scholarly journals Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems

Reproduction ◽  
2001 ◽  
pp. 601-610 ◽  
Author(s):  
MA Yaseen ◽  
C Wrenzycki ◽  
D Herrmann ◽  
JW Carnwath ◽  
H Niemann
Keyword(s):  
Growth Factor ◽  
Polyvinyl Alcohol ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Culture Systems ◽  
Igf I ◽  
Oviduct Fluid

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals 243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
P. Lonergan ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P.M. Moreira ◽  
B. Pintado ◽  
...  
Keyword(s):  
Growth Factor ◽  
Relative Abundance ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P&lt;0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P&lt;0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Serum Free ◽  
Epidermal Growth ◽  
Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P &lt; 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P &gt; 0.05), whereas the combination P4 + EGF was better than P4 alone (P &lt; 0.05). Blastocyst rate was not different (P &gt; 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P &lt; 0.05) blastocyst rate than control but it was lower (P &lt; 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 195
Author(s):  
R. Nishii ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Culture System ◽  
Calf Serum ◽  
Control Culture ◽  
Total Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Culture Systems ◽  
Individual Culture ◽  
Single Culture

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development

The effect of bovine embryo culture without proteins supplements until day 4 on transcription level of hyaluronan synthases, receptors and mtDNA content

Zygote ◽  
2009 ◽  
Vol 18 (2) ◽  
pp. 121-129 ◽  
Author(s):  
A.T. Palasz ◽  
P. Beltrán Breña ◽  
J. De la Fuente ◽  
A. Gutiérrez-Adán
Keyword(s):  
Embryo Culture ◽  
Receptor Gene ◽  
Blastocyst Stage ◽  
Low Molecular Weight ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Hyaluronan Synthases ◽  
Copy Numbers ◽  
Surface Active

SummaryThe effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration andin vitrodevelopment. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to ≤8-cell stage by day 4 were not affected by lack of BSA in the medium, but increased numbers of blastocysts developed in medium supplemented with BSA from days 1 or 4 with or without HA than in medium that had HA only. Addition of both HA and BSA at day 4 increased mtDNA copy numbers at the blastocyst stage. Data suggest that the addition of BSA and/or HA at 96 hpi increased expression ofRHAMMandHas2genes, but notCD44,Has1orHas3genes. Higher expression levels of Has2 than Has1 and the three isoforms indicate that high- rather than low-molecular-weight HA should be used for preimplantation bovine embryo culture.

scholarly journals 6ACETYLATION OF HISTONE H4 LYSINE-5 AND LYSINE-8 DURING DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 125
Author(s):  
W.E. Maalouf ◽  
R. Alberio ◽  
K.H.S. Campbell
Keyword(s):  
Dna Methylation ◽  
Cell Mass ◽  
Staining Intensity ◽  
Blastocyst Stage ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Histone H4 ◽  
Cell Stage ◽  
Intense Staining

The oocyte is remarkable in its ability to remodel the parental genomes following fertilization and to reprogram somatic nuclei as in nuclear transfer. While significant research has been carried out on DNA methylation patterns in the early embryo, increased interest in histone acetylation is more recent. The objective of this study was to characterize the pattern of acetylation of histone H4 lysine-5 (H4L5) and lysine-8 (H4L8) in the early pre-implantation bovine embryo. Bovine embryos were produced as previously described (Fouladi Nashta AA et al., 1998 Biol. Rep. 59, 255–262) and collected at different developmental stages, 1-cell (20h), 2-cell (30h), 4- and 8-cell (Day 2), 16-cell (Day 4), and blastocyst (Days 7–8) with an average of 6 embryos per group in two replicates. Embryos were fixed in 2.5% paraformaldehyde, 15min at room temperature (RT), stained with polyclonal rabbit antibodies against H4L5 (1:800) and H4L8 (1:600) residues (Serotec, UK) at 4°C overnight. A polyclonal swine anti-rabbit (1:200; Dako, Denmark) was used as secondary antibody for 40min at RT. Images were examined using a fluorescent microscope (Leica DMR, Germany). Image analysis and quantification were performed using Simple PCI software (Compix Imaging Systems, USA). Changes in intensities within and between different embryo stages were recorded as a ratio of red stain to blue counterstain. Data were corrected for confounding area and absorbance and analysed using a multivariate linear regression model. The intensity of staining for H4L5 appeared higher in 8-cell embryos than 2- and 4-cell embryos but not to a significant level (P≥0.05); 8-cell embryos also appeared higher in stain intensity than 16-cell but of borderline significance (P=0.073). Staining intensity decreased between the 8-cell and blastocyst stage (P≤0.05). In contrast, the intensity of acetylation staining for H4L8 residue decreased slightly between the 1- and 4-cell stages and then decreased significantly between the 4- and 8-cell stages (P≤0.05), increasing significantly by the 16-cell stage (P≤0.05). A significant decrease in staining intensity was observed at the blastocyst stage (P≤0.05). In blastocyst-stage embryos both lysine-5 and lysine-8 showed a differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. ICM cells showed intense staining and TE cells stained very weakly. The intensity results presented are cumulative of ICM and TE intensities, which explains the overall low levels of acetylation in blastocysts when compared to the earlier stages. Acetylation of H4L5 starts high in 1-cell embryo, as it is necessary for protamine replacement (Adenot et al., 1997 Development 124, 4615–4625), decreases when methylation is high and increases when methylation is low (as in the 8-cell stage which corresponds with zygotic gene activation). Acetylation of H4L8 decreases between the 1-and 8-cell stages; however, its association with changes in DNA methylation has yet to be determined.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

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