenges current practice in the context of infertility. The efficiency and safety of this treatment with regard to the reproductive system merits further investigation.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

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.

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