Robert E. Hinkley

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342.

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.

Non-propagated cortical reactions induced by the divalent ionophore A23187 in eggs of the sea urchin, Lytechinus variegatus☆

Abstract The divalent ionophore A23187 can induce a non-propagated discharge of cortical granules in sea urchin eggs resulting in the elevation of partial fertilization membranes. This occurs when unfertilized eggs (1) partially overlap the margin of a solid film of the ionophore; (2) touch a Sephadex bead previously infiltrated with the ionophore; or (3) are exposed to solutions of ionophore for short periods (15–30 sec). These results demonstrate that the release of cortical granules does not automatically induce the discharge of neighboring granules.

Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.

Comparative Effects of Halothane, Enflurane, and Methoxyflurane on the Incidence of Abnormal Development Using Sea Urchin Gametes as an In Vitro Model System

The incidence of sea urchin embryos developing abnormally after their exposure to equimolar concentrations of halothane, enflurane, and methoxyflurane has been determined. Halothane concentrations in the 0.6–1.25 mM range caused 18–96% of the embryos to undergo abnormal cleavage at the first cell division. This is important because embryos exhibiting atypical cleavage patterns at the first cell division eventually involute and die before reaching gastrulation. Over the same range of concentrations, enflurane and methoxyflurane have minimal effects on development. Hoi ever, when exposed to 2.5 mM methoxyflurane, nearly 40% of the cells did not fertilize. These results show that volatile anesthetic agents have decidedly different effects on development and suggest that the incidence of abnormal development may not correlate directly with the anesthetic potency of inhalational agents.

Fentanyl does not inhibit fertilization or early development of sea urchin eggs.

Sea urchin (Lytechinus variegatus) eggs have been used to study the effects of fentanyl on in vitro fertilization and early development as a model of human in vitro fertilization. Fentanyl did not affect fertilization or subsequent cell division when present in concentrations calculated to approximate or exceed those to which human ova would be exposed during clinical anesthesia. Lytechinus eggs exposed to fentanyl, then washed in fentanyl-free sea wafer before fertilization were also not affected and divided normally. The results suggest potential clinical utility of fentanyl during the harvesting of human ova for subsequent in vitro fertilization.

Inhibition of sperm motility by the volatile anesthetic halothane

Abstract The inhalational anesthetic halothane reversibly inhibits the motility of sea urchin sperm dose-dependently at concentrations up to 5 mM. Experiments with Triton X-100 extracted, trypsinized axonemes showed that halothane has no effect on the rate of axonemal disintegration in the presence of ATP. These results suggest that halothane inhibits flagellar activity by acting at a site other than the dynein ATPase component of the flagellum.

Further studies on dividing sea urchin eggs exposed to the volatile anesthetic halothane

Abstract Mitotic apparatus (MA)-isolation techniques and SDS-gel quantitation show that halothane, a widely used volatile anesthetic, inhibits the growth of the mitotic apparatus of echinoderm eggs in vivo, but has no detectable effect on the amount of actin associated with the cell cortex. These studies confirm and extend long standing observations on anesthetic-treated echinoderm eggs and support the hypothesis that anesthetics indirectly prevent cleavage by inhibiting the growth of mitotic asters.

Selective identification of sperm fused with the surface of echinoderm eggs by DNA-specific bisbenzimide (Hoechst) fluorochromes

When unfertilized echinoderm eggs are treated with the DNA‐specific bisbenzimide fluorochrome Hoechst 33342 and then fertilized with unlabeled sperm, a single spermatozoan bound to the egg surface becomes fluorescent. Several lines of evidence, including correlative scanning electron microscopic studies, indicate that the fluorescent sperm is, in fact, the fertilizing sperm which acquires fluorescence as a consequence of membrane fusion between the sperm and egg. Comparative studies show that several fluorochromes structurally related to H33342 can be used to selectively identify the fertilizing sperm at the egg surface and that H33258 possesses a distinct advantage when used to visualize the male and female pronuclei in eggs fixed prior to fluorochrome exposure. Finally, none of the fluorochromes tested in these studies have any discernible effect on development from the first cell division through the pluteus larva stage. These observations suggest that the fluorochrome‐transfer technique for identifying the fertilizing sperm may be useful in a wide variety of studies of gamete interaction as a simple and rapid cytological indicator for sperm‐egg fusion.

Retrograde transport of horseradish peroxidase in the rat's brain during halothane anesthesia

The horseradish peroxidase (HRP) method was used to study the effect of halothane anesthesia on retrograde transport in the central nervous system. Rats received neostriatal injections of 30% HRP (0.3 microl) and were placed in inhalation chambers perfused with air or air containing 1% halothane for up to 24 h. Subsequently, the brain were examined at rostro-caudal levels of the presence and distribution of HRP-containing cells. The number and distribution of HRP-labelled cells appearing at each cranial level were compared in control and halothane-anesthetized animals as a function of time. The results suggest that retrograde transport in the central nervous system is unaffected during halothane anesthesia.

On becoming a physician: Perspectives of students in combined baccalaureate‐MD degree programs

Students entering the first year of nine combined baccalaureate‐MD degree (CD) programs were administered the Matriculating Student Questionnaire (MSQ) of the Association of American Medical Colleges. A profile of the CD students’ perspectives on becoming a physician was compared with the profile of traditional medical students who responded to the MSQ. CD students chose medicine as a career goal earlier in life than their traditional counterparts. They also had more positive perceptions of medicine. Their decisions regarding future career interests in medicine differed in some respects from those of traditional students. Larger percentages of CD students were female, had Asian backgrounds, and had physician fathers. The views of CD students were partly related to the type of CD program they attended and to demographic/background characteristics, including age, which was suggested by preliminary comparisons between premedical students and CD students.