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1.
Sci Rep ; 12(1): 10540, 2022 06 22.
Article in English | MEDLINE | ID: mdl-35732675

ABSTRACT

In vitro production (IVP) of embryos in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or IVP. Fetal ultrasonographies were taken on gestation D55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle ≈ D280). IVP fetuses weighing ≥ 97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.


Subject(s)
Gene Expression Profiling , Insemination, Artificial , Animals , Cattle , Female , Fetus , Insemination, Artificial/veterinary , Pregnancy , Ultrasonography, Prenatal
2.
J Assist Reprod Genet ; 37(2): 417-426, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31838628

ABSTRACT

PURPOSE: We tested whether in vitro production (IVP) causes changes in DNA methylation in fetal liver and skeletal muscle and if exposure of cultured embryos to colony-stimulating factor 2 (CSF2) alters DNA methylation. METHODS: Female fetuses were produced by artificial insemination or transfer of an IVP embryo. Embryos were treated from days 5 to 7 after fertilization with CSF2 or vehicle. DNA methylation in fetal liver and skeletal muscle was determined by post-bisulfite adaptor tagging-based sequencing. The degree of DNA methylation for CpG sites in 50-bp windows of the promoter region 500 bp upstream of the transcriptional start site was compared between treatments. RESULTS: For liver, there were 12 genes (6% of those analyzed) in which DNA methylation was affected by treatment, with one 50-bp window per gene affected by treatment. For muscle, the degree of DNA methylation was affected by treatment for 32 windows (19% of the total windows analyzed) representing 28 distinct genes (23% of analyzed genes). For 19 of the 28 genes in muscle, the greatest deviation in DNA methylation was for the CSF2 group. CONCLUSION: Results are consistent with alterations in the methylome being one of the mechanisms by which IVP can result in altered fetal development and postnatal function in the resultant offspring. In addition, results indicate that maternally derived cell-signaling molecules can regulate the pattern of DNA methylation.


Subject(s)
DNA Methylation/genetics , Embryo Culture Techniques/methods , Embryonic Development/genetics , Epigenome/genetics , Animals , Blastocyst/metabolism , Cattle , Embryo, Mammalian/metabolism , Female , Fertilization in Vitro/methods , Gene Expression Regulation, Developmental/genetics , Insemination, Artificial , Pregnancy
3.
Arch Toxicol ; 83(11): 1001-7, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19593550

ABSTRACT

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 microM) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 muM can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.


Subject(s)
Insulin-Like Growth Factor I/pharmacology , Reactive Oxygen Species/metabolism , Vitamin K 3/toxicity , Vitamins/toxicity , Animals , Apoptosis/drug effects , Blastocyst/metabolism , Cattle , Dose-Response Relationship, Drug , Embryonic Development/drug effects , In Situ Nick-End Labeling , Vitamin K 3/administration & dosage , Vitamins/administration & dosage
4.
J Reprod Dev ; 55(1): 69-74, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18957823

ABSTRACT

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.


Subject(s)
Cattle , Cellular Senescence/physiology , Embryonic Development , Hot Temperature/adverse effects , Sex Ratio , Spermatozoa/physiology , Animals , Blastocyst/physiology , Cattle/embryology , Cells, Cultured , Cleavage Stage, Ovum/physiology , Ejaculation , Embryo, Mammalian , Female , Male , Pregnancy , Sperm Motility/physiology , Stress, Physiological/physiology
5.
J Reprod Dev ; 54(3): 217-20, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18277054

ABSTRACT

The occurrence of apoptosis in a fraction of blastomeres in the preimplantation embryo is well known but the consequences of this phenomenon for the developmental potential of the blastocyst has not been well established. Here we demonstrate that blastocysts with low amounts of activated group II caspase activity have increased potential for development to the hatched blastocyst stage. Bovine blastocysts produced in vitro were assayed using a non-invasive fluoregenic substrate that is cleaved by activated group II caspases (i.e., caspase-2, -3 and -7). Subsequently, blastocysts were cultured until Day 10 post-insemination and the proportion undergoing hatching determined. In Experiment 1, blastocysts were cultured without respect to stage of development (expanded or non-expanded); blastocysts classified as having low caspase activity had higher hatching rates than blastocysts with medium or high caspase activity. In Experiment 2, embryos were categorized as nonexpanded or expanded blastocysts. Caspase activity was lower and hatching rate higher for expanded blastocysts than for nonexpanded blastocysts. For nonexpanded blastocysts, embryos classified as having low caspase activity had higher hatching rates as compared to embryos with medium or high caspase activity. In conclusion, the capacity for blastocysts to undergo further development is related to degree of group II caspase activity. Conditions that enhance the incidence of apoptosis in blastocysts may reduce developmental competence. In addition, determination of caspase activity may be useful for selection of embryos for transfer into recipients.


Subject(s)
Blastocyst/cytology , Blastocyst/enzymology , Caspases/metabolism , Cattle , Embryo Transfer/veterinary , Animals , Apoptosis/physiology , Caspase 2/metabolism , Caspase 3/metabolism , Caspase 7/metabolism , Embryo Transfer/methods , Enzyme Activation/physiology , Female , Pregnancy , Zona Pellucida/enzymology
6.
Reproduction ; 133(6): 1129-37, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17636167

ABSTRACT

Heat shock and tumor necrosis factor-alpha (TNF-alpha) induce apoptosis through different mechanisms, with heat shock acting to cause mitochondrial depolarization and caspase-9 activation, while TNF-alpha acts through a receptor-mediated process to activate caspase-8. In some cells, however, TNF-alpha can also cause mitochondrial depolarization and caspase-9 activation. In the present study, we tested the hypothesis that heat shock at 41 degrees C and TNF-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism. Treatment of embryos with either heat shock (41 degrees C) or TNF-alpha increased the proportion of blastomeres that were TUNEL positive and the proportion of embryos exhibiting elevated caspase-9 activity. Furthermore, the caspase-9 inhibitor, z-LEHD-fmk, blocked the increase in TUNEL-positive nuclei caused by both heat shock and TNF-alpha. For embryos at day 6 after insemination, for example, the percent of blastomeres positive for TUNEL was 3.6% for control embryos, 11.1% for embryos cultured at 41 degrees C, and 15.1% for embryos cultured with 10 ng/ml TNF-alpha. In the presence of z-LEHD-fmk, the percent of cells positive for TUNEL was 3.7% for control embryos, 6.1% for embryos cultured at 41 degrees C, and 8% for embryos cultured with 10 ng/ml TNF-alpha. Although TNF-alpha did not cause a measurable increase in caspase-8 activity, there was a tendency (P = 0.07) for treatment of embryos with z-IETD-fmk, an inhibitor of caspase-8, to partly reduce the magnitude of the increase in TUNEL-positive cells caused by TNF-alpha. The percent of cells that were TUNEL positive was increased by TNF-alpha from 9.7 to 19.7% in the absence of inhibitor and from 13.0 to 15.6% in the presence of z-IETD-fmk. Results indicate that induction of apoptosis by both heat shock and TNF-alpha involve activation of caspase-9-dependent pathways. It is likely that TNF-alpha also activates apoptotic pathways involving caspase-8 but that the degree of activation is small and caspase-9-dependent pathways are required for full activation of apoptosis.


Subject(s)
Blastocyst/cytology , Cattle/physiology , Hot Temperature , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis/drug effects , Biomarkers/analysis , Blastocyst/drug effects , Blastocyst/enzymology , Caspase 9/analysis , Caspase 9/metabolism , Caspase Inhibitors , Embryonic Development/drug effects , Enzyme Activation , Female , In Situ Nick-End Labeling , Oligopeptides/pharmacology , Pregnancy , Tissue Culture Techniques
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