Three-Dimensional Reconstruction of Rabbit-Derived Pneumocystis carinii from Serial-Thin Sections I: Trophozoite

The highly complex ultrastructural morphology of the endomembrane system in Pneumocystis carinii led us to perform three-dimensional reconstruction from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The three-dimensional reconstruction of a small trophozoite made it possible to better understand the morphological relationship among organelles and to suggest cytophysiological hypotheses. By reconstructing other parasite stages, we gathered information about the evolution of organelles during the life cycle and about their physiology.     

Three-dimensional reconstruction of rabbit-derived Pneumocystis carinii from serial-thin sections. I: Trophozoite.

The highly complex ultrastructural morphology of the endomembrane system in Pneumocystis carinii led us to perform three-dimensional reconstruction from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The three-dimensional reconstruction of a small trophozoite made it possible to better understand the morphological relationship among organelles and to suggest cytophysiological hypotheses. By reconstructing other parasite stages, we gathered information about the evolution of organelles during the life cycle and about their physiology.     

Pelargonium embryogenesis: cytological investigations of organelles in early embryogenesis from the egg to the two-celled embryo

. Changes in the distribution of organelles and organelle-DNA in Pelargonium zonale from the mature egg cell stage to the first zygotic division during the early stages of embryogenesis were investigated using electron microscopy and fluorescence microscopy. The mature egg is a large, polarized bulbous-shaped cell, tapering toward its micropylar end. The wide chalazal region has a large nucleus that is surrounded by cytoplasm containing many giant mitochondria and large amyloplasts. The mitochondria contain a large amount of mitochondrial DNA and appear as long stretched rods or complex rings, sometimes consisting of several concentric or half-concentric circles in sections. The time from pollination to cell fusion is approximately 6-9 h and it is 20-24 h until the first zygotic division. The changes in the zygote and its organelles preparatory to division occur in 3 stages. At stage 1 (6-9 h after pollination), cell fusion occurs and the zygote begins to elongate. Many vacuoles of varying size appear surrounding the nucleus. At stage 2 (9-15 h), the zygote nucleus migrates to a central position in the cell and the mitochondria form a single ring that becomes either irregularly crushed or appears as long thin strings. Amyloplasts exhibit a gradual decrease in the number of starch grains. At stage 3 (15-20 h), the vacuoles disappear, except for a few that remain in the micropylar region, and cell size decreases. Mitochondria become short, fine strings or small rings. Amyloplasts with starch grains are no longer observed, but are transformed into large proplastids. Following the first division of the zygote, approximately equal-sized apical and basal cells are formed. Short rod-shaped or small ring-shaped mitochondria are randomly distributed near the nucleus of the apical cell, whereas mitochondria in the basal cell are long and rod-shaped. In the electron microscope, two types of plastids can be distinguished: dark oval plastids originating from the sperm cell, which are observed in both the apical and basal cell, and others with a less dense, amorphous matrix, believed to originate from egg amyloplasts, which are unevenly distributed in the micropylar region of the basal cell. Fluorometry using a video-intensified microscope photon counting system reveals that, correlated with changes in mitochondrial morphology, DNA amount within the mitochondrion decreases linearly during these stages.     

Genetic Architecture of Two Fitness-related Traits in Drosophila melanogaster: Ovariole Number and Thorax Length

In Drosophila melanogaster, ovariole number and thorax length are morphological characters thought to be associated with fitness. Maximum daily egg production in females is positively correlated with ovariole number, while thorax length is correlated with male reproductive success and female fecundity. Though both traits are related to fitness, ovariole number is likely to be under stabilizing selection, while thorax length appears to be under directional selection. Current research has focused on examining the sources of variation for ovariole number in relation to fitness, with a view towards elucidating how segregating variation is maintained in natural populations. Here, we utilize a diallel design to explore the genetic architecture of ovariole number and thorax length in nine isogenic lines derived from a natural population. The full diallel design allows the estimation of general combining ability (GCA), specific combining ability (SCA), and also describes variation due to reciprocal effects (RGCA and RSCA). Ovariole number and thorax length differed with respect to their genetic architecture, reflective of the independent selective forces acting on the traits. For ovariole number, GCA accounted for the majority (67.3%) of variation segregating between the lines, with no evidence of reciprocal effects or inbreeding depression; SCA accounted for a small percentage (3.9%) of the variance, suggesting dominance variation; no reciprocal effects were observed. In contrast, for thorax length, the majority of the non-error variance was accounted for by SCA (17.9%), with only one third as much variance (6.2%) due to GCA. Interestingly, RSCA (nuclear–extranuclear interactions) accounted for slightly more variation (7.5%) than GCA in these data. Thus, genetic variation for thorax length is largely in accord with predictions for a fitness trait under directional selection: little additive genetic variation and substantial dominance variation (including a suggestion of inbreeding depression); while the mechanisms underlying the maintenance of variation for ovariole number are more complex.     

Study on the concentration of circulating primordial germ cells (cPGCs) in early chick embryos

Experiments were conducted to elucidate the factor that influences the concentration of circulating primordial germ cells (cPGCs) in two-day old chick embryos. The concentration of cPGCs was observed to be highest at stage 14 (66.9 +/- 23.2 microliters) and decreased thereafter. However, considerable egg to egg variations in cPGC concentration, especially at stages 13, 14, 15, and 16 were observed. After conducting experiments to elucidate the source of egg to egg variation in cPGC concentration among embryos, it was revealed that there are hens that lay eggs which contain either constantly high (more than 80 PGCs/microliter) or constantly low (less than 30 PGCs/microliter) concentration of cPGCs. The results obtained from the present experiments showed that one of the major source of egg to egg variation in the concentration of cPGCs was due to the individual differences among females that produced the eggs.     

Genetic polymorphism at spinocerebellar ataxia 1 and 2 loci in Brazil

Dynamic mutation involves the expansion of a tandem arrayed DNA sequence that is polymorphic in the population. This mechanism is associated with neurological/neuromuscular disorders and the pathology depends on the extension of the repeated tract, with a specific threshold for each disease. We made a PCR-based characterization of allelic polymorphism of SCA1 and SCA2 loci in a sample of 200 pairs of chromosomes in a population in Rio de Janeiro and found 23 different alleles at the SCA1 locus, varying from 10 to 39 CAG repeats (mean 27.7 +/- 3.3, mode 28) and 10 different alleles ranging from 19 to 29 CAG (mean 22.1 +/- 1.0, mode 22) at the SCA2 locus. The level of heterozygosis was 53% (SCA1) and 8% (SCA2).     

Optimal offspring provisioning when egg size is "constrained": a case study with the painted turtle Chrysemys picta

Classic egg size theory predicts that, in a given environment, there is a level of maternal investment per offspring that will maximize maternal fitness. However, positive correlations among egg size and female body size are observed within populations in diverse animal taxa. A popular explanation for this phenomenon is that, in some populations, morphological constraints on egg size, such as ovipositor size (insects) or pelvic aperture width (lizards and turtles), limit egg size. Egg size may therefore increase with female body size due to body size‐specific constraints on investment per offspring, coupled with selection towards an optimal egg size. We use 17 years of data from a population of painted turtles Chrysemys picta to evaluate this hypothesis. In accordance with our predictions, we find that (1) morphological constraints on egg size are apparent only in relatively small females, similarly (2) egg mass exhibits a strong asymptotic relationship with female body size, suggesting egg mass is optimized only at large body sizes, (3) clutch size, not egg mass, varies with female condition, and (4) clutch size varies more than egg mass across years. Contrary to our predictions, we observe that (5) the egg mass‐clutch size tradeoff is not less pronounced at large body sizes. Our data do not fully support the traditional hypothesis, and recent models suggest that this hypothesis is indeed overly simplistic. When the selective environment of a female's offspring is influenced by her phenotype, optimal egg size may vary among maternal phenotypes. This concept can explain correlations among egg size and body size in many taxa, as well as the patterns observed in the present study. In this paradigm, a tight coupling of aperture width (or other ‘constraints’) and egg size may occur in small females, even when such morphological features are not causally related to variation in egg size. In this spirit, we question validity of invoking morphological constraints to explain covariation among egg size and female body size.     

Cytological Mechanism of Cytoplasmic Inheritance in Pinus tabulaeformis: II. Transmission of Male and Female Organelles During Fertilization and Proembryo Development

In an earlier report the ultrastructure and nucleoid organelles of male gamete in Pinus tabulaeformis Carr. have been described. Presently, the ultrastructure of the cytoplasm of the egg cell and pollen tube—immediately before fertilization and during cytoplasmic transmission of male gametophyte—has been described for the same species. The fate of parental plastids and mitochondria in the proembryo has also been followed. The mature egg cell contains a large amount of mitochondria, but seems to lack normal plastids. Most plastids have transformed into large inclusions. Apart from the large inclusions, there are abundant small inclusions and other organelles in the egg cell. During fertilization, pollen tube penetrates into the egg cell at the micropylar end and thereafter the contents are released. Plastid and mitochondrion of male origin are lacking near the fusing sperm-egg nuclei. The second sperm nucleus—not involved in karyogamy—remains at a site near the receptive vacuole. This nucleus is surrounded by large amount of male cytoplasm containing mixed organelles from the sperm cell, tube cell, and egg cell. At the free nuclear proembryo stage, organelles of male and female origin are visible in the perinucleus-cytoplasmic zone. Most of the mitochondria have the same morphological features as those in the egg cell. Some of the mitochondria appear to have originated from the sperm and tube cells. Plastids are most likely of male gametophyte origin because they have similar appearance as those of the sperm and tube cell. Large inclusions in the egg cell become vacuole-like. Paternal plastids have been incorporated into the neocytoplasm of the proembryo. In the cellular proembryo, maternal mitochondria are more abundant. Plastids resembling those of the sperm and tube cell are still present. These cytological results clearly show that in P. tabulaeformis , plastids are inherited paternally and mitochondria bipaternally. The cytological mechanism of plastid and mitochondrion inheritance in gymnosperm is discussed.     

Morphological restructuring of the cellular elements of the ventral tegmental area in selective damage to the catecholaminergic systems of the brain

Electron microscopical investigation of the ventral tegmental area at various time after administration of a specific neurotoxin 6-hydroxydopamine made it possible not only to reveal catecholamine reactive elements, but also those structural reorganizations in them, that are dependent on the lesion of the catecholaminergic systems, including certain changes in the receptor apparatus. Dark neurons with the change of different organelles preserve for a long time, up to 2.5 months. Reactive and destructive reorganizations of some axons, terminal buttons and postsynaptic poles of the ventral tegmental area are accompanied with certain functional disturbances, observed both experimentally and at a pathological process. The results of the work demonstrate that under conditions of the model experiments there is a possibility to study various stages of pathogenesis of a number of nervous-psychical diseases, connected or accompanied with disorders in certain mediator systems.     

The morphological and ontogenetic changes in the Protozoa during the transition to a sessile mode of life

The morphological adaptations of protozoans to sessile mode life and evolutionary changes in ontogeny are considered. There are main morphotypes of sessile protists: stalked organisms that attached to substrate by the extended base of body (basal disk), and unstalked organisms that are flatted on substrate. The origin of the morphotypes was independent in different taxa and involved nonhomologous structures. Adaptation to the sessile mode of life in the protists was connected with the progressive increase in the body size and intensity of organelle functions by polymerisation, subsequent division of function and change of functions. Evolution of adhesive organelles is characterised by growing intensity of their functions by allometric growth (usually without polymerisation), and in some cases with the subsequent division of functions and change of functions. The evolution manifests itself primarily in the organelles that provide interaction of cell with environment. The organelles that ensuring functioning of cell change due to correlations with the organelles of the first group. These two groups of organelles are similar to A.N. Sewertsoff's ecto- and endosomatic organs in multicellular organisms. The ontogeny of the sessile protists included three stages: formation of the migratory stage, distribution and choice of substrate and metamorphosis of the migratory stage after adhesion. As a rule there are no recapitulations on the first stage. The majority of structures tomotes or zoospores are inherited from the parent cell. Thus the present of some ancestral characteristics at the earlier stages of protistean ontogeny is display of the Baer's law. The main features of ontogeny evolution in sessile protists are the anaboly of the additional stages of life cycle, the displays of archallaxis or deviation during the migratory stage formation, and anaboly at the stage of buds morphogenesis after adhesion. At the last stage, the study of recapitulations is most perspective with the decision of phylogenetic problems in sessile protists.     

Lysosome-related organelles: a view from immunity and pigmentation

Lysosomes are ubiquitous organelles that carry out essential household functions. Certain cell types, however, contain lysosome-related organelles with specialized functions. Their specialized functions are usually reflected by specific morphological and compositional features. A number of diseases that develop due to genetic mutations, pathogen exposure or cell transformation are characterized by dysfunctional lysosomes and/or lysosome-related organelles. In this review we highlight adaptations and malfunction of the endosomal/lysosomal system in normal and pathological situations with special focus on MHC class II compartments in antigen presenting cells and melanosomes in pigment cells.     

鳙鱼ARISTICHTHYS NOBILIS早期发育的一些生物化学上的变化

本实验第一部分对鳙鱼卵及胚胎在不同的发育时期蛋白质合成的速度进行了研究,实验采用微量克氏定氮法,对鳙卵从未受精卵到受精卵、尾芽期和出膜期,共测定了十三个不同的发育时期。实验结果表明了在鱼的不同胚胎发育时期蛋白质合成速度有着明显的差异。 实验的第二部分对鳙鱼胚胎发育过程氨基酸组成和氨基酸含量进行了测定,其结果显示出鳙鱼卵和胚胎的各发育时期氨基酸的组分十分相似,但氨基酸的含量在各个时期是有差异的。     

General and specific combining ability from partial diallels of radiata pine: implications for utility of SCA in breeding and deployment populations

Variances for general combining ability (GCA) and specific combining ability (SCA) and the relationship between mid-parental GCA and SCA effects were estimated for tree diameter (DBH) from a series of 20 sets of 6×6 half-diallel mating experiments in radiata pine, planted at ten sites across Australia. Significant SCA variance for DBH was almost equal to GCA variance for the combined analysis of all ten sites. The importance of SCA variance varied among sites, from non-significant to SCA variance accounting for all genetic variation among full-sib families. Significant SCA × site interaction was detected among the ten sites. A significant and positive correlation between mid-parental breeding values and best linear unbiased predictions of the SCA effects was observed. About a quarter of extra genetic gain is achievable through use of SCA variance if selection is based on the best breeding values. To fully exploit genetic gain from SCA variance in a deployment population, positive assortative matings are required for the best parents. It is estimated that the additional deployment gain of 46.0% for ten sites combined, or 52.9% for four sites combined that had significant GCA as well as SCA effects, were achievable relative to gain from GCA only, if all SCA variance within this breeding population was exploited. For a breeding population, selection for breeding values may be sufficient due to positive correlations between breeding values and SCA values. For a deployment population to capture more SCA genetic gain, it is preferable to make more pair-wise mating for parents with higher breeding values.Communicated by O. Savolainen     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.     

Developmental significance of a cortical cytoskeletal domain in Chaetopterus eggs

The cortex of Chaetopterus eggs contains a cytoskeletal domain (CD) which includes a specific class of dense granular organelles and a large proportion of the maternal mRNA. This CD, along with its constituent dense granular organelles and mRNA, can be displaced to atypical locations in the egg by centrifugation. To investigate the developmental significance of the CD, we have examined the early development of egg and zygote fragments, prepared by centrifugation, which contained the CD, the nucleus, or both. Specifically, we prepared nucleate egg and zygote fragments depleted in the CD, and two-cell embryos in which the CD was present in only one cell. Nucleate centripetal egg fragments were both unable to develop after fertilization and were depleted in the CD, as shown by electron microscopy, acridine orange staining of cortical organelles, and hybridization with poly(U) and cloned DNA probes. In contrast, about 20-35% of the nucleate centripetal fragments derived from one-cell zygotes developed into swimming larva. Correlated with this improved success of development, we found that these zygotic centripetal fragments contained significant levels of the CD, using the same methods listed above. More effective removal of the CD from zygotic centripetal fragments by stratification prior to fragmentation virtually eliminated their ability to develop. The CD and associated components could be displaced into only one of the first two blastomeres by centrifugation of zygotes immediately prior to the first cleavage. Embryos containing the CD in only one blastomere continued to cleave, but formed defective larva. The results suggest that the cortical CD is necessary for normal embryonic development.     

THE FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

E75A and E75B have opposite effects on the apoptosis/development choice of the Drosophila egg chamber

The number of Drosophila egg chambers is controlled by the nutritional status of the female. There is a developmental checkpoint at stage 8, which is controlled by BR-C in the follicle cells along with ecdysteroid. During this period, developmental decision is made in each egg chamber to determine if it will develop or die. During nutritional shortage, inducing apoptosis in the nurse cells of stages 8 and 9 egg chambers reduces the number of egg chambers. We show that ecdysone response genes E75A and E75B are involved in inducing or suppressing apoptosis. It is thus possible that the E75 isoforms A and B are involved in the decision to develop or die in oogenesis. We have established part of the pathway by which ecdysone response genes control apoptosis of the nurse cells and hence select between degeneration or development of individual egg chambers at stages 8 and 9.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

Immunohistochemical localization of peroxisomal enzymes during rat embryonic development.

Peroxisomes are cytoplasmic organelles involved in a variety of metabolic pathways. Thus far, the morphological and biochemical features of peroxisomes have been extensively characterized in adult tissues. However, the existence of congenital peroxisomal disorders, primarily affecting tissue differentiation, emphasizes the importance of these organelles in the early stages of organogenesis. We investigated the occurrence and tissue distribution of three peroxisomal enzymes in rat embryos at various developmental stages. By means of a highly sensitive biotinyl-tyramide protocol, catalase, acyl-CoA oxidase, and ketoacyl-CoA thiolase were detected in embryonic tissues where peroxisomes had not thus far been recognized, i.e., adrenal and pancreatic parenchyma, choroid plexus, neuroblasts of cranial and spinal ganglia and myenteric plexus, and chondroblasts of certain skeletal structures. In other tissues, i.e., gut epithelium and neuroblasts of some CNS areas, they were identified earlier than previously. In select CNS areas, ultrastructural catalase cytochemistry allowed identification of actively proliferating organelles at early developmental stages in several cell types. Our data show that in most organs maturation of peroxisomes parallels the acquirement of specific functions, mainly related to lipid metabolism, thus supporting an involvement of the organelles in tissue differentiation.