Ack kinase regulates CTP synthase filaments during Drosophila oogenesis

The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.     

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Egg chambers from apterous⁴ (ap⁴), a female sterile mutant of Drosophila melanogaster, show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oöcyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oöcytes of ap⁴ flies. Within 12 hr after topical application of ZR515 to homozygous ap⁴ females the oöcyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oöcyte also shows some changes. In vitro studies in which ap⁴ ovaries were incubated in Schneider's Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oöcyte membrane.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.     

Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1

The Drosophila melanogaster histone lysine methyltransferase (HKMT) Eggless (Egg/dSETDB1) catalyzes methylation of Histone H3 lysine 9 (H3K9), a signature of repressive heterochromatin. Our previous studies showed that H3K9 methylation by Egg is required for oogenesis. Here we analyze a set of EMS-induced mutations in the egg gene, identify the molecular lesions of these mutations, and compare the effects on oogenesis of both strong loss-of-function and weak hypomorphic alleles. These studies show that H3K9 methylation by Egg is required for multiple stages of oogenesis. Mosaic expression experiments show that the egg gene is not required intrinsically in the germ cells for their early differentiation, but is required in the germ cells for their survival past stage 5 of oogenesis. egg is also required in germ stem cells for their maintenance, since egg⁻ germ stem cells initially survive but are not maintained as females age. Mosaic analysis also reveals that the early egg chamber budding defects in egg⁻ ovaries are due to an intrinsic requirement for egg in follicle stem cells and their descendents, and that egg plays a non-autonomous role in somatic cells in the germarium to influence the differentiation of early germ cells.     

Oogenesis in the female sterile (1)42 mutant of Drosophila melanogaster.

The sex-linked mutation fs(1)42 was induced by ethyl methane sulfonate. It has no effect on either the external morphology or longevity of adult hemizygotes or homozygotes. Heterozygotes and hemizygotes are fertile, but homozygotes are sterile. Egg chamber development proceeds through stages 8, and thereafter chambers degenerate. Dying follicle cells are seen in chambers at all positions in the ovarioles. Profollicle cells also die within germaria, and clusters of sister cystocytes take longer than normal to receive their coverings of follicle cells. Egg chambers in the vitellarium contain only about 60% the normal number of follicle cells, these generally have greater lateral dimensions, and their nuclei and nucleoli are also larger than normal. The follicular envelope of mutant chambers often contains gaps through which cystocytes send cytoplasmic projections. Abnormalities seen in development of the fs(1)42 oocyte are likely to be due to its envelope of defective follicle cells.     

瓜实蝇卵巢的发育阶段及分级

瓜实蝇是热带和亚热带地区最重要的经济害虫之一。本研究拟对卵巢发育进行分级,以判断田间捕获的瓜实蝇是否达到性成熟。室内饲养瓜实蝇成虫,对开始羽化至羽化后80 d的不同日龄成虫卵巢进行解剖,并记录了卵巢长度、卵巢宽度、卵巢指数（卵巢长度＆#215;卵巢宽度）和所含卵粒数。将瓜实蝇卵巢发育过程分为4期6个级别,分别为卵黄发生前期（Ⅰ级和Ⅱ级）、卵黄发生期（Ⅲ级和Ⅳ级）、抱卵期（Ⅴ级）、经产期（Ⅵ级）。随着卵巢的发育,Ⅰ～Ⅴ级卵巢长度和宽度迅速增长,卵巢发育成熟开始产卵以后,Ⅵ级卵巢长度和宽度逐渐减小。Ⅰ级和Ⅱ级卵巢长度与宽度大致相等,以后各期卵巢长度均大于宽度。Ⅲ级和Ⅳ级可见清晰的卵室,卵母细胞迅速增长,卵黄逐渐沉积。Ⅴ级可见清晰的卵粒,整个卵巢呈圆柱状。Ⅵ级开始排出卵子,所含卵粒数显著少于Ⅴ级;虽然此级卵巢长度大于Ⅳ级,但卵巢宽度、卵巢指数与Ⅳ级基本一致;Ⅵ级卵巢可见黄体,可结合黄体判定卵巢所处发育级别。本研究对评估粘虫板捕获的瓜实蝇雌虫生理日龄结构有潜在应用价值。     

Reproductive features of <Emphasis Type="Italic">Upeneus margarethae</Emphasis> (Mullidae), a species recorded in the coastal zone of Vietnam for the first time

The species Upeneus margarethae described recently from the western Indian Ocean is recorded in the coastal zone of Vietnam for the first time. The reproductive features of the representatives of the species from southern Central Vietnam (Nha Trang Bay), Central Vietnam (Da Nang), and North Vietnam (Ha Long Bay) are studied. Based on the analysis of maturity stages of female’s ovaries and gonadosomatic index, the fishes spawn over the entire year in Central Vietnam, but the spawning interruption is observed in North Vietnam during a winter season. In Nha Trang Bay, 50% of females reach sexual maturity at the body length (FL) 9.8 cm. Based on histological structure of ovaries and frequency distribution of oocyte diameter before the spawning, the oogenesis is continuous. Anomalous oocyte structure is registered in 20% of the females. Egg morphology is described for 3 h after activation of ovulated oocytes. A comparison of reproductive parameters in U. margarethae and U. tragula is conducted.     

Egg Load and Body Size of Lab-cultured Cotesia marginiventris

The egg load of lab-cultured Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), a solitary koinobiont endoparasitoid of noctuid caterpillars, was determined in this study. Information on egg load may provide clues to more efficient in vivo rearing of C. marginiventris. I tested the hypothesis that egg load, defined as the number of mature oöcytes (i.e., fully chorionated eggs) found in adult females, was related to body size. Cotesia marginiventris females possessed two ovaries and two ovarioles per ovary; mature eggs were found in ovaries and oviducts. Newly-emerged females held an average of 149 mature eggs. Immature eggs were slightly visible in the distal portions of the ovarioles; they were not counted. Egg load was marginally related to body size (i.e., hind tibia length). The results of this study suggest that (1) body size can sometimes predict egg load or potential fecundity of lab-cultured C. marginiventris and (2) an efficient rearing system that exploits the potential fecundity of C. marginiventris might involve using young females and allowing them to oviposit in new hosts, each day, for up to a week.     

Determination of DNA content in the nurse and follicle cells from wild type and mutant Drosophila melanogaster by DNA-Feulgen cytophotometry

DNA replication patterns in the nurse and follicle cells of wild type and a female sterile mutant, fs(1)1304, of Drosophila melanogaster have been studied by DNA-Feulgen cytophotometry, using a cell dispersal technique that allowed the measurement of DNA amounts in individual nuclei from egg chambers of known developmental stages. DNA-Feulgen values associated with various ovarian nuclei from egg chambers at different stages of development were used to assess a base line DNA content for ovarian tissues and to estimate the extent of DNA replication in the nurse cells and follicle cells of growing and mature egg chambers. Our data show that both the nurse and follicle cells undergo multiple cycles of endonuclear DNA replication and that there may be selective amplification as well as underreplication by portions of the genome in these highly polyploid, ovarian cells. Alternative models are proposed to account for the DNA replication patterns observed. Comparisons of DNA-Feulgen levels in wild type ovarian nuclei with those found for the fs(1)1304 mutant and its heterozygote in the balanced stock fs/FM3, show that equivalent DNA levels are present in follicle cell nuclei from all three types of females. Nurse cell nuclei in the homozygous fs stock, however, fail to achieve the same high DNA levels observed in both fs/FM3 and wild type nurse cell nuclei. Although the nuclei of follicle cells in ovaries from fs/fs females appear morphologically like those surrounding egg chambers in wild type ovaries, nurse cell nuclei from mutant females show a more compacted organization of their chromatin than found for nurse cell nuclei from wild type ovaries at similar developmental stages. Our findings suggest that a major effect of the fs(1)1304 mutation may be on the coiling behavior of chromatin and the conformation of DNA-protein moieties in both nurse cell and follicle cell nuclei. These changes in chromatin structure apparently are manifest by perturbations in DNA replication patterns and normal gene function in these biosynthetically active cells.     

Par-1 and Tau regulate the anterior-posterior gradient of microtubules in Drosophila oocytes

The formation of an anterior-posterior (AP) gradient of microtubules in Drosophila oocytes is essential for specification of the AP axis. Proper microtubule organization in the oocyte requires the function of serine/threonine kinase Par-1. The N1S isoform of Par-1 is enriched at the posterior cortex of the oocyte from stage 7 of oogenesis. Here we report that posterior restriction of Par-1 (N1S) kinase activity is critical for microtubule AP gradient formation. Egg chambers with excessive and ectopic Par-1 (N1S) kinase activity in the germline cells display phenotypes similar to those of egg chambers treated with the microtubule-depolymerizing drug colcemid: depolymerization of microtubules in the oocyte and disruption of oocyte nucleus localization. A phosphorylation target of Par-1, the microtubule-associated protein Tau, is also involved in oocyte polarity formation, and overexpression of Tau alleviates the phenotypes caused by ectopic Par-1 (N1S) kinase activity, suggesting that Par-1 regulates oocyte polarity at least partly through Tau. Our findings reveal that maintaining proper levels of Par-1 at correct position in the oocyte is key to oocyte polarity formation and that the conserved role of Par-1 and Tau is crucial for the establishment of an AP gradient of microtubules and for AP axis specification.     

Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.     

Morphology of skin incubation in Pipa carvalhoi (Anura: Pipidae)

South American Pipidae show a unique reproductive mode, in which the fertilized eggs develop in temporarily formed brood chambers of the dorsal skin after eggs have been deposited on the back of the female. We studied the skin incubation of Pipa carvalhoi using light microscopy and scanning electron microscopy. The skin consists of a stratified epithelium with a one‐layered stratum corneum, and the dermis. The dermis of the dorsal skin of nonreproductive and reproductive females lacks a distinct stratum compactum, which is typical for most anuran skins. The entire dermis shows irregularly arranged collagen bundles like a stratum spongiosum. Before egg laying, the skin swells, primarily by thickening and further by loosening of the middle zone of the dermis. In the epidermis, large furrows develop that are the prospective sites of egg nidation. The epidermis, which forms a brood chamber around the developing egg becomes bi‐layered and very thin and lacks a stratum corneum. Further, the dermis loosens and becomes heavily vascularized. Egg carrying females do not have mature oocytes in their ovaries indicating a slow down or interruption of egg maturation during this period. Similarities with the brood pouch of marsupial frogs are discussed. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Structure of ovaries of scale insects: ii. margarodidae (INSECTA,hemiptera, COCCINEA)

The paired, spindle-shaped ovaries of the second instar of the Polish cochineal, Porphyrophora polonica (L.) (Hemiptera: Coccinea) are filled with cystocytes that are arranged into rosettes. In the centre of each rosette, there is a polyfusome. During the third instar, cystocytes differentiate into oocytes and trophocytes (nurse cells) and ovarioles are formed. Ovaries of adult females are composed of about 300 ovarioles of the telotrophic type. Each of them is subdivided into a tropharium (trophic chamber) and vitellarium. The tropharium consists of trophocytes and arrested oocytes that may develop. The number of germ cells in the trophic chambers varies from 11 to 18 even between the ovarioles of the same ovary. The obtained results seem to confirm the concept of a monophyletic origin of the primitive scale insects (Archaeococcoidea).     

Inhibitory effects of oostatic hormone on ovarian maturation and ecdysteroid production in diptera

Extracts from post-vitellogenic ovaries of Musca domestica, Aedes atropalpus and Drosphila melanogaster were examined for the ability to inhibit the vitellogenic phase of growth in newly emerged A. atropalpus and D. melanogaster adult females. All extracts were inhibitory in A. atropalpus. D. melanogaster females were less sensitive than A. atropalpus females, only showing inhibition at a two-fold greater concentration of M. domestica extract. The titre of oostatic hormone in A. atropalpus ovaries correlated with the decline in ecdysteroid synthesis by these ovaries. A. atropalpus ovaries containing post-vitellogenic follicles were shown to inhibit ovarian ecdysteroid synthesis in vitro by vitellogenic ovaries of both A. atropalpus and D. melanogaster. D. melanogaster ovaries were not capable of eliciting such an inhibition in vitro, at least with the numbers of ovaries utilized in our experiments.     

Drosophila Fragile X Protein controls cellular proliferation by regulating cbl levels in the ovary

FMRP is an RNA binding protein linked to the most common form of inherited mental retardation, Fragile X syndrome (FraX). In addition to severe cognitive deficits, FraX etiology includes postpubescent macroorchidism, which is thought to result from overproliferation. Using a Drosophila FraX model, we show that FMRP controls germline proliferation during oogenesis. dFmr1 null ovaries contain egg chambers with both fewer and supranumerary germ cells. The mutant germaria contain a significantly increased number of cyclin E and PhosphoHistone H3 positive cells, suggesting that loss of FMRP leads to defects in cell cycle progression. BrdU incorporation and flow cytometry data suggest that, in addition to proliferation, germline endoreplication and ploidy are also affected by the loss of FMRP during ovary development. Here we report that FMRP controls the levels of cbl mRNA in the ovary and that reducing cbl gene dosage by half rescues the dFmr1 oogenesis phenotypes. These data support a model whereby FMRP controls germline proliferation by regulating the expression of cbl in the developing ovary.     

The study of the conditions for the fertilizationin vitro in maize

Suitable conditions for the fertilizationin vitro in maize have been studied. The germinating capacity of pollen in synthetic media was low; it was confirmed that it might be stimulated by supplementing agar with egg yolk. Application of pollen onto styles overhanging from the culture medium of excised ovaries was examined. After 5 days the styles could be cut off the ovaries, for the pollen tubes had already penetrated the embryo sacs. However, better results were obtained when cultivating ovaries along with segments of the maize cob. Solid media were more suitable for the development of kernels. Some of them germinatedin situ and gave rise to normal plants. From nucellar meristems of young kernels a callus could be derived which, on further cultivation, became green and regenerated shoots and roots. The cells of meristems exhibited a varying number of chromosomes.     

Ovarian tumors in Rbp9 mutants of Drosophila induce an immune response

The Drosophila protein, Rbp9, is homologous to human Hu, which is reported to be involved in small cell lung cancer. Rbp9 functions in cystocyte differentiation, and mutations in Rbp9 cause ovarian tumors. Here we show that the antimicrobial peptide, Attacin, is upregulated in Rbp9 mutants, especially in ovaries where tumors form. Upregulation seems to result from activation of the NF-kappaB pathway since we detected nuclear localization of Relish in Rbp9 mutant ovaries but not in wild type ovaries. Inactivation of NF-kappaB in the Rbp9 mutant allows prolonged survival of malformed egg chambers. We conclude that Drosophila initiates an anti-tumor defense response via activation of NF-kappaB.     

Testosterone-7alpha-3H uptake by the anlage of organs related to reproduction in female rate embryos in experiments in vivo and in vitro

The dynamics of testosterone-7alpha-3H (T-3H) uptake by the developing genital system organs, as well as by the suprarenals, brain cortex and muscles was studied in vivo experiments with the 14-20 days old female rat embryos. Besides, the T3H uptake by the reproductive tract tissues and muscles just after the isolation of embryos from the uterus and after 2 days cultivation in the diffusion chambers implanted in the abdominal cavity of the adult castrated rats was studied in in vitro experiments. Moreover, the T3H uptake by the embryonic tissues was determined in the absence of ovaries. The specific uptake was noted in all the tissues at all developmental stages under all experimental conditions. The selective T3H uptake was noted during the periods corresponding to the rudiments' morphogenesis carachteristic for males. In the absence of ovaries, the T3H uptake by the reproductive tract tissues in vitro experiments decreased, but after 2 days cultivation in the diffusion chambers increased. The possible participation of ovaries in the female sex differentiation and the bisexual nature of rudiments determined by the presence of receptors both to estro- and androgens are discussed.