Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, we studied the origin of oocyte and germ plasm-associated mitochondria during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton as it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. Our findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support our hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles.     

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard <Emphasis Type="Italic">Podarcis sicula</Emphasis>

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphonidae). I. Ovary structure and previtellogenic growth of oocytes

Glossiphonia heteroclita has paired ovaries whose shape and dimensions change as oogenesis proceeds: during early previtellogenesis they are small and club-shaped, whereas during vitellogenesis they broaden and elongate considerably. During early oogenesis (previtellogenesis), each ovary is composed of an outer envelope (ovisac) that surrounds the ovary cavity and is filled with hemocoelomic fluid, in which a single and very convoluted ovary cord is bathed. The ovary cord consists of germline cells, including nurse cells and young oocytes surrounded by a layer of elongated follicle cells. Additionally, follicle cells with long cytoplasmic projections occur inside the ovary cord, where they separate germ cells from each other. The ovary cord contains thousands of nurse cells. Each nurse cell has one intercellular bridge, connecting it to a central anucleate cytoplasmic mass, the cytophore (rachis); it in turn is connected by one intercellular bridge with each growing oocyte. Numerous mitochondria, RER cisternae, ribosomes, and Golgi complexes are transported from the nurse cells, via the intercellular bridge and cytophore, to the growing oocytes. Oogenesis in G. heteroclita is synchronous with all oocytes in the ovary in the same stage of oogenesis. The youngest observed oocytes are slightly larger than nurse cells, and usually occupy the periphery of the ovary cord. As previtellogenesis proceeds, the oocytes gather a vast amount of cell organelles and become more voluminous. As a result, in late previtellogenesis the oocytes gradually protrude into the ovary cavity. Simultaneously with oocyte growth, the follicle cells differentiate into two subpopulations. The morphology of the follicle cells surrounding the nurse cells and penetrating the ovary cord does not change, whereas those enveloping the growing oocytes become more voluminous. Their plasma membrane invaginates deeply, forming numerous broad vesicles that eventually seem to form channels or conducts through which the hemocoelomic fluid can easily access the growing oocytes.     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

Endopolyploidy in follicle epithelial cells around the nurse chamber during oogenesis in Chrysomya putoria (Diptera,Calliphoridae)

Feulgen-DNA values and nuclear areas were evaluated microspectrophotometrically for epithelial cells of the ovarian follicle during oogenesis in Chrysomya putoria. The aim was to investigate whether polyploidization occurred in the cells surrounding the nurse chamber and/or in those around the oocyte as well as whether different DNA amounts were found regarding the cell types considered. Four DNA endoreduplicative cycles could be demonstrated for the epithelial cells regardless of their localization on the follicle, during oogenesis. A small percentage of epithelial cells reaches a 32C degree. The nuclear area, however, did not increase at the same rate in cells covering the oocyte as in those covering the nurse chamber, in some of the oogenesis stages. The meaning of endopolyploidy for these cells is discussed, considering reports on relevance of secretory activities and their maintenance in polyploid cell systems.     

刘氏蝎蛉卵巢管结构和卵子发生（英文）

卵巢管结构及卵子发生过程在探讨昆虫系统发育关系中有重要意义, 深入研究长翅目昆虫卵巢管结构及卵子发生可为确定其在全变态类昆虫中的系统发育地位提供依据。本文利用光学显微镜和扫描、透射电子显微镜技术研究了刘氏蝎蛉Panorpa liui Hua卵巢管超微结构及卵子发生过程。结果表明：蝎蛉卵巢由12根多滋式卵巢小管组成, 每个卵巢小管分为端丝、生殖区和生长区。根据滋养细胞、卵母细胞及滤泡细胞的变化, 卵子发生过程可分为5个阶段：卵黄发生前早期、卵黄发生前中期、卵黄发生前后期、卵黄发生期及卵壳形成期。在卵黄发生期, 滋养细胞为卵母细胞提供养分后逐渐消亡, 而此时的卵母细胞可通过滤泡之间的细胞间隙从血淋巴中获取营养。在卵壳形成期间, 3种不同类型的滤泡细胞参与形成不同区域的卵壳, 从而形成不同花饰的卵壳表面。据此推测, 与其他目的滋养细胞数目相比, 每个卵室中2次有丝分裂形成3个滋养细胞可能是比较原始的特征, 表明长翅目昆虫可能是全变态类群中近基部的分支。     

Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida)

Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids.     

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Comparative Spiralian Oogenesis--Structural Aspects: An Overview

Considerable variety exists in ovarian structure and cellularinteraction in spiralians. During their development the eggsof Dwpatra cuprea, are associated with nurse cells; there areno follicle cells in this species. The nurse cells have prominentnuclei and connect to oocytes via cytoplasmic bridges throughwhich ribosomes, mitochondria and other inclusions pass. Morecommonly, follicle cells surround a portion of, or entire, oocytesin many species of spiralians. Usually, as in Ilyanassa, theyhave a well developed compliment of organelles. The structureand distribution of organelles within follicle cells impliesthat they are functionally active, but precisely in what mannerduring oogenesis is poorly understood. Other cell types, suchas Leydig and interstitial cells also seem to play a role inoogenesis. Within the oocyte, a host of components includingyolk, lipid, mitochondria, ribosomes, membranous cisternae,cortical granules, etc. are accumulated. Autosynthetic yolkformation is prevalent among spiralians. Surface differentiationincludes microvillar development. This may be uniform in someeggs or restricted to certain regions (e.g., the animal hemisphere)in other oocytes. Oocyte-follicle cell interactions change duringoogenesis. The topographical association of the oocyte withother ovarian cells influences subsequent animal-vegetal polarityand other ooplasmic differences. Examples of ooplasmic localizationsare discussed. Conventional EM has revealed no unusual corticalstructure in many oocytes although occasionally microtubulesand microfilaments are present.     

Distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

Mitochondria play a vital role during oocyte maturation, fertilization, and embryo development. In this study, confocal microscopy with the mitochondrial membrane potential-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) was used to investigate mitochondria distribution and activity of stage III zebrafish ovarian follicles. To support the mitochondrial origin of the fluorescence obtained by JC-1, a second mitochondrial probe, MitoTracker Green FM, was used. Cryo-scanning and transmission electron microscopy were also used to validate the distribution and localization of mitochondria obtained by mitochondrial staining. The mitochondrial probes were unable to penetrate the oocyte, and as a result it was not possible to observe stained mitochondria in the oocyte cytoplasm. However, mitochondrial staining of the granulosa cell layer surrounding the stage III zebrafish oocyte exhibited a contiguous aggregation pattern of mitochondria. Cryo-scanning electron microscopy studies also showed the oocyte surface to be covered by polygonal patterns of ridges of the same dimensions as the distributional arrangement of mitochondria in the granulosa cells. Though the results suggested the need for defolliculation to assess mitochondrial distribution and activity in the stage III zebrafish oocyte cytoplasm, the findings of this study will contribute to our understanding of oogenesis and folliculogenesis processes in fish.     

泥螺卵子发生的超微结构研究

利用透射电镜观察了泥螺卵子发生过程。结果表明 ,泥螺的卵子发生可划分为卵原细胞、卵黄发生早期、卵黄发生中期及卵黄发生后期卵母细胞 4个时期。卵原细胞核大而圆 ,胞质内分布有少量的线粒体和高尔基囊泡 ,细胞表面具微绒毛。卵黄发生早期的卵母细胞 ,胞质中各类细胞器发达 ,并出现数量较多的类朦胧子。卵黄发生中期的卵母细胞胞体迅速增大 ,核伸出伪足状突起 ,卵质中各种细胞器活动活跃 ,并参与形成卵黄粒和脂滴。此期还可观察到卵母细胞与滤泡细胞间的物质交换现象。卵黄发生后期的卵母细胞体积增至最大 ,细胞器数量减少。本文就卵黄发生前后卵母细胞内部构造的变化、意义及滤泡细胞与卵母细胞蛋白来源间的关系作了探讨     

Stage-specific apoptotic patterns during Drosophila oogenesis

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.     

The microfilament pattern in the somatic follicle cells of mid-vitellogenic ovarian follicles of Drosophila

The microfilament pattern in the somatic follicle cells of mid-vitellogenic stage 9 to 11 follicles of Drosophila was analyzed by staining F-actin with fluorescence-labeled phalloidin. During the analyzed stages of oogenesis, the follicular epithelium differentiates morphologically and functionally. These changes are also reflected at the organization of the microfilaments. At stage 10, they show no preferred orientation in the very thin follicle cells covering the nurse cells. In contrast, the microfilaments in the basal part of the columnar follicle cells covering the oocyte become organized in parallel bundles oriented perpendicular to the long axis of the follicle. During stages 10B/11 this organization is maintained at the nurse cell/oocyte border but becomes more sloppy towards the posterior pole of the follicle. The basal part of the follicle cells containing the microfilament bundles adheres so tightly to the basement membrane that this acellular layer cannot be separated mechanically from the epithelium. Indirect evidence from inhibition studies with cytochalasins and the effects of collagenase or pronase E added to the culture medium suggest that the microfilament bundles may promote increased adhesiveness of the follicle cells to the basement membrane. The possible functional implications of the microfilaments and their orientation are discussed.