The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.

Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor.

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta.

BACKGROUND: A sperm-specific phospholipase C zeta (PLCzeta) has been shown to trigger Ca(2+) oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLCzeta into oocytes, but it was unclear how much PLCzeta protein is required for development. Here we have injected cRNA encoding luciferase-tagged human PLCzeta (hPLCzeta-luc) into mouse oocytes and established the relationship between hPLCzeta-luc expression, Ca(2+) oscillations and development. METHODS: Mouse oocytes were injected with hPLCzeta-luc cRNA and a fluorescent Ca(2+)dye to monitor hPLCzeta-luc expression and Ca(2+) oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLCzeta-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr(2+). RESULTS: Repetitive Ca(2+) oscillations and oocyte activation were triggered by hPLCzeta over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLCzeta-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLCzeta expression. CONCLUSIONS: Human PLCzeta can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLCzeta-luc protein expression levels.

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Calcium oscillations, sperm factors and egg activation at fertilisation.

When an egg is fertilised by sperm, the first intracellular signalling event observed is a large transient increase in cytoplasmic free Ca2+ ions. Elevated Ca2+ is known to play a vital role as an intracellular messenger in all cells and the Ca2+ signal occurring in the egg at fertilisation triggers the subsequent events that mediate early embryo development. In mammalian eggs, the Ca2+ response is first observed as a Ca2+ wave that initiates near the point of sperm-egg fusion, spreads across the entire egg, and then continues as a series of intracellular Ca2+ oscillations. The way in which the fertilising sperm generates the Ca2+ response in the egg has been the subject of much debate over recent years. One proposal for which there is growing evidence suggests the mechanism of egg activation at fertilisation involves the introduction of a soluble sperm protein into the egg shortly after sperm-egg fusion.

Ca2+ oscillations and sensitization of Ca2+ release in unfertilized mouse eggs injected with a sperm factor.

The mechanisms by which a sperm factor caused Ca2+ oscillations in unfertilized mouse eggs was investigated by monitoring the fluorescence of intracellular Fluo-3. Injecting sperm extracts triggered Ca2+ oscillations that varied in number and frequency rather than amplitude. All the sperm factor-induced Ca2+ transients showed a characteristic rapid increase and decline. The oscillations persisted for variable times in eggs bathed in Ca2+ free media and were distinct in character from those caused by inserting pipettes containing inositol 1,4,5-trisphosphate (InsP3). InsP3-induced Ca2+ oscillations were always of high frequency but varied considerably in form and amplitude depending upon InsP3 concentration. The oscillations produced by injecting Ca2+ into unfertilized eggs were similar to those produced with low doses of InsP3 but different from those caused by sperm extract injection. After injection of sperm extracts further injection of InsP3, or Ca2+, produced high frequency large amplitude Ca2+ transients. It is concluded that the sperm factor mediates its effects by sensitizing Ca2+ release mechanisms.

Thimerosal causes calcium oscillations and sensitizes calcium-induced calcium release in unfertilized hamster eggs.

Calcium-induced-calcium-release (CICR) was assayed in unfertilized golden hamster eggs by injecting Ca2+ and monitoring Ca2(+)-dependent hyperpolarizing responses (HRs) and Ca2(+)-sensitive fluo-3 fluorescence. Incubating eggs in the sulfhydryl reagent thimerosal caused [Ca2+]i oscillations as monitored by Ca2(+)-dependent HRs and decreased approximately 10-fold the Ca2+ injection current required to generate an HR and cause a large intracellular Ca2+ increase. Thimerosal also enhanced the sensitivity of eggs to Ca2+ injection in a calcium-free medium. The effects of thimerosal on CICR were prevented by dithiothreitol and were not mimicked by injecting inositol 1,4,5-trisphosphate. The data suggest that thimerosal may be an alternative agent for studying CICR in caffeine-insensitive cells.

A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca2+ release in sea urchin egg homogenates.

Injection of sperm extracts triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilisation. Here, we show that addition of sperm extracts to sea urchin egg homogenates causes Ca2+ release and inositol 1,4,5-trisphosphate (InsP3) production. Furthermore depleting homogenates of phosphatidylinositol lipids using a phosphatidylinositol-specific phospholipase C blocked the sperm extract from causing InsP3 production and a Ca2+ rise. A response could be recovered by the addition of phosphatidylinositol 4,5-bisphosphate to either sperm extracts or egg homogenates. These data indicate that sperm extracts contain an InsP3-generating phospholipase C which may play a role in Ca2+ release at fertilisation.

Presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane.

We investigated the presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane, which was assessed by the hypo-osmotic swelling (HOS) test. We found no gross differences in the presence of oscillin in semen samples from men who presented with 70%, 40%, 25% or 2% of membrane-intact spermatozoa. By immunofluorescence, membrane-intact (HOS-positive) spermatozoa showed staining of a single band at the equatorial region, whereas over 80% of HOS-negative spermatozoa consistently showed a diffuse distribution of oscillin over the sperm head. However, some individuals presented with up to 50% of HOS-positive spermatozoa showing an aberrant localization of oscillin. We found a significant correlation rate (r=0.70, P < 0.05) between the percentage of HOS-positive spermatozoa with an equatorial oscillin localization and the fertilization rates achieved after intracytoplasmic sperm injection. These data suggest that the localization of oscillin in human spermatozoa might have an impact on egg activation and fertilization rates.

A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres.

A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.

Modified crossed leg raising test and sciatica.

A modification of the crossed leg raising test has been helpful in identifying patients with intervertebral disc disorders. Simultaneous flexion of the neck and elevation of the contralateral leg produced pain in the ipsilateral (presenting) sciatic notch in five patients with either free fragments or herniated disc found at operation. All patients were symptom-free postoperatively.

Deep vein thrombosis and pulmonary emboli in neurosurgical patients: a review.

This review examines the incidence, natural history, diagnosis, prophylaxis, and management of deep vein thrombosis (DVT) and pulmonary embolism (PE) in neurosurgical patients. Recent studies estimate the incidence of postoperative DVT detected by fibrinogen scanning in neurosurgical patients to be 29% to 43%. Specific factors that enhance the risk of venous thromboembolism include previous DVT, surgery, immobilization, advanced age, obesity, limb weakness, heart failure, and lower extremity trauma. Clinical diagnosis of venous thromboembolism is unreliable but can be augmented by noninvasive screening tests such as iodine-125-fibrinogen scanning, Doppler ultrasonography, and impedance plethysmography. As prophylactic measures, mini-dose heparin and external pneumatic compression of the legs have decreased the incidence of DVT in clinical studies of neurosurgical patients. However, no prophylactic measure has been convincingly shown to prevent PE in neurosurgical patients. Thrombi involving the popliteal, deep femoral, and iliac veins appear most likely to cause significant PE. Anticoagulation therapy constitutes standard management of DVT and PE; however, in neurosurgical patients the potential for precipitating intracranial or intraspinal hemorrhage may necessitate vena caval interruption. This appears to be an effective alternative to anticoagulation.

Modification of the Richmond subarachnoid screw for monitoring intracranial pressure. Technical note.

The authors describe a modification of a subarachnoid screw for monitoring intracranial pressure by hydrostatic coupling of the subarachnoid space to an external transducer. The device can be used in both children and adults and features more assured placement of the distal tip, increased stability, and enhanced safety on insertion.

Accessory nerve palsy following carotid endarterectomy. Report of two cases.

Two patients who had an accessory nerve palsy following carotid endarterectomy are presented. Both patients had high carotid bifurcations necessitating unusually high retraction and dissection. The ipsilateral accessory nerve was injured in the anterior cervical triangle in both cases. It is believed that vigorous lateral retraction of the superior aspect of the sternocleidomastoid muscle led to a stretch injury of the nerve. The symptoms completely resolved in both patients within 6 months.

Management of symptomatic deep venous thrombosis and pulmonary embolism on a neurosurgical service.

The authors present a retrospective analysis of the management of deep vein thrombosis (DVT) and pulmonary embolism (PE) in neurosurgical patients at the Massachusetts General Hospital from January, 1978, through June, 1982. There were 44 cases of DVT and 13 cases of PE. Management modalities included observation only, femoral vein ligation, inferior vena cava clipping, transvenous placement of an inferior vena cava filter or umbrella, and anticoagulation therapy. Six (75%) of eight patients with symptomatic DVT who were managed by observation alone had subsequent pulmonary emboli, and three (38%) died. Femoral vein ligation was followed by PE in one of four cases and led to significant leg swelling in two others. Neither observation alone nor femoral vein ligation can be recommended as routine management options. Partial inferior vena cava interruption with a De Weese clip, Kim-Ray Greenfield filter, or Mobin-Uddin umbrella all successfully prevented pulmonary emboli. The major problem associated with these methods was leg edema, which occurred in 47% of patients with clip placement, 25% with filter placement, and 21% with a Mobin-Uddin umbrella. Anticoagulation therapy was associated with a complication rate of 29% and a mortality rate of 15%. Fatal PE and paradoxical hypercoagulability with gangrene of a lower extremity were the causes of death. In one patient, hemorrhage into a glioblastoma occurred following discontinuation of anticoagulation therapy when the coagulation parameters were normal. The authors conclude that: 1) management with observation alone of patients with symptomatic DVT places the patient at risk for the development of life-threatening pulmonary emboli; 2) the safety and timing of therapeutic anticoagulation in postoperative neurosurgical patients or patients with tumors is unclear; and 3) partial interruption of the inferior vena cava with a transvenous filter successfully prevents PE and may represent a safer alternative to anticoagulation therapy.

Inadvertent middle cerebral artery embolism by a detachable balloon: management by embolectomy. Case report.

A case of middle cerebral artery embolism by a detachable intra-arterial balloon is presented. The balloon migrated after being detached in an effort to occlude the internal carotid artery proximal to an unclippable giant paraclinoid aneurysm. Volume expansion, induced hypertension, anticoagulation therapy, rapid middle cerebral artery embolectomy, and good collateral circulation are factors that may have contributed to the patient's complete recovery from hemiplegia.

Preoperative diagnosis of Lhermitte-Duclos disease by magnetic resonance imaging. Case report.

Lhermitte-Duclos disease is a benign, presumably hamartomatous lesion of the cerebellum which presents clinically as a mass lesion. Pathologically, it consists of thickening of both the molecular and granular cell layers of the cerebral cortex which enlarges the folia but allows for preservation of the gyral pattern of the cerebellar cortex. Preoperative diagnosis with computerized tomography and other studies has not been possible, and even at surgery the diagnosis may be missed because of the preservation of the gyral pattern. The sensitivity of magnetic resonance imaging allows recognition of the cortical nature of the mass lesion, and especially the gyral pattern within the mass lesion, providing a diagnostic image which is unlikely to be confused with any other pathological process in the cerebellum. Preoperative diagnosis of Lhermitte-Duclos disease allows surgeons to plan an appropriate decompressive procedure.

Spontaneous spinal subarachnoid hemorrhage and subdural hematoma. Report of two cases.

Two patients with spontaneous spinal subarachnoid hemorrhage are presented to emphasize the clinical and radiological features of this uncommon illness. Both had severe back pain at the onset. One patient had a subdural hematoma that compressed the conus medullaris and cauda equina, and was drained percutaneously; the other had clots in the subarachnoid space. The cerebrospinal fluid showed a polymorphonuclear pleocytosis that simulated septic meningitis. Complete spinal angiography failed to reveal a cause for the hemorrhages.

Spontaneous arteriovenous fistula of the superficial temporal artery.

A case of a nontraumatic arteriovenous fistula of the superficial temporal artery, documented on selective external carotid arteriography, is described in a 42-year-old man who was successfully treated by complete surgical excision. The clinical features, differential diagnosis, and treatment of arteriovenous fistulas of the superficial temporal artery are reviewed.