The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10?days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.     

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Amblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates.     

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.     

Der Einfluß von Li- und SCN-Ionen auf die Differenzierungsleistungen desAmbystoma-Ektoderms und ihre Veränderung bei kombinierter Einwirkung beider Ionen

Summary The development of the mouse oocyte during the primordial, primary and secondary follicular growth stages was studied by means of the electron microscope.During the early stages of oocyte maturation, mitochondrial multiplication takes place along with an apparent temporary transition from round to oval shape. The internal structure of many of the mitochondria is altered by separation of membranes of a crista to form a vacuole. This enlarges to pear-shaped configurations and gradually it forms so large a structure as to result in compression of adjacent cristae, thereby altering the entire appearance of the organelle.Dense round bodies encapsulated by a single membrane are found in the cytoplasm of oocytes of primary follicles near the periphery. The Golgi complex appears in primary follicle oocytes as an aggregation of vesicles. Gradually the number of lamellae in the complexes increase and these organelles become more peripherally located. The Balbiani yolk nuclei apparently is represented by a conglomeration of Golgi complexes and are present only in primordial and young primary follicle oocytes.The endoplasmic reticulum appears in the early stages only as rough-surfaced vesicles. At later stages individual cisternae become prominent. Apparently, a modified form of E. R. appears during maturation of the secondary follicle oocyte.Multivesicular complexes, each consisting of two components, small vesicles and larger vesicles enclosing microvesicles (multivesicular bodies), were commonly found during all stages of oocyte growth. The secondary follicle oocytes frequently contain multilamellar bodies. These are commonly found in juxtaposition to the multivesicular complexes and also near the egg periphery and occasionally near the nuclear envelope.This investigation was supported by a Public Health Service Research Career Program Award (5-K3-HD-5356-07) from the National Institute of Child Health and Human Development.     

Oogenesis in Xenopus laevis (Daudin). II. The induction and subcellular localization of tyrosinase activity in developing oocytes

Tyrosinase activity is increased at specific stages of development in Xenopus laevis oocytes in mature females by an injection of 1000 units of human chorionic gonadtropin (HCG). Enzyme activity is stimulated slightly in stage II oocytes, greatly (5- to 6-fold) in stage III and early stage IV oocytes, slightly in late stage IV, and not at all in stage V oocytes. Tyrosinase activity has been localized cytochemically in oocytes by the DOPA-reaction. The DOPA-reaction product is found in the distal cisterna of the Golgi complex and in an anastomosing network of smooth-surfaced tubules associated with the Golgi complex. No reaction product is found in the clustered elements of smooth endoplasmic reticulum which gives rise to the premelanosomes. Substantial melanization of the premelanosomes does not occur until the DOPA-positive Golgi complexes move into proximity with the premelanosomes at the oocyte periphery. Biochemical assay of the isolated melanin granules shows that premelanosomes isolated from stage III and IV oocytes contain significant tyrosinase activity. This activity appears to decrease in the later stages of melanization. It is concluded that the metabolic activities leading to the formation of oocyte pigmentation are stimulated by gonadotropins and the degree of response to the stimulation is quantitatively regulated according to parameters typical of the specific stage of oocyte development.     

Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals

Oogenesis in the anuran Xenopus laevis can be divided into six stages based on the anatomy of the developing oocyte. Stage I consists of small (50 to 100 μ) colorless oocytes whose cytoplasm is transparent. Their large nuclei and mitochondrial masses are clearly visible in the intact oocyte. Stage II oocytes range up to 450 μ in diameter, and appear white and opaque. Stage I and II are both previtellogenic. Pigment synthesis and yolk accumulation (vitellogenesis) begins during Stage III. Vitellogenesis continues through Stage IV (600 to 1000 μ), the oocytes grow rapidly, and the animal and vegetal hemispheres become differentiated. By Stage V (1000 to 1200 μ) the oocytes have nearly reached their maximum size and yolk accumulation gradually ceases. Stage VI oocytes are characterized by the appearance of an essentially unpigmented equatorial band. They range in size from 1200 to 1300 μ, are postivtellogenic and ready for ovulation. These stages of oocyte development have been correlated with physiological and biochemical data related to oogenesis in Xenopus.     

Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.     

Ultrastructural study of vitellogenesis and oogenesis of Metadena depressa (Stossich, 1883) Linton, 1910 (Digenea,Cryptogonimidae), intestinal parasite of Dentex dentex (Pisces,Teleostei)

The ultrastructural organization of the female reproductive system of Metadena depressa, digenean intestinal parasite of Sparidae (Dentex dentex), was investigated by electron microscopy. The vitellogenesis is divided into four stages: stage I, vitellocytes have a cytoplasm mainly filled with ribosomes and few mitochondria; stage II, beginning of the synthetic activity; stage III, active shell globule clusters synthesis; stage IV, mature vitellocytes are filled with shell globule clusters and generally contain several large lipid droplets. Glycogen granules are grouped at the periphery of the cell. The three stages of the oogenesis process take place in the ovary: stage I, oogonia are undifferentiated small cells located at the periphery of the organ; stage II, primary oocytes possess a higher nucleo-cytoplasmic ratio and a nucleus with a nucleolus and synaptonemal complexes indicating the zygotene-pachytene stage of the first meiotic division; stage III, mature oocytes are located in the proximal region of the organ and possess a cytoplasmic chromatoid body and cortical granules in a monolayer close to the periphery of the cell.     

The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material

The mitochondrial cloud is a prominent mass in the cytoplasm of previtellogenic oocytes of Xenopus laevis. It is shown here that the cloud contains both mitochondria and electron-dense granulofibrillar material (GFM). Using a combination of light microscopical, fluorescence, time-lapse filming, and electron microscopical techniques, the ontogeny of these components is reported and their fate is studied. It was found that the cloud is stationary in previtellogenic stages and fragments into islands of mitochondria and GFM during stage II (using the staging system of J. N. Dumont [1972) J. Morphol. 136, 153-180). These islands become localized in the peripheral cytoplasm at one pole of the stage III oocyte. By studying successive stages, GFM was followed through oogenesis and it was found localized only at the vegetal pole of stage IV and V oocytes. Furthermore, it was found that it bears a striking resemblance in position, appearance, and associations with mitochondria to the "germinal granules" of unfertilized eggs. Germinal granules have been shown by others to become incorporated into germ-line cells. It is concluded that the GFM is the precursor of this material and that the mitochondrial cloud is the site of its accumulation and localization in the previtellogenic oocyte.     

Daytime changes in oocyte development with relation to the tide for the Hawaiian saddleback wrasse, Thalassoma duperrey

Hourly variations in oocyte stages were characterized by size class and histological examination inassociation to the daytime tidal cycle for the Hawaiian saddleback wrasse, Thalassoma duperrey . Stage I (previtellogenic), stage II (vitellogenic) and stage III (hydrated) oocytes were identified as distinct clutches. During the autumn the profiles of oocytes stages showed rapid, group-synchronous development from stage I to stage III. Concurrent increases in percentages of both stage III oocytes and the gonadosomatic index (GSI) were positively correlated to and occurred I h before the high tide. Increases in stage I and stage II oocytes 1 h after high tide indicated development of new clutches following a decline in stage III oocytes, and the appearance of post-ovulatory follicles. The profile of stage II oocytes always exceeded 30% of the ovary. Lower GSI and percentages of stage III oocytes reflect significantly reduced reproduction in the summer: as in the autumn, both factors were significantly correlated, but neither variable showed a statistical relationship to the tide. Nevertheless, hydrated oocytes were found almost exclusively within 2 h of the high tide. The association of developmental changes with changes in tidal heights points to the importance of the tidal cycle or its underlying lunar influence as a predominant reproductive cue. These data show that developmental changes in oocytes occur more rapidly than observed in some more commonly studied temperate species which reproduce annually.     

The Synthesis and Localization of Envelope Glycoproteins in Oocytes of Xenopus laevis using Immunocytochemical Methods

The site of origin and the mode of differentiation of the coelomic envelope (CE) in growing oocytes of Xenopus laevis were studied using the rabbit antiserum raised against the isolated envelope from oviposited eggs. The antiserum preabsorbed with egg extracts reacted with most components of CE glycoproteins detectable by SDS-PAGE, and stained specifically the CE of full-grown (st. VI) oocytes using indirect immunofluorescence methods. Transmission electron microscopy employing IgG-conjugated colloidal gold demonstrated that the CE antigens were distributed throughout the whole cytoplasm of st. I oocytes, and began to be deposited around the oocyte surface at late st. I. During st. II to VI the density of CE antigens in the oocyte cytoplasm decreased markedly, while the deposition of extracellular CE antigens increased gradually in association with the formation of a fibrillar network. The CE antigens were observed in and around the highly extended oocyte microvilli during st. II to IV, but were never found in follicle cells at any stages of oocyte growth. On western blot analyses, the extracellular CE components appeared first at st. II, and increased both in amount and number of bands during st. III - V to attain a typical electrophoretic profiles of well-developed CE. The cytosols of growing oocytes, however, possessed several antigenic components which were distinct from those of extracellular CE, suggesting the occurrence of intracellular precursor molecules for CE. The CEs of st. IV oocytes defolliculated and cultured in [³H] leucine contained certain CE components that expressed the radiolabel on fluorography. These results indicate that in Xenopus laevis the oocyte is directly involved in the synthesis and assembly, as well as secretion of CE with least contribution by the follicle cells.     

Changes in the lipid inclusion/Sertoli cell cytoplasm area ratio during the cycle of the human seminiferous epithelium

The area occupied by Sertoli cell lipid inclusions--electron-lucent lipid vacuoles (LLV) and electron-dense lipid droplets (DLD)--at each stage of the cycle of the seminiferous epithelium was measured on electron micrographs in young adults and elderly men, and expressed as the ratio "area occupied by lipid inclusions/area occupied by the Sertoli cell cytoplasm". For LLV this ratio increased from stage I to stage III, and decreased from stage IV to stage VI in young adults. These results suggest that the development of LLV is synchronized with the spermatogenic process: the residual bodies released in stages I and II are phagocytized by Sertoli cells and transformed into LLV; the amounts of LLV decrease in the subsequent stages of the cycle and increase again when new residual bodies appear. In elderly men the ratio LLV/Sertoli cell cytoplasm was 1.9-2.9 times higher than in young adults at each stage of the cycle. This increase may be related to the increased germ-cell degeneration observed in ageing testes, DLD were less abundant than LLV and the DLD/Sertoli cell cytoplasm ratio did not undergo cyclic changes in young adults or elderly men.     

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle. This study suggests that spindle MTs and MFs during bovine oocyte meiosis are asymmetrically divided into the polar bodies.     

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     

Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality

The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction.     

Juvenile Hormone titre and vitellogenin gene expression related to ovarian development in primary reproductives compared with nymphs and nymphoid reproductives of the termite Reticulitermes speratus

To elucidate the reproductive cycle of termite queens, incipient colonies of Reticulitemes speratus (Isoptera: Rhinotermitidae) are established under laboratory conditions, and the transition of colony development is observed at 0.5, 1.5, 2.5, 3.5, and 7.5 months (stages I–V, respectively) after colony foundation. Ovarian development, vitellogenin gene expression and Juvenile Hormone (JH) titres are examined in the queens and in nonphysogastric nymphoids collected from natural colonies. A reproductive cycle in queens is observed, in which the oviposition rate is relatively higher during stages I and II, and then decreases during stages III and IV. Vitellogenic oocytes are not observed in the ovaries during stages III and IV, and the expression level of the vitellogenin gene is low, suggesting that egg production in queens is repressed during these stages. However, vitellogenin gene expression and egg deposition in queens resumes during stage V. Juvenile Hormone levels rise during the transition from nymphs to stage I queens, and elevated JH titres are observed also during stages III and IV. The decrease in JH titre in queens at stage II precedes the decline in vitellogenesis at stages III and IV. Thus, JH titre and vitellogenesis are correlated in an offset pattern. However, nonphysogastric nymphoid reproductives do not have vitellogenic oocytes in their ovaries, and their JH titre is two‐fold higher than that of queens, suggesting that elevated JH titre precedes vitellogenesis, as in queens.     

Myelin-like structures as a possible source of the smooth endoplasmic reticulum in early amphibian oocytes

A comparative study of amphibian oocyte ultrastructural organization has shown a significant accumulation of elements of the smooth endoplasmic reticulum in the oocyte cytoplasm at the third stage of development. The analysis of oocytes of two frog species, Xenopus laevis and Rana temporaria, at the first and second stages of their development enabled us to recognize in the cytoplasm of the oocyte some myelin-like structures (MLs) made of 30-40 densely packaged membranous layers and shaped as dense bodies. MLs are also present in the adjacent follicular cells and in the intercellular space. In the oocyte cytoplasm these structures are located near the nuclear envelope and other intracellular organelles. At the third stage of oogenesis, which is characterized by a high functional activity of the cells, MLs are seen to unwrap sequentially into double-layer membranes similar to the smooth endoplasmic reticulum cisternae. Intermediate steps of this process being also observed. It is supposed that MLs may play the role of membrane stocks to be used eventually for the formation of nascent endoplasmic membranes in the amphibian oocytes.