Cytoskeletal elements and intracellular transport

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.     

Cytoskeletal involvement in neuronal learning: a review

This paper introduces the ideas of neural networks in the context of currently recognized cellular structures within neurons. Neural network models and paradigms require adaptation of synapses for learning to occur in the network. Some models of learning paradigms require information to move from axon to dendrite. This motivated us to examine the possibility of intracellular signaling to mediate such signals. The cytoskeleton forms a substrate for intracellular signaling via material transport and other putative mechanisms. Furthermore, many experimental results suggest a link between the cytoskeleton and cognitive processing. In this paper we review research on intracellular signaling in the context of neural network learning.Abbreviations MT microtubule - MTs microtubules - ART adaptive resonance theory - RCE restricted coulomb energy - MAP microtubule associated protein - NO nitric oxide Correspondence to: J. Dayhoff     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Cytoskeletal dynamics and transport in growth cone motility and axon guidance

Recent studies indicate the actin and microtubule cytoskeletons are a final common target of many signaling cascades that influence the developing neuron. Regulation of polymer dynamics and transport are crucial for the proper growth cone motility. This review addresses how actin filaments, microtubules, and their associated proteins play crucial roles in growth cone motility, axon outgrowth, and guidance. We present a working model for cytoskeletal regulation of directed axon outgrowth. An important goal for the future will be to understand the coordinated response of the cytoskeleton to signaling cascades induced by guidance receptor activation.     

Cytoskeletal involvement during hypo-osmotic swelling and volume regulation in cultured chick cardiac myocytes

The membrane skeleton in spherical cardiac myocytes subjected to hypo-osmotic challenge was examined by laser scanning confocal microscopy. A distinct cortical layer intimately localized under the plasmalemma was revealed for spectrin and actin (including filamentous actin and alpha-sarcomeric actin). Desmin filaments were abundant and in close contact with the plasmalemma. During swelling and subsequent regulatory volume decrease (RVD) the structural integrity of these cytoskeletal elements remained intact, and the close association between actin and plasmalemma persisted as confirmed by double immunolabeling. Subplasmalemmal beta-tubulin labeling was sparse. Hypo-osmotic conditions disrupted the microtubules and depolymerized tubulin. Neither pretreatment with taxol nor with colchicine, resulted in any effect on cell volume regulation. The present results show that actin, desmin, and spectrin contribute to a subplasmalemmal cytoskeletal network in spherical cardiac myocytes, and that this membrane skeleton remains structurally intact during swelling and RVD. It is suggested that the integrity of this membrane skeleton is important for stabilization of the plasmalemma and the membrane-integrated proteins during hypo-osmotic challenge, and that it may participate in the regulation of the cell volume.     

Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment

Weekly injections of human chorionic gonadotropin (hCG; 1·5 IU g⁻¹ W ) induced spermiation in 87–100% farmed male European eels Anguilla anguilla over a 10-week period, producing 3 ml (100 g)⁻¹ milt volume, at 1˙4×10⁹ spermatozoa ml⁻¹ sperm mobility peaked in ninth week of treatment with 97% of males with motile sperm. Gonadotropin-treated males showed earlier but similar morphological changes to control males. Whereas higher sperm density was obtained 6 h after hCG administration, higher percentage of motile cells and longer sperm mobility were observed 24 h after the treatment indicating that this is the optimum time to obtain good sperm after the hormonal induction. Several monounsaturated fatty acids in sperm increased significantly from week 5 to 9 of treatment (when highest volume, density and mobility of milt were recorded), whereas eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 polyunsaturated fatty acids (PUFAs) were significantly reduced. Fatty acid levels returned to initial values after 13 weeks of treatment. These results are in agreement with the reduction of n-3 PUFAs recently observed in the ovary of female Japanese eel during artificial maturation, and an attempt is made to suggest a physiological explanation of its possible relation with sperm quality.     

Asymmetry of spermiation and sperm surface charge patterns over the giant acrosome in the musk shrew Suncus murinus

Spermatozoa of the shrew Suncus murinus, a mammal with abdominal testes, exhibit four unusual features: a giant acrosome; a dorsoventral asymmetry of their spermiation; a dorsoventral asymmetry of their head surface character; and also apparent surface maturity as they enter the epididymis. A Sertoli cell-periacrosomal cisternal complex envelops the giant acrosome during spermatid maturation. Spermiation is heraled by asymmetrical disorganization of the subplasmalemmal components of this complex and is completed by retraction of the Sertoli cell from the ventral and then the dorsal face of the spermatid head. This sequence or release is correlated with an asynchronous acquisition of negative surface charges on the spermatid head-demonstrable on glutaraldehyde-stabilized cells by the binding at pH 1.8 of positively charged colloidal particles of ferric oxide. Mature epididymal spermatozoa exhibit an asymmetry in the patterns of distribution of bound colloid over the dorsal vs. ventral surfaces of the sperm head, as well as regional differences between the tail midpiece and principal piece. Surface distributions of anionic residues and lectin (Con A)-binding sites characteristic of mature Suncus spermatozoa are demonstrable within the testis, unlike the situation in most nannals where distinct modifications of the sperm surface occur during epididymal passage.     

F-actin involvement in guinea pig sperm motility

Sperm motility is a must for natural fertilization to occur. During their travel through the epididymis, mammalian spermatozoa gradually acquire the ability to move. This is accomplished through a sliding movement of the outer doublet microtubules of the axoneme which is energized by the dynein ATPase. Within its complex structure, the mammalian sperm flagellum contains F-actin and thus, we decided to test in the guinea pig sperm flagellum the role of F-actin in motility. During maturation, capacitation, and the acrosome reaction, a gradual decrease of the relative concentration of F-actin was observed. Motility increased as spermatozoa became able to fertilize. Gelsolin, phalloidin, and KI inhibited sperm motility. Gelsolin canceled sperm motility within 20 min of treatment while 0.6 M KI had immediate effects. Phalloidin diminished hyperactive sperm motility slightly. All three compounds significantly increased the relative concentration of F-actin. Latrunculins are conventional drugs that destabilize the F-actin cytoskeleton. Latrunculin A (LAT A) did not affect sperm motility; but significantly increased F-actin relative concentration. The results suggested that in guinea pig spermatozoa, randomly severing F-actin filaments inhibits flagellar motility; while end filament alteration does not. Thus, specific filament regions seem to be important for sperm motility.     

Characterization of normal spermiation and spermiation failure induced by hormone suppression in adult rats

At the end of spermatogenesis, elongated spermatids are released from supporting Sertoli cells via the process termed spermiation. Previous studies have shown that spermiation failure occurs after hormone suppression, in which spermatids are retained instead of releasing. However, the molecular mechanisms involved in spermiation and spermiation failure are largely unknown. The aims of the present study were, first, to characterize the ultrastructural events associated with normal spermiation and spermiation failure using light and electron microscopy and, second, to investigate the localization of cell adhesion-associated (beta1-integrin and cadherins) and junction-associated molecules (integrin-associated kinase [ILK], beta-catenin, and espin) during these processes. Four adult Sprague-Dawley rats received testosterone and estradiol implants and FSH antibody (2 mg kg-1 day-1) for 7 days to suppress testicular testosterone and FSH and to induce spermiation failure. Four rats treated with saline were used as controls. After testosterone and FSH suppression, spermiation at the ultrastructural level appeared to be normal until the final disengagement of the spermatids from Sertoli cells (stage VIII), at which stage a large number of retained spermatids were noted. Immunohistochemical localization of espin showed that during spermiation, removal of the ectoplasmic specialization (ES) occurred 30 h before spermatid disengagement, suggesting that non-ES junctions mediate the spermatid-Sertoli cell interaction before and during disengagement. beta1-Integrin and beta-catenin remained associated with spermatids after ES removal and until disengagement; however, ILK was removed along with the ES. Though detectable, N-cadherin was not associated with the spermatid-Sertoli cell junction. After testosterone and FSH suppression, beta1-integrin, but not N-cadherin or beta-catenin, remained associated with spermatids that failed to spermiate. In conclusion, hormone suppression-induced spermiation failure is caused by defects in the disengagement of spermatids from the Sertoli cell, and this process likely is mediated by beta1-integrin in an ILK-independent mechanism.     

Timing of sperm transport, sperm penetration and cleavage in the rat

Times of sperm entry into the oviduct from the uterus, into the ampulla from the isthmus; of sperm penetration into oocytes, and of cleavage, were determined using three mating regions. Time intervals and their errors of estimation were calculated. Spermatozoa were first found in the isthmus of the oviduct no earlier than 15 minutes after coitus, but required four hours to ascend the oviduct to the ampulla. The rate of sperm arrival was equal to the rate of sperm penetration, i.e., about 3 sperms/hour. Time of cleavage in vivo was 20.6 hours after sperm penetration in ad libitum mated animals. In culture, oocytes cleaved at exactly the same time as in vivo. Delaying sperm arrival to the site of fertilization (by delaying mating) shortened the time interval between median time of sperm penetration and median time of cleavage. It was concluded that the time of cleavage of the oocyte reflects primarily the time of sperm penetration, but is also influenced by the postovulatory age of the oocyte.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

Components in seminal plasma regulating sperm transport and elimination

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.     

Inhibition of sperm transport and fertilization in superovulating ewes

In Experiment 1, all ewes were treated with follicle stimulating hormone (FSH-P) to induce superovulation. Ewes came into natural estrus or were treated with prostaglandin F(2)alpha (PGF(2)alpha) or 6-methyl-17-acetoxyprogesterone (MAP) to regulate the time of estrus. The ewes were mated during estrus and necropsied 3 h after mating. Regulation of estrus with either compound reduced the number of sperm recovered from the cervix, uterus, and oviducts and increased the proportions of sperm recovered from the cervix and uterine body that were immotile, dead, or had disrupted membranes. In Experiment 2, all ewes were in natural estrus. They either ovulated naturally or were superovulated, and ewes in each group were necropsied at 3 or 23 h after mating. Superovulation reduced the number of sperm in oviducts, uterus, and anterior segments of the cervix at both time intervals and increased the proportions of sperm that were immotile, dead, or had disrupted membranes. In Experiment 3, of 3x2 design, ewes were in either natural estrus or estrus regulated with PGF(2)alpha or with MAP; they ovulated naturally or were superovulated. Ewes were necropsied 3 d after mating and ova were examined. Both regulation of estrus and superovulation reduced the proportion of ova that were fertilized and reduced the number of accessory sperm attached to fertilized ova.     

Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare

Our objective was to determine whether the concentration of cooled sperm inseminated influenced sperm transport and intensity of the uterine inflammatory reaction 2, 4 and 24h after insemination. Experimental subjects were 189 estrous mares with a dominant follicle > or =35 mm in diameter and no bacterial growth or neutrophils detected in uterine smears. Each mare was randomly assigned to receive one of the following intrauterine treatments (volume, 20 mL): insemination with 5x10(6) mL(-1) or 25x10(6) mL(-1) or 50x10(6) mL(-1) sperm diluted in 3 mL seminal plasma (SP) and 17 mL skim milk; seminal plasma or skim milk extender. Mares in a control group received no intrauterine treatment. Mares were slaughtered 2, 4 or 24h after insemination or infusion. Oviducts were separated from the uterus, and uterus and oviducts were then flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected for further histopathological examination. The grade of uterine fibrosis and the amount of neutrophils in the stratum compactum were evaluated. A sample of each tubal flushing was examined for sperm count, and a sample of each uterine flushing was examined for PMN count. It was concluded that compounds in the insemination dose provoked a uterine inflammatory response, which was more rapid and intense as sperm concentration increased. In contrast, sperm transport through 4h after insemination was not influenced by sperm concentration.     

Effect of coitus on sperm transport and fertilization in rabbits

The number of spermatozoa into the Fallopian tubes and levels of in vivo fertilization were measured following different treatment. The results show that copulation by the artificially inseminated animal has a strong influence on the transport of the rabbit spermatozoa and "in vitro" fertilization. This constitutes some more evidence about the physiological role played by the utero-tubal junction.     

Intraperitoneal insemination, sperm transport and capacitation in the pig

Intraperitoneal insemination was studied in a total of 58 pigs, both to ascertain the success of this route of sperm deposition with the eventual use of frozen-thawed boar semen in mind and to estimate the timing of capacitation in the absence of uterine exposure of spermatozoa. Ovulation was controlled in mature gilts, and 5–20 ml freshly collected semen containing approximately 10⁸ spermatozoa per ml introduced through the peritoneum either by means of mid-ventral laparotomy or using a 3.5-in (ca. 9 cm) × 18-gauge hypodermic needle.Embryo development to the morula and blastocyst stage appeared chronologically and cytologically normal after intraperitoneal insemination, but the timing of semen deposition was critical: optimal levels of fertilization (60%) arose from insemination in the 12 h preceding ovulation. Fertility was never comparable to that found after natural mating due to the inefficiency of sperm transport into the oviducts and the absence of significant sperm reservoirs. The timing of sperm capacitation after intraperitoneal insemination was not reduced when compared with that found after insemination directly into the oviducts, indicating a negligible contribution of peritoneal exposure to this process. Spermatozoa were not phagocytosed in the oviducts, but rather descended to the uterus at the same time as the developing embryos or degenerating eggs, the sperm flagellum usually being separated from the head by this stage.