Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere.
01 Apr 1994-Biology of Reproduction (Society for the Study of Reproduction)-Vol. 50, Iss: 4, pp 715-724
TL;DR: Two distinct culture environments are characterized, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro, and survival of the embryo under a reduced oxygen atmosphere is investigated.
Abstract: The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.
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TL;DR: The quality of blastocysts was equivalent, independently of fertilization or coculture systems, for ovine zygotes derived from in vivo (IVOF) or in vitro fertilization (IVF) on sheep oviductal cells.
Abstract: The development and quality of ovine zygotes derived from in vivo (IVOF) or in vitro fertilization (IVF) were compared after coculture on sheep oviductal cells. The same criteria were used to evaluate the coculture of IVF zygotes in CZB medium for 2 d followed by 199 medium for 6 d (CZB-199 coculture) or in 199 medium for 8 d (199 coculture). A higher overall developmental rate to blastocyst stages was obtained with IVOF (65.7%) than with IVF (23.2%) zygotes. More IVF zygotes reached blastocyst stages in CZB-199 (36.7%) than in 199 coculture (22.9%). The morphological aspect did not differ significantly between IVOF and IVF or between 199 and CZB-199 blastocysts. Histological examination revealed no significant difference in the pyknotic and mitotic indices and mean number of cells in the trophoblast and in the inner cell mass of hatched blastocysts between IVOF and IVF or between CZB-199 and 199 cocultures. According to criteria used in this study, the quality of blastocysts was equivalent, independently of fertilization or coculture systems. The use of CZB medium during the first cleavages increases the proportion of blastocysts.
3 citations
TL;DR: The results suggest that granulosa cells may be a primary site of PTHrP production and release in bovine ovary, and Supplementation of serum-free cSOFMaa oocyte maturation medium with PTHR resulted in a concentration-dependent increase in development to the blastocyst stage in vitro.
Abstract: Parathyroid hormone related protein (PTHrP) and its receptor have well-established roles in the development and regulation of many tissues, including bone and mammary gland. The objectives of this study were: (1) to characterize the distribution of mRNAs encoding parathyroid hormone (PTH)-related protein (PTHrP) and receptor (PTHR) in bovine ovary; (2) to characterize the distribution of PTHrP and PTHR polypeptides in bovine ovary; (3) to examine the influences of PTHrP (1-141) treatment during bovine oocyte maturation in vitro on blastocyst development. mRNAs encoding PTHrP and PTHR were detected by in situ hybridization methods in oocytes, and granulosa cells in all follicles from primordial to large antral. PTHrP and PTHR polypeptides displayed distinct distribution patterns with PTHrP polypeptides primarily confined to oocytes from primordial to large antral follicles. PTHrP polypeptides were detectable but at a reduced level in ovarian stroma and in granulosa and thecal layers. PTHR polypeptides were detected in oocytes of all follicular stages but were predominantly found in ovarian stroma, granulosa and theca follicular layers. Supplementation of serum-free cSOFMaa oocyte maturation medium with PTHrP (1-141) resulted in a concentration-dependent increase in development to the blastocyst stage in vitro. The results suggest that granulosa cells may be a primary site of PTHrP production and release. Oocytes from all follicular stages stained strongly for PTHrP polypeptides and PTHrP enhanced development to the blastocyst stage in vitro.
3 citations
Cites methods from "Preimplantation development of in v..."
...All sections 107 109 Oocyte Collection, Insemination and Embryo Culture: 110 Cumulus oocyte complexes (COCs), were collected by razor blade slashing of slaughterhouse 111 ovaries within 4 h of removal from the animal (Wiemer et al., 1991;Watson et al., 1994)....
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TL;DR: The availability of high numbers of easily accessible embryos has led to a substantial advance in knowledge of their physiology and widened the range of experimental models that can effectively be used in developmental studies, especially since, in some cases, models using these species may be more relevant to human embryology than those using rodents.
3 citations
Dissertation•
01 Jan 2003
TL;DR: The presence of BRL cell and OOEC even in the absence of heparin significantly improved the fertilization and embryo development suggesting their capacitation and sperm viable ability.
Abstract: Title : IN VITRO MATURATION, FERTILIZATION, EMBRYO CULTURE AND ACTIVATION TREATMENTS OF OVINE OOCYTES IN DIFFERENT CULTURE SYSTEMS Name of the student : AKSHAYA KUMAR KUNDU Degree for which submitted : Ph.D. (Veterinary Physiology) Name of the Chairman : Dr.A.Thangavel, Ph.D., Year and University : 2003 Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051. There is a rapid development in technologies to produce large number of mammalian embryos, in vitro at low cost for research and commercial purposes, still then in vitro developmental capacity of ovine oocytes is significantly lower than that of in vivo system. Generation of a lot of data may be useful to optimize the requirements for oocyte development, in vitro. This study was aimed to improve ovine oocyte in vitro maturation, fertilization, embryos development and activation of the ovine oocytes to produce parthenogenic embryos. The cumulus oocyte complexes (COCs) were harvested by slicing method from slaughterhouse-derived sheep ovaries. The COCs were matured in different in vitro culture systems, viz., 1. maturation of oocytes in TCM-199, SOF and Ham's F-10 media, 2. supplementation of IGF-I (100 ng/ml), EGF (10 ng/ml) and IGF-I (100 ng/ml) + EGF (10 ng/ml), 3. supplementation of cysteamine @ 50, 100, 200 m and 4. supplementation of s-mercaptoethanol (s-ME) (12.5, 25, 50 M) to the oocyte maturation medium. The cauda epididymal sperm from slaughterhouse-derived testes were utilized for in vitro insemination of the oocytes. The oocytes and sperms were co-incubated over the BRL cell and OOEC monolayers in the fert-TALP medium with and without heparin. The ovine embryos were cultured in different culture systems viz: 1. embryo culture in TCM-199, SOF and Ham's F-10 media, 2. supplementation of IGF-I (100 ng/ml), EGF (10 ng/ml) and IGF-I (100 ng/ml) + EGF (10 ng/ml) to the IVC medium, 3. embryo co-culture with BRL, Vero, BRL/Vero mixed cell and OOEC monolayers and 4. supplementation of Hb (1 g/ml), L-NAME (1 M) and Hb (1 g/ml) + L-NAME (1M) to the IVC medium. The matured ovine oocytes were activated by ethanol, calcium ionophore and thimerosal to produce parthenogenic embryos. The results were observed as follows. TCM-199 and SOF media were found suitable for in vitro ovine oocyte development. IGF-I, EGF and their combination accelerated the oocyte maturation leading to increased fertilization rate and improved embryo development. The presence of cysteamine and s-ME during IVM were found beneficial for ovine oocyte development. The presence of BRL cell and OOEC even in the absence of heparin significantly improved the fertilization and embryo development suggesting their capacitation and sperm viable ability. The SOF medium showed better competence for ovine embryo culture over TCM-199 and Ham's F-10 media. IGF-I, EGF and their combination in IVC medium enhanced the embryo development rate and increased cell number in blastocysts. BRL, Vero, BRL/Vero mixed cell and OOEC monolayers significantly improved the in vitro embryo development. The activated parthenogenic embryos produced were found to be quantitative but not qualitative compared to the IVF oocytes.
2 citations
TL;DR: The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients, and generated direct candidates of blastocySt-specific genes that determine the fate of the embryo after transfer.
Abstract: Early embryonic mortality is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows. The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients. Biopsies (3 to 40% of the intact embryo) were taken from IVP Day 7 blastocysts (n = 98) and 60 to 70% was transferred to recipients (2-year-old Simmental heifers) after re-expansion. Based on the success of pregnancy, biopsies were pooled in 3 groups: those resulting in no pregnancy (G1), resorption (G2), and calf delivery (G3). Gene expression analysis of these groups of biopsies was performed using a home-made bovine pre-implantation-specific array (with 219 clones) and bovine cDNA array (BlueChip) (with 2000 clones) (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Approximately 3 µg of amplified RNA from pooled biopsies (10 biopsies each) was used as a template in reverse transcription reactions incorporating amino-modified dUTPs into labeled cDNA using the CyScribeTM post-labelling kit (Amersham Bioscience, Freiburg, Germany). The synthesized cDNAs from all 3 groups were differentially labeled using Cy3 and Cy5 dyes. Slides were scanned using a GenePix 4000B scanner and images were analyzed using GenePix Pro Version 4.0 software (Axon Instruments, Union City, CA, USA). Data were analyzed using Significant Analysis for Microarray (SAM) software (http//www.stst.stanford.edu/tips/SAM). Quantitative real-time PCR was used to confirm the differentially expressed genes revealed by microarray experiments. Results revealed that 52 genes were differentially regulated between G1 and G3, and 58 genes were differentially regulated between G2 and G3. Biopsies resulting in calf delivery were enriched with genes necessary for implantation (COX-2 and CDX2), signal transduction (PLAU), polyamine biosynthesis (ODC1), response to oxidative stress (TXN), growth factor (BMP15), and placenta-specific 8 (PLAC8). Biopsies that ended up with resorption were enriched with transcripts involving protein phosphorylation (KRT8) plasma membrane (OCLN), and glucose metabolism (PGK1, AKR1B1). Biopsies resulting in no pregnancy were enriched with transcripts involving inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes that determine the fate of the embryo after transfer.
2 citations
Cites background from "Preimplantation development of in v..."
...The timing of zygotic gene activation, or competence to sustain appreciable transcriptional activity in bovine embryos may be controlled temporally by a time dependent mechanism referred to as zygotic clock rather than by developmental stage (Nothias et al.1995, Watson et al. 1999)....
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...However, after one to four cleavage divisions, based on the species, the maternal phase gradually looses its development control (Telford et al. 1990, De Sousa et al. 1998, Watson et al. 1999)....
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...Similarly, disruption of sodium/potassium transporting ATPase (Na/K-ATPase) gene expression by antisense oligonucleotide inhibition disrupted blastocyst formation; these results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation (Watson et al. 1999)....
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References
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TL;DR: In this article, a general class of regression models for ordinal data is developed and discussed, which utilize the ordinal nature of the data by describing various modes of stochastic ordering and this eliminates the need for assigning scores or otherwise assuming cardinality instead of ordinality.
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Abstract: One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.
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TL;DR: This communication describes the successful culture of one-cell to eight-cell sheep ova and one- cell and eight- cell cattle ova to the morula and blastocyst stages and reports a high embryo survival after transfer of cultured Ova to recipient animals.
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