Increased expression of pro-inflammatory cytokines at the fetal-maternal interface in bovine pregnancies produced by cloning.

PROBLEM: A significant rate of spontaneous abortion is observed in cattle pregnancies produced by somatic cell nuclear transfer (SCNT). Major histocompatibility complex class I (MHC-I) proteins are abnormally expressed on the surface of trophoblast cells from SCNT conceptuses. METHOD OF STUDY: MHC-I homozygous compatible (n = 9), homozygous incompatible (n = 8), and heterozygous incompatible (n = 5) pregnancies were established by SCNT. Eight control pregnancies were established by artificial insemination. Uterine and trophoblast samples were collected on day 35 ±1 of pregnancy, the expression of immune-related genes was examined by qPCR, and the expression of trophoblast microRNAs was assessed by sequencing. RESULTS: Compared to the control group, trophoblast from MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL1A, IL2, IL10, IL12B, TBX21, and TNF, while GNLY expression was downregulated. The MHC-I homozygous incompatible treatment group expressed increased levels of IFNG, IL1A, and IL2 while the MHC-I homozygous compatible group did not differentially express any genes compared to the control group. In the endometrium, relative to the control group, MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL10, IL12B, and TNF, while GATA3 expression was downregulated. The MHC-I homozygous incompatible group expressed decreased amounts of CSF2 transcripts compared with the control group but did not have abnormal expression of any other immune-related genes. MHC-I incompatible pregnancies had 40 deregulated miRNAs compared to control pregnancies and 62 deregulated microRNAs compared to MHC-I compatible pregnancies. CONCLUSIONS: MHC-I compatibility between the dam and fetus prevented an exacerbated maternal immune response from being mounted against fetal antigens.

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

PROBLEM: 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. METHOD OF STUDY: Quantitative reverse transcription- polymerase chain reaction (PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5'-aza-deoxycytidine was used to assess the role of methylation in gene regulation. RESULTS: MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator (CIITA), and STAT1. The MHC-Ia genes and BoLA-NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of BoLA-NC2, -NC3, and -NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold. CONCLUSION: DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC.

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

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.

BACKGROUND: DNA 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. RESULTS: We 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. CONCLUSIONS: Our analysis suggests that Lin28 pseudogenes do not acquire 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.

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.

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 (Ca(2+)(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 (Ca(2+)(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 Ca(2+) 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.

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.

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 microg/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 from colcemid-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.

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.

Nicotine induces multinuclear formation and causes aberrant embryonic development in bovine.

The present study was designed to investigate the effects of nicotine on development of bovine embryos derived from parthenogenetic activation (PA) and in vitro fertilization (IVF). Nicotine caused disfigured secondary meiotic spindle structures and affected embryonic development in a dose-dependent manner. Concentrations at 0.01-0.5 mM resulted in cleavage and blastocyst rates similar to the controls for both PA and IVF embryos. Nicotine at 2.0 and 4.0 mM significantly decreased the cleavage rates and none of the embryos developed beyond the 16-cell stage. Nicotine might disrupt the polymerization of microfilaments leading to impaired chromosome alignment or segregation, and induce the formation of polynuclei with a variety of abnormal nuclear structures such as 2-6 nuclei, 2-4 metaphase plates, 2-4 sets of anaphase/telophase plates, and the co-existence of polynuclei and 2-4 sets of anaphase/telophase plates. Nicotine adversely affected blastocyst chromosomal composition. Fifty-six to 70% of the IVF blastocysts and 71-88% of the PA blastocysts were polyploid and/or mixoploid after culture in 0.2-1.0 mM nicotine-containing media, which were higher (P < 0.05 or P < 0.01) than the controls. Cell numbers of the nicotine-cultured blastocysts were significantly lower than the control. In conclusion, nicotine induced disfigured spindles and irregular chromosome alignment and possibly impaired cytokinesis, which lead to decreased quality of the yielded blastocysts.

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes.

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.

Effect of nicotine on in vitro maturation of bovine oocytes.

The putative effect of nicotine on maturation and the chromosomal complement of bovine oocytes were investigated in the present study. Cumulus-enclosed oocytes were incubated in maturation medium with 0, 0.5, 1.0, 2.5, 5.0, and 10.0 mmol concentrations of nicotine. The results indicated that: (1) nicotine affected cumulus cell expansion in a dose-dependent manner and the perivitelline space failed to form when concentrations were equal to or greater than 5.0 mmol; (2) oocytes treated with 0.5 and 1.0 mmol nicotine concentrations resulted in maturation rates (83.3% and 85.9%, respectively) which was similar to the control (86.2%), whereas treatment with 2.5 and 5.0 mmol concentrations significantly decreased maturation rates to 70.2% and 26.7%, respectively; (3) nicotine at or over 2.5 mmol caused extremely irregular meiotic spindles and interrupted microfilament organization; (4) chromosomal analyses of oocytes with PB1 showed that oocytes derived from 0.5 and 1.0 mmol nicotine groups had haploid complements similar to the control (87-90%), but when the concentrations were increased to 2.5 and 5.0 mmol the haploid state was significantly reduced to around 70%; (5) oocytes at GVBD (germinal vesicle breakdown) and metaphase I stages were less affected by nicotine at 5.0 and 10.0 mmol concentrations than GV-stage oocytes; (6) maturation rates of the short-term nicotine-treated oocytes could be improved when subsequently incubated in normal maturation medium. Prolonged culture of nicotine-pretreated oocytes resulted in self-activation and some oocytes formed 1 or 2 pronuclei. In conclusion, nicotine affects bovine oocyte cumulus cell expansion, maturation rate, and chromosomal complement in a dose-dependent and an oocyte-stage-dependent manner.

Nicotine alters bovine oocyte meiosis and affects subsequent embryonic development.

The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development.

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

The developmental competence of bovine nuclear transfer embryos derived from cow versus heifer cytoplasts.

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos. In order for nuclear transfer to be successful, a differentiated donor cell must be reprogrammed and restored to a totipotent state. This reprogramming is probably accomplished by factors within the oocyte cytoplasm. This study indicates that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared to heifer oocytes based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages. Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes and resulted in higher pregnancy retention at 90 and 180 days and a greater number of term deliveries. Following delivery more calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos.

Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media.

Treatment of in vitro matured bovine oocytes with colcemid results in a membrane protrusion that contains maternal chromosomes, which can be easily removed by aspiration. Four experiments were designed to evaluate the overall and temporal effects of conditioned medium (CM) by bovine cumulus cells on development of nuclear transfer (NT) bovine embryos and to examine the chromosomal composition and allocation of inner cell mass (ICM) and trophectoderm (TE) of the subsequent blastocysts. The nuclear transfer embryos were cultured in various CR1aa media conditioned by preculture with bovine cumulus cells. Development to the blastocyst stage in BSA-containing CM (BCM) and serum-containing CM (SCM) were similar to co-culture group (24-30%). The 24 hr-conditioned BCM yielded higher blastocyst development than 48 and 72 hr-conditioned BCM. Temporary exposure of embryos to BCM and SCM followed by CR1aa was also studied. Morula and blastocyst development were not different among the groups cultured in BCM for 72, 96, and 168 hr, but were significantly higher (P < 0.01) than groups exposed to BCM for 24 and 48 hr, respectively. Blastocyst development in SCM for 24 hr (29%), 96 hr (25%), and 168 hr (27%) were much higher (P < 0.05) than those in SCM for 48 hr (12%) and 72 hr (10%). The analyses of chromosomal composition of the resulting blastocysts indicate approximately 80% of the blastocysts cultured in CR1aa with co-culture or groups initially exposed to BCM for 24 hr followed by culture in CR1aa were diploid. However, the incidence of diploidy were only 36-60% in SCM-cultured groups and groups cultured in BCM beyond 48 hr. Conditioned media did not affect the allocation of ICM and TE in the blastocyst. No difference was found in the ratio of inner cell mass to total cells in co-culture, BCM or SCM groups (0.424, 0.441, and 0.473, respectively). In conclusion, bovine cumulus cell-CM and CR1aa with co-culture supported comparable development and blastocyst ICM:total cell ratio of bovine NT embryos. However, CM affected the blastocyst chromosomal composition and induced higher mixploidy.