Effects of Forskolin on Fine Structures of Medaka Follicles

Effects of forskolin (FK), which stimulates production of 17α, 20β-dihydroxy-4-pregnen-3-one and estradiol-17β, on the fine structure of preovulatory follicles of Oryzias latipes were examined. Granulosa cells incubated in culture medium containing FK exhibited dislocation of the nucleus from the chorion side to the basement membrane side, vesiculation of conspicuous dilated endoplasmic reticulum (ER) with electron-dense material and Golgi lamellae, and development of large oval mitochondria with an electron-dense matrix. Moreover, a thin vesicular layer adherent to the outermost layer of the chorion was found in all immature oocytes at the end of incubation in the presence of FK. Intercellular junctions between granulosa cells and the oocyte gradually decreased during incubation in the presence of FK, and were finally lost with closure of the radial canals in the chorion at the end of the incubation. On the other hand, intrafollicular oocytes that were first incubated with FK for 10 hr, matured normally when they were incubated an additional 8 hr in plain medium. In granulosa cells of these follicles, the dilated ER and vacuolated Golgi lamellae were no longer detectable. These observations suggest that the development of dilated ER and vacuolated Golgi lamellae is characteristic of granulosa cells induced by FK.     

Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes

Freeze fracture and lanthanum tracer experiments have shown that gap junctions exist throughout folliculogenesis between granulosa cells and growing mouse oocytes (Anderson and Albertini, J. Cell Biol.71, 680–686, 1976). The following lines of experimentation in the present study suggest that metabolic cooperativity exists between granulosa cells and their enclosed oocytes, i.e., gap junctions are functional, and that in most cases examined, greater than 85% of the metabolites present in follicle-enclosed oocytes were originally taken up by the granulosa cells and transferred to the oocyte via gap junctions: (1) When incubated with various radiolabeled compounds, follicle-enclosed oocytes contained more intracellular radioactivity than did oocytes with no attached granulosa cells (denuded oocytes); (2) for two radiolabeled ribonucleosides examined, the distribution of phosphorylated metabolites in follicle-enclosed oocytes resembled that of granulosa cells and differed significantly from that in denuded oocytes; (3) pulse-chase experiments with radiolabeled ribonucleosides revealed that during the chase period more radioactivity became associated with the follicle-enclosed oocyte; (4) treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and oocytes; and (5) a series of control experiments using (a) medium conditioned by granulosa cells and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not permitted demonstrated that contact between follicle cells and oocytes was necessary for observing metabolic cooperativity. Metabolic cooperativity was also found between follicle cells and oocytes in the two culture systems which support growth of mouse oocytes in vitro. The fact that oocytes do not grow well, if at all, in the absence of follicle cells and the large contribution of nutrients apparently furnished to the oocyte by the granulosa cells is consistent with the concept that gap junction mediated metabolic cooperativity between follicle cells and their enclosed oocytes is vital for mammalian oocyte growth.     

Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes

One of the currently popular hypotheses for the regulation of meiotic resumption in mammalian oocytes proposes that the preovulatory surge of luteinizing hormone causes down-regulation of follicular gap junctions, which in turn disrupts transfer of a meiotic arrester from the somatic cells into the oocyte. The present study has investigated this hypothesis by examining the integrity of membrana granulosa cell gap junctions during the period of irreversible commitment to maturation of golden Syrian hamster oocytes in vivo. Our results have revealed a significant progressive decrease in the fractional area of cell surface occupied by gap junction membrane with increasing percentage of oocytes irreversibly committed to mature (1.946% and 0.921% fractional gap junction area at 0% and 100% oocytes irreversibly committed to mature, respectively, P less than 0.05). This net loss of membrana granulosa cell gap junctions from the cell surface was accompanied by a significant decrease in density of gap junction particles, whether they were arranged in rectilinear or non-rectilinear packing patterns. Furthermore, the number of gap junction particles per unit area of surface membrane scanned also underwent a significant progressive decrease with increasing percentage of oocytes irreversibly committed to mature. These data with the hamster are consistent with the hypothesis that down-regulation of membrana granulosa cell gap junctions may be of central importance in the regulation of gonadotropic stimulation of meiotic resumption in mammalian oocytes.     

Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma.

Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.     

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.     

Medaka Oocytes Rotate Within the Ovarian Follicles During Oogenesis

The purpose of the current investigation was to ascertain whether medaka oocytes rotate within the follicle. Isolated medaka follicles were incubated in modified L15 Medium for 3 hr at 26°C. During incubation, movement of oocytes within follicles held on slides under a microscope was recorded by a video cassette recorder. Within the follicle, the surface of which was marked with carbon particles, the movement of the intrafollicular oocyte was traced by dislocation of its attaching and non-attaching filaments on the chorion. Pre-vitellogenic oocytes exhibited rotation around the predetermined animal-vegetal axis, accompanied by rotation at a slightly oblique angle to the axis. The velocity of oocyte rotation was about 40–48 μm hr⁻¹ and was similar among oocytes of different stages between the pre-vitellogenic and early vitellogenic phases of oogenesis. Rotation was inhibited by cytochalasin B treatment. Also, it was not observed in oocytes surrounded only by the granulosa cell layer when the thecal cell layer and the basement membrane were removed from the follicle. In oocytes with a thick chorion, rotation was also inhibited by impaling the oocytes with a glass needle at a right angle to the animal-vegetal axis of the oocyte. These results provide evidence that growing medaka oocytes rotate primarily around their animal-vegetal axis and at a slightly oblique angle to the axis. That the rotation of medaka oocytes may depend upon the movement of the granulosa and the thecal cells within the follicles was discussed.     

Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

Mammalian oocytes grow within ovarian follicles in which the oocyte is coupled to surrounding granulosa cells by gap junctions. We report here that growing oocytes isolated from mouse preantral follicles are incapable of recovering from an experimentally induced acidosis, and that oocytes acquire the ability to manage acid loads by activating Na(+)/H(+) exchange during growth. By contrast, granulosa cells from similar preantral follicles possess substantial Na(+)/H(+) exchange capacity, which is attributable to the simultaneous action of two Na(+)/H(+) exchanger isoforms: NHE1 and NHE3. Granulosa cells were also found to possess a V-type H(+)-ATPase that drives partial acidosis recovery when Na(+)/H(+) exchange is inactivated. By monitoring intracellular pH (pH(i)) in small follicle-enclosed oocytes, we found that the oocyte has access to each of these acidosis-correcting activities, such that small follicle-enclosed oocytes readily recover from acidosis in a manner resembling granulosa cells. However, follicle-enclosed oocytes are unable to access these activities if gap-junction communication within the follicle is inhibited. Together, these experiments identify the NHE isoforms involved in regulating oocyte pH(i), indicate that gap junctions allow granulosa cells to exogenously regulate oocyte pH(i) against acidosis until the oocyte has acquired endogenous pH(i) regulation, and reveal that granulosa cells possess multiple mechanisms for carrying out this function.     

Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes

Ovine cumulus-enclosed oocytes collected from antral follicles (3-5 mm in diameter) were cultured in vitro with 2 x 10(6) granulosa cells/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle-stimulating hormone-luteinizing hormone (FSH-LH; -10 micrograms/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6-7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus-oocyte complexes (COCs) were incubated in CD (5 micrograms/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.     

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)     

Characteristics of invasive cells found in between zona pellucida and oocyte during follicular atresia in mice

During the process of follicular atresia, cells are observed to invade the zona pellucida (invasive cells) where they presumably play an important role in eliminating degraded oocytes. Although our preliminary studies have suggested that these cells may originate from granulosa cells and not from macrophages, a detailed morphological analysis of the cells has not been conducted. The objective of this study was to characterize the cells more precisely by electron microscopy and immunohistochemistry, using sexually immature mice. The results show that the invasive cells were first observed within advanced primary (non-antral) atretic follicles. The cells frequently contained cytoplasmic lysosome-like granules after passing through the zona pellucida. F4/80 and Mac-1, reported as macrophage-specific antibodies, were reactive with the cells in most cases, but some immunonegative invasive cells were also observed. The ultrastructural features of the invasive cells were quite similar to those of granulosa cells, not macrophages. Gap junctions, which are typical cytoplasmic structures of epithelial cells, were frequently identified between neighbouring cells. Although direct evidence indicating a contribution by the cells to the elimination of degenerated oocytes was not obtained, our results strongly suggest that the invasive cells originated from granulosa cells surrounding the zona pellucida, and that they may have a macrophage-like cell function for the elimination of oocytes from atretic follicles in mice.     

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH(i)), did not exhibit the pH(i)-regulatory HCO(3)(-)/Cl(-) and Na(+)/H(+) exchange activities found in fully-grown oocytes, and had low pH(i). However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH(i) increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH(i) is instead maintained at approximately 7.2 throughout oocyte development, and the growing oocyte exhibits HCO(3)(-)/Cl(-) exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO(3)(-)/Cl(-) exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH(i) may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO(3)(-)/Cl(-) exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO(3)(-)/Cl(-) exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH(i) is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.     

Development of primordial and prenatal follicles from undifferentiated somatic cells and oocytes in the hamster prenatal ovary in vitro: effect of insulin

Fetal hamster ovaries were cultured for up to 16 days in the presence or absence of various dosages of insulin to evaluate the induction of folliculogenesis in vitro. In the absence of insulin, a few primordial follicle-like structures appeared by the 4th day, and distinct primary follicles (stage 1) appeared by the 12th day of culture. The organelles in the oocytes and adjacent granulosa cells developed along with follicular growth. Moreover, gap junctions between the oocyte and somatic cell plasma membrane also developed as early as 8 days in culture. In the presence of 0.2 microg/ml insulin, primary follicles developed after 8 days, and approximately 4% secondary follicles with 2-3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 microg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.     

Fine structure of the micropylar cell and its change during oocyte maturation in the chum salmon,Oncorhynchus keta

In the ovarian follicle, the micropylar cell (MPC) is distinguished from neighboring granulosa cells by its larger cell size and its thick cytoplasmic process. The micropylar cell body fits into a shallow depression (micropylar vestibule) on the outer surface of the egg envelope; its process extends through the micropylar canal, which extends from the bottom of the vestibule through the full thickness of the zona pellucida interna. At its distal end, the cell process expands into a bulb which fits into an indentation of the ooplasmic surface immediately beneath the inner opening of the micropylar canal. Intermediate and desmosomelike junctions establish an intimate association between MPC process and oocyte. Various kinds of organelles and inclusions in the MPC show a characteristic pattern of cytoplasmic distribution; rough endoplasmic reticulum with markedly dilated cisternae is found exclusively in the main cell body, while microtubules and thin filaments are observed in the cytoplasmic process. Immediately before or during the breakdown of the germinal vesicle in the intrafollicular oocyte, the cytoplasmic process of the MPC gradually decreases in length and begins to withdraw from the micropylar canal. At the same time, the ooplasmic surface protrudes outward to form a papilla in the canal. The intimate MPC-oocyte association disappears during formation of the ooplasmic papilla. Hydration of the oocyte apparently occurs at the final stage of maturation and probably participates in papilla formation. Although the MPC undergoes degenerative changes as ovulation draws near, it remains attached to the inner surface of the granulosa cell layer even after its association with the oocyte has completely disappeared. We speculate that the micropyle develops during fish oogenesis through the combined activity of the MPC and neighboring granulosa cells. It appears that the cell body of the micropylar cell and nearby granulosa cells exert mechanical pressure on the external surface of the growing oocyte and thus participate in formation of the micropylar vestibule. The cytoplasmic process of the MPC evidently forms a passive barrier to deposition of material for the egg envelope in the animal pole, thereby resulting in formation of the micropylar canal.     

Light- and electron-microscopic observations on the relationship between prelampbrush oocytes and surrounding granulosa cells in the laying Japanese quail (Coturnix coturnix japonica).

Transmission electronmicroscopic (TEM) observations demonstrated that the most superficial region of quail oocytes during the prelampbrush stage differs locally from the deeper ooplasm and is an active zone which forms exooplasmic cones, ridges or knob-like protrusions in the direction of/or in the granulosa cells. This exooplasm, in which no mitochondria were seen, is separated from the endooplasm, by a narrow interrupted filamentous layer. Using a lipid-preserving method of fixation, morphological evidence was found for the transport of lipid material from the granulosa cells into the exooplasm of the oocyte. Open intercellular bridges between exooplasm and granulosa cell cytoplasm were also seen. Differences between the electronmicroscopic aspect of clear and dark granulosa cells have been described.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes

Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with ³H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of ³H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG. During coculture, cattle cumulus cells were closely associated with the basement membrane, but no gap junctions were formed among heterologous granulosa cells. These results suggest that an inhibitory factor secreted by pig granulosa cells is not species specific and it can act in vitro without the mediation of gap junctions. © 1993 Wiley-Liss, Inc.     

Mouse oocyte development in vitro with various culture systems

These experiments were designed to determine whether or not hormones are required for the growth of mouse oocytes and to assess the possible role of companion granulosa cells in oocyte growth. To approach these problems, four systems for the culture of oocytes, either alone or in association with granulosa cells, were utilized: (1) isolated oocyte culture, (2) isolated oocyte-ovarian cell coculture, (3) isolated follicle culture, and (4) ovarian organ culture. Oocytes from 8-day-old B6D2F₁ mice failed to grow in isolated oocyte culture. Addition of follicle-stimulating hormone (FSH), 17β-estradiol (E₂), or serum to the medium failed to prevent oocyte degeneration or to promote oocyte growth. On the other hand, oocytes in isolated follicle culture or in organ culture grew significantly in defined medium. The results showed that oocytes grown in isolated follicle culture under defined conditions and in the absence of gonadotropins resemble oocytes grown in vivo in terms of their ultrastructural characteristics, with the exception of enlarged mitochondria. In addition, these oocytes were shown to exhibit some normal functional characteristics in terms of their increased levels of CO₂ evolution from exogenous pyruvate, and the ability of the fully grown oocytes to initiate meiotic maturation when freed from granulosa cells. It was concluded that gonadotropins are not necessary for oocyte growth and that gonadotropins are not required to potentiate the spontaneous meiotic maturation of oocytes which occurs after their isolation from granulosa cells. The results indicated that association of granulosa cells and oocytes was necessary for oocyte growth. However, isolated oocytes in coculture with ovarian cells failed to grow. Addition of FSH or E₂ to the cocultures failed to promote oocyte growth or delay oocyte degeneration. It was concluded that, under the culture conditions used, granulosa cells must be in contact with the oocyte, perhaps by means of specialized cell junctions, for oocyte growth to occur.     

Heat stress induces distinct responses in porcine cumulus cells and oocytes associated with disrupted gap junction and trans-zonal projection colocalization

Cumulus cells (CCs), the granulosa cells surrounding the oocytes, play critical roles in oocytes maturation through intercellular communication by extending trans-zonal projections (TZPs) to contact oocytes via gap junctions (GJs). The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with GJ/TZP colocalization remain unclear. In this study, porcine cumulus-oocyte complexes (COCs) were subjected to HS at 41.5°C for 24 hr during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate, and early embryo developmental potentials. CCs and oocytes isolated from COCs demonstrated distinct responses to HS. The messenger RNA abundance of heat shock protein-related genes and mitochondrial DNA-encoded genes, together with ATP content, were significantly increased in CCs, yet decreased in oocytes, despite activation of caspase 3 detected in both CCs and oocytes. Similar changes were observed when denuded oocytes and isolated CCs subjected to HS separately, except mitochondria reactive oxygen species (mROS). In heat-stressed COCs, mROS was significantly increased only in oocytes. However, when isolated CCs and denuded oocytes were heat-stressed separately, mROS was significantly increased only in CCs. Moreover, F-actin, a TZP marker, and its colocalization with a GJ protein connexin-45, were significantly reduced in heat-exposed COCs. These results indicate that HS induces distinct responses in porcine CCs and oocytes in association with disrupted GJ and TZP colocalization.