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: It is found that forskolin significantly increased STC gene expression and secretion by both rat and bovine TICs, an effect that was only replicated by human (h) chorionic gonadotropin (CG).
Abstract: Stanniocalcin (STC) is a recently discovered mammalian hormone that is widely distributed in many tissues. In rodents the STC gene is most highly expressed in ovary, specifically in androgen-producing thecal and interstitial cells. In addition, ovarian levels of expression rise 15-fold over pregnancy. The objective of this study was to develop a primary culture system for ovarian thecal-interstitial cells (TICs) to identify factors governing STC production and release. We used highly purified primary cultures of rat and bovine TICs, the purity of which was routinely assessed with antigenic and enzymatic markers. The functionality of cells was assured by their responsiveness to LH in the form of progesterone release. We found that forskolin significantly increased STC gene expression and secretion by both rat and bovine TICs, an effect that was only replicated by human (h) chorionic gonadotropin (CG). Coincubation of TICs with hCG and phosphodiesterase inhibitors further increased STC secretion, whereas coincubation of TICs with hCG and protein kinase A inhibitors attenuated hCG-stimulated release. Intriguingly, ovarian STC proved to be substantially larger than the 50-kDa homodimer produced in most other tissues. These results indicate that ovarian STC is physically distinct, a feature that could explain its presence in serum during pregnancy and lactation.
29 citations
TL;DR: This in vitro model is capable of demonstrating a true invasion of embryo within the endometrial stroma and may be suitable in studies related to early embryo implantation.
Abstract: One of the limitations in embryo implantation research is the lack of an available in vitro model that faithfully replicates embryo-uterine interactions. In previous studies, embryos were cultured on a monolayer of either uterine epithelial cells or extracellular matrix substratum on which embryos could adhere and outgrow. However, these models failed to display embryonic invasion, primarily because of the shortage of critical structural and molecular supports that are available in vivo. In the present study, we used intact mouse uterine endometrium collected on Day 4 of pregnancy and placed in contact with blastocysts to initiate coculture experiments in a defined medium at the airliquid interface. The culture medium was composed of Ham F12/Dulbecco modified Eagle medium (1:1), 30% fetal calf serum, 63.5 nmol/L of progesterone, 7.14 nmol/L of estradiol-17b, 100 mg/ml of insulin, and 20 ng/ml of epidermal growth factor, whereas the incubation condition was mixed air of 50% oxygen, 5% carbon dioxide, and 45% nitrogen with a humidity of greater than 90% at 378C. Our observations from 24 h of culture clearly demonstrated that embryos were capable of attachment to the uterine endometrium and displayed partial invasion into the endometrial stroma. Interestingly, no outgrowth of trophoblasts on the surface of uterine endometrium was seen, but embryos exhibited a pole-specific attachment. Overall, this model is capable of demonstrating a true invasion of embryo within the endometrial stroma and may be suitable in studies related to early embryo implantation. embryo, implantation
25 citations
Cites background from "Preimplantation development of in v..."
..., 95%) was necessary for endometrial culture; otherwise, it might interrupt the embryonic metabolism by producing excessive free radicals, which results in cellular damage and death of the embryos [22, 23]....
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TL;DR: The co-culture system with bovine oviduct epithelial cells used for the development of bovines zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.
Abstract: There are convincing arguments to suggest that the success of early reproductive events is reliant on a satisfactory dialogue between gametes-embryo and the oviduct epithelium. The aim of this study was to develop and characterize an in vitro model to study these interactions. Cattle zygotes produced in vitro were cultured in either SOF or TCM-199 in the presence or absence of bovine oviduct cell monolayers (BOEC), under 20% or 5% O2 . The embryonic development rate and its quality (cell numbers, cryosurvival) were evaluated, as were the BOEC contents in 11 candidate transcripts (real-time PCR) at different time points. A BOEC co-culture did indeed increase the rate of development in both media under 5% O2 (41 vs 27% and 28 vs 10% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). The effect of BOEC on the developmental rate was more pronounced under 20% O2 (35 vs 6% and 27 vs 4% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). BOEC significantly increased the embryonic cell count in TCM-199 (122.5 ± 11.1 vs 70.3 ± 9.6; p < 0.05) and embryonic cryosurvival in both media. The expression levels of SOD, FGF2 and TGF-β1 in BOEC remained steady during culture, although mRNA levels of OGP, C3, PGR and ESR2 were clearly reduced, suggesting a dedifferentiation of BOEC during culture. However, SSP1 and GPX4 transcripts were slightly increased during culture, this rise becoming significant by the end of the culture period. In conclusion, our co-culture system with bovine oviduct epithelial cells used for the development of bovine zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.
25 citations
Journal Article•
TL;DR: It was concluded that GTE at concentrations of 0.3 mg ml -1 IVM medium improvement the in vitro maturation and embryo development of sheep COC's to blastocyst stage.
Abstract: This study was carried out to investigate the nuclear maturation and embryo development after in vitro fertilization of sheep cumulus oocyte complexes (COCs), either in green tea leaves extract (GTE) supplemented tissue culture medium (TCM-199) at different concentrations (0, 0.3, 0.6 and 1.2 mg ml -1 ). Oocytes of a control group were matured in a maturation medium without GTE (0 mg GTE ml -1 ). After maturation, half of oocytes were fixed and stained to evaluate the nuclear maturation. The rest of oocytes were fertilized in vitro with fresh semen then cultured for 9 days for assessment of the oocytes developmental capacity. The results showed that supplementation of GTE resulted in a significantly (p≤0.05) increasing of oocytes developed to metaphase II (M II) when compared to control group, exception group V that no significant difference compared to group I. Group II showed higher significant M II than other treatments groups. Addition of GTE to IVM medium resulted in significantly improvement of blastocyst formation with 0.3 mg ml -1 concentration than 0.6, 1.2 mg ml -1 , and control group. It was concluded that GTE at concentrations of 0.3 mg ml -1 IVM medium improvement the in vitro maturation and embryo development of sheep COC's to blastocyst stage. Also, addition of GTE at 0.6 mg ml -1 to IVM medium had little benefit in increasing
21 citations
Cites background from "Preimplantation development of in v..."
...This can influence on the early embryo development of mouse, hamster, and bovine embryos (Blondin et al., 1997; Harvey et al., 2002; Watson et al., 1994), sperm motility and axonemal protein phosphorylation (Aitken et al., 1993), and block of in vitro two cell embryos (NasrEsfahani and Johnson,…...
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TL;DR: The results of these experiments indicate that maturation, fertilization, and developmental rates of ovine oocyte matured in the portable incubator are similar to those of oocytes matured in a conventional incubator.
21 citations
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