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.     

Ovarian nests in cultured Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula comprised of previtellogenic oocytes

Ovarian nests in the ovaries of sexually maturing Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula were investigated. They comprised early previtellogenic, diplotene stage oocytes and somatic cells. In the nucleoplasm, these oocytes contained chromatin in the form of grains, threads and lampbrush chromosomes, primary nucleoli and multiple nucleoli. Two stages of oocytes in nests were distinguished by differences in the distribution of mitochondria with distorted cristae and lipid droplets in the ooplasm. These stages were as follows: pre‐early stage 1 (PE 1) and early stage 1 (EP 1) previtellogenic oocytes. In PE 1 oocytes few mitochondria with distorted cristae and lipid droplets were distributed randomly. The ooplasm of PE 1 oocytes was not differentiated into homogeneous and granular compartments. In EP 1 oocytes, mitochondria with distorted cristae were more numerous and grouped in the vicinity of the nucleus, lipid droplets accumulated near these mitochondria. In the nucleoplasm of EP 1 oocytes several low electron‐dense spherical bodies, possibly Cajal bodies, were present.     

Astrocytes shed large membrane vesicles that contain mitochondria, lipid droplets and ATP

Various cells types, including stem and progenitor cells, can exchange complex information via plasma membrane-derived vesicles, which can carry signals both in their limiting membrane and lumen. Astrocytes, traditionally regarded as mere supportive cells, play previously unrecognized functions in neuronal modulation and are capable of releasing signalling molecules of different functional significance. In the present study, we provide direct evidence that human fetal astrocytes in culture, expressing the same feature as immature and reactive astrocytes, release membrane vesicles larger than the microvesicles described up to now. We found that these large vesicles, ranging from 1–5 to 8 μm in diameter and expressing on their surface β1-integrin proteins, contain mitochondria and lipid droplets together with ATP. We documented vesicle content with fluorescent-specific dyes and with the immunocytochemistry technique we confirmed that mitochondria and lipid droplets were co-localized in the same vesicle. Scanning electron microscopy and transmission electron microscopy confirmed that astrocytes shed from surface membrane vesicles of the same size as the ones detected by fluorescence microscopy. Our results report for the first time that cultured astrocytes, activated by repetitive stimulation of ATP released from neighboring cells, shed from their surface large membrane vesicles containing mitochondria and lipid droplets.     

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly (P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher (P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group.In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.     

Possible participation of mitochondria in lipid yolk formation in oocytes of paddlefish and sturgeon

The ovary of paddlefish and sturgeons (Acipenseriformes) is composed of discrete units: the ovarian nests and ovarian follicles. The ovarian nests comprise oogonia and numerous early dictyotene oocytes surrounded by somatic prefollicular cells. Each ovarian follicle consists of a spherical oocyte and a layer of follicular cells situated on a thick basal lamina, encompassed by thecal cells. The cytoplasm of previtellogenic oocytes is differentiated into two distinct zones: the homogeneous and granular zones. The homogeneous cytoplasm is organelle-free, whereas the granular cytoplasm contains numerous organelles, including mitochondria and lipid droplets. We have analyzed the cytoplasm of early dictyotene and previtellogenic oocytes ultrastructurally and histologically. In the cytoplasm of early dictyotene oocytes, two morphologically different types of mitochondria can be distinguished: (1) with well-developed cristae and (2) with distorted and fused cristae. In previtellogenic oocytes, the mitochondria of the second type show various stages of cristae distortion; they contain and release material morphologically similar to that of lipid droplets and eventually degenerate. This process of mitochondrial transformation is accompanied by an accumulation of lipid droplets that form a single large accumulation (lipid body) located in the vicinity of the oocyte nucleus (germinal vesicle). The lipid body eventually disperses in the oocyte center. The possible participation of these mitochondria in the formation of oocyte lipid droplets is discussed. This work was supported by funds from the research grant BW/IZ/2005 to M.Ż. An erratum to this article can be found at http://dx.doi.org/. An erratum to this article can be found at     

Histological changes during ovarian maturation in the tarnished plant bug,Lygus lineolaris (Palisot de beauvois) (Hemiptera : Miridae)

Histological changes during the first gonotropic cycle in the telotrophic ovarioles of Lygus lineolaris (Hemiptera : Miridae) were studied by light and transmission electron microscopy. Each oocyte goes through a gonotrophic cycle that lasts for ca 7 days during which time 3 distinct stages are observed: previtellogenic, vitellogenic and choriogenic. In the previtellogenic stage, oocytes descend into the vitellarium and increase in size, while maintaining contact with the trophic core by means of nutritive cords. During vitellogenesis, ovarioles are characterized by the development of intercellular spaces in the follicular epithelium and numerous microvilli on the oocyte surface. Yolk granules are incorporated by pinocytosis and the granules coalesce, resulting in large yolk droplets. The trophic core supplies ribosomes, mitochondria and lipid to the oocytes, and its morphology remains unchanged throughout the gonotropic cycle. Vitellogenesis ends with the formation of vitelline membrane on the oocyte surface. During choriogenesis, an egg shell consisting of an exo- and endochorion is formed on the surface of the vitelline membrane. With the completion of choriogenesis, the mature oocyte is ready to be ovulated. During the gonotropic cycle, the oocyte increases in size 10–12-fold, while the germarium remains unchanged in size.     

Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.     

Structure of ovaries and oogenesis in the snow scorpionfly boreus hyemalis (LINNE)(MECOPTERA : BOREIDAE)

The ovaries of the snow scorpionfly, Boreus hyemalis (Mecoptera : Boreidae) are panoistic and comprise 7–8 ovarioles. Each ovariole consists of a terminal filament, elongated vitellarium, and ovariole stalk (=pedicel) only ; in adult specimens, functional germaria are absent. Five consecutive stages of oogenesis i.e., early, mid- and late previtellogenesis, vitellogenesis, and choriogenesis have been distinguished in imagines. Oocyte nuclei (=germinal vesicles) of previtellogenic oocytes contain numerous polymorphic multiple nucleoli (or nucleolar masses), endobodies, and chromatin aggregations. Next to the nuclear envelope, large accumulations of nuage material are localized. The ooplasm of late previtellogenic oocytes is differentiated into transparent (perinuclear) and opaque (peripheral) regions. Ultrastructural investigations have revealed that within the latter, abundant ribosomes as well as mitochondria, elements of endoplasmic reticulum, Golgi complexes, annulate lamellae, symbiotic bacteroids, lipid droplets and distinctive accumulations of membrane-free clathrin-like cages are present. Early- and mid previtellogenic oocytes are invested with flat somatic cells that gradually transform into a follicular epithelium. In the vicinity of 3-cell junctions, neighbouring follicular cells are joined by narrow intercellular bridges. During late previtellogenesis, numerous microvilli develop on the oocyte surface. They interdigitate with morphologically similar but less frequent microvilli of the follicular cells. Concurrently, first endocytotic vesicles appear in the cortical ooplasm. In the context of presented results, the phylogenetic relationships between mecopterans (boreids) and fleas are discussed.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Cytochemical and fine structural analysis of oogenesis in the gastropod, Ilyanassa obsoleta

An analysis of differentiating oocytes of the gastropod, Ilyanassa obsoleta, has been made by techniques of light and electron microscopy. Early previtellogenic oocytes are limited by a smooth surfaced oolemma and are associated with each other by maculae adhaerentes. Previtellogenic oocytes are also distinguished by a large nucleus containing randomly dispersed aggregates of chromatin. Within the ooplasm are Golgi complexes, mitochondria and a few cisternae of the rough endoplasmic reticulum. When vitellogenesis begins, the oolemma becomes morphologically specialized by the formation of microvilli. One also notices an increase in the number of organelles and inclusions such as lipid droplets. During vitellogenesis there is a dilation of the saccules of the Golgi complexes and cisternae of the endoplasmic reticulum. Associated with the Golgi complexes are small protein-carbohydrate yolk precursors encompassed by a membrane. These increase in size by fusing with each other. The “mature” yolk body is a membrane-bounded structure with a central striated core and a granular periphery. At maturity a major portion of the ooplasmic constituents such as as mitochondria and lipid droplets occupy the animal region while the bulk of the population of yolk bodies are situated in the vegetal hemisphere. The follicle cells incompletely encompass the developing oocyte. In addition to the regularly occurring organelles, follicle cells are characterized by the presence of large quantities of rough endoplasmic reticulum and Golgi complexes whose saccules are filled with a dense substance. Associated with the Golgi saccules are secretory droplets of varied size. Amongst the differentiating oocytes and follicle cells are Leydig cells. These cells are characterized by a large vacuole containing glycogen. A possible function for the follicle and Leydig cells is discussed.     

Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes

Summary— To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10 000 g for 5 min at 20°C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100–300 μm, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between ?35 and ?65 mV, and the membrane resistances were in the 500 kΩ to 1 MΩ range. Under voltage clamp conditions, the oocytes exhibited Ca²⁺-activated Cl^? currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.     

泥螺卵黄发生过程中线粒体的变化

利用透射电镜（TEM）技术研究了泥螺卵黄发生过程中线粒体的形态结构的变化特点,结果表明,从卵黄发生早期到晚期,卵母细胞内线粒体经历了从外部形态到内部结构的一系列变化。卵黄合成初期的卵母细胞内,线粒体多,结构典型,仅部分线粒体外膜破裂,嵴 和内膜逐渐消失,卵黄发生中期,线粒体基质空泡化,嵴和内膜消失,腔内充满颗粒状物质,最后演变成卵黄颗粒,随着卵母细胞的发育,卵黄颗粒的数量和直径逐渐增加,卵黄发生后期,卵质中胞器不发达,细胞质中充满卵黄颗粒,在卵黄颗粒之间仅有少量线粒体存在,提供细胞代谢所需的能量,此外,对线粒体在卵黄形成中的功能,去向及行为变化等 进行了讨论。     

Ultrastructural observation of oogenesis in the crustacea amphipoda Orchestia gammarellus (Pallas)

The oogenesis of the Crustacea Amphipoda Orchestia gammarellus can be divided in five stages taking into consideration both the oocyte ultrastructure and the physiology of the ovary. The primary oogonium (12 μm in diameter) is lodged within the germinative zone: after division, the daughter cell (or secondary oogonium) leaves this area and enters meiotic prophase. Stage I is represented by the oocyte with visible chromosomes (12–18 μm in diameter) the cytoplasmic ultrastructure of which is comparable to that of the oogonium. Stage II or previtellogenesis is characterized by a considerable growth of the oocyte (18–80 μm in diameter) which becomes enriched in ribosomes and vesicles of the rough endoplasmic reticulum; the oocyte does not yet contain any vitelline reserve (proteinaceous and lipid). Stage III or primary vitello-genesis (80–160 μm in diameter) is typified by the synthetic activity of the rough endoplasmic reticulum, corresponding to an endogenous accumulation of proteinaceous yolk. Stage IV or secondary vitellogenesis (160–800 μm in diameter) only appears during the period of reproduction; by means of endocytosis the oocyte accumulates yolk spheres in addition to lipid droplets, the origin of which is uncertain; towards the end of vitellogenesis, cortical granules become a feature that is noted for the first time in Crustacea. The last stage or maturation (800 μm in diameter) starts right before or immediately after the exuviation of the female and ends with fertilization.     

Histo- and cytophysiology of the lactating mammary gland of the African elephant (Loxodonta africana)

The lactating mammary gland of the African elephant (Loxodonta africana) has been studied with a panel of morphological techniques focusing on (1) the functional changes during the secretory process, (2) proliferative process [by application of proliferating cell nuclear antigen (PCNA) immunohistochemistry] and apoptotic phenomena [by use of the TUNEL technique] in the individual lobules, and (3) components of milk and milk-fat-globule membrane. In the lactating gland, the lobules are variably differentiated; within a lobule, however, the alveoli are usually similarly differentiated. The morphology of their alveoli suggests a classification of the lobules into types 1–3. Lobules of type 1 are composed of immature tubular alveoli with mitotic figures and numerous PCNA-positive nuclei; advanced type 1 alveoli contain abundant glycogen and specific secretory granules. Lobules of type 2 are further subdivided. In type 2a lobules, the epithelial cells of the alveoli form tall apical protrusions, which in part are occupied by small lipid droplets and which are pinched off in an apocrine fashion. The number of lysosomes varies considerably. Type 2b is the most common type, with striking basal membrane foldings, abundant rough endoplasmic reticulum cisterns, large Golgi apparatus, numerous mitochondria, lipid droplets, and protein vesicles with 30- to 90-nm-wide casein micelles. The lipid droplets are pinched off with minimal amounts of cytoplasm. Type 2c is composed of alveoli with a cuboidal epithelium and few signs of secretory activity. Increasing expression of peanut-agglutinin-binding sites parallels the maturation and differentiation of the glandular cells. Type 3 lobules are marked by numerous TUNEL-positive nuclei and large lipid droplets and are apparently degenerating structures. Cytokeratin (CK) 14 is usually present in the myoepithelial cells; CK 19 and CK 7 mark ductal and immature alveolar epithelia. Milk protein content varies between 2.6% and 6.3%, and casein micelles range from 35 to 90 nm in diameter. The diameter of intra-alveolar milk fat globules ranges from 5 to 25 µm and the membranes bear a filamentous surface coat composed of membrane-anchored mucins; gel-electrophoretic analysis of these mucins from different individuals demonstrates the presence of mucin MUC 1, which is expressed with considerable genetic heterogeneity.     

Sex pheromone gland of the female European corn borer moth,Ostrinia nubilalis (Lepidoptera,Pyralidae): ultrastructural and biochemical evidences

The sex pheromone gland of the female European corn borer moth, Ostrinia nubilalis was studied using light and electron microscopy. The pheromone gland is formed by hypertrophied epidermal cells at the mid-dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10. Active glandular cells contain extensive apical membrane foldings, a single nucleus, many free ribosomes, numerous mitochondria, microtubules and lipid droplets. Smooth endoplasmic reticulum is scanty. In young moths, the glandular cells are smaller in size, the microvilli at the apical membrane are poorly developed and the cytoplasm contains fewer mitochondria, microtubules, and no lipid droplets. The surrounding unmodified epidermal cells are small cuboidal or squamous cells. These cells have ill-defined apical membrane foldings and do not contain lipid droplets in the cytoplasm and the overlying cuticle. Fatty acids analyses revealed the presence of the sex pheromone components, (E)-11-tetradecenyl acetate, and their immediate precursors, methyl (E)-11- and methyl (Z)-11-tetradecenoate, only in the dorsal portion of the cylindrical intersegmental membrane. Results of the present study show that the sex pheromone gland of O. nubilalis is restricted to the dorsal aspect of the intersegmental membrane between segments 8-9/10 and is not a ring-gland.     

Oogenesis in pig ovaries during the prenatal period: ultrastructure and morphometry

Oogenesis in fetal pig ovaries comprises the successive changes from the primordial germ cells to the dictyotene oocytes in primordial ovarian follicles. In this study the observations were carried out with an electron microscope and stereological analysis was performed. At the ultrastructural level there are no differences between the primordial germ cells and oogonia, but oogonia are connected with the intercellular bridges. The onset of the dictyotene phase was accompanied by the changes in the cytoplasm of oocytes. Near the nucleus, the yolk nucleus is formed containing numerous Golgi bodies, endoplasmic reticulum (ER), mitochondria and granules. ER proliferates in contact with the external leaflet of the nuclear envelope forming the narrow ER cisterns. Between the nuclear envelope and ER cisterns, the vesicles with grey content are visible. The proliferating ER forms numerous concentric cisterns around the nucleus. Next, the most external cisterns fragment, detach, and then form the cup-like structures. These structures separate the distinct areas of cytoplasm-compartments, which contain mitochondria, ribosomes and lipid droplets. The cells of cortical sex cords of the ovary, which encloses the oocyte, form the follicles. The volume of oocytes in forming follicle increases due to the increase in the number of the cell inclusions: lipid droplets, vacuoles and yolk globules. In the oocytes of primordial ovarian follicles, the compartments are transformed into the yolk globules, which are encountered by a sheath of ER cisterns and the grey vesicles; they contain the mitochondria, lipid droplets and light vacuoles. The role of the compartments and yolk globules as metabolic units is discussed in comparison with similar structures of the mature eggs of pigs and other mammal species.     

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.     

Ultrastructural study of oogenesis in Phoronopsis harmeri (Phoronida)

Temereva, E.N., Malakhov, V.V. and Yushin, V.V. 2011. Ultrastructural study of oogenesis in Phoronopsis harmeri (Phoronida). —Acta Zoologica (Stockholm) 92 : 241–250. The successive stages of oogenesis in Phoronopsis harmeri were examined by electron microscopy methods. During the oogenesis, each oocyte is encircled by vasoperitoneal (coelomic) cells forming a follicle. The previtellogenic oocytes are small cells which accumulate ribosomes for future synthesis; their cytoplasm contains characteristic clusters of mitochondria and osmiophilic particles resembling a germ plasm of other metazoans. The cytoplasm of the vitellogenic oocytes includes numerous mitochondria, cisternae of the rough endoplasmic reticulum, Golgi bodies and annulate lamellae. The synthesis of three types of inclusions was observed: strongly osmiophilic granules (lipid droplets) as a prevalent component, distinctly larger granules surrounded by membrane (proteinaceous yolk) and numerous large vesicles with pale flocculent content. No inclusions which could be unequivocally interpreted as the cortical granules were detected. The surface of the vitellogenic oocytes is covered by microvilli which increase in number and length during development. The oogenesis in Phoronida may be interpreted as follicular because of close association of oocytes with the vasoperitoneal tissue. However, well‐developed synthetic apparatus together with a strongly developed microvillous surface and absence of endocytosis indicate a clear case of autosynthetic vitellogenesis. Thus, in phoronids, there is a combination of simply developed follicle and autosynthesis that, apparently, is plesiomorphic character.     

Ultrastructure and function of follicle cell in the ovary ofBranchiostoma belcheri

The ultrastructure of follicle cells in the ovary at different developmental stages ofBranchiostoma has been observed in detail with a transmission electron microscope. The results indicate that only one kind of follicle cell exists with structural features related to steroid hormone biosynthesis: (i) oval or round mitochondria with tubules; (ii) smooth surfaced endoplasmic reticulum; (iii) several large lipid droplets in the cytoplasm; (iv) a well developed Golgi complex and tubular rough surfaced endoplasmic reticulum, as can be found in mammalian theca interna cells. In addition, as steroid hormone synthesizing cells, they obviously play an important role in the phagocytosis of relict gametes and cellular debris and may have a nutritive function for the oocytes. They can produce abundant secretory granules in stages III-IV ovaries. In mature ovaries they transform into flat epithelial cells with numerous microfilaments which may play a role in ovulation.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.