Analysis of the spatial organization of the XL chromosome attachment site in nurse cell nuclei of the malaria mosquito <Emphasis Type="Italic">Anopheles atroparvus</Emphasis>

The spatial position of the site of XL chromosome attachment to the nuclear envelope of ovarian nurse cells relative to the oocyte has been analyzed in the malaria mosquito Anopheles atroparvus van Thiel. The XL chromosome attachment sites in the oocyte-nurse cell system of this species have been demonstrated to be orderly arranged, with the attachment sites in two out of three nurse cells in the same layer identically oriented relative to the oocyte.     

The interrelation of the polytene chromosomes of generative tissues in Drosophila melanogaster]

The principles of three dimensional organization of primary and secondary orders polytene chromosomes in ovarian nurse cells of Drosophila melanogaster were elucidated. Contrary to somatic tissues, no joining of chromosome arms into local chromocentre was discovered. The chromosomes are separated in the nuclear space and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites, the arms of autosomes (especially primary polytene chromosomes) being separated in the area of attachment. Polytenized XR arm of the X chromosomes were discovered. The architecture of chromosomes discovered in ovarian nurse cells is tissue-specific and differs considerably from the organization of polytene chromosomes of somatic tissues.     

missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila

In Drosophila, a single oocyte develops within a 16-cell germline cyst. Although all 16 cells initiate meiosis and undergo premeiotic S phase, only the oocyte retains its meiotic chromosome configuration and remains in the meiotic cycle. The other 15 cells in the cyst enter the endocycle and develop as polyploid nurse cells. A longstanding goal in the field has been to identify factors that are concentrated or activated in the oocyte, that promote meiotic progression and/or the establishment of the oocyte identity. We present the characterization of the missing oocyte gene, an excellent candidate for a gene directly involved in the differentiation of the oocyte nucleus. The missing oocyte gene encodes a highly conserved protein that preferentially accumulates in pro-oocyte nuclei in early prophase of meiosis I. In missing oocyte mutants, the oocyte enters the endocycle and develops as a polyploid nurse cell. Genetic interaction studies indicate that missing oocyte influences meiotic progression prior to pachytene and may interact with pathways that control DNA metabolism. Our data strongly suggest that the product of the missing oocyte gene acts in the oocyte nucleus to facilitate the execution of the unique cell cycle and developmental programs that produce the mature haploid gamete.     

Tissue-Specific Differences in the Spatial Interposition of X-Chromosome and 3R Chromosome Regions in the Malaria Mosquito Anopheles messeae Fall.

Spatial organization of a chromosome in a nucleus is very important in biology but many aspects of it are still generally unresolved. We focused on tissue-specific features of chromosome architecture in closely related malaria mosquitoes, which have essential inter-specific differences in polytene chromosome attachments in nurse cells. We showed that the region responsible for X-chromosome attachment interacts with nuclear lamina stronger in nurse cells, then in salivary glands cells in Anopheles messeae Fall. The inter-tissue differences were demonstrated more convincingly in an experiment of two distinct chromosomes interposition in the nucleus space of cells from four tissues. Microdissected DNA-probes from nurse cells X-chromosome (2BC) and 3R chromosomes (32D) attachment regions were hybridized with intact nuclei of nurse cells, salivary gland cells, follicle epithelium cells and imaginal disсs cells in 3D-FISH experiments. We showed that only salivary gland cells and follicle epithelium cells have no statistical differences in the interposition of 2BC and 32D. Generally, the X-chromosome and 3R chromosome are located closer to each other in cells of the somatic system in comparison with nurse cells on average. The imaginal disсs cell nuclei have an intermediate arrangement of chromosome interposition, similar to other somatic cells and nurse cells. In spite of species-specific chromosome attachments there are no differences in interposition of nurse cells chromosomes in An. messeae and An. atroparvus Thiel. Nurse cells have an unusual chromosome arrangement without a chromocenter, which could be due to the special mission of generative system cells in ontogenesis and evolution.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

Synthesis and phosphorylation of ecdysteroids during ovarian development in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.     

Development of the oocyte and its accessory cells of the polychaete,Diopatra cuprea (Bosc)

The oocyte-nurse cell complex of the polychaetous annelid, Diopatra cuprea, has been explored by various methods of light microscopy and by the technique of electron microscopy. Early in its development the complex appears as a string of cells floating within the coelomic cavity. As this string of cells develops, the volume of one cell (approximately the middle one) increases greatly; while that of the remaining cells, referred to as nurse cells, increase slightly. Due to this differential growth, the two opposing strands of nurse cells are displaced to one side of the oocyte. Nurse cells are joined to one another by cytoplasmic bridges. Cytoplasmic bridges also exist between the strands of nurse cells and the oocyte. The presence of numerous ribosomes within the bridges between the oocyte and nurse cells encourages us to suggest that this organelle may be transferred to the oocyte via this route. The transported ribosomes may be used by the maturing oocyte, or they may be stored by the egg to be utilized during embryogenesis. Moreover, we believe that the nurse cells are not involved in the production of the protein-carbohydrate yolk bodies for we think that these are elaborated by the endoplasmic reticulum in collaboration with certain Golgi complexes of the oocyte.     

Oogenesis in Hydra: nurse cells transfer cytoplasm directly to the growing oocyte

Oogenesis in Hydra occurs in so-called egg patches containing several thousand germ cells. Only one oocyte is formed per egg patch; the remaining germ cells differentiate as nurse cells. Whether and how nurse cells contribute cytoplasm to the developing oocyte has been unclear. We have used tissue maceration to characterize the differentiation of oocytes and nurse cells in developing egg patches. We show that nurse cells decrease in size at the same time that developing oocytes increase dramatically in volume. Nurse cells are also tightly attached to oocytes at this stage and confocal images of egg patches stained with the fluorescent membrane dye FM 4-64 clearly show large gaps (10 microm) in the cell membranes separating nurse cells from the developing oocyte. We conclude that nurse cells directly transfer cytoplasm to the developing oocyte. Following this transfer of cytoplasm, nurse cells undergo apoptosis and are phagocytosed by the oocyte. These results demonstrate that basic mechanisms of alimentary oogenesis typical of Caenorhabditis and Drosophila are already present in the early metazoan Hydra.     

Peculiarities of the organization of egg chambers in carabid ground beetles and their phylogenetic implications

The ovaries of 31 species of the coleopteran familyCarabidae have been studied by light and electron microscopy. Ovarioles of all the examined insects are of the polytrophic type. In the majority of the species a constant number of nurse cells per egg chamber has been observed. However, several species do not obey the 2(n) rule and the number of nurse cells varies considerably even in the consecutive egg chambers of the same ovariole. In spite of the differences, the number of intercellular bridges connecting nurse cells to the oocyte is fixed and species specific. InCarabidae seven types of egg chambers have been characterized regarding the number of divisions, the number of nurse cells and the way the nurse cells are bridged to the oocyte. Some phylogenetic implications are considered.     

Comparative developmental physiology and molecular cytology of the polytrophic ovarian follicles of the blowfly Sarcophaga bullata and the fruitfly Drosophila melanogaster

1. The ovarian follicles of Sarcophaga and Drosophila consist of one oocyte and 15 nurse cells, the whole being surrounded by follicle cells. Although oocyte and nurse cells are genetically identical sibling cells, and although they are interconnected by cytoplasmic bridges, their physiology is very different. 2. The DNA content of the oocyte nucleus (germinal vesicle) never exceeds 4C, while values of polyploidisation up to 1024C have been measured in the nurse cells, this being dependent on their position within a follicle. 3. The nurse cell nuclei very actively synthesize RNA, while the germinal vesicle is almost completely inactive in this respect. 4. It has been possible to visualise the major cytoskeletal elements in the different ovarian cell types. Cellular markers of polarity and dorsoventral asymmetry have been described. 5. Electrophysiological measurements have been performed to find out whether or not the self-electrophoresis principle may be involved in polarised transport between nurse cells and oocyte. 6. Most of the vitellogenin is synthesized by the fat body but some follicle cells also synthesize small amounts. 7. The role of 20-OH ecdysone in the induction of vitellogenin synthesis in the fat body, as well as the presence of met-enkephalin like immunoreactivity in the gonads is well established in both species. Not so clear is the exact role of juvenile hormones and the nature of brain factors controlling ovarian development. 8. Drosophila has the advantage of its well documented genetics while the larger species Sarcophaga is preferable for the study of (electro-) physiological and cell biological mechanisms.     

Mutations in supernova,indicate that this gene is required for the division of germ line cells in Drosophila

Mutations in supernova, previously shown to uncouple chromosome replication from segregation during cleavage in Drosophila embryos, also sanctions extra divisions of cystoblasts and spermatoblasts. This leads either to the formation of egg chambers which contain more than fifteen nurse cells or testes which have an excess of spermatocytes. In maturing egg chambers two potential oocytes may be specified in which case they are often ectopically located and connected with surrounding nurse cells by four ring canals. However, a typical oocyte nucleus is not always present and these chambers usually become necrotic and degenerate. The nurse cells are of variable size, but are still interconnected by a system of ring canals. They all possess a polyploid nucleus. Sequestering of maternal mRNA's from the nurse cells into the potential oocyte(s) takes place but there is no localization of this maternal information within the oocyte probably because of defective microtubule assembly. Many spermatocytes fail to complete meiosis so that bundles of spermatids are reduced in size and the males have reduced fertility. It is proposed that this gene is indirectly involved in regulating the timing of mitotic divisions in both cystoblasts and spermatoblasts through its interference with microtubule assembly which is consistent with its role during embryogenesis.     

Systemic reorganization of the architechtonics of polytene chromosomes in onto- and phylogenesis of malaria mosquitoes. Structural features regional of chromosomal adhesion to the nuclear membrane

Principles of organization of chromocenter in salivary gland cells and zones of chromosome attachment to nuclear envelope in ovarian nurse cells were determined. It was shown that blocks of centromeric heterochromatin (alfa-heterochromatin) have no direct connection with nuclear envelope. Such connections are ensured by beta-heterochromatin. Homologous chromosome regions were shown to be of different morphology and nature of chromosome-membrane links in different mosquito species. A map of polytene chromosomes of ovarian nurse cells in Anopheles messeae Fall, was established. No differences were found in band quantity of these chromosomes as compared to salivary gland chromosomes.     

POLARIZED INTERCELLULAR BRIDGES IN OVARIAN FOLLICLES OF THE CECROPIA MOTH

Fluorescein-labeled rabbit serum globulin was injected into vitellogenic oocytes of the cecropia moth. Though the label spread throughout the ooplasm in less than 30 min, it was unable even after 2 h to cross the complex of intercellular bridges connecting the oocyte to its seven nurse cells. After injection into a single nurse cell, fluorescence was detected in the oocyte adjacent to the bridge complex within 3 min and had spread throughout the ooplasm in 30 min. Here also, the cell bodies of the six uninjected nurse cells remained nonfluorescent. Four of the nurse cells are not bridged directly to the oocyte but only through the apical ends of their siblings. Unidirectional movement must therefore occur in the apical cytoplasm of the nurse cells, as well as in the intercellular bridges. The nurse cells of healthy follicles had an intracellular electrical potential -40 mV relative to blood or dissecting solution, while oocytes measured -30 mV. A mV difference was also detected by direct comparison between a ground electrode in one cell and a recording electrode in the other. Three conditions were found in which the 10 mV difference was reduced or reversed in polarity. In all three cases fluorescent globulin was able in some degree to cross the bridges from the oocyte to the nurse cells.     

The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190 kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions.     

Interspecies differences of co-orientation of primary polytene chromosomes in ovarian nurse cells in Drosophila melanogaster, D. simulans and D. mauritiana]

Essential differences in the architecture of primary polytene chromosomes in ovarian nurse cells between Drosophila melanogaster, D. simulans and D. mauritiana were discovered. The individual chromosomes of D. melanogaster (as well as their arms) are separated significantly from each other and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites. D. simulans has the combination of the centromeric parts of the chromosomes X, 2, 3 into the "elongated" chromocentre, and in addition, the arms of chromosomes are also separated and attached to the nuclear envelope. Unlike the rest of the chromosomes, D. mauritiana has the chromosome 3 with firm combinated arms and the large heterochromatic block in the centromere, without being attached to the nuclear envelope.     

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Molecular genetic analysis of the X-chromosomal nuclear envelope attachment region in nurse cells of the malaria mosquitoes Anopheles messeae Fall

DNA of the X-chromosomal nuclear envelope attachment region was isolated from malaria mosquito Anopheles messeae Fall. nurse cells by chromosome microdissection. A DNA library of the region was constructed using a plasmid vector. DNA sequencing revealed gene fragments, tandem repeats, and a great variety of transposable elements (TEs). The X-chromosomal nuclear envelope attachment region was concluded to correspond in molecular organization and cytogenetics to diffuse intercalary heterochromatin.