In-situ characterization of porcine fibroblasts in response to silver ions by Raman spectroscopy and liquid scanning transmission electron microscopy.

Since silver ion is known for its antimicrobial function, most of the research has focused mainly on toxicity effects rather than the role of silver ion in general biology and the behind mechanism of actions of silver ion in mammalian cells. Moreover, a conventional in vitro approach to estimate the effects of silver ion on cells does not provide information about the biochemical changes and might accompany artifacts due to invasive and destructive sample preparation processes. In the present study, in-situ real time approaches were applied to evaluate the impact of silver ion (0.57, 1.34, 1.96, 2.33 mg/L) on fibroblast cells. Raman spectroscopy analysis showed that Raman peak intensities of proteins and nucleic acids significantly increased in the cells after exposure to silver ion for 21 h, especially at relatively higher levels 1.34, 1.96, and 2.33 mg/L. Raman peak at 1585 cm-1 and liquid scanning transmission electron microscopy energy-dispersive x-ray spectroscopy (STEM-EDS) analysis revealed the fate of silver ion that was taken up by the cell and reduced into metallic silver accumulating in the cell as silver nanoparticles. These results suggest cells were undergoing different activities such as enhanced metabolic activities rather than cell apoptosis or cell death. Additionally, Raman spectroscopy predicted the level of silver ion exposed to the cell at 2.11 ± 0.38 and 1.73 ± 0.26 mg/L by the PLS prediction model, compared with the results measured by inductively coupled plasma mass spectrometry (ICP-MS), 2.14 ± 0.07 and 1.87 ± 0.07 mg/L respectively, suggesting Raman spectroscopy can provide a new and fast approach to determine and measure the concentration of silver ion or probably other tested molecules treated to the cell for the future research.

Effects of electrical biostimulation and silver ions on porcine fibroblast cells.

The medical applications of electrical biostimulation and silver ions have been evaluated in laboratory experiments and clinical studies for more than two decades. Their effects on preventing infection and promoting wound healing have been described. However, little is known about the role of electrical biostimulation and/or silver ion on changes in cellular transcriptome dynamics. To our knowledge, few studies have been conducted to investigate the potential of electrical biostimulation and silver ions in cell reprogramming. Besides, it is essential to assess any possible adverse effects or potential benefits of the silver ions on mammalian cells to address its safety concerns and to improve silver medical products. In this study, we investigated transcriptomic changes in porcine fibroblast cells in response to electrical biostimulation in the presence of silver ions. Exposed cells presented distinct morphological changes after treatment, which was mainly due to the exposure of silver ions rather than the electrical current itself. Gene expression analyses suggested that electrical biostimulation and silver ions did not increase the expression of pluripotency genes. Interestingly, a set of genes related to cellular metabolic processes were differentially expressed after cells were exposed to electrically generated silver ions for 21 hours. We found that 2.00 mg/L of electrically generated silver ion caused an increase of ATP generation and an increase of the total pool of NAD+ and NADH, while ROS production did not change. Aside from toxic effects, the results reported herein demonstrate the alternative effects of silver ions on mammalian cells, especially an oxidative phosphorylation burst. To our knowledge, this response of mammalian cells to silver ions has not been described previously. Although the function of this burst is not understood, it may lead to alterations in cellular activities such as metabolic remodeling and cell reprogramming, and/or serve an as-yet unknown function in neutralization or detoxification of the silver ions within the cells.

Serial Culture Is Critical for In Vitro Development of Parthenogenetic Embryos in the Golden Syrian Hamster.

Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.15%) or HECM-10 (63.82%). None of the cleaved embryos developed beyond the two-cell stage when cultured in either of the two media. However, some of the embryos overcame the two-cell block and developed to the blastocyst stage (26.45%) when they were first cultured in HECM-10 for 24 hours and then in HECM-9 (serial culture media HECM-10-9) for 72 hours. Blastocyst development was further significantly (66.2%) improved when embryos were cultured in HECM-10 supplemented with ethylenediaminetetraacetic acid for 24 hours, then in HECM-9 supplemented with glucose for 72 hours (serial culture media HECM-11a-b). Hamster oocytes activated with ionomycin, ethanol, or a combination of the two treatments would develop to the blastocyst stage in serial culture media HECM-11a-b, whereas none of the spontaneously activated oocytes cleaved (0% vs. 86.93%, p < 0.05). DNA and microtubule configurations of spontaneously activated and AA oocytes were assessed by immunocytochemical staining and fluorescence microscopy. The results indicate that serial culture and the method of activation are critical for overcoming the in vitro developmental block of hamster parthenogenetic embryos. This study is the first to report blastocyst development from parthenogenetically activated hamster oocytes.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.

Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

SGO1 maintains bovine meiotic and mitotic centromeric cohesions of sister chromatids and directly affects embryo development.

Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV) to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s) of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.

Decreased expression of CD9 in bovine oocytes after cryopreservation and the relationship to fertilization capacity.

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity.

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.

Effect of cryopreservation on acetylation patterns of lysine 12 of histone H4 (acH4K12) in mouse oocytes and zygotes.

PURPOSE: to determine the effect of cryopreservation on acH4K12 in oocytes and their respective zygotes. METHODS: AcH4K12 in fresh or vitrified-warmed oocytes and their respective zygotes at 70 min-12 h post-fertilization were assessed using fluorescent staining. RESULTS: 1. AcH4K12 levels increased significantly in vitrified oocytes compared to controls. 2. Respective zygotes derived from vitrified oocytes had abnormal chromatin distribution or acH4K12 patterns before and after pronuclear formation. CONCLUSION: Cryopreservation alters AcH4K12 patterns in oocytes, which subsequently affect the chromatin distribution and acH4K12 in fertilized oocytes.

In vitro development and chromosomal configuration of bovine somatic cloned embryos with nonenucleated metaphase II oocytes.

This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MII spindle, 94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MII spindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9%) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs. 23.1%; 27.5 vs. 67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n, 2n/8n, and 2n/4n/8n. (6) Nuclear transfer preceding the oocyte enucleation experiment indicated that prolonged presence of oocyte nuclei induced abnormal DNA configuration and reduced in vitro development of transferred somatic nuclei, but short time presence of oocyte nuclei did not affect the in vitro development of cloned embryos. We conclude that oocyte MII spindles induce donor cell PCC, the developmental capacity of cloned embryos reconstituted with nonenucleated oocytes is inferior to those with enucleated oocytes, and that all such derived blastocysts are polyploidy.

Changes in acetylation on lysine 12 of histone H4 (acH4K12) of murine oocytes during maternal aging may affect fertilization and subsequent embryo development.

OBJECTIVE: To compare acH4K12 levels in oocytes during mouse aging and then assess how such changes might affect the developmental potential of oocytes. DESIGN: Experimental animal study. SETTING: State key laboratory and university research laboratory. ANIMAL(S): Kunming white strain mice. INTERVENTION(S): Oocytes obtained from TSA treated group or aging mouse group were fertilized and the formation of pronuclei and subsequently developmental potential in vitro or in vivo were assessed. MAIN OUTCOME MEASURE(S): AcH4K12 levels in oocytes were assessed using fluorescence staining, and confocal microscopy and oocyte developmental potentials were determined by in vitro or in vivo methods. RESULT(S): The AcH4K12 levels in oocytes statistically significantly increased during mouse aging. When histone acetylation of oocytes of young mice was artificially increased by trichostatin A (TSA) treatment, the acH4K12 levels in male and female pronuclei in fertilized oocytes showed statistically significant changes. About 38.9% of TSA-treated oocytes failed to form pronuclei or formed morphologically abnormal pronuclei 6 hours after fertilization, which statistically significantly decreased the blastocyst rate of TSA-treated oocytes when compared with the control group (41.5% vs. 60.5%). A similar reduction in blastocyst development was also observed when oocytes collected in older mice were compared with younger mice (17.3% vs. 69.4%). CONCLUSION(S): The AcH4K12 levels in oocytes statistically significantly increased during the aging process in mice, and such changes may affect the acetylation patterns and morphology of pronuclei during fertilization and lead to a reduction in oocyte developmental potential.

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.

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.

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.

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.

Specific integrin subunits in bovine oocytes, including novel sequences for alpha 6 and beta 3 subunits.

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of alphaV, alpha6, alpha4, alpha2, ss1, and ss3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine alpha6 and beta3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins.

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.