Benjamin R. Sessions

Abnormal levels of transcript abundance of developmentally important genes in various stages of preimplantation bovine somatic cell nuclear transfer embryos.

Based on microarray data comparing gene expression of fibroblast donor cells and bovine somatic cell nuclear transfer (SCNT) and in vivo produced (AI) blastocysts, a group of genes including several transcription factors was selected for evaluation of transcript abundance. Using SYBR green-based real-time polymerase chain reaction (Q-PCR) the levels of POU domain class 5 transcription factor (Oct4), snail homolog 2 (Snai2), annexin A1 (Anxa1), thrombospondin (Thbs), tumor-associated calcium signal transducer 1 (Tacstd1), and transcription factor AP2 gamma (Tfap2c) were evaluated in bovine fibroblasts, oocytes, embryos 30 min postfusion (SCNT), 12 h postfertilization/activation, as well as two-cell, four-cell, eight-cell, morula, and blastocyst-stage in vitro fertilized (IVF) and SCNT embryos. For every gene except Oct4, levels of transcript were indistinguishable between IVF and SCNT embryos at the blastocyst stage; however, in many cases levels of these genes during stages prior to blastocyst differed significantly. Altered levels of gene transcripts early in development likely have developmental consequences downstream. These results indicate that experiments evaluating gene expression differences between control and SCNT blastocysts may underestimate the degree of difference between clones and controls, and further offer insights into the dynamics of transcript regulation following SCNT.

Oolemma Receptors and Oocyte Activation

At fertilization the sperm triggers a series of intracellular calcium oscillations that are pivotal to oocyte activation and development. Although the biological significance of the characteristic intracellular calcium (Ca2+i) oscillations is not fully understood, calcium ions are known to be involved in cortical granule release and in controlling cell cycle progression. Two different hypotheses attempt to explain how sperm initiate (Ca2+i) oscillations in mammalian oocytes. One hypothesis is that spermatozoa interact with a receptor located in the plasma membrane of the oocyte, which results in induction of pathways leading to activation. This receptor is coupled to a GTP-binding protein or to have tyrosine kinase activity and have the ability to induce activation of phospholipase C (PLC). In turn, PLC stimulates the hydrolysis of phosphatidyl inositol (4,5)–bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), a common Ca2+ releasing compound. Most studies used to develop the mammalian model of oocyte activation have been performed in the mouse. There is a paucity of information from other mammalian models. The predominant mouse model of oocyte activation is that there is a soluble factor (PLC-zeta) delivered to the cytosol after fertilization that induces oocyte activation. However, as data in other mammals is collected, substantial evidence is beginning to support the existence of other more complex oocyte activation pathways in both murine and non-murine systems. Indeed, activation may involve redundant processes, each of which acting alone may be able to induce aspects of oocyte activation. Recent findings demonstrate the involvement of receptors that are known to associate in large, multimeric complexes. This fact leads one to speculate that the process of oocyte activation by the sperm cell is a highly complex and elaborate process that likely involves many more players than perhaps was initially expected.

Colcemid-Treatment of Heifer Oocytes Enhances Nuclear Transfer Embryonic Development, Establishment of Pregnancy and Development to Term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO2 in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods ☆

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

Enucleation of Demecolcine-Treated Bovine Oocytes in Cytochalasin-Free Medium: Mechanism Investigation and Practical Improvement

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.

Why clone horses and mules

The ability to produce offspring from animals otherwise incapable of reproduction has important implications in the equine. Routinely, male equines are electively castrated to produce animals (geldings) that are more easily managed and handled. These animals are then much more conveniently and safety used in various competitions such as cutting, reining, polo, roping, and racing. The ability to recover the genetics of animals that either prematurely die or die prior to sufficient impact of valuable genomics is an important consideration in the horse.

Nicotine combined with okadaic acid or taxol adversely affects bovine oocyte maturation and subsequent embryo development

OBJECTIVE To investigate the effects of nicotine in combination with okadaic acid (OA) or taxol on bovine oocyte maturation and subsequent embryonic development. DESIGN Prospective randomized study. SETTING University research laboratory. PATIENT(S) Bovine ovaries, oocytes, and embryos. INTERVENTION(S) Oocyte maturation and subsequent embryo development in the presence of nicotine in combination with okadaic acid and taxol. MAIN OUTCOME MEASURE(S) Haploid composition, cleavage rate, IVF and parthenogenetic embryo development, blastocyst cell number. RESULT(S) The results showed that nicotine and OA or nicotine and taxol significantly decreased oocyte maturation rates. Combinations of nicotine (10 or 50 micromol/L) and OA (0.01 or 0.05 micromol/L) did not affect oocyte haploid composition; however, taxol alone or combined with nicotine caused a decrease in haploid composition compared with control. Parthenogenetic activation of oocytes that were matured in nicotine, OA, or taxol resulted in blastocyst development rates of 12%-17%, which were not different from the control. Various combinations of nicotine and OA or nicotine and taxol significantly lowered (3%-8%) blastocyst development compared with control. The average cell number of blastocysts derived from nicotine + OA- and nicotine + taxol-treated oocytes was lower than all other treatment groups and control. For oocytes fertilized in vitro, oocytes matured in OA, taxol, or combinations of nicotine and OA or taxol-containing media resulted in significantly lower cleavage rates and blastocyst development compared with the control group and the 10 micromol/L nicotine treatment group. The IVF embryos cultured in nicotine + OA-containing medium had a significantly lower blastocyst development rate. CONCLUSION(S) Combinations of nicotine with OA or taxol adversely affect oocyte maturation and subsequently result in poor blastocyst development.

Genetic and epigenetic regulation of major histocompatibility complex class I gene expression in bovine trophoblast cells

The regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC‐Ia) and non‐classical MHC class I (MHC‐Ib) genes are poorly understood.

Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning

ABSTRACT Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4+ lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8+, FOXP3+, and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

DNA methylation of the LIN28 pseudogene family

BackgroundDNA methylation directs the epigenetic silencing of selected regions of DNA, including the regulation of pseudogenes, and is widespread throughout the genome. Pseudogenes are decayed copies of duplicated genes that have spread throughout the genome by transposition. Pseudogenes are transcriptionally silenced by DNA methylation, but little is known about how pseudogenes are targeted for methylation or how methylation levels are maintained in different tissues.ResultsWe employed bisulfite next generation sequencing to examine the methylation status of the LIN28 gene and four processed pseudogenes derived from LIN28. The objective was to determine whether LIN28 pseudogenes maintain the same pattern of methylation as the parental gene or acquire a methylation pattern independent of the gene of origin. In this study, we determined that the methylation status of LIN28 pseudogenes does not resemble the pattern evident for the LIN28 gene, but rather these pseudogenes appear to acquire methylation patterns independent of the parental gene. Furthermore, we observed that methylation levels of the examined pseudogenes correlate to the location of insertion within the genome. LIN28 pseudogenes inserted into gene bodies were highly methylated in all tissues examined. In contrast, pseudogenes inserted into genomic regions that are not proximal to genes were differentially methylated in various tissue types.ConclusionsOur analysis suggests that Lin28 pseudogenes do not aquire patterns of tissue-specific methylation as for the parental gene, but rather are methylated in patterns specific to the local genomic environment into which they were inserted.