Vesicle Migration Research Articles

Involvement of 26S Proteasome in Oocyte Maturation of Goldfish Carassius auratus

Abstract Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, inhibited the activity of 26S proteasome in goldfish oocytes and arrested the oocytes at two distinct stages of maturation. One was prior to the migration of germinal vesicle (GV) toward the animal pole, and the other was between the attachment of GV to the oocyte plasma membrane and GV breakdown (GVBD). The first DFP-sensitive period corresponded to the period during which the activity of proteasomes increased, but maturation-promoting factor (MPF) was still inactive. During the second DFP-sensitive period, MPF was abruptly activated, although the proteasome activity detectable in the oocyte cytosol extracted by ultracentrifugation reached the lowest level during this period. These results suggest the requirement of 26S proteasome for at least two stages of oocyte maturation, the early stage before GV migration and the later stage, including MPF activation immediately before GVBD.

Microinjection of anti-alpha-tubulin antibody (DM1A) inhibits progesterone-induced meiotic maturation and deranges the microtubule array in follicle-enclosed oocytes of the frog, Rana pipiens.

Microinjection of anti-alpha-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog, Rana pipiens. DM1A (46 nl; 10 mg/ml) injection significantly increased the ED50 value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46 nl; 2.5 mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46 nl; 7.5 mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparently depolymerised by DM1A (46 nl; 10 mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell; however, apparent sparse bundles of microtubules arranged in an approximately 600 microns shell were associated with the injectate region 24 h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm = -31.8 mV) compared with control (immature) DM1A-injected cells (Vm = -44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm = -13.9 mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in the Rana follicle-enclosed oocyte.

The role of tank-treading motions in the transverse migration of a spheroidal vesicle in a shear flow

The behavior of a spheroidal vesicle, in a plane shear flow bounded from one side by a wall, is analysed when the distance from the wall is much larger than the spheroid radius. It is found that tank treading motions produce a transverse drift away from the wall, proportional to the spheroid eccentricity and the inverse square of the distance from the wall. This drift is independent of inertia, and is completely determined by the characteristics of the vesicle membrane. The relative strength of the contribution to drift from tank-treading motions and from the presence of inertial corrections, is discussed.

Reproductive Conditions of the Atlantic Sturgeon (Acipenser oxyrinchus) in the Hudson River

Ninety-four prespawning adult Atlantic sturgeon (Acipenser oxyrinchus) were sampled in the Hudson River for age, sex, body size, gonad weight, fecundity, mature oocyte size, and plasma concentrations of gonadotropins, sex steroids, and vitellogenin during the spring spawning migrations in 1992 and 1993. In males, the age and total length ranged from 12 yr to 19 yr and from 133 cm to 204 cm and in females from 14 yr to 36 yr and from 197 cm to 254 cm. The majority of males were 13–16 yr old, and females were 16–20 yr old. Some females had residual atretic ovarian bodies, presumably remaining from a previous spawning and indicating iteroparity. Pre-ovulatory condition was recognized by migration of the germinal vesicle or by germinal vesicle breakdown and by significantly elevated plasma gonadotropins, progesterone, and vitellogenin. All pre-ovulatory females were captured upriver from Hudson River kilometer 136. Individual fecundity ranged from 0.4 million to 2.0 million eggs and oocyte diameter from 2.4 mm to 2.9 mm, and both characters exhibited a significant (p<0.05) positive relationship with female body size. Iteroparous females, tentatively identified by the presence of atretic bodies remaining in the ovary from a previous spawning, had significantly (p<0.05) higher fecundity and produced larger eggs, compared with females spawning presumably for the first time.

Organization of Cytoplasmic Microtubules during Maturation of Goldfish Oocytes

Abstract The organization of cytoplasmic microtubules during hormone-induced meiotic maturation of goldfish oocytes in vitro was examined by confocal immunofluorescence microscopy using an anti-tubulin antibody. The microtubule network was well distributed in fully grown immature oocytes. Once goldfish oocytes resumed meiotic maturation by a proposed maturation-inducing hormone of this species (17α, 20β-dihydroxy-4-pregnen-3-one, 17α, 20β-DP), cytoplasmic microtubules serially re-organized. Soon after the onset of the germinal vesicle (GV) migration towards the animal pole, the former microtubule network disappeared, followed by the appearance of a long perinuclear tail with high ordered microtubules extending from the vegetal surface of the GV. Incubation of fully grown immature follilces in colcemid, an inhibitor of tubulin polymerization, caused the disappearance of microtubules. However, this treatment did not prevent either the 17α, 20β-DP-induced migration of the GV or GVBD. Coincident with the brea...

Xenopus oocyte maturation: cytoplasm alkalization is involved in germinal vesicle migration.

In Xenopus laevis oocytes a transient increase in intracellular pH has been reported to occur during progesterone-induced maturation. Using a cytological approach, we have systematically analyzed germinal vesicle breakdown and meiotic spindle formation in various experimental conditions either preventing or promoting pHi changes. Injection of a neutral buffer (MOPS pH 6.9) induced a cytosolic acidification of 0.3 pH unit and inhibited by 30% the formation of the maturation white spot after progesterone exposure; in oocytes displaying a white spot, only half showed a spindle, often located far from the plasma membrane. Similar results were observed with a Na-free medium which prevents oocyte alkalization. Injection of an alkaline buffer (Tris pH 9) was able to induce migration of the germinal vesicle in 25% of the oocytes in the absence of progesterone, but failed to induce GVBD. Taken together, these results suggest that the increase in pHi observed during maturation may be involved in the migration of the germinal vesicle towards the plasma membrane. We also incubated oocytes in the presence of procaine, a weak base often used to artificially alkalize the oocyte cytoplasm. The changes induced by exposure to procaine were different from those resulting from alkaline buffer injection. Indeed procaine promoted GVBD, as well as spindle formation and chromosome condensation. However these events appeared without migration of the germinal vesicle, suggesting that the expected alkalization did not occur.

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17β (E2), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-tri-hydroxy-4-pregnen-3-one (20β-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20β-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E2 and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20β-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E2 and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20β-S and oocyte GVBD by white perch follices. DHP and 20β-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20β-S in the control of FOM in white perch and white bass.

Manipulation of the seasonal reproductive cycle in winter flounder, Pleuronectes americanus, using a gonadotropic hormone releasing hormone

A gonadotropic hormone-releasing hormone analogue ([D-Ala6, Pro9-NHEt]LHRH, GnRH-A) was administered to the winter flounder, Pleuronectes americanus (Walbaum) at different stages of the reproductive cycle from September 1988 to July 1989. Both males and females responded to releasing hormone treatment throughout the year except for a brief summer period in post-spawned fish when the gonads were regressed. In females, GnRH-A markedly increased plasma estradiol-17β and testosterone levels and accelerated growth of the ovaries during the period of early gonadal recrudescence beginning in August. In prespawning females, GnRH-A treatment increased plasma testosterone levels in association with a stimulation of germinal vesicle migration and ultimately ovulation. During the fall, GnRH-A treatment of males increased plasma androgen levels (testosterone and 11-ketotestosterone) and stimulated growth of the testes. In February, a time when spermiation is initiated in only half of the males, the proportion of spermiating males was increased by treatment with releasing hormone, indicating that spermiation can be advanced in some males by ca. 2 mo.

Antagonistic effects of xenobiotics on steroid-induced final maturation of Atlantic croaker oocytes in vitro

The antagonistic effects of Kepone (chlordecone) and o,p'-DDD on maturation-inducing steroid (17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S) induced final maturation of Atlantic croaker ( Micropogonias undulatus). Oocytes were investigated in an in vitro germinal vesicle breakdown (GVBD) bioassay. Exposure to Kepone or o,p'-DDD (100 nM-100 μM) prevented the majority of the oocytes from completing GVBD in response to 20β-S; many of them were arrested at the lipid coalescence or germinal vesicle migration stages when exposed to high xenobiotic concentrations (100 μM). In addition, clearing of the ooplasm, oil droplet formation and hydration were incomplete in xenobiotic-exposed oocytes that underwent GVBD. The xenobiotics inhibited GVBD in a concentration-dependent manner. One minute exposure to either of the xenobiotics was sufficient to block GVBD. Washing the follicle-enclosed oocytes after xenobiotic exposure restored their ability to undergo GVBD in response to 20β-S. These results suggest that o,p'-DDD and Kepone exert antagonistic effects on 20β-S action, possibly by competing for the ovarian 20β-S membrane receptor.

Plasma Levels of Gonadal Steroids during Final Oocyte Maturation of Striped Bass, Morone saxatilis L.

Levels of estradiol-17β (E 2), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were measured by radioimmunoassay (RIA) in blood plasma of striped bass undergoing final oocyte maturation (FOM). Females were captured just prior to, or in the early stages of, FOM and induced to complete maturation and ovulation with injected human chorionic gonadotropin, synthetic salmon gonadotropin-releasing hormone analogue (sGnRHa; [ d-Arg 6-Pro 9 NEt]-sGnRH), sGnRHa plus the dopamine receptor antagonist, domperidone (DOM), or OVAPRIM, a commercial preparation of sGnRHa + DOM. Their plasma levels of immunoreactive DHP and 20β-S were significantly greater at ovulation relative to the time of hormone injection, whereas the plasma levels of E 2 and T were greatest at injection and decreased by ovulation and 24 hr thereafter. Plasma levels of 20β-S, but not DHP, were sustained at high levels after ovulation. Fish injected only with DOM did not undergo FOM, its associated changes in plasma steroid levels, or ovulation. In females captured at various natural stages of FOM, plasma levels of 20β-S and DHP were low during germinal vesicle migration (GVM), peaked coincident with germinal vesicle breakdown, and then decreased near the time of ovulation. Plasma levels of E 2 and T were greatest during GVM and decreased as DHP and 20β-S levels increased. Analyses of conjugated versus free plasma steroids showed 64-79% of the various hormones to be in the free fraction. RIA of plasma fractionated by reversed-phase HPLC showed that half of the 20β-S immunoreactivity coeluted with 5β-pregnan-3α.17,20β,21-tetrol, a putative 20β-S metabolite with 99.7% cross-reactivity in the 20β-S RIA. These results indicate that striped bass follow the typical profile of changing plasma steroid levels seen in other teleosts during FOM, with a clear shift from C 18 and C 19 steroids to C 21 steroids. They suggest that both DHP and 20β-S, both potent inducers of striped bass FOM in vitro, may play a role in regulating FOM in this species.

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglionin vivo following stimulation-induced exhaustion

Cat superior cervical ganglia (SCG) were tetanically stimulated in vivo at 30-100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 microns-2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 microns-2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.

Development of the pellicle and thecal plates following ecdysis in the dinoflagellateGlenodinium foliaceum

The ultrastructure and development of the amphiesma of the dinoflagellateGlenodinium foliaceum was studied using conventional electron microscopy and immunocytochemistry. Ecdysis (shedding of the flagella, the outer two membranes of the cell, and the thecal plates) was induced by centrifugation. The cells were resuspended and the thickening of the pellicle and the development of the new thecal vesicles and plates was studied over a 9 h period. After ecdysis, the thin pellicle which underlay the thecal plates in the motile cells thickens to form a complex structure of four distinct layers: an outer layer of randomly oriented fibrils, a 50 nm layer of fibrils oriented perpendicular to the dense layer, the dense layer which has a trilaminate structure, and a wide inner homogeneous layer. The new thecal vesicles form in these pelliculate cells by the migration of electron translucent amphisomal vesicles over the layer of peripheral microtubules to a position directly under the plasmalemma. The thecal vesicles then flatten and elongate. A discontinuous pellicular layer appears within them. Subsequently, the thecal vesicles widen and are filled with a fibrillogranular substance overlying the pelliculate layer. The thecal plates form on top of this fibrillogranular material. By this time, most cells have escaped from the pellicle and are motile. At first, the outer thecal vesicle membrane is continuous with the inner thecal vesicle membrane at the sutures, but when this connection is broken, the dense pelliculate layers become continuous across the suture as does the inner thecal vesicle membrane. At ecdysis, this membrane becomes the new plasmalemma of the cell. Cells at each stage of pellicle thickening and thecal development were labelled with a polydonal antiserum raised against the 70 kDa epiplasmic protein ofEuglena acus. This antiserum labelled both the thecal plates of the motile cells and the inner homogeneous layer of the pellicle of ecdysed non-motile cells. No other amphiesmal structure was labelled, nor was any intracellular compartment.

Effects of acrylamide on germinal vesicle migration and dissolution in oocytes of the frog, Rana pipiens

We have demonstrated that when Rana oocytes are treated with 10 m M acrylamide, germinal vesicle migration (GVM) is promoted while meiosis reinitiation by progesterone is inhibited. A number of other specific alterations result from the acrylamide treatment: (i) A dense band of fibrillar material appears adjacent to the oolemma in acrylamide-treated oocytes. Furthermore, (ii) the fibrillar material reacts with an intermediate filament antibody using immunogold techniques applied to transmission electron microscopy. Moreover, (iii) acrylamide inhibits progestogen-induced annulate lamellae breakdown. In addition, (iv) the cortical mitochondria-rich layer appears to be thickened by acrylamide, which also (v) affects oocyte microvillar retraction and organization. Finally, (vi) electrophysiological measurement of membrane voltage indicates that acrylamide does not significantly affect cell viability during the incubation period used in this study. In summary, acrylamide exerts profound effects on the physiological event of GVM, and these are consistent with the hypothesis that changes in the cytoskeleton are a contributing factor in meiosis reinitiation.

Steroids involved with final oocyte maturation in the winter flounder

A number of androgens and progestogens including 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17β-hydroxy-5β-androsten-3-one (5β-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5β-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3β,17α-hydroxy-5-pregnen-20-one (17-PE) and 3β,17α,20β-dihydroxy-5-pregnene (17,20-PE) were found during later oocyte stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (<0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5β-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5β-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studied to date.

Metabolism of progestogens during in vitro meiotic maturation of follicle‐enclosed oocytes of the goldfish (Carassius auratus)

AbstractProgestogens are known to elicit meiotic maturation of fish oocytes in vitro. Exogenous 17α‐hydroxyprogesterone (HP), as assessed by radioimmunoassay (RIA), decreased exponentially to less than 10% of the initial level in media during a 48 hr incubation with large (i.e., 1 mm dia.) ovarian follicles of goldfish undergoing meiotic maturation in vitro. During follicle incubation, media, into which [3H]‐HP was introduced, showed exponential showed exponential reduction of [3H]‐HP with a concomitant increase in the number of [3H]‐HP metabolites. Analysis of follicle extracts revealed that small amounts of [3H]‐HP were taken up by the follicles early in the incubation (i.e., 3 hr) and converted to several metabolites; the major one of these, which accumulated in the follicle, comigrated with 17α, 20β dihydroxy 4‐pregnen 3‐one (DHP). Autoradiography of extracts run on TLC revealed an increasing number of metabolites with time, i.e., 9 major spots for media, 6 major spots for follicles incubated 24 hr with [3H]‐HP. Three of the more prominent metabolites, DHP, 3α, 17α‐dihydroxy5β‐pregnan 20‐one, and 5β‐pregnan3α, 17α, 20β‐triol, were identified tentatively by comigration with standards on TLC and crystallization to constant specific activity. HPLC of extracts revealed a selective accumulation of DHP in follicles over time i.e., this steroid was the major metabolite peak associated with follicles by 24 hr incubation. Nevertheless, the process of oocyte maturation had already been initiated before the peak level of DHP had been reached. By contrast, HP diminished with time in follicles despite the relatively high levels occurring in the media. This research indicates that a complex steroid milieu at the level of individual, fully grown, ovarian follicles is produced in vitro from exogenous HP; significant HP metabolism occurs by 6 hr incubation, well before the process of oocyte maturation (i.e., germinal vesicle migration and dissolution) has begun.

Ultrastructure of sperm development and mature sperm morphology in three species of commensal bivalves (Mollusca: Galeommatoidea).

Ultrastructural features of the ovotestes, spermatogenesis, and the mature sperm are described for three galeommatid bivalves, Divariscintilla yoyo, Divariscintilla troglodytes, and Scintilla sp., from stomatopod burrows in eastern Florida. All three species yielded similar results except with respect to mature sperm dimensions. The ovotestis contains three types of somatic cells within the testicular portion: flattened myoepithelial cells defining the outer acinal wall; underlying pleomorphic follicle cells containing abundant glycogen deposits; and scattered, amoeboid cells containing lysosomal-like inclusions which are closely associated with developing sperm. Early spermatogenesis is typical of that reported from other bivalves. In contrast, the late stages of spermiogenesis involve the migration and gradual rotation of the acrosomal vesicle, resulting in a mature acrosome tilted about 70° from the long axis of the cell. The mature sperm possesses an elongated, slightly curved nucleus; a subterminal, concave acrosome with a nipple-like central projection; five spherical mitochondria and two centnoles in the middlepiece; and a long flagellum. The rotational asymmetry and the presence of perimitochondrial glycogen deposits in these sperm are unusual in the Bivalvia and may be associated with fertilization specializations and larval brooding common among galeommatoideans.

LIGHT‐INDUCED CHANGES IN THE VESICULAR APPARATUS OF Phycomyces SPORANGIOPHORES DURING THE PHOTOTROPIC RESPONSE

Abstract— We studied the ultrastructure of laterally illuminated stage I sporangiophores of Phycomyces blakesleeanus to determine whether the phototropic response had any structural correlate. Stimulated sporangiophores conserved the distinctive division into the four longitudinal zones described for state I, and showed no significant differences in the distribution of major organelles in the sides proximal and distal to the light source. On the other hand, apical vesicles were asymmetrically distributed, being present in lower numbers in the faster growing (distal) side of the sporangiophore. We suggest that this asymmetrical distribution reflects the differential utilization of vesicles necessary to sustain the corresponding growth rate of each side. Illumination therefore, must affect the rate of vesicle migration and utilization, more than their rate of synthesis.

Characterization of membrane receptor activity for 17α,20β,21-trihydroxy-4-pregnen-3-one in ovaries of spotted seatrout ( Cynoscion nebulosus)

The proposed maturation-inducing substance (MIS) of spotted seatrout ( Cynoscion nebulosus) is 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S). In this study, we characterized the binding of radioactive 20β-S to plasma membranes from the ovaries of spotted seatrout. Bound 20β-S was isolated by filtration of membrane suspensions and quantified by measurement of the radioactivity content of the filters. The saturable component of 20β-S binding reached equilibrium within 5 min at 0°, showed linearity with membrane concentration, and was pH dependent (optimum, 7.5–7.8). Scatchard analyses suggested a single class of high-affinity ( K D , 10 −9 M), low-capacity (10 −13–10 −12 mol/g ovary) binding sites for 20β-S. High levels of saturable binding were found in membrane preparations from the ovary, testis, and liver, but not from the gills. 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-P) showed relatively little affinity for the 20β-S binding site. However, this steroid was converted to a compound immunologically and chromatographically similar to 20β-S by intact ovarian follicles, a finding which may explain its previously reported high potency in an in vitro oocyte maturation bioassay. Conversely, although reportedly a weak inducer of oocyte maturation, progesterone readily displaced 20β-S from its binding site. Thus, progesterone appears to be a relatively inactive ligand with high affinity for the 20β-S receptor. The concentration of 20β-S binding sites in ovaries was significantly higher during final oocyte maturation (germinal vesicle migration) than at earlier stages of development. These results strongly suggest that the 20β-S binding activity characterized in our study represents authentic MIS receptors. A distinct, soluble binding site for 17α,20β-P was also identified in seatrout ovaries, but its biological function remains unclear. A hypothesis is presented for the significance of this 17α,20β-P binding site.

Structure and Behaviour of Organelles in Living Pollen Tubes ofLilium longiflorum

The morphology and the movement of organelles, in particular of the endoplasmic reticulum (ER), have been studied using video-enhanced microscopy in living pollen tubes of Lilium longiflorum Thunb. The patterns of the cytoplasmic streaming in the pollen grains and tubes generally correspond with the descriptions by Iwanami (1956). We found, however, that the organelles move as single and independent elements reflecting highly individual characteristics. The apical zone of the pollen tube, the so-called 'clear' zone, is populated by numerous intensively vibrating organelles of small size, most probably Golgi vesicles. The migration of vesicles to the apical plasma membrane of the growing pollen tube is not a smooth flow of particles, but a turbulent process. Spherical bodies with a high contrast, probably polysaccharide (p-) particles and/or lipid drops, and some mitochondria are also visible in the extreme tip. Their penetration into the apex seems to be inhibited by the dense mass of agitated vesicles. The small vacuoles located in the central zone are very motile; they change their shape continuously. The tonoplast, therefore, must be very plastic and, to a certain extent, mechanically resistant. The vegetative nucleus and the generative cell maintained a stable shape despite constant collisions with the organelles of the vigorously streaming cytoplasm. The thin cytoplasmic layer in the vacuolate zone contains tubular ER (diameter about 0-3 /urn) arranged in complex closed networks. The tubules are permanently moving. Gentle undulations alternated with sudden dramatic distortions. Rapid fusions or divisions of tubules are common. This dynamic behaviour is taken as proof of the plasticity of the ER tubules and the high fluidity of the membranes of the ER. Organelles preferentially move along the ER tubules in a saltatory manner. It is assumed that the ER tubules are associated with cytoskeletal elements.

Fate of gap junctions in isolated adult mammalian cardiomyocytes.

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.