Cytoskeletal and contractile proteins in coelomic oocytes,unfertilized and fertilized eggs of discoglossus pictus (Anura)

The distribution of actin, myosin, and tubulin has been investigated in coelomic oocytes, unfertilized and fertilized eggs of Discoglossus pictus utilizing: (1) immunofluorescence; (2) electron microscopy; (3) incubation with heavy meromyosin (HMM), and (4) SDS-polyacrylamide gel electrophoresis (PAGE). In coelomic oocytes, the germinative area (GA) has long, irregular microvilli containing microfilaments. In the rest of the oocyte, the microvilli are shallow. During the transit of the oocyte in the oviduct, a dimple forms by the invagination of the GA. A palisade of microfialment bundles is present in the finger-shaped microvilli of the dimple and extends for about 10 μm in the cytoplasm. In the rest of the egg, microvilli are absent and only random filaments appear in the cortex. Following HMM incubation, the dimple microfilaments are decorated with arrowheads pointing toward the bulk of the cytoplasm. SDS-PAGE of egg extracts shows bands co-migrating with actin (43K), pyruvate kinase (57K), and phosphorylase (94K). As result fertilization, the pattern of microfilament bundles in the dimple disappers in parallel with the dimple invergination itself. Generally, the entire oocyte cortex is positive to immunofluorescent staining with anti-actin, antimyosin, and antitubulin antibodies. However, the pattern of distribution and intensity of immunofluorescent staining changes for each antiserum, during different stages. It is concluded that a contractile system is present in Discoglossus eggs, and it is particularly developed in the dimple. The dimple is probably a major compartment for the storage of unpolymerized tubulin.     

Study of the interaction between rabbit cardiac contractile and regulatory proteins. An <Emphasis Type="Italic">in vitro</Emphasis> motility assay

A series of experiments was performed in an in vitro motility assay with reconstructed thin filaments to obtain pCa-force relationships for cardiac isomyosins V1 and V3. Two concentrations of each isomyosin (200 and 300 microg/ml) on the surface of a flow cell were tested. Isometric force was estimated as the amount of actin-binding protein, alpha-actinin, stopping thin filament movement. It was found that the amount of alpha-actinin stopping the movement at saturating calcium concentration for V3 was twice higher than for V1 at both concentrations of isoforms. Hill coefficients of cooperativity (h) were determined for pCa-force relationships. The value of h did not differ significantly for isoforms at 300 microg/ml of protein (h was 1.56 for V1 and 1.54 for V3). However, the Hill coefficient was higher for V3 isoform at 200 microg/ml (h = 2.00 and 1.76 for V3 and V1, respectively). Importantly, the Hill coefficient increased for both isoenzymes when their concentrations were decreased. The connection between Hill coefficient and cooperative interactions between cardiac contractile and regulatory proteins is analyzed in detail.     

Fine structure of the spermatozoon in the mite Parasitus niveus (Mesostigmata,Acari)

The spermatozoa of the mite, Parasitus niveus, are rod-shaped cells possessing a very elongated and zig-zag shaped nucleus. The cytoplasm is filled by so-called “striated bodies” and mitochondria. The plasmalemma forms five complicated structures, called stiff bands. In the peripheral cytoplasm lie flattened canaliculi and flattened cisternae. The morphology of the spermatozoa is compared with that of other mite spermatozoa described in the literature.     

Subcellular localization of calcium deposits in the noble crayfish Astacus astacus spermatophore: Implications for post‐mating spermatophore hardening and spermatozoon maturation

The freshly ejaculated spermatophore of crayfish undergoes a hardening process during post‐mating storage on the body surface of female. The ultrastructural distribution of calcium deposits were studied and compared in freshly ejaculated and post‐mating noble crayfish spermatophores, using the oxalate‐pyroantimonate technique, to determine possible roles of calcium in post‐mating spermatophore hardening and spermatozoon maturation. Small particles of sparsely distributed calcium deposits were visible in the wall of freshly ejaculated spermatophore. Also, large amount of calcium deposits were visible in the membranes of the freshly ejaculated spermatozoon. Five minutes post‐ejaculation, granules in the spermatophore wall appeared as porous formations with numerous electron lucent spaces. Calcium deposits were visible within the spaces and scattered in the spermatophore wall matrix, where smaller calcium deposits combined to form globular calcium deposits. Large numbers of the globular calcium deposits were visible in the wall of the post‐mating spermatophore. Smaller calcium deposits were detected in the central area of post‐mating spermatophore, which contains the sperm mass, and in the extracellular matrix and capsule. While the density of calcium deposits decreased in the post‐mating spermatozoon membranes, numerous small calcium deposits appeared in the subacrosomal zone and nucleus. Substantial changes in calcium deposit distribution in the crayfish spermatophore during post‐mating storage on the body of female may be involved in the processes of the spermatophore hardening and spermatozoon maturation. J. Morphol. 277:445–452, 2016. © 2016 Wiley Periodicals, Inc.     

Sperm-egg interactions in the shrimp Rhynchocinetes typus

Sperm-egg interaction in Rhynchocinetes typus was studied with the phase-contrast and scanning electron microscopes. R typus spermatozoa present in the vas deferens have the shape of a round-headed nail. After contact with seawater it is possible to observe the unfolding of the rays or stellate arms, giving the spermatozoon the appearance of an inverted umbrella. From the center of the flat face of the umbrella emerges a spike with longitudinal striations. Ovarian eggs and spermatozoa were mixed in vitro by agitating them for two minutes in Millipore-filtered seawater. The first gamete contact was established by the spermatozoon through the tip of the spike, which exerted a lytic action on the egg envelopes. After the rigid spike was completely inside the egg, the rays became aligned parallel to each other and began to enter the eggs. Toward the final stages of ray entry, it was possible to observe fusion of the ray membranes with one another, and later the fusion process continued toward the tip of the radial spines. Concomitantly, the egg surface that surrounds the sperm swelled in a circular fashion and formed a fertilization cone. After the spermatozoon entry was complete, a scarlike mark appeared at the place on the egg surface through which penetration occurred. The whole penetration process was completed within 45-60 minutes.     

Ultrastructure of the head of ctenomys maulinus spermatozoon with special reference to the nucleus

The spermatozoa of the neotropical hystricomorph rodent Ctenomys maulinus have been examined cytochemically and under the transmission electron microscope. The head is flattened dorsoventrally. At the caudal end of the head there is a process oriented parallel to the tail. This process corresponds to a cylindrical extension of the nucleus, which constitutes a unique feature among mammals.     

Elbow flexor evoked twitch contractile properties in untrained men and women and male bodybuilders

The influence of elbow joint angle on elbow flexor isometric evoked twitch contractile properties was assessed in 15 young women (F), 18 young men (M) and 11 male bodybuilders (BB). Measurements were made at elbow joint angles of 1.31, 1.57, 1.83, 2.09, 2.36, 2.62 and 2.88 rad (3.14 rad =180° =full extension). The largest peak twitch torque values [mean (SE) N · m] in F [3.77 (0.20)], M [10.38 (0.68)] and BB [11.38 (1.05)] occurred at 2.88 rad. Peak torque was progressively smaller at smaller joint angles, but the decline from 2.88 to 1.31 rad was greater in M (68%) and BB (76%) than F (59%). Thus, the magnitude of intergroup differences in peak twitch torque (PT) was joint angle dependent. Twitch time to peak torque (TPT) was influenced in a complex way by joint angle in the three groups; BB had the lowest values at small joint angles but the highest values at the largest angles. Half-relaxation time (HRT) generally increased from the smallest to largest joint angles in a pattern that did not differ significantly among the three groups. Maximum rates of twitch torque development and relaxation showed the same pattern of results as PT; indicating that these time-related measures were more sensitive to joint angle effects on PT than on TPT or HRT. The results of this study indicate that careful consideration should be given to the selection of joint angles in the measurement of evoked twitch contractile properties for the purpose of making group comparisons or investigating the effects of interventions such as training.     

The ultrastructure of the spermatozoon and spermiogenesis in Cryptocotyle lingua (digenea: Heterophyidae)

During spermiogenesis two lateral flagellar processes and a median process arising from the apex of the zone of differentiation, fuse to form the elongated unipartite spermatozoon. Two axial units, therefore, with the ‘9+1’ pattern of microtubules are incorporated into the spermatozoon. The nucleus, in the head region, contains dense lamellar subunits arranged in a spiral in the long axis. These are formed by condensation of the chromatin during spermiogenesis. The single elongated mitochondrion, resulting from early fusion of small mitochondria, extends through the head and middle regions of the spermatozoon. Peripheral microtubules, present originally in the zone of differentiation, are arranged in straight dorsal and ventral rows, along the length. β glycogen particles accumulate in the spermatozoa after they have separated from the residual cytoplasm. Spermatozoa are present in the testes on the second day after infection of the bird host and accumulate in the vesicula seminalis from the third day onwards.     

Regulation of contractile vacuole formation and activity in Dictyostelium

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV–plasma membrane fusion and discharging. disgorgin⁻ cells produce large CVs due to impaired CV–plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin⁻ large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.     

The morphology of the squirrel spermatozoon: A highly complex male gamete with a massive acrosome

This study describes the morphology of the spermatozoon from the cauda epididymidis of representative members of two squirrel subfamilies, the Sciurinae and Callosciurinae, as determined by fluorescent, scanning, and transmission electron microscopy. All species examined possess a massive apical segment of the sperm acrosome. It varied markedly in the extent of its caudal flexion but was always much larger, and more complex, than that of the spermatozoon of most other rodents so far documented, although somewhat similar to that of some hystricomorph species. Because this sperm form appears to be present within at least two of the three major living clades of Rodentia, it is possible that it is the ancestral condition within this mammalian order. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Influence of spawning on swimming performance and muscle contractile properties in the short-horn sculpin

The total distance travelled during the first two kinematic stages of the escape response of short-horn sculpin was significantly greater in post spawning fish (0·41 L) than in gravid fish (0·23 L). The maximum velocity of the snout during the C-bend was significantly higher (5·6 L s⁻¹) in postspawning fish than in gravid fish (3·8 L s⁻¹). To investigate some of the mechanisms underlying changes in swimming performance, the contractile properties of fast muscle fibres were determined in fish of similar body length. The rate of tetanic force relaxation (time from last stimulus to 50% peak force) was 34% faster in gr avid than in postspawning fish. Maximum contraction velocity, determined by the slack-test method, was significantly higher in gravid than in postspawning fish (6·8 v . 5·9 muscle lengths s⁻¹). In contrast, both maximum isometric stress and power output (determined from the force–velocity relationship) were >50% higher in fibres from postspawning than from gravid fish, even though myofibrillar protein and water contents were similar (120 mg g⁻¹ wet mass and 86% of body mass, respectively). The results show that swimming performance and the contractile properties of fast muscle fibres vary with the reproductive cycle in short-horn sculpin acclimated to the same photoperiodic and temperature regime.     

Distribution of intramembranous particles and filipin-sterol complexes in the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.     

Giardia spp.: Distribution of contractile proteins in the attachment organelle

Giardia spp. trophozoites isolated from rat small intestine were examined by light microscopy, electron microscopy, SDS-gel electrophoresis, and immunocytochemistry. In SDS-gels of protein extracts of isolated Giardia spp. trophozoites protein bands corresponding to myosin, α-actinin, and actin were identified by comigration with avian myofibril proteins and molecular weight standards. Actin was specifically identified in SDS-gels by immunoautoradiography. Immunostaining for actin, α-actinin, myosin, and tropomyosin in trophozoites was demonstrated in the periphery of the ventral disc in an area corresponding to the lateral crest. Electron-dense fibrillar was observed in the lateral crest of the ventral disc by electron microscopy. Immunostaining for actin and α-actinin was also observed in the area of the median body, a microtubular organelle, and in electron-dense fibrillar material associated with the intracellular axonemes of the posterior-lateral flagella. The localization of these contractile proteins in the ventral disc suggests that they may play an important role in the mechanism of trophozoite attachment.     

Fine structure of the mature spermatozoon of rhynchocinetes typus,crustacea decapoda

Rhynchocinetes typus spermatozoa obtained from the vas deferens have the shape of a round-headed nail. The head measures 30 μm in diameter and 14 μm of height. At the center of the flat face of the head emerges a single rigid spike of 53 μm in length. Cross sections of this spike show that it has a wall of 0.4 μm in thickness and a core of 0.6 to 0.8 μm. The outer surface of the spike has a longitudinal striation. When the spermatozoa are placed in sea water it is possible to observe the unfolding of rays. The number of rays in different spermatozoa of the same individual varies from 9 to 13. Each ray is formed by a channel-like sheath that contains a rigid rod that occupies about 1/3 the length of the ray. This rod has a transverse striation with a periodicity of 185A. The rays are bound among them by a thin membranous sheet that is highly folded in vas deferens spermatozoa. At the distal end of each ray there is a rigid spine of 50 μm in length. The nucleus is coplanar to the radial plane and it extends through the rays. The structure and ultrastructure of R typus spermatozoa depart from that reported for spermatozoa of other Caridea species.     

The induction of contractile roots in Gladiolus grandiflorus

The number of contractile roots formed in gladioli was inversely related to the depth of the planted corm. Below a certain depth, no contractile roots were produced. Large corms did not produce contractile roots at any planting depth but produced two or more small corms. Depth perception is a function of two independent mechanisms, namely, temperature fluctuations in the root-initiation zone and the amount of light perceived mainly by the upper sheath leaf, the length of which varies with planting depth. Various growth substances applied to the leaves or corms did not induce contractile roots in dark-grown plants but roots were induced by indole-3-butyric acid in both small and large corms grown at constant temperatures and light. Abscisic acid retarded the formation of contractile roots under inductive conditions.Abbreviations CR contractile root(s) - IBA indole-3-butyric acid     

Monitoring dimeric status of IZUMO1 during the acrosome reaction in living spermatozoon

The acrosome reaction (AR) is indispensable for successful spermatozoon-oocyte fusion. Recent studies have indicated that sperm IZUMO1 gradually gathers in the equatorial segment (EQ), which is the initiation site of sperm-egg fusion, after the AR. In addition, by examining the binding process of oocytes and Izumo1-expressing cultured cells to reconstitute the early steps of fertilization, we previously demonstrated that robust IZUMO1-dependent adhesion specifically occurs at the contact site along with the dimerization of IZUMO1. However, when IZUMO1 dimerizes after the AR in living spermatozoon is unknown. Here, we report dynamics of IZUMO1 dimerization during the AR in spermatozoa by combining transgenic mice and time-lapse imaging using a set of bimolecular fluorescence complementation (BiFC) probes. Surprisingly, dimeric IZUMO1 was already formed at the acrosomal cap region before the AR and redistributed into the EQ after the AR. We categorized the translocation of the dimer into two types: Type 1, the near-simultaneous appearance of BiFC signals with IZUMO1-mCherry; and Type 2, the delayed formation of dimer in the EQ. Those findings suggest that, before encountering oocytes, spermatozoa are prepared to boost their affinity with JUNO.     

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae,Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.