Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons.

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day-70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos.

Porcine conceptus Oct-4 mRNA expression during peri-implantation development.

OCT-4 is a transcriptional regulator of pluripotent cells throughout embryonic and germ cell lineage development prior to cellular differentiation during murine, bovine and porcine peri-implantation development. In contrast to murine OCT-4 expression, bovine and porcine expression is detected in both the inner cell mass and trophoblast. Delayed down regulation of OCT-4 gene expression in farm species may be a consequence of the lengthened period of peri-implantation. Expression of OCT-4 mRNA has not been characterized during conceptus attachment to the uterine surface in the pig. The objective of the present study was to determine conceptus OCT-4 mRNA expression during porcine peri-implantation development from days 10-17 of gestation. Total RNA was extracted from multiple pools of conceptuses collected on days 10, 12, 13, 15 and 17 of pregnancy. Quantitative RT-PCR was utilized to assay conceptus OCT-4 mRNA synthesis. Day of conceptus development significantly affected (p < 0.001) OCT-4 mRNA expression. Conceptus expression of OCT-4 was greatest on days 10 and 12 of pregnancy being approximately 7.7- and 11.6-fold greater compared to expression on days 15 and 17, respectively. Results from the present study suggest that down regulation of OCT-4 may be critical in trophoblastic expansion and uterine epithelial attachment during establishment of pregnancy in the pig.

Parthenogenetic activation of porcine oocytes after nuclear transfer.

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.

Preimplantation genetic diagnosis: prevention of serious genetic disorders.

Couples who are at high risk of passing a severe debilitating genetic disorder on to their offspring now have an option for preventing their future child from being affected by the disorder. The new field in medical genetics, preimplantation genetic diagnosis (PGD), involves testing single cells biopsied from in-vitro derived preimplantation stage (approximately 8-cell) preembryos and assessing each of them as to whether it is affected or not. Thus, PGD dramatically reduces the risk of a couple having a child afflicted with a genetic disorder by diagnosing an affected preembryo before it is transferred to the mother for implantation and establishment of pregnancy. This preventive procedure allows parents who are known carriers of a severe genetic disease to have unaffected children.

Diacylglycerol-enhanced electrical activation of porcine oocytes matured in vitro.

This study was undertaken to examine the effect of second messengers added to the electroporation medium on electric pulse-induced artificial activation of meiotic Metaphase II porcine oocytes. Six separate experiments evaluated second messengers added to electroporation medium. When added to electroporation medium, neither phospholipase C (PLC: 0 to 2.5 Units/ml), D-myo-inositol triphosphate (IP(3): 0 to 10,000 muM), nor guanosine 5'-O-(3-thiotriphosphate; GTP-gamma-S: 0 to 100 muM) had any effect (P> 0.05) on activation rates. However, addition of 1,2-dioctanoyl-sn-glycerol (DiC(8)) increased activation rates in a dose-dependent response. At a level of 1,000 muM, DiC(8) resulted in a higher activation rate (P< 0,05) than 0.0, 0.1, 1 or 10 muM of DiC(8) with a pulse, and the 1,000 and 10,000 muM of DiC(8) no-pulse control groups. Effects of DiC(8) (1,000 muM) and IP(3) (100 muM) in combination or individually were investigated. At 1,000 muM, DiC(8) caused a higher rate of activation (P< 0.05) than 100 muM IP(3), but the result was not different from DiC(8) + IP(3). In another experiment, no difference (P> 0.05) was observed between DiC(8), GTP-gamma-S and IP(3), but DiC(8) + GTP-gamma-S + IP(3) + PLC yielded a higher (P< 0.05) activation rate than PLC or the rate of the controls. No significant development (blastocyst) was observed after 5 days of culture in any of the experiments. Protein profiles of activated oocytes, determined by 1D SDS-PAGE, were characteristic of pronuclear-stage embryos. These data indicate that the addition of DiC(8) to the electroporation medium synergistically enhances the rate of activation of electrically stimulated in vitro-matured porcine oocytes.

Electroporation of inositol 1,4,5-triphosphate induces repetitive calcium oscillations in murine oocytes.

The purpose of these experiments was to determine the effect of electroporation of IP3 into the cytosol of murine secondary oocytes and evaluate any alterations in [Ca2+]i resulting from Ca2+ release from intracellular stores. In addition, we evaluated the effect of ethanol (ETOH) on the release of Ca2+ from intracellular stores. Oocytes were loaded with the Ca2+ indicator fluo-3 by incubation in 100 microliters drops of medium containing 2 microM fluo-3/AM for 60 min at 37 degrees C. Changes in fluorescence were monitored by use of an inverted microscope which had been connected to a spectrofluorometer. Fluorescent intensity measurements were acquired for a minimum of 416 sec time span or up to 1,248 sec, with integration readings of 1 sec duration obtained every 2 sec throughout the measurement period. The experimental design consisted of comparing the rise in [Ca2+]i of fluo-3 loaded secondary oocytes subjected to electroporation in PBS and Ca(2+)-free PBS, each containing 25 microM IP3, to that elicited by PBS and Ca(2+)-free PBS containing a final concentration of 7% ETOH. Non-pulsed control secondary oocytes were placed in PBS + 25 microM IP3 during monitoring of [Ca2+]i fluorescence. Pulsed control secondary oocytes were placed in Ca(2+)-free PBS, subjected to electroporation pulse, and monitored for [Ca2+]i fluorescence. Electroporation of IP3 was accomplished by placing fluo-3 loaded secondary oocytes between the electrodes of a glass slide fusion chamber which had been overlaid with 300 microliters of PBS + 25 microM IP3 or Ca(2+)-free PBS + 25 microM IP3.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo.

These studies were conducted to identify the point during the 4-cell stage at which the porcine embryo begins to control development. Reproductive tracts of gilts were flushed 48 h after the onset of estrus to obtain 1- and 2-cell embryos. To determine the duration of the 4-cell stage in vitro, development of 29 embryos was timed from cleavage to the 4-cell stage and from cleavage to the 8-cell stage. The average duration of the 4-cell stage was 50.5 h. The duration of the 4-cell stage was positively correlated (p < 0.01) with culture time in vitro before cleavage to the 4-cell stage. DNA content was determined by using the Feulgen's reaction and quantified with micro-densitometry. Staining units (SU; density x area) were calculated at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, and 36 h post-cleavage to the 4-cell stage (P4C). Results revealed a possible G1 phase (< 2 h) with DNA synthesis starting within 2 h P4C. DNA synthesis was completed by 16 h P4C, and was followed by an extended G2 phase. Embryos were evaluated for uptake and incorporation of [35S]methionine and for qualitative changes in protein profiles specific to time points during the 4-cell stage (2, 10, 14, 16, 18, 24, 30, and 40 h P4C). Methionine uptake and incorporation into protein followed similar patterns, both decreasing until 16-18 h P4C, followed by a steady increase through the 4-cell stage. Protein profiles revealed qualitative changes beginning at 14 and 16 h P4C.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.

Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development.

This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca(2+)-free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV.cm-1, 99 microsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrofusion-induced intracellular Ca2+ flux and its effect on murine oocyte activation.

These experiments were designed to monitor influx of extracellular Ca2+ into the murine ooplasm following a 1.56 kV.cm-1 direct current (DC) electrofusion pulse and subsequently to determine its effect on rate of activation. Pulse media consisted of non-electrolyte (0.3 M mannitol) and electrolyte (phosphate-buffered saline; PBS) media each containing 0.0, 0.05, or 0.9 mM Ca2+ (groups T1-T3 and T4-T6, respectively). Cumulus-free oocytes were incubated in 100 microliters drops of PBS containing 2 microM of the calcium indicator fluo-3/AM for 60 min at 37 degrees C. Fluo-3/AM-loaded oocytes were equilibrated for 7 min in assigned treatment media (T1-T6) prior to application of DC pulse. Change in fluorescent intensity was monitored for 6.5 min after DC pulse by photon counting spectrofluorometry. Fluorometric measurements demonstrate a dramatic rise in intracellular free Ca2+ (Ca2+i) following DC pulse is associated with Ca2+ ion concentration in the pulse medium. Significantly (P less than 0.01) higher Ca2+i levels were observed when 0.9 mM Ca2+ was added to the pulse medium (T3 and T6) compared with pulse medium containing lower Ca2+ ion concentrations (T1, T2, T4, and T5; P greater than 0.05). Differences (P less than 0.01) were observed in peak Ca2+i levels 18 sec after pulse with mean percent change in fluorescence of 5.1%a, 33.9%b, 112.7%c, 1.2%a, 9.3%a, and 99.9%c for T1-T6, respectively (values with different superscripts are significantly different at P less than 0.01). Increased oocyte membrane permeability to Ca2+ ion after DC pulse was observed for a minimum of 5 min after delivery of the 1.56 kV.cm-1 pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of microinjection and two types of electrical stimuli on bovine sperm-hamster egg penetration.

These experiments were designed to test the effects of an electrofusion and an electroporation pulse on bovine sperm-hamster egg development. In experiment 1, single motile sperm were injected into the perivitelline space of each egg. A 4,500 V/cm, 30 microseconds fusion pulse (FP) was applied while sperm-egg membrane contact was maintained. It was observed that single motile sperm were rendered immotile immediately after FP application whereas nonpulsed single motile sperm remained motile for up to 36 h postinjection. In addition, both motile and sonicated spermatozoa were injected directly into the ooplasm prior to receiving an FP to determine whether the FP was detrimental to sperm viability. In experiment 2, to induce the acrosome reaction, an 1,150 V/cm electroporation pulse was applied to washed bovine sperm suspended in TALP medium containing 5 mM Ca2+. Treated and nontreated sperm were coincubated with zona-free hamster ova, and sperm-pentrating ability was measured. Results from experiment 1 indicate that FP failed to induce sperm-egg fusion (0/69). FP did not, however, inhibit decondensation or pronuclear formation of sperm injected into hamster egg ooplasm. Single motile sperm injected into the ooplasm resulted in development of both pulsed (19/28) and nonpulsed (21/28) groups. Sonicated tail-free sperm heads injected into the ooplasm resulted in no detectable difference between treated (18/30) and nontreated (19/30) groups. In experiment 2, treatment of sperm with electroporation pulse +5 mM Ca2+ increased zona-free hamster ova penetration scores over nontreated sperm within bulls (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Early embryonic development in vitro by coculture with oviductal epithelial cells in pigs.

This experiment was designed to evaluate the ability of three different somatic cell cultures to promote development of early cleavage stage pig embryos. A total of 245 2-cell, 4-cell, 8-cell, and 16-cell pig embryos were cocultured for 5 days with porcine oviductal epithelial cells (POEC), porcine fetal fibroblast monolayer (PEF), a combined POEC and PEF coculture system (PEF-POEC), or Dulbecco's Modified Eagle Medium alone (DMEM). Embryos were collected at slaughter from the reproductive tracts of superovulated prepubertal gilts. Embryos were recovered, evaluated, and randomly placed in one of the four treatment groups. POEC were recovered from oviductal flushes, washed, and placed in 24-well plates. PEF were obtained from 30-day to 60-day fetuses and established in culture. Finally, PEF-POEC consisted of a confluent monolayer of PEF in the bottom of 24-well plates also containing a Costar semipermeable membrane chamber with POEC in it. Embryos were evaluated every 24 h to determine stage of development. More (p less than 0.05) embryos developed to blastocysts in POEC (70% and 54%, respectively) and PEF-POEC (67% and 61%, respectively), than in either DMEM (16% and 2%, respectively) or PEF (27% and 23%, respectively). However, development of embryos did not differ (p less than 0.05) for POEC and PEF-POEC. These data indicate the presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.