Ultrastructural and cytochemical studies on the germarium of Dicrocoelium dendriticum (Plathelminthes,Digenea)

Summary The unpaired germarium of Dicrocoelium dendriticum contains many female germ cells at different stages of maturation and is enveloped by a fibrous basal lamina-like structure and a multilayered cytoplasmic sheath whose origins and functions are discussed. The maturation process of primary oocytes occurs completely within the prophase of the first meiotic division. It has been divided into three stages, as previously suggested for monogeneans. Stage I corresponds to oogonia and early oocytes which are located in the distal germinative area of the gonad. These cells are characterized by a high nucleo/cytoplasmic ratio and a poorly differentiated cytoplasm. Stage II corresponds to maturing oocytes grouped in the central area of the gonad and exhibiting long synaptonemal complexes and a prominent nucleolus. The main feature of cytoplasmic differentiation is the increase in the number of RER and Golgi complex which are involved in the production of small electron-dense granules. Stage III corresponds to mature oocytes located in the proximal area of the germarium near the origin of the oviduct. In this stage, the granules become regularly distributed in a monolayer in the peripheral ooplasm and make contact with the oolemma. They show a distinctive complex structure, are composed of proteins and glycoproteins and do not contain polyphenols. Their possible role as cortical granules is discussed in relation to chemical composition and previous studies on other Plathelminthes. Neither yolk globules nor glycogen are present in the oocytes.Abbreviations I oogonium and early oocyte - II growing oocyte - III mature oocyte - cg cortical granule - cs cytoplasmic sheath - db dense body - ecm extra cellular matrix - ER endoplasmic reticulum - fl fibrous extracellular layer - gc Golgi complex - m mitochondria - N nucleus - nu nucleolus - RER rough endoplasmic reticulum - sc synaptonemal complex     

Ultrastructural study on hepatic melanin in Xenopus laevis

Summary The livers of Xenopus laevis, grouped by chronological age (0.5,2 and 3 yrs), were studied electron microscopically. Ultrastructurally most of the melanin granules in the mature female liver showed an-internal structure similar to the melanin granules of the oocytes. The hepatic melanin granules of immature females and of all males were pleomorphic and failed to show the characteristic internal structure similar to those of the oocytes. The oocyte is the probable source of most of the hepatic melanin of the mature female.     

Histochemical nature of cortical granules in the human egg

Summary The distribution and histochemical nature of cortical granules have been studied in the human egg. They are distributed as small granules adjacent to the plasma membrane of growing oocytes, and consist of carbohydrates and possibly some protein. The cortical granules of the human egg have been compared and contrasted to those in Amphioxus, fishes, and amphibians.     

Histochemical observations on the cortical granules in the eggs of Indian lizards

Summary The development, distribution and histochemical nature of cortical granules have been investigated in growing eggs of two species of Indian lizards (Hemidactylus flaviviridis Rüppel and Uromastix hardwickii). Numerous cortical granules develop in the peripheral ooplasm of growing oocytes and are finally arranged in the cortical cytoplasm of the egg. They consist of a carbohydrate-protein complex; most of the carbohydrate component is an acid mucopolysaccharide. The cortical granules in the eggs of lizards have been compared and contrasted to those in Amphioxus, fishes, amphibians, and mammals.     

The female gonad in two species of Microplana (Platyhelminthes,Tricladida, Rhynchodemidae): Ultrastructural and cytochemical investigations

The female gonad of the land planarians Microplana scharffi and Microplana terrestris consists of two small germaria located ventrally in the anterior third of the body and of two ventro‐lateral rows of oblong vitelline follicles distributed between the intestinal pouches. Both these structures are enveloped by a tunica composed of an outer extracellular lamina and an inner sheath of accessory cells. Oocyte maturation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be correlated with the production of egg granules with a fenestrated/granular content of medium electron density, about 4–5 μm in diameter, which remain dispersed in the ooplasm of mature oocytes. On the basis of cytochemical tests showing their glycoprotein composition, and their localization in mature oocytes, these egg granules have been interpreted as yolk. In the vitelline follicles, vitellocytes show the typical features of secretory cells with well‐developed rough endoplasmic reticulum and Golgi complexes involved in the production of eggshell globules and yolk. The eggshell globules, which appear to arise from repeated coalescences of two types of Golgi‐derived vesicles, contain polyphenols and, when completely mature, they measure about 1–1,2 μm in diameter and show a meandering/concentric content pattern as is typical of the situation observed in most Proseriata and Tricladida. Mature vitellocytes also contain a large amount of glycogen and lipids as further reserve material. On the basis of the ultrastructural features of the female gonad and in relation to the current literature the two species of rhynchodemids investigated appear to be closely related to the freshwater planarians belonging to the family Dugesiidae. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation

Morphological changes in the growing and maturing oocytes of Patiria ( Asterina ) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.     

The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae)

Zara, F.J., Gaeta, H.H., Costa, T.M., Toyama, M.H. and Caetano, F.H. 2011. The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae). —Acta Zoologica (Stockholm) 00 :1–13. Several studies use macroscopic patterns of the ovarian development in crustaceans. Here, we examined the relationship between ovary histochemistry, changes in gonadosomatic and hepatosomatic indices against the macroscopic pattern of the ovarian development in Callinectes danae. Animals were collected in the south coast of São Paulo State, Brazil. Ovaries were macroscopically classified as juvenile, rudimentary, developing, intermediary, mature, and rudimentary ovigerous. Samples were fixed in 4% paraformaldehyde, processed for historesin, and stained with HE, protein, and neutral and acid polysaccharides detection. The juvenile oocytes are not enclosed by follicular cells and have fewer yolk nuclei being less intense in PAS reactivity than rudimentary oocytes. Developing oocytes show yolk granules and thick follicular cells. Yolk granules were positive for proteins and neutral polysaccharides. The intermediary stage is marked by a qualitative increase in yolk granules and the onset of chorion formation. In mature oocytes, cytoplasm is completely filled by yolk granules and the chorion is completely formed. Ovigerous ovaries have several atretic follicles and large quantities of hemocytes in the process of tissue reorganization. In C. danae, the changes in cell, goandosomatic and hepatosomatic indices coinciding with macroscopic observations and any combination of different macroscopic stages in a single pattern should be avoided.     

Asymmetrically distributed ecdysteroid-related antigens in follicles and young embryos of Drosophila melanogaster

Summary We have produced monoclonal and polyclonal antibodies against an antigen that is asymmetrically distributed in mature oocytes of Drosophila melanogaster. During late oogenesis and early embryogenesis the antigen undergoes dramatic changes in its cellular localization: until about 2.5 h before completion of oogenesis it is homogeneously distributed in the cytoplasm, then it becomes localized in granules that are more numerous in posterior than in anterior peripheral positions of the ooplasm. The germ plasm is void of the antigen. Shortly after egg deposition the antigen is released from the granules and forms a shallow temporary gradient in the egg. Later during embryogenesis the antigen is associated with the yolk-containing cytoplasm. At the syncytial blastoderm stage it is also detected in the peripheral nuclei. Preliminary evidence suggests that the antigen is an ecdysteroid-related molecule. Five different anti-ecdysone antisera were found to bind to the same antigen or to an antigen with the same localization as our monoclonal antibody. In pattern mutants affecting anteroposterior polarity, the described asymmetrical distribution of the antigen is abnormal. In the mutant BicD, for example, which leads to the formation of two abdomina of opposite polarity, the antigen-containing granules are distributed homogeneously in mature oocytes.     

Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley-Liss, Inc.     

Oocyte-follicle cell differentiation in two species of amphineurans (Mollusca), Mopalia mucosa and Chaetopleura apiculata

Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs. As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies. In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.     

Do fast increasing food conditions promote the midsummer decline of Daphnia galeata?

Ovaries from mature giant red shrimp Aristaeomorpha foliacea were investigated histochemically and ultrastructurally. Four growing stages of the oocytes were distinguished: premeiosis stage, previtellogenetic stage, early vitellogenic stage and late vitellogenic stage. In addition, occasional resorptive oocytes were found. Oogonia and premeiotic oocytes were found in germinative zones. Previtellogenic and vitellogenic oocytes were localized in maturative zones. As vitellogenesis proceeded, oocytes showed a progressive development in the number of lipid droplets as well as in the extension of RER, constituted of dilated cisternae, uniformely scattered throughout the cytoplasm. The RER produced yolk granules and a lampbrush-like substance. The latter was released under the oolemma and constituted a characteristic cortical zone. The oolemma did not develop microvilli or micropinocytotic vesicles to incorporate yolk precursors. Thus, the protein yolk appeared to be of endogenous origin. Few somatic cells were found around the oocytes, but they never gave place to a continuous epithelial layer around oocytes, thus it is not possible to speak of ovarian follicle. The cytoplasm of these mesodermal-oocyte associated cells (MOAC) was characterized by a typical steroidogenic apparatus. Few resorptive immature oocytes were found inside late vitellogenic oocytes. Since the ovaries were packed with late vitellogenic oocytes and the few immature oocytes were hardly detectable, oocyte maturation occurred in a synchronous way.     

A morphologic,cytochemical, and chromatographic analysis of lipid yolk formation in the oocytes of the dog

The oocytes of carnivorous mammals are distinguished by the presence of large amounts of a lipid, yolk like material. In the oocytes of the dog, lipid yolk formation marks one of the earliest indications of occyte maturation. In early primary oocytes, the yolk bodies are scattered within the ooplasm, while in later stages they are in discrete clusters. Lipid yolk material appears to be formed by at least two mechanisms. Throughout most of oogenesis the oocyte contains scattered dense granular bodies that become vacuolated by droplets of lipid material and may be transformed, by this process, into lipid yolk bodies. These granular bodies are highly reactive for acid phosphatase and are positive for glycoprotein with the PA-CA-methenamine technique. In addition, other glycoprotiein-rich yolk bodies appear to arise from many of the small dictyosomes. In secondary follicles these two mechanisms often appear to act conjointly with the dense vacuolated granules coalesing with the larger yolk bodies. Small yolk bodies are intensely reactive for glycoprotein, becoming less reactive as they enlarge and mature. The developing yolk bodies are often associated with the acid phosphatase-positive granules. The peripheral portions of the larger yolk bodies are faintly reactive for both acid phosphatase and glycoprotein. All reactivity is lost in mature yolk bodies. Thin layer chromatography of the total lipids extracted from isolated oocytes reveals a pattern that is consistent among dogs of the same and of different breeds. The most abundant lipid fraction from each dog oocyte extraction stains strongly for glycolipid.     

Ultrastructural Studies on Gametogenesis of the Prawn Palaemon serratus (Pennant). I. Oogenesis

The oogenesis of the common prawn P. serratus is described. The reproductive cycle is annual and there is little evidence for a second spawning period. Oogenesis under the electron microscope follows a typical crustacean pattern and has been divided into seven stages according to the structure of the oocytes. The production of yolk is mainly intra-oocytic, with a small extra-oocytic contribution. Yolk, in the form of small granules, arises within the lamellar elements of the rough endoplasmic reticulum. These were then transformed into finely granular yolk bodies. Lipid droplets, as in other crustacean species, arise de novo in the cytoplasm and there is no association between lipids and cell organelles.     

Ultrastructural studies on the formation of yolk granules in the statoblast of a fresh-water bryozoan,Pectinatella gelatinosa

The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.     

Ultrastructural and cytochemical aspects of the germarium and the vitellarium in Syndesmis patagonica (Platyhelminthes,Rhabdocoela, Umagillidae)

The cytoarchitecture of the female gonad of the endosymbiont umagillid Syndesmis patagonica has been investigated using electron microscopy and cytochemical techniques. The female gonad consists of paired germaria and vitellaria located behind the pharynx in the mid‐posterior region of the body. Both the germaria and the vitellaria are enveloped by an outer extracellular lamina and an inner sheath of accessory cells which contribute to the extracellular lamina. Oocyte maturation occurs completely during the prophase of the first meiotic division. Oocyte differentiation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be involved in the production of round granules, about 2–2.5 μm in diameter, with a homogeneous electron‐dense core surrounded by a granular component and a translucent halo delimited by a membrane. These egg granules migrate to the periphery of mature oocytes, are positive to the cytochemical test for polyphenol detection, are unaffected by protease and have been interpreted as eggshell granules. The mature oocytes also contain a small number of yolk granules, lipid droplets, and glycogen particles scattered throughout the ooplasm. The vitellaria are branched organs composed of vitelline follicles with vitellocytes at different stages of maturation. Developing vitellocytes contain well‐developed rough endoplasmic reticulum and small Golgi complexes involved in the production of eggshell and yolk globules. Eggshell globules are round, measure 4–5 μm in diameter, and have a mosaic‐like patterned content which contains polyphenols. The yolk globules, 2–3 μm in diameter, show a homogeneous protein content of medium electron density, devoid of polyphenols, and completely digested by protease. The mature vitellocytes also contain glycogen as further reserve material. The presence of polyphenolic eggshell granules in the oocytes and of polyphenolic eggshell globules with a mosaic‐like pattern in the vitellocytes have been considered apomorphic features of the Rhabdocoela + Prolecithophora. J. Morphol. 275:703–719, 2014. © 2014 Wiley Periodicals, Inc.     

Ultrastructure of oogenesis in imposex females of <Emphasis Type="Italic">Babylonia areolata</Emphasis> (Caenogastropoda: Buccinidae)

During a tributyltin (TBT)-exposure experiment, the ultrastructural features of oogenesis have been examined in TBT-induced imposex females of Babylonia areolata and compared with those of the normal female. The results obtained from such experiment demonstrates that B. areolata exhibits a low to moderate intensity of imposex because all VDSI values are never higher than 3. Ultrastructures of germ cell development including oogonia, pre-vitellogenic, early vitellogenic, late vitellogenic and mature oocytes show that oogenesis in imposex female is similar to that of normal females except for the presence of numerous lipid droplets in the cytoplasm of the oocytes and the follicle cells in imposex females, indicating the degeneration of their oocytes. Vitellogenesis in B. areolata involves both auto- and heterosynthetic processes that resemble those of the basal gastropods and the pulmonates. In addition, the presence of cortical granules and microvilli are unique structures of this species.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

Ultrastructural aspects of the germovitellarium of two prorhynchids (Platyhelminthes,Lecithoepitheliata)

Summary

The female gonad of two fresh-water prorhynchids, Geocentrophora baltica and Prorhynchus stagnalis, has been investigated by means of conventional electron microscopy and cytochemical techniques. Both species have an unpaired germovitellarium located under the gut; accessory cells surround the germovitellarium of G. baltica. The germovitellarium consists of a restricted germinative area where early differentiating oocytes and vitellocytes are randomly associated, and an extensive growth area with follicular organization. Each follicle consists of a single alecithal oocyte surrounded by numerous vitellocytes. The main features of oocyte differentiation are the accumulation of lipid droplets and the appearance of Golgi complexes and small bodies possibly representing secondary lysosomes. Vitellocytes show features typical of secretory cells, including well-developed rough endoplasmic reticulum (RER) and Golgi complexes which are involved in the production of type A and type B inclusions, hi both species, type A inclusions appear first, have a glycoprotein content, do not contain polyphenols, and become localized in the peripheral cytoplasm of mature vitellocytes; they have been interpreted as eggshell forming granules. Type B inclusions are larger, have a proteinaceous content with a different structure in the two species examined, and remain scattered in the cytoplasm of mature vitellocytes; they are considered to be yolk. The finding of eggshell forming granules without polyphenols in prorhynchids contrasts with the condition in most platyhelminths that have a sclerotized eggshell formed through a tanning process of polyphenolic substances. The small bodies in the oocytes and the eggshell granules in the vitellocytes of Lecithoepitheliata differ from those observed in prolecithophorans, which have oocyte and vitellocyte inclusions similar to those of the Rhabdocoela.     

The ultrastructure of oogenesis and yolk formation in Labidocera aestiva (Copepoda: Calanoida)

Yolk formation in the oocytes of the free-living, marine copepod, Labidocera aestiva (order Calanoida) involves both autosynthetic and heterosynthetic processes. Three morphologically distinct forms of endogenous yolk are produced in the early vitellogenic stages. Type 1 yolk spheres are formed by the accumulation and fusion of dense granules within vesicular and lamellar cisternae of endoplasmic reticulum. A granular form of type 1 yolk, in which the dense granules within the cisternae of endoplasmic reticulum do not fuse, appears to be synthesized by the combined activity of endoplasmic reticulum and Golgi complexes. Type 2 yolk bodies subsequently appear in the ooplasm but their formation could not be attributed to any particular oocytic organelle. In the advanced stages of vitellogenesis, a single narrow layer of follicle cells becomes more developed and forms extensive interdigitations with the oocytes. Extra-oocytic yolk precursors appear to pass from the hemolymph into the follicle cells and subsequently into the oocytes via micropinocytosis. Pinocytotic vesicles fuse in the cortical ooplasm to form heterosynthetically derived type 3 yolk bodies.     

DNA methylation establishment of CpG islands near maternally imprinted genes on chromosome 7 during mouse oocyte growth

The genome methylation is globally erased in early fetal germ cells, and it is gradually re‐established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S‐group (60–100 μm), M‐group (100–140 μm), and L‐group (140–180 μm). The fully grown germinal vesicle (GV)‐stage and metaphase II (M2)‐stage mature oocytes were also collected. These oocytes were used for single‐cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII‐stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later‐stage growing oocytes, fully grown GV oocytes, and mature MII‐stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes.