181 The effects of aging in the oocyte induced by trichostatin A

2020 ◽  
Vol 32 (2) ◽  
pp. 218
Author(s):  
H. A. Arena ◽  
E. Hicks ◽  
M. Mentler ◽  
B. D. Whitaker
Keyword(s):  
Embryonic Development ◽  
Trichostatin A ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Treatment Groups ◽  
Early Maturation ◽  
Stage Of Development ◽  
Developmental Characteristics ◽  
Effects Of Aging

Successful fertilization and embryonic development decreases for an aged oocyte that is not fertilized during the optimal window of time after ovulation. If fertilization of an aged oocyte does occur, the developing embryo is prone to have a delay through meiosis and a decrease in cleavage and blastocyst formation. Trichostatin A (TSA) prohibits the breakdown of the germinal vesicle during meiosis. The objective of this study was to create an invitro model to study the effects of aging in pig oocytes. Oocytes (n=1489) were matured with or without TSA (100ngmL−1) for 24 or 48h followed by an additional 16h of maturation without TSA. A portion of the oocytes were evaluated for stage of meiosis (MI and MII) before maturation (n=95), after the first part of maturation (OMI; 24 or 48h, n=363), or after completing maturation (OMII; 16h, n=230). The remaining oocytes (n=801) were fertilized for 6 to 8h and potential embryos developed. A portion of the embryos were evaluated for fertilization characteristics 12h after IVF (n=400). Remaining embryos (n=401) were evaluated for cleavage by 48h and blastocyst formation by 144h, after IVF. Data were analysed using ANOVA and Tukey's test. Oocytes matured without TSA for 64h had a significantly higher (P<0.05) percentage of oocytes at the MI stage of meiosis compared with all other treatment groups (16.00±6.80), and oocytes matured without TSA for 40h had a significantly higher (P<0.05) percentage of oocytes at the MII stage of meiosis compared with all other treatment groups (14.7±11.30). The results (Table 1) indicate that supplementation of TSA to OMI significantly decreased fertilization penetration rates compared with not supplementing TSA for 24h. However, the percent of embryos cleaved by 48h after IVF was significantly higher in oocytes matured for 40h compared with those matured for 64h. The percent of embryos reaching the blastocyst stage of development by 144h after IVF was significantly lower in oocytes matured for 64h compared with those matured for 40h without TSA. In conclusion, these results suggest that supplementation of TSA during early maturation delays meiosis and decreases fertilization, and increasing maturation time causes a decrease in early embryonic development. By supplementing TSA during early maturation and increasing the length of maturation by 24h, we have created an invitro model to study the effects of aging in pig oocytes. Table 1.Fertilization and developmental characteristics of oocytes1 Amount of TSA added during OMI (ngmL−1) Length of OMI (h) Oocytes penetrated (%) Polyspermicoocytes2 (%) Oocytes with MPN2 (%) Cleavage (%) Day 7 blastocyst (%) 0 24 83.00±3.80a 15.70±4.00 75.90±4.70 77.20±4.20a 27.70±4.20a 0 48 72.00±4.50ab 20.80±4.80 76.40±5.00 58.00±5.00bc 12.00±5.00b 100 24 65.00±4.80b 30.80±5.80 73.80±5.50 68.00±4.70ab 21.00±4.70ab 100 48 56.00±5.00b 30.40±6.20 62.50±6.50 46.00±5.00c 8.00±5.00b a,bMeans with different superscripts in the same column differ (P<0.05). 1TSA, trichostatin A; OMI, oocyte maturation 1. 2Percent of the number of oocytes penetrated.

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.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

2017 ◽  
Vol 29 (1) ◽  
pp. 185
Author(s):  
L. R. Madzhie ◽  
M. A. Raseona ◽  
L. P. Nethenzheni ◽  
O. Ajao ◽  
M. L. Mphaphathi ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Culture Media ◽  
Uv Light ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Murine Embryos

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

Follicle size indicates oocyte maturity and blastocyst formation but not blastocyst euploidy following controlled ovarian hyperstimulation of oocyte donors

2020 ◽  
Vol 35 (3) ◽  
pp. 545-556
Author(s):  
David H McCulloh ◽  
Nino Kutchukhidze ◽  
Tea Charkviani ◽  
Tengiz Zhorzholadze ◽  
Tamar Barbakadze ◽  
...  
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Oocyte Maturity ◽  
Follicle Diameter ◽  
Follicle Size ◽  
Oocyte Donors ◽  
The Relationship

Abstract STUDY QUESTION Is there is an association between follicle size and the quality of oocytes retrieved from them as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy? SUMMARY ANSWER Although follicle size is a valuable predictor of oocyte maturity and is a significant predictor of the ability of a fertilized oocyte to become a quality blastocyst, the ploidy of each quality blastocyst is not related to the size of the follicle from which its oocyte was retrieved. WHAT IS KNOWN ALREADY It is unclear whether the oocytes within larger follicles are the best oocytes of the cohort. Although there have been studies examining follicle size in relation to embryo quality, there has been no study relating the incidence of euploidy in embryos to follicle size. STUDY DESIGN, SIZE, DURATION The purpose of this study was to examine follicle sizes and the oocytes from those follicles (and the embryos that result from those oocytes) to see if there is an association between follicle size and the quality of oocytes as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy. Follicle sizes for oocytes were assessed both as diameters (mm) and as Z values (expressed as their size relative to the mean and standard deviation of that donor’s follicular cohort). Comparisons were made using cumulative histograms, rolling averages and receiver operator characteristic (ROC) curves and its AUC. PARTICIPANTS/MATERIALS, SETTING, METHODS Twenty-two oocyte donors (ages: 24.5 ± 3.5 years) whose recipients would use ICSI for insemination were enrolled in this study. Follicles were aspirated one-at-a-time to be certain that the aspirated oocyte was from the same follicle measured. The follicle measurement (size) was noted in the embryology records. Oocytes were cultured individually throughout their time in the embryology laboratory so that follicle sizes could be uniquely associated with each oocyte. Oocytes and embryos were analyzed according to the size of the follicle from which the oocyte was retrieved. MAIN RESULTS AND THE ROLE OF CHANCE Three hundred seventeen oocytes (96.1%) had an associated follicle size. Of the oocytes with follicle sizes, 255 (80.4%) had a polar body (MII), and 60 (18.9%) were immature: 31 (9.8%) with a visible germinal vesicle (GV stage) and 29 (9.1%) with neither a polar body nor a visible germinal vesicle (MI). The incidence of MII oocytes was significantly associated with larger follicle size using either mm (ROC’s AUC = 0.87; P &lt; 0.0001) or Z values (ROC’s AUC = 0.86; P &lt; 0.0001). Among MII oocytes there was no association with follicle size for the appearance of 228 oocytes with two pronuclei (2 PN). Among 2 PN’s, the development of 94 quality blastocysts that underwent trophectoderm biopsy (TE Bx) exhibited a significant association with larger follicles using either mm (ROC’s AUC = 0.59; P = 0.01) or Z values (ROC’s AUC = 0.57; P = 0.01). The use of follicle diameter as a feature to distinguish between fertilized oocytes that would ultimately become blastocysts versus those that would not become blastocysts resulted in an enrichment for blastocyst formation from 20 to 40%. Of the 94 quality blastocysts, 51 were determined by next generation sequencing (NGS) to be euploid.Although oocyte maturity and the incidence of blastocyst formation were associated with follicle size, the incidence of euploidy among biopsied blastocysts was not. Follicles measured by two different methods (mm or Z values) led to predominantly the same conclusions. LIMITATIONS, REASONS FOR CAUTION This study investigated the relationship between follicle size and measures of oocyte/embryo quality when donors were treated similarly. Therefore, this study does not investigate the effects of triggering and retrieving oocytes when the follicle cohorts are of different sizes or lead follicles are of different sizes. Although no association was found between follicle size and euploid blastocysts, the fact that blastocyst ploidy is not entirely dependent upon oocyte ploidy (e.g. aneuploidies derived from mitotic errors or from the fertilizing sperm) makes it difficult to infer the relationship between follicle diameter and oocyte ploidy. WIDER IMPLICATIONS OF THE FINDINGS It is confirmed that follicle diameter is predictive of oocyte maturity. However, once oocyte maturity is known, the diameter of the follicle from which the oocyte was retrieved is not instructive. Embryos generated through fertilization and development of the mature oocytes from any observed follicle diameter were equally likely to become euploid blastocysts. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by ReproART: Georgian American Center for Reproductive Medicine. None of the authors declare any actual conflicts of interest. D.H.M. received compensation from ReproART, Biogenetics Corporation and the Sperm and Embryo Bank of New York and honoraria and travel funding from Ferring Pharmaceuticals and from Granata Bio. S.M. received compensation from Cooper Genomics and an honorarium and travel funding from Ferring Pharmaceuticals. L.C. is the founder of LTD Ovamedi, the organization that represents Cooper Genomics in Georgia, and received travel funding from the European Society for Human Reproduction and Embryology. TRIAL REGISTRATION NUMBER N/A.

scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

302 EFFECT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2015 ◽  
Vol 27 (1) ◽  
pp. 240
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Intracellular Ros ◽  
Sperm Penetration

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on in vitro porcine oocytes maturation and subsequent embryonic development after pathenogenetic activation (PA) and in vitro fertilization (IVF). We investigated nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, sperm penetration (SP) analysis, and subsequent embryonic development after PA and IVF. Each concentration (0, 1, 10, and 100 ng mL–1) of GDF8 was added in maturation medium during process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± s.e.m. After 44 h of IVM, no significant difference was observed on nuclear maturation from the different concentration (0, 1, 10, and 100 ng mL–1) of GDF8 treatment groups (85.5, 85.9, 89.4, and 87.6%, respectively) compared with the control (P > 0.05). The 10- and 100-ng mL–1 GDF8-treated groups showed a significant (P < 0.05) decrease in intracellular ROS levels compared with other groups. The embryonic developmental competence after PA was affected with GDF8 treatment during IVM. The 10- and 100-ng mL–1 treatment groups showed significantly (P < 0.05) higher cleavage rates (67.5 and 69.1%, respectively) compared with control group (53.7%). The 10- and 100-ng mL–1 treatment groups also showed significantly (P < 0.05) higher blastocyst formation rates (50.5 and 52.7%, respectively) compared with other groups (34.5 and 35.8%). The IVF embryonic developmental competence also was affected with GDF8 treatment during IVM. The 10-ng mL–1 treatment group showed a significantly (P < 0.05) higher blastocyst formation rates and total cell number compared with control (21.5 and 131.3 v. 15.0 and 92.6%, respectively). Also, in the sperm penetration assessment, the 10- and 100-ng mL–1 treatment groups showed higher mono spermy ratio and fertilization efficiency (32.7 and 27.1, 32.0 and 26.5 v. 22.6 and 19.7%, respectively) than control, which was significant (P < 0.05). In conclusion, the treatment with 10 ng mL–1 of GDF8 during IVM improved the PA and IVF porcine embryo developmental competence by decreasing the intracellular ROS levels.This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.

The effects of N-acetyl-L-cysteine supplementation on in vitro porcine oocyte maturation and subsequent fertilisation and embryonic development

2012 ◽  
Vol 24 (8) ◽  
pp. 1048 ◽  
Author(s):  
B. D. Whitaker ◽  
S. J. Casey ◽  
R. Taupier
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
The Other ◽  
Success Rates ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Intracellular Glutathione ◽  
Porcine Oocyte

The effects of supplementation with 1.5 mM n-acetyl-l-cysteine (NAC) during in vitro oocyte maturation were studied. Oocytes were supplemented with 1.5 mM NAC during maturation for 0 to 24 h, 24 to 48 h, or 0 to 48 h then subjected to IVF and embryo development. Oocytes were evaluated after maturation for intracellular glutathione concentration, superoxide dismutase and glutathione peroxidase activities and DNA fragmentation. Fertilisation and embryonic development success were also evaluated. There was no effect of treatment on intracellular glutathione concentrations, enzyme activities or fertilisation success rates. Supplementing NAC during maturation significantly decreased (P < 0.05) the percentage of oocytes with fragmented DNA compared with no NAC supplementation. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of oocytes with male pronuclei than for oocytes from the other treatment groups. There was no difference in the percentage of embryos cleaved by 48 h after IVF between treatment groups. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of embryos reaching the blastocyst stage by 144 h after IVF compared with the other treatment groups. These results indicate that supplementation of the oocyte maturation medium with 1.5 mM NAC, specifically during the last 24 h, improves male pronucleus formation and blastocyst development in pigs.

215 COMPARISON BETWEEN CONVENTIONAL DIRECT TRANSFER FREEZING AND VITRIFICATION FOR THE CRYOPRESERVATION OF IN VIVO EMBRYOS FROM BRAHMAN CATTLE

2007 ◽  
Vol 19 (1) ◽  
pp. 224 ◽  
Author(s):  
J. H. Pryor ◽  
C. R. Looney ◽  
D. Walker ◽  
G. E. Seidel, Jr ◽  
J. F. Hasler ◽  
...  
Keyword(s):  
Animal Health ◽  
Blastocyst Stage ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Multiple Ovulation ◽  
Stage Of Development ◽  
Experimental Media ◽  
Commercial Company ◽  
Brahman Cattle

There is a need to develop an efficient cryopreservation technique for Brahman cattle embryos that would lead to improved success in the propagation of this breed. The objective of this study was to compare the post-thaw pregnancy rates in recipient cows after nonsurgical transfer of Brahman in vivo-derived embryos frozen in ethylene glycol (EG) and by a new vitrification (VT) method. Prior to the initial in vivo study, hatching rates were recorded 72 h post-thaw on 3 treatment groups (No treatment: 43/55, 78%; EG: 39/83, 47%; and VT: 33/103, 32%) of IVF embryos to determine if development could be achieved. This warranted Phase I: In vivo embryos were produced by multiple ovulation and nonsurgical embryo collections from Brahman cows 7 days post-estrus and AI. Embryos in morula/blastocyst stage of development were graded and maintained in holding medium (Vigro; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) after collection, until randomly allocated for EG or VT protocols. Vitrification was performed using experimental media provided by a commercial company (V1: Vigro + 5 M EG; V2: Vigro + 7 M EG + 0.5 M Galactose + Ficoll; and Diluent: Vigro + 0.5 M Galactose) and by direct plunging into LN2 (Walker et al. 2005 Reprod. Fertil. Dev. 17, 153). Freezing was performed at 0.5�C min-1 from -6�C to -32�C, using a commercially available medium (Vigro Freeze Plus; Bioniche). All embryos were packaged in sterile 0.25-mL plastic straws to allow for direct transfer (DT). Embryos were stored in LN2, and later thawed and nonsurgically transferred to synchronized recipient cows on Day 7 of their cycle. EG straws were air-thawed 5 s and then thawed in 30�C H2O for 10 s. VT straws were air-thawed 10 s and then thawed in 37�C H2O for 20 s prior to shaking them down to mix columns. Pregnancy results evaluated by ultrasound on Day 35 for the presence of a fetus from 4 replicates were 27% for EG (3/11) and 8% for VT (1/13) embryos. Problems with VT straws cracking during air thawing led to a second trial (Phase II) which changed the direct plunging technique for VT to a vapor freeze of 1 to 15 min prior to plunging into LN2. Pregnancy results from 16 transfers were 38% for EG (3/8) and 50% for VT (4/8) embryos, with no straws cracking. Although the number of observations is small, the results obtained showed that VT-DT is satisfactory for producing pregnancies comparable to EG-DT. Further research is required to confirm the results obtained in this preliminary study, and to test whether this method will allow the successful production of pregnancies under different environmental and field conditions.

234 PARTHENOGENETIC ACTIVATION OF DOMESTIC CAT OOCYTES USING STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 196 ◽  
Author(s):  
C. Wang ◽  
K. Lee ◽  
S. Koh ◽  
Z. Machaty
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Blastocyst Formation ◽  
Formidable Challenge ◽  
Significant Difference

Cloning domestic cats is useful in comparative medicine programs as it may provide insight into unique disease mechanisms and facilitate investigation of new therapeutic options. It is also believed to be beneficial for the conservation of precious animal models. However, as in many species, low birth rates after nuclear transfer remain a formidable challenge. One potential reason for the low efficiency is poor embryo development following activation of the reconstructed oocytes. The number of methods available to induce a transient increase in the oocytes' cytosolic free calcium level to stimulate development is rather limited. Although strontium has been reported to successfully activate the developmental program of mature mouse and rat oocytes, it was without effect in all other species studied. Here we investigated the effect of strontium on mature cat oocytes. Oocytes collected from the cat ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mm cysteine, 0.1 mm cysteamine, 1 IU mL–1 eCG, 2 IU mL–1 hCG, 25 ng mL–1 epidermal growth factor (EGF) for 24 h. For intracellular calcium measurements, mature oocytes were incubated in the presence of 2 µ m fura-2Am, a calcium indicator dye, and 0.02% pluronic F-127 for 40 min. Individual oocytes were transferred into calcium-free HEPES, and SrCl2 was added to the medium at a final concentration of 20 mm. Changes in the intracellular free calcium levels were then monitored using an InCyt Im2™ fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA). Preimplantation embryonic development was also evaluated by incubating the oocytes with 20 mm SrCl2 in calcium-free HEPES medium supplemented with 7.5 µg mL–1 cytochalasin B for 6 h. Control oocytes were activated by two 20-µs-long, 100 kV cm–1 direct current pulses and incubated in the presence of 7.5 µg mL–1 cytochalasin B for 6 h. After activation, the oocytes were cultured in FOCM for 6 days. At the end of the culture period, embryonic development was recorded; the nuclear number of the embryos was also determined after staining with Hoechst 33342. Data were subjected to one-way ANOVA, and differences between treatments were analyzed using the Tukey test. We found that strontium triggered a transient rise in the intracellular free calcium concentration in all oocytes tested (N = 20). Strontium treatment also induced cleavage in 49.7% (92/185) of the oocytes, while 4.9% (9/185) of the activated oocytes developed to the blastocyst stage. In the electroporated group, cleavage frequency was 57.1% (104/182) and blastocyst formation was 8.8% (16/182). Data analysis showed that there was no significant difference between the two groups in terms of cleavage frequency and blastocyst formation. This is the first study to demonstrate that strontium can induce cytoplasmic calcium increase in cat oocytes and trigger development up to the blastocyst stage. The results also indicate that SrCl2 may be useful for oocyte activation during cat nuclear transfer. Additional studies are needed to determine whether SrCl2 can trigger development more effectively than current activation techniques.

127 REGULATION OF H3K27me3 AND H3K4me3 DURING INITIAL PORCINE EMBRYONIC DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 222
Author(s):  
Y. Gao ◽  
V. Hall ◽  
P. Hyttel
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Histone Methylation ◽  
Blastocyst Stage ◽  
Limb Bud ◽  
High Expression ◽  
Cell Stage ◽  
Stage Of Development ◽  
Low Levels ◽  
Genome Activation

During mammalian development, gene expression is partly regulated by posttranslational modifications of histones. In particular, H3K27me3 and H3K4me3 are involved in transcriptional repression and activation, respectively. In this study, we examined the global levels of H3K27me3 and H3K4me3, as well as the expression levels of their specific methylases and demethylases during porcine pre-implantation embryonic development. Global histone methylation was analyzed by immunocytochemical quantification within in vivo porcine embryos at 1-cell (Day 1), 4-cell (Day 3), morula (Day 5), and late blastocyst (containing the epiblast; Day 9). The numbers of embryos analyzed for H3K27me3 at the 1-cell, 4-cell, morula, and late blastocyst stage were 7, 8, 6, and 5, respectively, and for H3K4me3 at these four stages were 7, 6, 6, and 5, respectively. At the same developmental stages, mRNA expression of methylases (EZH2, EED, and SUZ12, three core components of PRC2) and demethylases (JMJD3 and UTX) of H3K27me3 was performed on pooled embryos (n = 10), as well as expression of methylases (MLL1 and ASH1L) and demethylase (RBP2) of H3K4me3, by comparative RT-PCR. Expression was compared with pooled embryos from the limb bud stage (Day 21). GAPDH was used as the reference gene, and expression was normalized to Day 21 embryos. Our results show that the levels of global histone methylation of H3K27me3 and H3K4me3 decrease gradually from 1-cell to morula, but both were increased in late blastocysts. The levels of H3K27me3 methylase (EZH2, EED, and SUZ12) transcripts increased from 1-cell to late blastocyst stage. Low expression of the H3K27me3 demethylase JMJD3 was found at 1-cell stage and high expression at the 4-cell stage from when it decreased gradually to the late blastocyst. UTX expression was low but peaked at the 4-cell stage. Expression of H3K4me3 methylase MLL1, was low, whereas ASH1L expression was high at the 4-cell stage. RBP2, a demethylase of H3K4me3, was highly expressed at the late blastocyst stage. In conclusion, at the major genome activation (the 4-cell stage), H3K27me3 and H3K4me3 have decreased to moderate levels, which apparently balance each other with respect to gene repression and activation allowing for genome activation. At the 4-cell stage the activation of H3K4me3 is favored as a consequence of low levels of H3K27 methylases and high levels of H3K27 demethylases combined with high levels of H3K4 methylases and low levels of H3K4 demethylases. Interestingly, at the late blastocyst stage of development, high expression of H3K27me3 methylases and the H3K4me3 demethylase, RBP2, are observed, indicating repression of gene expression, which is counterintuitive to accelerating development. We speculate other factors, such as microRNA or other kinds of epigenetic mechanisms, might play a critical role at this developmental stage. Thus, further research is required to explain these phenomena occurred during early porcine development.

scholarly journals 268PREGNANCIES FROM FROZEN IVF CATTLE EMBRYOS USING SEX-SORTED AND UNSORTED SPERM

2004 ◽  
Vol 16 (2) ◽  
pp. 254 ◽  
Author(s):  
R.C. Fry ◽  
C.R. Earl ◽  
F.K. Hollinshead ◽  
D. Wild ◽  
W. Lindemans
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Blastocyst Stage ◽  
Control Group ◽  
Adult Cattle ◽  
Treatment Groups ◽  
Transfer Programs ◽  
Stage Of Development ◽  
User Friendly

Previously we demonstrated that sex-sorted sperm could produce IVF embryos from juvenile and adult cattle at rates similar to those for unsorted sperm (Fry et al., 2003 Theriogenology 52, 198). In this study we investigated the pregnancy rates of recipient cattle following the transfer of frozen/thawed IVF embryos generated from young heifers using sex-sorted and unsorted sperm. COCs collected from FSH-stimulated Senepol or Beefex heifers by TVR were matured, fertilized with either sex-sorted or unsorted Senepol sperm and cultured for 6 days under our standard laboratory conditions (Fry et al., 2003 Theriogenology 59, 446, Earl et al., 1997 Theriogenology 47, 255). Embryos reaching the blastocyst or expanded blastocyst stage of development were frozen by the CL-V method of vitrification. Briefly, embryos were equilibrated for 5–10min in HEPES-199 media containing 20% FCS (HM), placed in HM containing 10% EG, 10% DMSO for approximately 2 minutes and then in HM containing 20% EG, 20% DMSO for between 20–60sec (Vatja et al., 1997 Cryoletters 18, 191). Vitrification was achieved by collecting between 5–10 IVF embryos in a 3-μL droplet and securing this droplet to a coded CL-V holder. The droplet was vitrified using the CL-V kit (Lindemans et al., 2004 Theriogenology in press) and then sealed in a precooled ‘‘straw’’ for storage in liquid nitrogen. To thaw, the ‘‘straw’’ with specimen was removed from storage;; the specimen droplet was withdrawn from the ‘‘straw’’ and placed directly into HM containg 0.2M sucrose (SM). After approximately 5–10min each embryo was assessed, loaded into a tomcat catheter in SM and transferred surgically into a recipeint cow within 10–15min of thaw. Of 129 Brahman and Brahman cross cows receiving 2 injections of 125μg cloprostenol 11 days apart, 60 exhibited oestrus 2–4 days after the second injection and 53 were deemed suitable for embryo transfer. Pregnancy was determined by ultrasound on Day 40. No difference in pregnancy rate was found between treatment groups (P&gt;0.05; Table 1). The low submission rate (60/129) and pregnancy rate for the in vivo control group indicate that the fertility of the recipient cows may have been compromised by the drought conditions predominating in Central Queensland. Notwithstanding, the CL-V method for the vitrification of IVF embryos produced by either sex-sorted or unsorted sperm gave similar and very promising pregnancy results of around 40%. This provides new opportunities for the rapid banking of large numbers of sexed IVF embryos generated from elite cattle by TVR for user friendly embryo transfer programs. Table 1 Pregnancies from IVF embryos derived from sex-sorted and unsorted sperm and frozen by the CL-V method of vitrification, or from in vivo embryos frozen in glycerol

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