TSG-6 is concentrated in the extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and inter-alpha-inhibitor binding.

During development of ovarian follicles in mammals, cumulus cells and the oocyte form a mucoelastic mass that detaches itself from peripheral granulosa cell layers upon an ovulatory surge. The integrity of this cumulus-oocyte complex (COC) relies on the cohesiveness of a hyaluronan (HA)-enriched extracellular matrix (ECM). We previously identified a serum glycoprotein, inter-alpha-inhibitor (IalphaI), that is critical in organizing and stabilizing this matrix. Following an ovulatory stimulus, IalphaI diffuses into the follicular fluid and becomes integrated in the ECM through its association with HA. TSG-6 (the secreted product of the tumor necrosis factor-stimulated gene 6), another HA binding protein, forms a complex with IalphaI in synovial fluid. The purpose of this study was to investigate whether TSG-6 is involved in the ECM organization of COCs. Immunolocalization of TSG-6 and IalphaI in mouse COCs at different ovulatory stages was analyzed by immunofluorescence and laser confocal microscopy. IalphaI, TSG-6, and HA colocolized in the cumulus ECM. Western blot analyses were consistent with the presence of both TSG-6 and TSG-6/IalphaI complexes in ovulated COCs. These results suggest that TSG-6 has a structural role in COC matrix formation possibly mediating cross-linking of separate HA molecules through its binding to IalphaI.

Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

Inter-alpha-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes.

This report characterizes the effects of excess hyaluronan (HA) upon the expansion of the cumulus oocyte complex (COC) within intact follicles and upon ovulation and oocyte viability in mice. Covalent linkage between heavy chains of the inter-alpha-inhibitor (IalphaI) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of IalphaI to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of IalphaI heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of IalphaI heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct.

The ovarian blood follicle barrier is both charge- and size-selective in mice.

This report characterizes the permeability and selectivity properties of the ovarian blood-follicle barrier. Proteins of similar size but opposite net charge possess strikingly different permeabilities with respect to this barrier. Inter-alpha-inhibitor (I alpha I, 220 kDa, pI approximately 6.2) is excluded from the follicle until an ovulatory stimulus, whereas immunoglobulin G (IgG, 155 kDa, pI approximately 6.5-7.0) passes into the follicle without an ovulatory stimulus. However, cationization of I alpha I results in its influx into the follicle in the absence of an ovulatory signal. Conversely, anionization of IgG results in its exclusion from the follicle unless an ovulatory stimulus (hCG administration) is provided. Molecular size also plays a role in blood-follicle barrier selectivity. For example, cationization of alpha 2-macroglobulin (pI approximately 8.5; 700 kDa) fails to facilitate its entry into unstimulated follicles. Conversely, negatively charged BSA (pl approximately 4.5; 66 kDa) passes freely into unstimulated follicles. These studies support the hypothesis that the blood-follicle barrier is size-selective but that charge sign and density play a role in the permeability of this barrier to proteins within an intermediate size range.

Establishment and characterization of a conditionally immortalized smooth muscle/myometrial-like cell line.

A novel smooth muscle/myometrial-like cell line, SMU1-10, has been generated from the uterus of a H-2Kb-tsA58 transgenic mouse carrying a thermolabile SV40 large T-antigen gene. These cells grow continuously when maintained at the permissive temperature (33 degrees C) for the SV40 large T-antigen but stop dividing when placed at the non-permissive temperature (39 degrees C) and ultimately die within 3 weeks. All of the SMU1-10 cells produce smooth muscle alpha-actin (SMAA) at both 33 degrees C and 39 degrees C. A subset of the cells also contain smooth muscle gamma-actin (SMGA), a hallmark of smooth muscle differentiation, and the fraction of cells staining for this actin increases from about 1% when maintained for three days at 33 degrees C to as much as 30% at 39 degrees C over the same length of time. However, the appearance of SMGA in SMU1-10 cells appears to be regulated mainly at a post-transcriptional level since in situ hybridization indicates that all cells contain SMGA mRNA at both 33 degrees C and 39 degrees C. SMU1-10 cultures also contain smooth muscle myosin heavy chain (SM-MHC) and SM22 alpha, both of which are only found in smooth muscle of the adult mouse. Three additional smooth muscle (myometrium)-related markers, connexin 43, the thromboxane A2 receptor, and the progesterone receptor also are present in these cells. At the nonpermissive temperature for SV40 large T-antigen, the both level of SMGA mRNA and the number of cells staining for this actin are significantly increased in the presence of progesterone, a process that is similar to the upregulation of SMGA in the myometrium late in pregnancy. Overall, SMU1-10 cells provides a potentially useful in vitro model system to study smooth muscle/myometrial differentiation.

Covalent linkage between proteins of the inter-alpha-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells.

The direct interaction of hyaluronic acid (HA) and proteins of the inter-alpha-inhibitor family plays a critical role in organization and stabilization of the expanding cumulus extracellular matrix (cECM) following an ovulatory stimulus. Despite similarities in the morphology of cumulus oocyte complexes (COCs) expanding in vivo and in vitro, we find that the cECM of COCs which expand within intact follicles are more elastic and resistant to shear stress than the cECM of those stabilized in vitro. Western blot analysis shows that only the heavy chains of inter-alpha-inhibitor are incorporated into the cECM and appears to be covalently linked to HA after stabilization in vivo while intact inter-alpha-inhibitor is bound to the HA-enriched cECM by a non-covalent mechanism in in vitro stabilized COCs. However, purified pre-alpha-inhibitor and HA can form covalent linkage in the presence of granulosa cells or with granulosa cell-conditioned medium. In addition, COCs resistance to shear stress is also enhanced by coincubation with granulosa cells. Upon formation of the apparent covalent linkage between heavy chains and HA in culture medium, the light chain (bikunin) is concomitantly released into the medium as a complex with chondroitin sulfate moieties of inter-alpha-inhibitor supporting the possibility that HA may replace the chondroitin sulfate linkage to the heavy chains. We speculate that a factor(s) secreted by granulosa cells within the follicle may catalyze a transesterification reaction resulting in an exchange of chondroitin sulfate with HA at the heavy chain/chondroitin sulfate junction followed by release of chondroitin sulfate-bikunin into the follicular fluid. It is also possible that the consequent further stabilization of the cECM through the covalent interaction of HA and heavy chains of inter-alpha-inhibitor may play an important role in the process of ovulation.

Diabetes-mediated decreases in ovarian superoxide dismutase activity are related to blood-follicle barrier and ovulation defects.

The activity of nitric oxide (NO) has been implicated as an important mediator in ovarian function, including the regulation of the blood-follicle barrier and ovulation. Both clinical and experimental diabetes have been found to impair endothelial-dependent vascular activity through the inactivation of NO. It has also been shown that diabetes-induced inactivation of NO can be rescued with administration of insulin as well as with free radical scavengers such as superoxide dismutase (SOD). In this study, we report decreases in both ovulation and the activity of the blood-follicle barrier in insulin-dependent diabetic prepubertal mice treated with exogenous gonadotropic hormones. Moreover, these ovarian defects are rescued with administration of insulin, nitroprusside, L-arginine, and the free radical scavengers citiolone and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). We found that the activity of Cu/Zn SOD in the ovaries of diabetic mice used in this study is decreased by 48% compared to that in nondiabetic mice. In contrast, inhibition of SOD activity in nondiabetic mice induces defects in the blood-follicle barrier and ovulation similar to those observed in diabetic mice. Lastly, we report the localization of endothelial nitric oxide synthase, inducible NOS, Cu/Zn SOD, and the LH receptor to the same population of endothelial cells surrounding the preovulatory follicle, supporting our hypothesis that the signaling of ovarian NO within the ovarian microvasculature at the time of ovulation may be compromised in these diabetic mice as a consequence of the loss of the protective activity of Cu/Zn SOD.

Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide.

Although initially described over 30 years ago, the blood-follicle barrier has remained a biological enigma. In this study, we characterize the blood-follicle barrier with respect to its regulation of intrafollicular inter-alpha-trypsin inhibitor protein (I alpha I) influx after an ovulatory stimulus. We have found that I alpha I is localized within the ovarian vasculature but is excluded from the follicular compartment until an ovulatory stimulus is given. Within minutes after an ovulatory dose of human chorionic gonadotropin, I alpha I is localized within the follicular fluid of responding follicles where this protein becomes associated with and stabilizes the newly synthesized hyaluronic acid-rich cumulus extracellular matrix. Analysis of this process has shown that intravenous injection of sodium nitroprusside or excess substrate for nitric oxide synthase, L-arginine, mimics the effect of gonadotropic hormones on the influx of I alpha I into the follicular compartment of preovulatory follicles. Moreover, intravenous injection of specific nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine, inhibits gonadotropin-mediated intrafollicular influx of I alpha I and also inhibits ovulation in the mouse.

Steroid hormone regulation of rat myometrial gap junction formation: effects on cx43 levels and trafficking.

The formation of myometrial gap junctions coincides with onset of labor in many mammalian species, including humans. The assembly of gap junction protein into functional gap junction plaques is a final step in a cascade that begins with estrogen-dependent expression of the connexin43 (cx43) gene and continues with synthesis of cx43 in the rough endoplasmic reticulum (RER) and transport to the Golgi, followed by its trafficking to the plasma membrane and its assembly into functional gap junctions. Moreover, in several models of preterm labor in rats, precocious synthesis, trafficking, and assembly of cx43 follow an increase in the estrogen:progesterone ratio. The actions of these steroids on cx43 expression, gap junction formation, and labor led us to consider whether or not the cascade of cx43 expression and gap junction assembly typical of preterm and term labor would be disrupted by manipulations that inhibit labor through experimental reduction of the estrogen:progesterone ratio. Ovariectomized and non-ovariectomized pregnant rats were treated with minimal doses of progesterone or the anti-estrogenic compound ICI 182780 over a time course sufficient to inhibit labor. We found that cx43-positive gap junction formation was prevented in all animals treated with ICI 182780 or progesterone but that the mechanism by which this disruption occurred was different in anti-estrogen- and progesterone-treated animals. We found that ICI 182780 significantly inhibited the typical rise in myometrial cx43 concentrations normally observed just before labor. In contrast, it was surprising to find that significant cx43 was synthesized in myometrium of progesterone-treated intact and ovariectomized animals even though labor was inhibited. However, we found that the trafficking of myometrial cx43 from the Golgi and assembly into gap junctions at the plasma membrane were suppressed in these progesterone-treated animals, providing further support for the hypothesis that it is not synthesis of cx43 per se but trafficking of cx43 to the plasma membrane and its assembly into gap junctions that are required for effective synchronized myometrial contractions typical of labor.

Proteins of the inter-alpha-trypsin inhibitor family stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid.

We have previously identified a glycoprotein of the inter-alpha-trypsin inhibitor family of proteins as a serum factor responsible for the stabilization of the expanding cumulus mass. In this study, the mechanism of interaction of this cumulus extracellular matrix stabilizing factor (cESF) with hyaluronic acid (HA) has been explored. It was found that the pH optimum for binding of cESF and HA is 7 and that binding is sensitive to ionic strength. The dissociation constant is about 1.9 x 10(-8) M in 10 mM sodium phosphate buffer (pH 7.2). Circular dichroism studies show that cESF contains about 24% alpha-helical and 42% beta-sheet structure. Gross conformational changes in cESF, however, are not detected in the presence of HA. We also found that modification of lysine residues of cESF with citraconic anhydride greatly reduced its binding with HA and completely abolished its cumulus stabilizing activity, and deblocking lysine residues restored its capacity to bind with HA and its cumulus matrix stabilizing activity. This evidence supports the hypotheses that cESF stabilizes the expanding cumulus extracellular matrix by directly binding with HA and that cESF may serve as a structural protein to organize the formation of the cumulus extracellular matrix. Our evidence also supports the view that binding of cESF and HA is through a stereo-specific charge interaction. Putative binding sites of cESF that interact with HA are postulated.

Sequential effects of follicle-stimulating hormone and luteinizing hormone on mouse cumulus expansion in vitro.

LH and FSH play critical roles in the development and function of the cumulus oophorus of the mammalian follicle, but the significance of cumulus LH receptors and the nature of the functional interaction between LH and FSH in relation to the process of cumulus expansion are not well understood. Controversy may arise from contradictory results of cumulus LH receptor assays, possibly as a consequence of relative sensitivities of the assays utilized, limited availability of tissue of specific developmental stages, or limitations on the access of radiolabeled ligands to cumulus LH receptors in the intact follicle. It has also been difficult to understand the basis for the apparent difference in gonadotropin requirements for cumulus expansion in vitro and in vivo; FSH seems to be required for expansion in vitro and LH initiates expansion in vivo. In this study, we show that expansion of mouse cumulus-oocyte complexes (COCs) in vitro occurs to a degree comparable to that seen in vivo when COCs are challenged with highly purified FSH (hpFSH) and highly purified LH (hpLH) together or when treatment with hpFSH precedes treatment with LH. Saturation radioreceptor assays revealed that FSH up-regulates cumulus cell LH receptors in the absence of LH and that LH may stimulate expansion upon binding to these acutely formed receptors. A model of cumulus expansion describing effects of both FSH and LH in vivo is proposed.

Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass.

Gonadotropin-stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2-cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation.

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.

Identification of a factor in fetal bovine serum that stabilizes the cumulus extracellular matrix. A role for a member of the inter-alpha-trypsin inhibitor family.

Fetal bovine serum (FBS) has been widely used in the culture of cumulus-oocyte complexes, since it is believed that FBS stabilizes the expanding cumulus extracellular matrix. In this study, we have identified a factor in FBS that is responsible for this effect. FBS was first fractionated by stepwise precipitation with ammonium sulfate followed by high performance liquid chromatography (HPLC) on a gel filtration column. Active fractions, as determined by their ability to stabilize the cumulus extracellular matrix in a bioassay system, were further purified by HPLC on DEAE ion-exchange and gel filtration columns. The purified factor was exhibited as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr 150,000 and a single peak on a C-8 reverse-phase HPLC. The sequence of the first 14 NH2-terminal amino acids was determined by Edman degradation, revealing significant sequence identity of the bovine serum factor with the light chain shared by members of the inter-alpha-trypsin inhibitor family. Western blot analysis using anti-human I alpha I IgG shows that the antibody reacts positively with the purified factor, and immunodepletion of FBS with anti-I alpha I antibody agarose eliminated its ability to stabilize the extracellular matrix. This factor is found in mouse follicular fluid collected 6 h following human chorionic gonadotropin injection to stimulate ovulation, but not in unstimulated mice. Anti-I alpha I-positive epitopes were also localized within the cumulus extracellular matrix of mouse preovulatory follicles in immunocytochemical preparations using anti-human I alpha I IgG, supporting the possibility that this molecule or molecules may diffuse into follicular fluid after an ovulatory stimulus to act as structural linkers that ensure normal cumulus expansion, through stabilization of the cumulus extracellular matrix thus supporting the process of ovulation.

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.

Olfactory bulb granule cell aggregates: morphological evidence for interperikaryal electrotonic coupling via gap junctions.

Anaxonic interneurons of the granule cell type in the mammalian main olfactory bulb (MOB) are characterized by prominent membrane specializations, which include reciprocal, interdendritic chemical and electrical synapses; however, the latter are thought to be restricted to the external plexiform layer (EPL) and connect granule-mitral- and granule-tufted-cell dendrites (Landis et al., 1974). The present study focuses on interperikaryal membrane specializations between tangentially oriented aggregates of granule microneurons in the lamina granularis interna (IGL). Both infraprimate (Rattus norvegicus, Gerbillus perpallidus) and primate species (Callithrix jacchus) were studied using the following methods: (1) transmission electron microscopy (TEM), (2) freeze-fracture analysis, (3) light and TEM immunohistochemistry using affinity-purified antibodies directed against the connexin-32 or connexin-43 carboxy tail fragment of the gap-junction protein (GJP), and (4) intracellular Lucifer yellow injections in fixed tissue (LYF technique). Freeze-fracture replicas of the MOB-IGL showed that adjacent granule cell perikarya have numerous particle aggregates on the cytoplasmic membrane; in terms of their structure and arrangement, such particles are characteristic of gap junctions. The existence of junctional membranes was substantiated by application of antibodies against GJP demonstrating punctate immunoreactivity, frequently confined to the interperikaryal plasmalemmae of granule cells in the IGL and their dendritic processes in the EPL. Upon TEM analysis, GJP-like immunoreactivity was additionally found in membranous organelles, including Golgi apparati and associated vesicular components. In order to test the permeability of identified membrane specializations, the LYF technique was used, which resulted in bright fluorescence of the perikaryal and dendritic components of the transsomatically injected neuron and staining of neighboring neurons with similar morphology. These findings imply that small molecules can diffuse across the interperikaryal membrane specializations. The existence of gap junctions between granule cell perikarya suggests that there is a significant, low-resistance electrical transmission between aggregated granule cells. This coupling might permit synchronization of neural discharge among small aggregates of these neurons. Gap junctions between granule cells may also serve signaling functions associated with the protracted period of granule cell development.

Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass.

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex.

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.