The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro

2002 ◽  
Vol 14 (7) ◽  
pp. 443 ◽  
Author(s):  
F. Figueiredo ◽  
G. M. Jones ◽  
G. A. Thouas ◽  
A. O. Trounson
Keyword(s):  
Culture Medium ◽  
Preimplantation Embryo ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Sulfate Proteoglycan ◽  
Preimplantation Embryo Development ◽  
Cell Numbers ◽  
Mouse Preimplantation Embryo

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte–macrophage colony-stimulating factor.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 137
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
D. Hermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Cell Numbers ◽  
Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

scholarly journals Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy

2019 ◽  
Vol 116 (28) ◽  
pp. 14105-14112 ◽  
Author(s):  
Lei Huang ◽  
Berhan Bogale ◽  
Yaqiong Tang ◽  
Sijia Lu ◽  
Xiaoliang Sunney Xie ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Inner Cell Mass ◽  
Culture Media ◽  
False Negative ◽  
Cell Mass ◽  
False Negative Rate ◽  
Critical Examination ◽  
Preimplantation Genetic Testing ◽  
Embryo Mosaicism

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

scholarly journals The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity

Open Biology ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 170210 ◽  
Author(s):  
Aleksandar I. Mihajlović ◽  
Alexander W. Bruce
Keyword(s):  
Cell Fate ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Cell Mass ◽  
Cell Fate Decision ◽  
Preimplantation Embryo Development ◽  
Cell Lineages ◽  
Mouse Preimplantation Embryo ◽  
Fate Decision ◽  
Mouse Preimplantation Embryo Development

During the first cell-fate decision of mouse preimplantation embryo development, a population of outer-residing polar cells is segregated from a second population of inner apolar cells to form two distinct cell lineages: the trophectoderm and the inner cell mass (ICM), respectively. Historically, two models have been proposed to explain how the initial differences between these two cell populations originate and ultimately define them as the two stated early blastocyst stage cell lineages. The ‘positional’ model proposes that cells acquire distinct fates based on differences in their relative position within the developing embryo, while the ‘polarity’ model proposes that the differences driving the lineage segregation arise as a consequence of the differential inheritance of factors, which exhibit polarized subcellular localizations, upon asymmetric cell divisions. Although these two models have traditionally been considered separately, a growing body of evidence, collected over recent years, suggests the existence of a large degree of compatibility. Accordingly, the main aim of this review is to summarize the major historical and more contemporarily identified events that define the first cell-fate decision and to place them in the context of both the originally proposed positional and polarity models, thus highlighting their functional complementarity in describing distinct aspects of the developmental programme underpinning the first cell-fate decision in mouse embryogenesis.

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