Inositol transport in preimplantation rabbit embryos: effects of embryo stage,sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [3H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol-1 in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 mmol-1 [corrected] for Km were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.     

Embryonic genotype and inbreeding affect preimplantation development in cattle

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.     

The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality

A series of Ca(2+) oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca(2+) signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca(2+) increase, with ethanol that causes a single large Ca(2+) increase, or with Sr(2+) that causes Ca(2+) oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stagecompared tothose activated by Sr(2+) orethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr(2+)-activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca(2+) increase because they developed to blastocyst stages at high rates when co-treated with Sr(2+) or ethanol. Embryos activated by either Sr(2+) or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr(2+) leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca(2+) signal does not affect initial parthenogenetic events, but can influence later gene expression and development.     

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.     

Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 x 10(4) copies of macaque mtDNA and 1.3 x 10(6) copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.     

The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage

The capacity of the preimplantation embryo to undergo apoptosis in response to external stimuli is developmentally regulated. Acquisition of apoptosis does not occur in the cow embryo until between the 8- and 16-cell stages. The purpose of the present experiments was to determine the mechanism by which apoptosis is blocked in the bovine two-cell embryo. Heat shock (41 degrees C for 15 h) did not increase activity of caspase-9 or group II caspases (caspase-2, -3, and -7) in two-cell embryos but did in day 5 embryos. Exposure of embryos to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to depolarize mitochondria resulted in activation of caspase-9 and group II caspases at both stages of development. For day 5 embryos, CCCP also increased the proportion of blastomeres that underwent DNA fragmentation as determined by the TUNEL assay. In contrast, CCCP did not increase TUNEL labeling when applied at the two-cell stage. In conclusion, failure of heat shock to increase caspase-9 and group II caspase activity in the two-cell embryo indicates that the signaling pathway leading to mitochondrial depolarization and caspase activation is inhibited at this stage of development. The fact that CCCP treatment of two-cell embryos induced caspase-9 and group II-caspase activity indicates that caspase activation is possible following mitochondrial depolarization. However, since CCCP did not increase TUNEL labeling of two-cell embryos, actions of group II-caspases to activate DNases is inhibited.     

Short communication: lack of stage-specific embryonic antigen-1 expression by bovine embryos and primordial germ cells

The objective of this study was to determine whether stage-specific embryonic antigen-1, a cellular marker commonly used to identify murine undifferentiated embryonic cells, is also a useful marker for bovine pluripotent cells. Expression of stage-specific embryonic antigen-1 was examined by indirect immunohistochemistry on bovine preimplantation embryos and on primordial germ cells contained in the genital ridge. Expression of stage-specific embryonic antigen-1 was not observed in any of the cleavage-stage bovine embryos examined, including one-cell, two-cell, four-cell, eight-cell, morula, and blastocyst stages, nor in tissue sections of bovine genital ridges collected from embryos on d 34, 37, and 40 of gestation. As expected, expression of stage-specific embryonic antigen-1 was detected on murine preimplantation embryos and on murine teratocarcinoma cells. Results of this study indicate that, unlike in the mouse, stage-specific embryonic antigen-1 is not a useful cellular marker for pluripotent bovine embryonic cells or bovine primordial germ cells.     

Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes after in vitro fertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result of de novo synthesis at the onset of differentiation of the pluripotent blastomeres.     

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.     

Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators

The present study was undertaken to examine the effects of various transition metal ion chelators, both polyaminocarboxylates (including nitrilotriacetate (NTA), ethylenediaminediacetate (EDDA), ethyleneglycolbistetraacetate (EGTA), ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA)) and non-polyaminocarboxylates (dipicolinic acid and deferoxamine), on the development in vitro of one-cell ICR strain mouse embryos to the four-cell and blastocyst stages. The order of stability constants of polyaminocarboxylates for transition metal ions such as zinc, copper and iron is as follows: NTA < or = EDDA < EGTA < EDTA < DTPA. Addition of 10 or 100 micromol polyaminocarboxylates x l(-1) to the medium significantly enhanced the development of most one-cell embryos (66-88%) beyond the two-cell stage compared with that (< 25%) in medium without polyaminocarboxylates. Although EDDA, EDTA and DTPA at 10 micromol x l(-1) induced the development of most one-cell embryos to the four-cell stage and beyond, a higher concentration (100 micromol x l(-1)) of NTA and EGTA was required to obtain a similar result. Therefore, the ability of polyaminocarboxylates to overcome the two-cell block is not correlated with their potency to chelate transition metal ions. In contrast, the non-polyaminocarboxylates dipicolinic acid and deferoxamine, at 10 and 100 micromol x l(-1), did not have the same effect. Taken together, the results indicate that the ability of polyaminocarboxylates to overcome the two-cell block in embryo development is due to some common feature or features other than the ability to chelate transition metal ions.     

Growth hormone,insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alpha IR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alpha IR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, GH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.     

Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro

The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.     

Apoptosis in rabbit embryos produced by fertilization or nuclear transfer with fibroblasts and cumulus cells

In this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts, the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.     

Manipulation of antioxidant status fails to improve fertility of lactating cows or survival of heat-shocked embryos

Experiments were conducted to test whether enhancement of antioxidant status could improve fertility and milk yield in dairy cows and resistance of cultured embryos to heat shock. Three experiments in three herds were performed to evaluate the effect of multiple intramuscular injections of 500 mg of vitamin E and 50 mg of selenium at 8 to 21 d before expected calving and at 30 and 80 d postpartum on reproduction of lactating Holstein cows. Vitamin E and selenium injections did not improve reproductive function or milk yield in any of the studies. The predicted 305-d milk yield (averages of least-squares means across treatments) were: 9478, 7073, and 10,204 kg projected 305-d milk for experiments 1, 2, and 3, respectively. Percentages of cows pregnant at first service were 30, 16, and 24% in experiments 1, 2, and 3, respectively. Three studies were performed to test whether vitamin E improved development of cultured bovine embryos exposed to heat shock. Heat shock of 41 degrees C at the two-cell stage reduced development to the blastocyst stage, but culture with 100 microM vitamin E did not reduce effects of heat shock on impaired development. For example, 9 h at 41 degrees C reduced blastocyst development from 51.2 +/- 3.3% to 3.4 +/- 3.3% in the absence of vitamin E and from 54.0 +/- 3.3% to 5.2 +/- 3.3% in the presence of vitamin E. Development of morulae to the blastocyst stage was not compromised by culture at 41 degrees C for 9 h. Additionally, there was no overall effect of vitamin E on morula development. In conclusion, multiple injections of vitamin E and selenium at the administered levels did not improve postpartum fertility nor milk yield of lactating Holstein cows in three different herds, and there was no direct thermoprotective effect of vitamin E for cultured, heat-shocked embryos.     

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.