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PubMed1992

In vitro development of zygotes from prepubertal gilts after microinjection of DNA.

The effect of pronuclear microinjection of DNA and culture in excised mouse oviducts on the development of porcine zygotes was assessed in this study. Precocious ovulation was induced in prepubertal gilts with pregnant mare's serum gonadotrophin and hCG. Zygotes received either pronuclear microinjection of buffer alone, buffer containing a DNA construct, or no microinjection. Zygotes were cultured in vitro in either modified Krebs-Ringer bicarbonate medium (KRB) for 144 h or in mouse oviduct (MO) explant culture with KRB for 48, 72, 96, or 120 h. Pronuclear microinjection of DNA resulted in a lower (P less than .05) cleavage index (CI) than did buffer or no microinjection (CI 2.16 +/- .10 vs 2.80 +/- .13 and 2.93 +/- .10). The CI loss was greatest for DNA-injected zygotes at the two-cell stage of development. Coculture of zygotes in MO resulted in a higher CI (P less than .01) than did culture in KRB. Culture in MO for 72 h was the most beneficial system compared with MO for 48, 96, or 120 h (P less than .05; CI 3.25 +/- .12 vs 2.66 +/- .18, 2.79 +/- .14, and 2.40 +/- .14, respectively). Microinjection of DNA, not merely the mechanical procedure, was detrimental to early zygote development and may be the cause of low pregnancy rates.

Animals↗

PubMedDate not supplied

Expression of thermotolerance following microinjection of poly(A)RNA isolated from thermotolerant CHO cells.

Poly(A)RNA was isolated from thermotolerant cells and microinjected into recipient non-tolerant Chinese hamster ovary (CHO) cells. The injected cells expressed thermotolerance to a subsequent test heat treatment both in terms of the end-points of colony formation (cell survival) and resumption of protein synthesis after test heating (translational labelling). The magnitude of thermotolerance expression was dependent on the experimental end-point (increase up to 3.8-fold for translational labelling and approximately 2-fold for survival) and on the time between microinjection and the test heat treatment. Control experiments showed that poly(A)RNA from non-tolerant cells did not alter the heat response of microinjected cells. Proteins corresponding to the poly(A)RNA from thermotolerant cells were analysed by in vitro translation and by labelling of microinjected cells, followed by SDS-PAGE. In vitro translations showed high levels of transcripts for classical heat-shock proteins (HSP 70/72, 89, 110) in poly(A)RNA from thermotolerant versus control cells. However, proteins synthesized in intact cells showed no detectable differences when cells were microinjected with poly(A)RNA from thermotolerant versus control cells, or not injected at all. In principle the data show that microinjection of specific poly(A)RNA fractions can be used for defining the contribution of individual gene products to the cellular heat response.

Animals↗

PubMed1991

Microinjection of a p21ras antibody into PC12 cells inhibits neurite outgrowth induced by nerve growth factor and basic fibroblast growth factor.

The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21ras protein.

Adrenal Gland Neoplasms↗

PubMed1993

Microinjection of bovine embryos with a foreign gene and its detection at the blastocyst stage.

This study was designed to measure the survival and development of pronuclear stage bovine zygotes after microinjection with DNA and either culture in vitro or incubation in the sheep oviduct and to determine the percentage of embryos that retain exogenous DNA at the blastocyst stage. In vitro matured and fertilized oocytes were examined for pronuclear development 18 to 20 h after coincubation with sperm. An ovine somatotropin gene construct was microinjected into ova with visible pronuclei. Microinjected ova and unmanipulated ova from the same in vitro derived pool were cocultured with oviductal epithelial cells or incubated in the sheep oviduct and cocultured, respectively, for 7 d. Blastocysts were subjected to polymerase chain reaction analysis for detection of exogenous DNA. The percentage of cleaved embryos that formed blastocysts was similar for microinjected and unmanipulated cultured ova. The percentage of blastocysts recovered from the sheep oviduct that hatched was higher than for blastocysts that developed in coculture. Fourteen of 26 (54%) blastocysts that developed from microinjected ova were positive for the exogenous DNA by polymerase chain reaction. The implications are discussed for polymerase chain reaction detection of the exogenous gene construct in a relatively high proportion of microinjected blastocysts.

Animals↗

PubMed1999

Microinjection of an antibody against HSP 72 in keratinocytes to study acute UV injury.

First, a method of microinjection of antibodies in primary human keratinocytes in culture was established. Second, in acute UV irradiation, the physiological role of heat shock protein (HSP) 72 in keratinocytes was studied with this method. Primary human keratinocytes in culture were injected with "controls" as fluorescent dyes, phosphate buffered saline (PBS), an irrelevant secondary antibody and an antibody against a protein with known protective function in UV erythema, HSP 72. UV irradiation was applied and survival, colony forming and immunohistochemistry for injected and non-injected keratinocytes were evaluated in a time course. Puncturing the plasma membrane with injections of "controls" as FITC, PBS and the IgG anti-mouse antibody did not result in reflux of injected material or any alteration in morphology or colony-forming ability for 24 h. Keratinocytes injected with an mAb to HSP 72 without UV irradiation survived microinjection for up to 12 days, while surprisingly, more than double of injected and irradiated ones died after 12 h compared to not injected and irradiated ones. Moreover, microinjection of the antibody to HSP 72 in the nucleus resulted in a loss of the immunohistochemical labeling for HSP 72 in these cells after 12 h. Microinjection of the "controls" did not harm the survival, forming of colonies and expression of HSP 72 in keratinocytes for 24 h. In contrast, microinjection of an mAb against HSP 72 led to an increase in cell death after UV irradiation, confirming that HSP 72 is important for UV protection. Microinjection of antibodies in human keratinocytes in culture might allow the study of the physiological role of some proteins.

Antibodies, Monoclonal↗

PubMed2002

[Construction of transgenic mice carrying enhanced green fluorescent protein gene by seminiferous tubule microinjection].

OBJECTIVE: To study the feasibility of establishing transgenic mice carrying enhanced green flourescent protein (EGFP) gene by means of seminiferous tubule microinjection. METHODS: The vector expressing enhanced green fluorescent protein under the control of human cytomegalovirus immediate-early promoter (pCMV-EGFP) was selected and mixed with liposome in vitro. Microinjection at different doses of the liposome-entrapped plasmid DNA into the seminiferous tubules of male mice at different ages was performed to establish transgenic mice, which were made to mate with female mice at least 40 d after the microinjection. Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis, and the frozen sections of different tissues from 2 of the founders mice were prepared for fluorescence microscopic observation. RESULTS: Among the 41 mice receiving the microinjection, 32 survived and retained their mating ability and fertility, and among their 382 offspring 133 were positive for EGFP DNA as demonstrated by PCR, 15 of which were confirmed by Southern blotting analysis. The age of the mice or the doses of microinjection they received was not shown to impact the integration of EGFP gene, and fluorescence microscopy failed to detect significant EGFP expression in the tissues of the founder mice (P>0.05) in comparison with normal mice. CONCLUSION: Seminiferous tubule microinjection is simple and practicable to implement gene transfer in mice.

Animals↗

PubMed1985

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts.

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.

Cell Fractionation↗

PubMed1995

Microinjection of baclofen in the ventromedial medulla of rats: antinociception at low doses and hyperalgesia at high doses.

Neurons of the nucleus raphe magnus (NRM) and adjacent nucleus reticularis gigantocellularis pars alpha (NGCp alpha) receive a tonic inhibitory input from gamma-aminobutyric acid (GABA)-ergic neurons that is mediated by GABAA receptors. However, comparatively little is known about the role of GABAB receptors in these nuclei. The present study examined the effects on nociceptive threshold of microinjection of a wide dose range (0.1-150 ng) of the GABAB receptor agonist, baclofen hydrochloride (BAC), in the NRM, NGCp alpha or the nucleus reticularis giganto-cellularis (NGC). Microinjection of low doses of R(+)-BAC (0.1-1.0 ng) in the NRM or the NGCp alpha, but not the NGC increased response latencies in the tail flick test. As the dose of BAC was increased to 30.0 to 50.0 ng, response latencies in the tail flick test diminished to control values. Microinjection of the highest dose, 150 ng, in either the NRM, NGCp alpha or NGC significantly decreased response latencies in the tail flick test. Response latencies in the hot plate test were not increased by microinjection of 0.1 to 5.0 ng of R(+)BAC in the NRM, NGCp alpha or NGC. Although hot plate latency was increased after microinjection of 30.0 to 50.0 ng of R(+)-BAC in these nuclei, the effect was confounded by the occurrence of motor dysfunction. The effects of BAC were stereospecific as nociceptive threshold and motor function were not altered by microinjection of either 0.5 ng or 150 ng of the less active stereoisomer, S(-)-BAC. The biphasic effect of R(+)-BAC suggests the existence of multiple mechanisms by which GABAB receptors modulate the activity of neurons in the ventromedial medulla. It is proposed that the antinociception produced by low doses of R(+)-BAC results from disinhibition (activation) of neurons in the NRM and NGCp alpha as a consequence of the presynaptic inhibition of inhibitory GABAergic and/or noradrenergic inputs. The decrement in antinociceptive effect and hyperalgesia produced by higher doses of R(+)-BAC may result from a presynaptic inhibition of excitatory inputs to the NRM and NGCp alpha, postsynaptic hyperpolarization of neurons in these nuclei or the unmasking of a descending facilitatory pathway originating from the NRM, NGCp alpha and NGC.

Animals↗

PubMed1984

Non-opiate analgesia induced by carbachol microinjection into the pontine parabrachial region of the cat.

These studies investigated the effect of microinjection of the cholinergic agonist carbamylcholine (carbachol) into various sites of the dorsolateral pontine tegmentum of the cat. Carbachol microinjection into an area surrounding the lateral half of the brachium conjunctivum (parabrachial region, PBR) produced profound suppression of nociceptive responses. In the dorsal part of PBR, carbachol microinjection produced no generalized sensory, emotional or motor deficits, indicating that nociceptive transmission was primarily affected. Carbachol microinjection into the ventral part of PBR resulted in slight suppression of motor responses in addition to profound nociceptive suppression. Carbachol-produced analgesia (CPA) observed within PBR blocked supraspinally as well as spinally integrated responses normally elicited by either phasic or tonic noxious stimuli. Atropine sulfate, but not mecamylamine hydrochloride, significantly antagonized CPA, indicating that muscarinic receptors mediate this phenomenon. The opiate antagonist naloxone, systemically administered either prior to or after carbachol microinjection, did not reliably attenuate CPA. Microinjection of morphine into the sites from which CPA had previously been obtained did not produce significant effects on nociceptive responses. Thus, opiate mechanisms appear not to be necessary either for the activation of this system or for the production of the resultant analgesia. These findings indicate that the neural population examined in the present study is anatomically and pharmacologically distinct from previously identified opiate-mediated pain inhibitory systems. Results are discussed in light of other recent evidence indicating the existence of endogenous non-opiate pain inhibitory systems.

Animals↗

PubMed1984

Localization of HMG chromosomal proteins in the nucleus and cytoplasm by microinjection of functional antibody fragments into living fibroblasts.

We have used microinjection and cell fractionation to localize the chromosomal high mobility group proteins (HMG) in human fibroblasts. Electrophoretic analysis of nuclear and cytoplasmic fractions from the fibroblasts indicates that the concentration of HMG-1,2 in the cytoplasm is 2.9 times larger than in the nucleus indicating that the majority of the cellular HMG-1,2 is present in the cytoplasm. In contrast, HMG-17 remains predominant in the nuclear fraction. We conclude that the cellular distribution of HMG-1,2 is significantly different from that of HMG-17. To avoid possible artifacts due to cell fractionation, fluoresceinated HMG-1 and HMG antibodies were microinjected into living fibroblasts. The cellular distribution of the injected proteins was monitored using fluorescent microscopy. Fluoresceinated HMG-1 microinjected into the cytoplasm moves very rapidly into the nucleus and concentrates in the nucleolus of living human fibroblasts. However, some control non-nuclear proteins also migrated into the nucleus raising the possibility that exogenous injected proteins do not always distribute in the same pattern as the endogenous proteins. The localization of microinjected F(ab)2 fragments derived from anti-HMG-1 was compared to that of microinjected F(ab)2 derived from anti-histones. Whereas the anti-histone F(ab)2 when injected into the cytoplasm migrated into the nucleus, the anti-HMG-1 F(ab)2 remained in the cytoplasm. Microinjection of anti-HMG-17 and anti-histone inhibited transcription in living cells, anti-HMG-1,2 did not. We conclude that HMG-1,2 proteins are present in both the nucleus and cytoplasm of living fibroblasts.

Antibodies↗

PubMed1995

In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration.

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.

Journal Article↗

PubMed1981

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.

Cell Fusion↗

PubMed1986

Microinjection of kainic acid into the hypothalamus of golden hamsters prevents vasopressin-dependent flank-marking behavior.

The purpose of this study was to define the vasopressin-sensitive area in the anterior hypothalamus-medial preoptic area (AH-MPOA) of the golden hamster that is involved in the expression of flank-marking behavior. Male hamsters implanted with guide cannulae stereotaxically aimed at various sites in the AH-MPOA were microinjected initially with 0.1 ng of arginine vasopressin (AVP) in a volume of 10 nl. Hamsters that flank-marked in response to these injections were subsequently microinjected into the same sites with kainic acid (0.2 microgram/20 nl; n = 10) or an equal volume of 1 M NaOH as a vehicle control (n = 10). Four days later hamsters were tested for odor-induced flank marking by placing them into the recently vacated home cage of other hamsters and for flank marking in response to the microinjection of AVP. Animals treated with kainic acid exhibited significantly (p less than 0.01) fewer AVP and odor-induced flank marks as compared to the number of flank marks observed prior to treatment. There was no significant reduction in the number of flank marks in hamsters microinjected with the NaOH vehicle. In another group of hamsters, microinjection of kainic acid (0.2 microgram/20 nl) into the 3rd ventricle (n = 4) and other sites of the hypothalamus (n = 4) did not significantly alter odor-induced flank marking. The locations of the microinjection sites indicate that the neurons sensitive to AVP and involved in the expression of flank-marking behavior are found in the ventromedial area of the AH-MPOA extending from the caudal border of the suprachiasmatic nucleus to the rostral limit of the supraoptic nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Communication↗

PubMed1988

Hypothalamic microinjection of alpha 2-adrenoceptor agonists causes greater sympathoinhibition in spontaneously hypertensive rats on high NaCl diets.

We tested the hypothesis that in NaCl-sensitive spontaneously hypertensive rats (SHR-S) maintained on high NaCl diets, sympathoinhibitory neurons in the anterior hypothalamic area display increased responsiveness to alpha 2-adrenergic receptor stimulation, giving rise to exaggerated depressor responses. Clonidine (0.6-2.5 micrograms) was microinjected directly into the anterior hypothalamic area of SHR-S maintained for 2 weeks on high (8%) and normal (1%) NaCl diets, and blood pressure and heart rate responses were monitored. Controls were NaCl-resistant SHR (SHR-R) and normotensive Wistar-Kyoto (WKY) rats. Clonidine microinjection into the anterior hypothalamic area resulted in dose-dependent rapid-onset depressor and bradycardic responses that were significantly greater in SHR-S fed on a high NaCl diet. In SHR-R and WKY rats, the high NaCl diet had no significant effect on blood pressure or heart rate responses to clonidine. Further studies demonstrated that the response to microinjection of clonidine into the rat anterior hypothalamic area was location-specific, since injections into surrounding hypothalamic nuclei gave a longer latency in the onset of either depressor and bradycardic responses or pressor and tachycardic responses. The response of clonidine was blocked by concurrent microinjection of the selective alpha 2-adrenergic antagonist rauwolscine but not by the alpha 1-adrenergic antagonist prazosin, confirming that the response was alpha 2-adrenoceptor-specific. Microinjection of the selective alpha 2-agonist guanabenz into the anterior hypothalamic area produced depressor and bradycardic responses in SHR-S and Sprague-Dawley rats, while microinjection of the selective alpha 1-agonist phenylephrine into the anterior hypothalamic area had no effect on either blood pressure or heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Characterization of susceptibility to audiogenic seizures in ethanol-dependent rats after microinjection of gamma-aminobutyric acid (GABA) agonists into the inferior colliculus, substantia nigra or medial septum.

The relative anticonvulsant potential of the gamma-aminobutyric acid (GABA) agonist, muscimol, was compared after microinjection into either the inferior colliculus, substantia nigra or medial septum of ethanol-dependent rats. Bilateral microinjection of muscimol (10-30 ng) into the inferior colliculus 15 to 60 min before testing suppressed all sound-induced seizure components (wild running, clonus and tonus) in rats withdrawn from ethanol for 6.5 to 8.5 hr. However, forelimb tremors were not altered. Audiogenic seizures were suppressed for at least 3 hr after muscimol (30 ng). In the medial septum and substantia nigra, microinjection of muscimol (30-100 ng) only partially reduced the tonic component of audiogenic seizures and exerted no effect on the frequency of wild running or clonus. GABA (10 micrograms) and two other GABA agonists [4,5,6,7-tetrahydroisoxazolo[5, 40c]pyridin-3-ol (THIP), 300 ng and chlordiazepoxide, 10-30 micrograms], microinjected into the inferior colliculus, also reduced audiogenic seizure susceptibility. However, 1, 3-butanediol, which suppresses ethanol withdrawal seizures after peripheral administration in rats, was inactive. The relative proconvulsant potential of the GABA antagonist, bicuculline methiodide, also was compared after microinjection into either the inferior colliculus, substantia nigra or medial septum of ethanol naive rats. In each animal, audiogenic seizure-like wild running, clonus and tonus were evoked by microinjecting bicuculline methiodide into the inferior colliculus at the rate of 6.0 ng/6 min. However, these reactions did not occur when bicuculline methiodide was applied at a slower rate (1.8 ng/6 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Acoustic Stimulation↗

PubMed1995

Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.

Animals↗

PubMed1991

Subzonal microinjection of mouse spermatozoa: insufficient sperm motility might induce phagocytosis.

Acrosome-reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty-one percent of the microinjected oocytes reached a morphologically apparent two-cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.

Animals↗

PubMed1992

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.

Animals↗