Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes

In the oocytes of many animals, the germinal vesicle (GV) relocates from the center to the periphery of the oocyte upon meiosis reinitiation, which is a prerequisite to the formation of meiotic spindles beneath the cell surface in order for meiosis to succeed. In the present study, we have investigated nuclear positioning using sea-cucumber oocytes. Upon meiosis reinitiation, the GV relocates to the cell periphery beneath a surface protuberance. After GV breakdown, polar bodies were extruded from the top of the protuberance, which we therefore called the animal pole process. The GV relocation was inhibited by nocodazole but not by cytochalasin. Immunofluorescent staining and electron microscopy of microtubular arrays revealed that: (i) in immature oocytes, two centrosomes were situated beneath the animal pole process far apart from the GV, anchoring to the cortex via astral microtubules; (ii) upon meiosis reinitiation, microtubular bundles were newly formed between the centrosomes and the GV; and (iii) the microtubular bundles became short as GV migration proceeded. These observations suggest that microtubules and centrosomes participate in GV relocation. A very large mass of annulate lamellae, having a 20-microm diameter, was found in the vegetal pole of the oocytes.     

Control of microtubule nucleating activity in the cytoplasm of maturing mouse oocytes.

Taxol, a drug which promotes microtubule assembly, was used to assess the microtubule nucleating activity of pericentriolar material (PCM) in mouse oocytes prevented from undergoing germinal vesicle breakdown (GVBD), compared with oocytes allowed to proceed normally through GVBD and also in nucleate and anucleate oocyte fragments. Both immunofluorescence staining and ultrastructural analysis reveal that taxol induces aster formation in the cortex of oocytes undergoing GVBD, while formation of a continuous sheet of microtubule bundles parallel to the membrane is induced in metabolically GV-arrested oocytes. Since taxol also induces the formation of asters in anucleate as well as in nucleate oocyte fragments, provided they are not treated with activators of protein kinases A or C, it is concluded that microtubule nucleating activity is related to the acquisition of Maturation Promoting Factor (MPF) and does not require mixing between the nucleoplasm and cytoplasm.     

Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation.     

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro-tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley-Liss, Inc.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Displacement of the mitotic apparatus which induces ectopic polar body formation or parthenogenetic cleavage in starfish oocytes.

When the mitotic apparatus (MA) at meiosis I and II in starfish oocytes was detached from the animal pole and translocated to the other cortex, MA induced polar body formation, which indicates reattachment of MA to the cortex. MA attachment was so strong that MA at meiosis II was frequently broken into two parts during detachment and from the remnant part remaining at the cortex an aster derived and a nucleus derived from the detached part. When they were apart until the cleavage stage, the oocyte divided into the aster-containing and nucleus-containing blastomeres and, further, only the former blastomere divided repeatedly. This result indicates that the centrosome in the peripheral aster, which presumes to be discarded into the second polar body, always has the capacity of duplication but the centrosome in the inner aster, which stays in the oocyte interior, has not the capacity and confirms our previous report ( Saiki and Hamaguchi (1998) Dev. Biol. 203, 62-74). Furthermore, it is found by observing meiotic MA formation that this peculiar centrosome delivery at meiosis II is ensured by the fact that the attachment of the aster staying in the oocyte interior to the cortex occurs earlier than centrosome duplication.     

Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation

Cortical granules (CGs) undergo a substantial change in distribution in the mouse oocyte cortex during meiotic maturation. In order to determine the mechanism of their change in distribution near the time of ovulation, CG density, total number per oocyte, and domain areas were quantitated. CGs were visualized microscopically by Lens culinaris agglutinin-biotin and Texas red-strepavidin fluorescence as well as by electron microscopy. Immature germinal vesicle stage (GV) oocytes from adult mice had a continuous cortical localization with some interior granules. Mature oocytes had an asymmetric cortical distribution with a CG-free domain, overlying the meiosis II metaphase spindle, occupying 40% of the cortex. The mean CG densities of the granule-occupied cortex of mature oocytes and the entire cortex of GV oocytes were 43 and 34 CGs/100 micron 2, respectively. The mean total numbers of CGs/oocyte were 4127 (mature) and 7440 (GV), and staining was absent in fertilized oocytes with two pronuclei. Calcium ionophore (A23187)-activated mature oocytes had a mean total number of 1235 CGs, some of which may have been in the process of exocytosis. The first polar body had few CGs, and thus was unlikely to account for the difference in CG number between GV and mature oocytes. The smaller total number and higher density of CGs in mature mouse oocytes suggests that both exocytosis and redistribution are plausible mechanisms for the development of the CG-free domain. Prefertilization exocytosis could account for the locus of sperm penetration which others have reported to occur in the hemisphere opposite the meiotic spindle in the mouse.     

银鲫卵母细胞体外诱导成熟技术的建立

研究以银鲫为材料, 根据银鲫(Carassius auratus gibelio)卵母细胞生发泡(Germinal vesicle, GV)边移程度及剥离GV中减数分裂前期染色体的凝集状态, 将银鲫Ⅳ时相的卵母细胞分为GV0、GV1、GV2和GV3四个时期; 并进一步比较了分别处于这4个时期银鲫卵母细胞体外诱导培养的成熟率、卵裂率和孵化率。结果表明, GV1期之后的卵母细胞均可有效进行体外诱导成熟, 可正常受精发育, 由于GV1期卵母细胞有较长时间用于显微操作, 因此GV1期卵母细胞被选为进行体外诱导的最早时期的卵母细胞。以GV1期卵母细胞为研究材料, 摸索了银鲫卵母细胞体外诱导成熟的适宜条件: 取GV1期的Ⅳ时相卵母细胞, 放置于pH 8.5、加有1 μg/mL孕酮激素(17α, 20β-dihydroxy-4-pregnen-3-one, DHP)的格氏平衡盐溶液(Gey’s balanced salt solution, GBSS)中, 在23℃培养箱中体外诱导12h后, 将滤泡膜剥离后再进行人工体外授精, 其所获胚胎的孵化率可达55.5%。此外, 将体外转录合成的带GFP标签的h2af1o mRNA注射到GV1期卵母细胞, 发现经显微操作和体外诱导后不仅可以通过GFP绿色荧光信号活体观察GVBD、受精、卵裂和早期胚胎发育的全过程, 而且诱导成熟的卵子仍可正常受精和胚胎发育。研究建立的银鲫卵母细胞体外诱导成熟技术为银鲫和其他鱼类卵母细胞发育过程研究及其相关基因和细胞显微操作提供了技术平台。     

The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.     

Snoods: a periodic network containing cytokeratin in the cortex of starfish oocytes.

An extensive fibrous cytoskeletal component in the cortical cytoplasm of oocytes of the starfish Pisaster ochraceus reproducibly stains with anticytokeratin antibody and hence contains cytokeratin. The large-meshed network resembles a snood (hair net). Snood fibers form loops and branches throughout the cortex of a premeiotic oocyte, except at the animal pole where they emanate from a nonstaining zone surrounding the centrosomes. By immunofluorescence microscopy of isolated cortices and electron microscopy of isolated cortices and intact oocytes, snood fibers exhibit complex striations with a periodicity of approximately 0.75 micron. Snoods are not colocalized with the cortical arrays of microtubules and are unaffected by drugs that disrupt microtubules or microfilaments. Stimulation of oocyte maturation by 1-methyladenine causes snoods to disappear, presumably by disassembly, about halfway to the time of germinal vesicle breakdown. They do not reappear during meiosis, fertilization, or development to the two-cell stage, and their functional importance, if any, during oogenesis or development remains to be elucidated.     

Cytoskeletal organization in the oocyte,zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura)

Ovulation occurs in Sminthopsis macroura approximately 160 hr after administration of 1.3 IU PMSG, and yields significantly more oocytes than does spontaneous ovulation (P = 0.001). Germinal vesicle (GV)-stage oocytes have a thin cortical rim of microfilaments, which is disrupted by exposure to cytochalasin D. After GV breakdown, the first meiotic spindle forms subcortically and parallel to the oolemma. It rotates during anaphase and telophase to extrude the first polar body. This rotation is associated with a local cortical concentration of microfilaments, which is extruded in the first polar body. The second meiotic spindle is orthogonal to the surface, and extrusion of the second polar body is not associated with obvious local changes in cortical actin, resulting in a polar body containing little polymerized actin. The sites of second polar body emission and sperm entry are always in the half of the oocyte opposite the concentrating yolk mass, and are within 60° of each other in most oocytes. During the concentration and eccentric movement of the yolk, microfilaments condense around it. During yolk expulsion, these microfilaments become continuous with those located subcortically. During early cleavage, the cytocortex of the zygote, but not of the extruded yolk mass, stains heavily for polymerised actin. Multiple sites of pericentriolar material are detectable in the cytoplasm of some secondary unfertilized oocytes which, in the presence of taxol, generate large cytasters and pseudospindle structures. After fertilization, a large aster is formed in association with the sperm entry point and serves as the center of an extensive cytoplasmic network of microtubules which surrounds but does not enter the yolk mass. Taxol treatment generates small cytasters within this meshwork and promotes selective stabilization of some periyolk microtubules opposite to the sperm aster. © 1995 Wiley-Liss, Inc.     

Lipid Content and Composition during the Oocyte Development of Two Gorgonian Coral Species in Relation to Low Temperature Preservation

Our previous studies have suggested that chilling sensitivity of coral oocytes may relate to their relatively high lipid intracellular content and lipid composition. The distribution of lipids during the oocyte development was determined here for the first time in two gorgonian species (Junceella juncea and Junceella fragilis). The main lipid classes in the two gorgonian oocytes were total lipid, wax ester, triacylglycerol, total fatty acid, phosphatidylethanolamine and phosphatidylcholine. The results indicated that early stage oocytes of J. juncea and J. fragilis were found to have increased lipid content than late stage oocytes. The content of wax ester was significantly higher in the early stage oocytes of two gorgonian corals (51.0±2.5 and 41.7±2.9 μg/mm(3)/oocyte) than those of late stage oocytes (24.0±1.4 and 30.4±1.2 μg/mm(3)/oocyte, respectively). A substantial amount of phosphatidylethanolamine and total fatty acid was detected at each stage of oocyte development in two gorgonian ranges from 107 to 42 μg/mm(3)/oocyte and 106 to 48 μg/mm(3)/oocyte, whilst low levels of phosphatidylcholine were found in two gorgonian oocytes. The levels of total lipid in the late stage oocytes of J. juncea were significantly higher than those of J. fragilis. The observed differences may partially be related to different habitat preferences as higher lipid levels in J. juncea, a deeper-water coral species exposed to lower temperature seawater, might relate to adjustments of cell membranes in order to increase membrane fluidity.     

Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.     

Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification

The purpose of this study was to determine the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the open pulled Straw method (OPS). We evaluated the effects of pre-treating the oocytes with 1 microM Taxol on chromosome organization, spindle morphology, cortical granule distribution and the ability of fertilized oocytes to develop to the blastocyst stage. After calf or cow oocyte vitrification without Taxol, significantly higher proportions of spindle abnormalities in the form of abnormal spindle structures or dispersed or decondensed chromosomes were observed compared to fresh control oocytes. In contrast, when we compared calf oocytes pre-treated with Taxol before vitrification with control calf oocytes, similar percentages of oocytes showing a normal spindle morphology were observed. The percentages of oocytes with a peripheral cortical granule (CG) distribution increased when the oocytes were pretreated with Taxol and vitrified, while oocytes vitrified without Taxol pre-treatment gave rise to higher cortical distribution percentages. Cleavage and blastocyst rates were significantly lower for vitrified versus untreated oocytes, both in cow and calf oocytes. Significantly higher cleavage rates were obtained when calf and cow oocytes were vitrified with Taxol. Pre-treatment with Taxol before cow oocyte vitrification yielded significantly higher blastocyst rates. Calf oocytes, however, were unable to develop to the blastocyst stage, irrespective of previous Taxol treatment. These results indicate that the pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by the cryopreservation process, and potentially improves the subsequent development of vitrified bovine oocytes. Summary sentence: Pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and potentially improves the development of vitrified bovine oocytes.     

Sea urchin oocytes possess elaborate cortical arrays of microfilaments, microtubules, and intermediate filaments

Extensive arrays of microfilaments, microtubules and cytokeratin-type intermediate filaments were detected in the cortex of Strongylocentrotus droebachiensis oocytes using fluorescently labeled antibodies on both cortex and whole mount preparations. All three filament systems undergo dramatic structural reorganization during meiotic maturation of the egg. Microfilaments form a dense meshwork within the cortex of the oocyte. After meiosis, the filaments rearrange and shorten, resulting in a more loosely organized network. Both cortical microtubules and microtubules associated with a microtubule-organizing center are observed within the oocyte. After meiosis, the number and length of the cortical microtubules gradually diminish. A microtubule organizing center is found situated between the germinal vesicle and the plasma membrane in many oocytes. A network of filaments extends from the microtubule organizing center and radiates peripherally toward the germinal vesicle, presumably marking the animal pole. Cytokeratin-like intermediate filaments form a reticular network within the oocyte cortex, then solubilize during meiosis. In whole mounts of oocytes there is a single focal center of cytokeratin staining from which filaments radiate. Indirect immunofluorescence experiments, using anti-tubulin and anti-cytokeratin antibodies simultaneously, reveal the intermediate filament focal center to be localized within the microtubule organizing center. These results demonstrate the presence of a complex cortical cytoskeleton in premeiotic eggs of the sea urchin, Strongylocentrotus droebachiensis.     

In vivo changes in oocyte germinal vesicle related to follicular quality and size at mid-follicular phase during stimulated cycles in the cynomolgus monkey

Histological examination of gonadotrophin stimulated Macaca fascicularis ovaries removed at mid-follicular phase showed that germinal vesicles (GV) could exhibit different configurations in follicles greater than 1000 microns in diameter. We describe 3 types of nuclear organization called GV1 (dispersed and filamentous chromatin), GV2 (clumped and filamentous chromatin) and GV3 (perinucleolar chromatin condensation). Gonadotrophin stimulation and follicular atresia induced modifications in GV chromatin dispersion. Such modifications were of a higher degree in the case of atresia which could even induce in vivo germinal vesicle breakdown (GVBD). Our findings were as follows. The frequency of GV1 oocytes was always low, but was higher in healthy than in atretic follicles, whereas GV3 oocytes were more frequent in atretic compared to healthy follicles; the oocytes which resumed meiosis in vitro were most probably those which were at the GV3 stage at the time of recovery; GV nuclear changes were related to follicle size and quality, but not to oocyte size. The mean follicular size increased from GV1 to GV3 oocyte stages whatever the follicle quality; the nucleus was often observed in a peripheral position even in GV1 oocytes; zona pellucida appearance was related to GV stage and follicle quality and was more often observed to be abnormal or absent in case of GV3 oocytes included in atretic follicles. Oocyte nuclear modifications therefore appear to be a prerequisite to resumption of meiosis.     

Germinal vesicle migration and dissolution in Rana pipiens oocytes: effect of steroids and microtubule poisons

Germinal vesicle migration (GVM) as evidenced by the appearance of the germinal vesicle at the animal pole surface was induced by nocadazole and demecolcine (colcemid). Nocodazole significantly lowered the progesterone ED50 for germinal vesicle dissolution (GVD). Both demecolcine and nocodazole enhanced centrifugation-induced GVM (i.e., lowered ooplasmic viscoelasticity) after 6-h incubation, and both potentiated the effect of progesterone in this assay. Estradiol, by contrast, inhibited GVM induced by demecolcine in both follicle-enclosed and denuded oocytes. Estradiol was also found to inhibit the normal enhancement of centrifugation-induced GVM by demecolcine or progesterone. Taxol was found to have effects that were generally opposite to those of demecolcine and nocodazole. Taxol inhibited centrifugation-induced GVM either alone or in the presence of progesterone. In addition, taxol significantly increased the progesterone ED50 for GVD induction. Taken together the available data support the hypothesis that microtubules play a role in maintaining the internal position of the germinal vesicle in the prematuration oocyte and that changes occur in the oocyte cytoskeleton during maturation.     

The Mechanical Structure of the Cytoplasm of the Echinoderm Egg Determined by "Gold Particle Method" using a Centrifuge Microscope

A method was developed to investigate the mechanical structure of the cytoplasm based on the movement of an intracellular gold particle subjected to centrifugal acceleration (the gold particle method). The movement of the particle in the cell was observed and recorded with a new centrifuge microscope of stroboscopic type (13). In eggs and oocytes of the echinoderms, Clypeaster japonicus, Asterias amurensis , and Asterina pectinifera , the particle moved in the cytoplasm by an applied centrifugal acceleration in the centrifugal direction, but the course was not exactly straight and the velocity fluctuated during the movement, suggesting the existence of a network structure in the cytoplasm. In fertilized eggs, the movement of the particle by the centrifugal acceleration was impeded by the structures of the sperm aster and the cleavage diaster. The apparent viscosity of the cytoplasm in fertilized eggs changed in parallel to the development of the sperm aster and the mitotic diaster in the cell. These results indicate that the asters are really rigid structures in the cell as previously shown by the magnetic particle method (8).     

Effect of forskolin on the spontaneous maturation and cyclic AMP content of rat oocyte-cumulus complexes

Forskolin (0-100 microM), a reversible stimulator of the catalytic subunit of adenylate cyclase, induced dose-response increases in the % germinal vesicle (GV) (ID50 = 2.68 microM, where ID50 is the dose of forskolin which maintained the meiotic arrest at the germinal vesicle stage, determined cytogenetically, of 50% cultured oocytes), and the cAMP content (determined by RIA) of cumulus-enclosed oocytes and cumulus masses. A significant positive correlation was established between the amount of cAMP within the cumulus mass and that in the corresponding enclosed oocyte (r = 0.78). In contrast, neither the % GV nor the cAMP content of cumulus-free oocytes was affected by the drug. The arresting action of forskolin upon cumulus-enclosed oocytes was dependent upon the presence of adherent cumulus cells, and was transient and fully reversible. The gradual decrease in the % GV of cumulus-enclosed oocytes cultured from 4 to 12 h in 25 microM-forskolin (84, 54 and 22% GV at 4, 8 and 12 h, respectively) was accompanied by a drastic fall in intra-oocyte cAMP at 8 h (41.2 +/- 2.5 and 1.1 +/- 0.6 fmol/oocyte at 4 and 8 h, respectively), while the cumulus cell cAMP content remained constant (135.3 +/- 14.7 and 145.9 +/- 28.7 fmol/cumulus at 4 and 8 h, respectively). Moreover, heterologous metabolic coupling, assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte, significantly decreased. These results show that cumulus cell cAMP is transferred to the rat oocyte where it appears to play a pivotal role in the regulation of meiosis and that the rat oolemma does not appear to possess active catalytic subunits of adenylate cyclase in an amount adequate to stimulate measurably cAMP synthesis.