Action of the chorionic gonadotropin hormone on the gap junctions network in the antral follicles of rat

A morphometrical study of gap junctions has been realized in rat antral follicle after an injection of 20 I. U. of hCG. A clear and rapid reduction of the gap junctions number and of the membranous surface they are occupying has been demonstrated whatever the follicle region one consider. The significance of those gap junction modifications is discussed with respect to resumption of meiotic maturation.     

Ultrastructural modifications of the follicular epithelium accompanying the onset of vitellogenesis in the whirligig beetle,Gyrinus natator

Summary The ultrastructure of the follicle cells during previtellogenesis and early vitellogenesis have been studied. In previtellogenesis follicle cells are columnar with numerous bundles of microtubules located along the lateral plasma membranes. Oocyte-follicle cell gap junctions are not found in this stage. At the onset of vitellogenesis, the bundles of microtubules disappear and are replaced by an apically located ring of microtubules. The modification of microtubular cytoskeleton is not followed by the development of intercellular spaces between the follicle cells. Concurrently, numerous gap junctions are formed between specialized follicle cell processes and oocyte microvilli, which are arranged in characteristic cone-shaped aggregations. It is suggested that cytoskeletal changes and formation of heterologous gap junctions, occurring at the onset of vitellogenesis, are induced by juvenile hormone.     

THE APPEARANCE AND STRUCTURE OF INTERCELLULAR CONNECTIONS DURING THE ONTOGENY OF THE RABBIT OVARIAN FOLLICLE WITH PARTICULAR REFERENCE TO GAP JUNCTIONS

Lanthanum tracer and freeze-fracture electron microscope techniques were used to study junctional complexes between granulosa cells during the differentiation of the rabbit ovarian follicle. For convenience we refer to cells encompassing the oocyte, before antrum and gap junction formation, as follicle cells. After the appearance of an antrum and gap junctions we call the cells granulosa cells. Maculae adherentes are found at the interfaces of oocyte-follicle-granulosa cells throughout folliculogenesis. Gap junctions are first detected in follicles when the antrum appears. In early antral follicles typical large gap junctions are randomly distributed between granulosa cells. In freeze-fracture replicas, they are characterized by polygonally packed 90-Å particles arranged in rows separated by nonparticulate A-face membrane. A particle-sparse zone surrounds gap junctions and is frequently occupied by small particle aggregates of closely packed intramembranous particles. The gap junctions of granulosa cells appear to increase in size with further differentiation of the follicle. The granulosa cells of large Graafian follicles are adjoined by small and large gap junctions; annular gap junctions are also present. The large gap junctions are rarely surrounded by a particle-free zone on their A-faces, but are further distinguished by particle rows displaying a higher degree of organization.     

Differential modulation of rat follicle cell gap junction populations at ovulation

The hypothesis proposed in the late 1970s that meiotic resumption in mammalian oocytes might result from the disruption of gap junction communication between follicle cells and the oocyte has not been supported by metabolic cooperation experiments which demonstrate that exogenous tracer transfer from the cumulus oophorus to the oocyte does not decrease until several hours after germinal vesicle breakdown (GVBD). Since these studies utilized isolated cumulus-oocyte complexes for their measurements, however, they excluded from consideration the possible effect of separation of the cumulus oophorus from the membrana granulosa which was required for this assay. We considered the possibility that the disruption of cumulus junctions within the intact follicle could mimic this experimental manipulation and previously reported that cumulus gap junctions were dramatically down-regulated during the period of GVBD in vivo. In the present study, we have utilized quantitative morphometric techniques to analyze the responses of other gap junction populations in intact preovulatory rat follicles to an ovulatory stimulus and demonstrate now that membrana granulosa, cumulus, and cumulus-oocyte gap junctions are down-regulated at different times and rates during the preovulatory period. Although membrana gap junctions are down-regulated during the period of meiotic resumption, their loss is not as rapid or as complete as in the cumulus oophorus. Cumulus-oocyte gap junctions are down-regulated after meiosis resumes but during the same period other investigators have demonstrated a reduction in metabolite transfer between the cumulus oophorus and the oocyte. Our results are interpreted to suggest that the cumulus oophorus may regulate the conduction of meiosis inhibitory signals between the membrana granulosa and the oocyte.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.     

间隙连接蛋白在卵泡发育过程中的作用

间隙连接广泛分布于各种组织细胞中,由其构成的通道允许小分子信号物质在相邻细胞间直接传递,在细胞间的通讯方面起着非常重要的作用。间隙连接由连接蛋白(Cx)组成,目前已经发现Cx家族有20多个成员[1],它们在相邻细胞间组成同种或异种间隙连接,调控着细胞的增殖和分化。在哺乳动物卵泡发育过程中,卵母细胞与周围的颗粒细胞之间形成的缝隙连接,介导胞间通讯,对生殖细胞迁移、卵母细胞减数分裂能力恢复、颗粒细胞分层、卵泡成腔、黄体形成、促性腺激素信号传递有非常重要的调节作用。本文根据近年来相关的研究报道,对卵泡发育过程中间隙连接的作用进行综述。     

Gap junction amplification in rat ovarian granulosa cells: I. A direct response to follicle-stimulating hormone

The ability of follicle-stimulating hormone (FSH), the estrogens, estradiol-17β and diethylstilbestrol, and the estrogen antagonists, clomiphene and enclomiphene citrate to affect the growth and internalization of hypophysectomized rat granulosa cell gap junction membranes was compared in ovarian follicles assigned to one of four follicle size classes (60–149, 150–249, 250–319, and 320–450 μm diameter). In the absence of exogenous hormone stimulation, atresia prevents follicle growth beyond 320 μm in diameter but surface gap junction membrane increases throughout this early follicle growth. Internalization of gap junction membrane is first detected at the 150- to 249-μm follicle stage and also increases with follicle size. Therefore, growth and turnover of gap junction membrane occur at a basal rate in the absence of gonadotropin or steroid hormone stimulation. Estrogen and estrogen antagonist injections result in no significant differences in the amount of surface or internalized junction membrane in the three smallest follicle size classes when compared to the untreated hypophysectomized animals. However, estrogen but not estrogen antagonists rescues growing follicles from atresia and permits their further growth into the 320- to 450-μm follicle size class. As a result of the additional follicle growth, both surface and internalized junction membrane increase beyond that seen in the largest follicles from hypophysectomized animals. In contrast to other treatments, FSH stimulation promotes amplification of gap junction membrane in all size classes and, like estrogen, rescues follicles from atresia and promotes their entry into the 320- to 450-μm follicle size class. Surface gap junction membrane is amplified two- to fourfold over other treatments in the first three follicle size classes, but reaches maximal levels in the 250- to 319-μm follicles. The internalized junction membrane which first appears in the 150- to 249-μm size class is dramatically increased over other treatments in the 250- to 319- and 320- to 450-μm size classes. These studies indicate that exogenous estrogen stimulation promotes gap junction growth indirectly by sustaining the basal rate of junction synthesis in follicles rescued from atresia. In contrast, exogenous FSH stimulation directly amplifies the developmental sequence of gap junction growth and turnover. During early follicle growth, FSH stimulation preferentially promotes increases in surface gap junctions while internalization of surface junctions is increased during later follicle growth.     

Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures

The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.     

Development of gap junctions in normal and mutant ovaries of Drosophila melanogaster

An ovarian follicle of Drosophila consists of an oocyte, 15 nurse cells, and hundreds of follicular epithelial cells. A freeze-fracture analysis of the surfaces between glutaraldehyde-fixed ovarian cells showed that all three cell types were interconnected by gap junctions. This is the first report of gap junctions between adjacent nurse cells, between nurse cells and oocytes, and between follicle cells and oocytes in Drosophila. Since we did not observe intramembranous particle clumping into crystalline patterns and since structurally different gap junctions occurred at different times in development and at different cell-cell interfaces, it is unlikely that fixation artifacts influenced particle distribution in our experiments. A computer-assisted morphometric analysis showed that the extent, size, and morphology of gap junctions varied with development and that these junctions can cover up to 9% of the cell surfaces. To test the role of gap junctions in follicular maturation, we studied ovaries from flies homozygous for the female sterile mutation fs(2)A17, in which follicles develop normally until yolk deposition commences. During the development of mutant follicles, gap junctions became abnormal before any other morphological aspect of the follicle. These studies show that gap junctions are available to play an important role in coordinating intercellular activities between all three cell types in ovarian follicles of Drosophila.     

Intercellular junctions in the adrenal medulla: A comparative freeze-fracture study

Observations of freeze-fractured specimens revealed that intercellular junctions in adrenal medulla are different in nature and number according to species. Only gap junctions of diverse size exhibiting characteristic loop-like configurations were found in hamster chromaffin cells. In addition to such gap junctions, polymorphic focal tight junctions occasionally combined with particle clusters or small gap junctions were found in guinea-pig. So far, no intercellular junctions were found in rat. Discussion is focused on the possible function of these junctions, in keeping with their presumably high lability.     

Gap junctions between microvilli of an oocyte and follicle cells in the teleost (Plecoglossus altivelis)

In the teleost, Plecoglossus altivelis, intercellular junctions between microvilli of an oocyte and follicle cells were studied by electron microscopy. Microvilli, which were radiated from an oocyte and arrived at the surface of follicle cells, established contact with follicle cells. These contact areas appeared to be a seven-layered membrane with an overall thickness of about 18 microns by standard fixation. In freeze-fracture replicas, many small aggregates of intramembraneous particles were revealed on the cleavage faces of cytoplasmic membranes of follicle cells. These morphological evidences suggest that in the teleost gap junctions exist between the oocyte and follicle cells, especially on the surface of follicle cells.     

Heterologous gap junctions between oocytes and follicle cells of an insect,Dysdercus intermedius,and their potential role as ion current pathways

Summary In telotrophic insect ovaries, the oocytes develop in association with two kinds of supporting cells. Each ovary contains five to seven ovarioles. An ovariole consists of a single strand of several oocytes. At the apex of each ovariole is a syncytium of nurse cells (the tropharium), which connects by strands of cytoplasm (the trophic cords) to four or more previtellogenic oocytes. In addition, each oocyte is surrounded by an epithelium of follicle cells, with which it may form gap junctions. To study the temporal and spatial patterns of these associations, Lucifer yellow was microinjected into ovaries of the red cotton bug, Dysdercus intermedius. Freeze-fracture replicas were examined to analyze the distribution of gap junctions between the oocyte and the follicle cells. Dye-coupling between oocytes and follicle cells was detectable early in previtellogenesis and was maintained through late vitellogenesis. It was restricted to the lateral follicle cells. The anterior and posterior follicle cells were not dye-coupled. Freeze-fracture analysis showed microvilli formed by the oocyte during mid-previtellogenesis, and the gap junctions became located at the tips of these. As the microvilli continued to elongate until late vitellogenesis, gap junction particles between them and follicle cell membranes became arranged in long arrays. The morphological findings raise questions about pathways for the intrafollicular phase of the ion currents known to surround the previtellogenic and vitellogenic growth zones of the ovariole.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Fate of annular gap junctions in the papillary cells of the enamel organ in the rat incisor

Summary To investigate the mechanisms whereby annular gap junctions in the papillary cells of the enamel organ are degraded intracellularly, continuously growing rat incisors were examined by electron microscopy of routine thin sections as well as for the cytochemical localization of inorganic trimetaphosphatase activity. Routine thin-section analysis revealed small flat or undulated gap junctions, hemi-annular gap junctions between an invaginated cell process and a cell body, and fully internalized cytoplasmic annular gap junctions. Both hemi-annular and annular gap junctions usually contain various organelles and/or inclusions, such as mitochondria, endoplasmic reticulum, ribosomes, vesicles, and lysosomes in the cytoplasm confined by the junctional membranes. Annular gap junctions are sometimes fused with vesicular or tubulovesicular structures. Cytochemistry of inorganic trimetaphosphatase activity revealed an intense enzymatic reaction within a system of tubular structures and round or oval dense bodies. Both structures are believed to correspond to primary lysosomes. A part of the Golgi apparatus also shows a weak reaction. Although hemi-annular gap junctions never show enzymatic reaction, annular gap junctions sometimes contain reaction products throughout their interior cytoplasm and inclusions. Fusion of annular gap-junctional membranes with reaction-positive tubular structures is also observed. In one instance, revealed in serial sections, an annular gap junction was encircled entirely by a reaction-positive structure. These results suggest that cytoplasmic annular gap junctions are formed by endocytosis of hemi-annular gap junctional membranes from the cell surface and then degraded intracellularly by lysosomal enzymes.     

The follicle cell-oocyte interaction in ovarian follicles of the stick insect Bacillus rossius (Rossi): (Insecta: Phasmatodea)

Developing ovarian follicles of Bacillus rossius have been examined ultrastructurally in an attempt to understand how inception of vitel-logenesis is controlled. Early vitellogenic follicles are characterized by a thick cuboidal epithelium that is highly interlocked with the oocyte plasma membrane. Gap junctional contacts are present both at the follicle cell/oocyte interface and in between adjacent follicle cells. In addition, microvilli of follicle cells protrude deeply into the cortical ooplasm of these early vitellogenic oocytes. With the onset of vitellogenesis, wide intercellular spaces appear in the follicle cell epithelium and at the follicle cell/oocyte interface. Gap junctions become progressively reduced both on the follicle cell surface and on the oocyte plasma membrane. Microvilli from the two cell types no longer interlock. From a theoretical standpoint each of the two structural differentiations present at the follicle cell/oocyte interface—gap junctions and follicle cell microvilli—could potentially trigger inception of vitellogenesis. Gap junctions might permit the passage of a regulatory molecule, transferring from follicle cells to oocyte, which would control the assembly of coated pits on the oocyte plasma membrane. Alternatively cell interaction via microvilli might induce the appearance of coated pits, thus creating a membrane focus for vitellogenin receptors. Both possibilities are discussed in relation to current literature.     

A detergent-independent procedure for the isolation of gap junctions from rat liver

In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.     

Ultrastructural characteristics of the follicle cell-oocyte interface in the oogenesis of Ceratophrys cranwelli

In this work we carried out an ultrastructural analysis of the cell interface between oocyte and follicle cells during the oogenesis of the amphibian Ceratophrys cranwelli, which revealed a complex cell-cell interaction. In the early previtellogenic follicles, the plasma membrane of the follicle cells lies in close contact with the plasma membrane of the oocyte, with no interface between them. In the mid-previtellogenic follicles the follicle cells became more active and their cytoplasm has vesicles containing granular material. Their apical surface projects cytoplasmic processes (macrovilli) that contact the oocyte, forming gap junctions. The oocyte surface begins to develop microvilli. At the interface both processes delimit lacunae containing granular material. The oocyte surface has endocytic vesicles that incorporate this material, forming cortical vesicles that are peripherally arranged. In the late previtellogenic follicle the interface contains fibrillar material from which the vitelline envelope will originate. During the vitellogenic period, there is an increase in the number and length of the micro- and macrovilli, which become regularly arranged inside fibrillar tunnels. At this time the oocyte surface exhibits deep crypts where the macrovilli enter, thus increasing the follicle cell-oocyte junctions. In addition, the oocyte displays coated pits and vesicles evidencing an intense endocytic activity. At the interface of the fully grown oocyte the fibrillar network of the vitelline envelope can be seen. The compact zone contains a fibrillar electron-dense material that fills the spaces previously occupied by the now-retracted microvilli. The macrovilli are still in contact with the surface of the oocyte, forming gap junctions.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion.