Cumulus cells surrounding oocytes with high developmental competence exhibit down-regulation of phosphoinositol 1,3 kinase/protein kinase B (PI3K/AKT) signalling genes involved in proliferation and survival.

STUDY QUESTION: Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome? SUMMARY ANSWER: Eleven genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome. WHAT IS KNOWN ALREADY: The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs. STUDY DESIGN SIZE, DURATION: The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERß, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples. MAIN RESULTS AND THE ROLE OF CHANCE: Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway. LIMITATIONS REASONS FOR CAUTION: Small sample size and retrospective design. WIDER IMPLICATIONS OF THE FINDINGS: The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals. STUDY FUNDING/COMPETING INTEREST(S): Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest.

Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms.

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H2O2 and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

Impact of vitrification on the mitochondrial activity and redox homeostasis of human oocyte.

STUDY QUESTION: Do the extreme conditions of vitrification affect mitochondrial health and reactive oxygen species (ROS) levels of human oocytes? SUMMARY ANSWER: Vitrification of discarded human oocytes shifts the intracellular redox potential towards oxidation but does not alter the mitochondrial potential or intracellular ROS levels. WHAT IS KNOWN ALREADY: Recent studies have reflected increased ROS levels in warmed young oocytes and have highlighted the temporal dynamic loss of mitochondrial potential that could, therefore, lead to a decrease in ATP production, impairing embryo development. Mitochondrial function can also be evaluated in vivo by the FAD/NAD(P)H autofluorescence ratio, which reflects the respiratory chain activity and is considered as a marker of the intracellular redox state. STUDY DESIGN, SIZE, DURATION: A total of 629 discarded Metaphase II (MII) oocytes collected from June 2013 to April 2014 were included in this control (fresh oocytes, n= 270) versus treatment (vitrified oocytes, n= 359) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Discarded MII oocytes were donated to research by young (<27 years old) and reproductively aged (>36 years old) women who underwent ovarian stimulation for IVF at a university-affiliated private fertility clinic. Redox state was assessed by measuring the FAD/NAD(P)H autofluorescence ratio, while ROS and mitochondrial activity were reported by in vivo labelling with carboxy-H2DCFDA and JC-1, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Young and aged oocytes showed high and similar survival rates (81.8 versus 83.1%, not significant). Confocal microscopy revealed that the FAD/NAD(P)H ratio was significantly higher in vitrified oocytes than in fresh oocytes, suggesting a significant shift towards the oxidized state in oocytes after vitrification, regardless of the maternal age. Mitochondrial distribution was not affected by vitrification. Furthermore, it was not possible to resolve any difference in mitochondrial potential using JC-1 potentiometric dye or in reactive oxygen species (ROS) production (assessed with H2-DCFDA staining) between fresh and vitrified oocytes. Therefore, measurement of intracellular redox potential by autofluorescence imaging may be a more sensitive method to assess oxidative stress or mitochondrial demise in human oocytes because it showed a higher resolving power than JC-1 staining and displayed less variability than H2-DCFDA staining. LIMITATIONS, REASONS FOR CAUTION: Owing to sample availability, MII discarded oocytes (in vitro matured oocytes and unfertilized oocytes 20 h after ICSI) were included in the study. These discarded oocytes do not necessarily reflect the physiological condition of the MII human oocyte. WIDER IMPLICATIONS OF THE FINDINGS: Although vitrified oocytes yield comparable clinical outcomes compared with fresh oocytes, lower cleavage and blastocyst rates can be observed during in vitro culture. Data here obtained suggest that the redox state of human oocytes could be affected by vitrification. Therefore, the importance of adding protective antioxidant molecules to the vitrification solution and to the post-warming culture medium to improve embryo cleavage deserves some research. STUDY FUNDING/COMPETING INTERESTS: This research project was supported by the Valencian Government (Val+i+D program, M.N.-C.), INCLIVA Foundation for health research (G.S.-A.) and by the University of L'Aquila and Regione Abruzzo ('Reti per l'Alta Formazione' - P.O.F.S.E. Abruzzo 2007-2013 G.D.E.). No conflicts of interest were declared.

Regular and Moderate Exercise Counteracts the Decline of Antioxidant Protection but Not Methylglyoxal-Dependent Glycative Burden in the Ovary of Reproductively Aging Mice.

Population aging results in urgent needs of interventions aimed at ensuring healthy senescence. Exercise often results in healthy aging, yet many molecular mechanisms underlying such effects still need to be identified. We here investigated whether the age-dependent accumulation of oxidative and methylglyoxal- (MG-) related molecular damage could be delayed by moderate exercise in the mouse ovary, an organ that first exhibits impaired function with advancing age in mammals. CD1 female mice underwent two- or four-month treadmill-based running through the transition from adult to middle age, when ovaries show signs of senescence, and markers of protection against reactive oxygen species (ROS) and MG were measured. The long-term exercise reduced the protein oxidative damage in the ovaries (P < 0.01), and this was linked to the preservation of the glutathione peroxidase protection against ROS (P < 0.001), as well as to the increased glutathione availability (P < 0.001). Conversely, even though the age-related deactivation of the MG-targeting systems was partially prevented by the long-term running programme (P < 0.001), exercised mice were not protected from the age-dependent glycative burden. In summary, lately initiated regular and moderate exercise limited some changes occurring in the ovaries of middle-aged mice, and this might help to develop nonpharmacological cointerventions to reduce the vulnerability of mammalian ovaries towards redox dysfunctions.

Possible involvement of advanced glycation end products in periodontal diseases.

Periodontal diseases are considered as multifactorial conditions initiated by infection with pathogenic bacteria, promoted by inflammation and immune response against bacteria and modified by different environmental and genetic factors. Recently, interest in periodontal diseases has been increasing due to the awareness that the hyperinflammatory status associated with this disorder could impose a significant increase of reactive oxygen species (ROS) relevant to numerous systemic diseases driven by a pro-oxidant profile. A highly complex interplay occurs between oxidative stress and AGEs (Advanced Glycation End products), a group of heterogeneous compounds that form constantly under physiologic conditions, although their rate of formation is markedly increased in hyperglycemia and oxidizing conditions. Starting from the most relevant hypotheses on the pathogenesis of periodontal diseases, the present review outlines its relationship with oxidative stress and inflammation response in order to make a critical evaluation of the potential role of AGEs in periodontal deterioration. Although direct evidence for the presence of AGEs in the periodontal ligament is still lacking, valuable approaches based on the use of periodontal cells along with genetic and biochemical studies in animal models and chronic periodontal patients support a potential role for protein glycation in the aetiology and severity of this disease. Following a review of the current literature, the present study highlights the need for further investigation on the presence of AGEs in the periodontal ligament as a means for the comprehension of the pathogenic mechanisms underlying periodontal diseases in order to develop prevention and treatment modalities for this dysfunction.

Female reproductive dysfunction during ageing: role of methylglyoxal in the formation of advanced glycation endproducts in ovaries of reproductively-aged mice.

Reproductive dysfunction with ageing has been so far extensively characterized in terms of depletion of ovarian follicles and reduced ability to produce gametes competent for fertilization. Nevertheless, molecular mechanisms underlying this process are still poorly understood. In the present study we addressed the hypothesis that methylglyoxal (MG), a major precursor of Advanced Glycation Endproducts (AGE), may contribute to molecular damage occurring during ovarian ageing. Our results showed that the biochemical activity of glyoxalase 1, the main component of the MG scavenging system, is significantly decreased in ovaries from reproductively-aged mice in comparison with the young group. This effect was associated with decreased expression at protein and RNA level of this enzyme and increased intraovarian level of MG. MG-arginine adducts argpyrimidine as detected with a specific antibody was found to accumulate with ageing in specific ovarian compartments. Separation of ovarian proteins by 2D gels and Western blotting revealed an approximate 30-fold increase in the extent of protein glycation in aged ovaries along with the appearance of eight argpyrimidine modified proteins exclusive for the aged group. In conclusion, the present results show that impaired MG detoxification causing relevant damage to the ovarian proteome might be one of the mechanisms underlying reproductive ageing and/or ageing-like ovarian diseases.

Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice.

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.

Serum CA125 level modifications in women undergoing repeated IVF cycles.

In the present paper, serum CA125 modifications in patients undergoing their first IVF cycle were compared with those of patients in their second attempt. A significant increase of this marker was detected in each group of patients at day 14 after embryo transfer. However, the level of CA125 monitored in the patients in their second attempt was significantly higher than that determined in patients undergoing their first ovarian stimulation. This condition does not influence either ovarian response or oocyte and embryo quality. Moreover similar IVF outcome was obtained. Therefore we propose that patients undergoing repeated assisted reproductive technology (ART) cycles may suffer from ovarian surface epithelial damage and/or altered cellular growth rate.

Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells.

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women > or =38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes.

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.

Gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk, Unio elongatulus, binds to and induces acrosome reaction in human spermatozoa through a protein kinase C-dependent pathway.

In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewisa-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.

Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) signal in mouse oocyte activation.

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.

Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol.

Calmodulin-dependent protein kinase II (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mumol KN-93 l-1 produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mumol KN-93 l-1 was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation.

An evaluation of propofol toxicity on mouse oocytes and preimplantation embryos.

Mouse biological assays were used to investigate potential adverse effects of propofol on the oocyte's competence to fuse with spermatozoa and on the embryo's ability to develop to the blastocyst stage. Cumulus-enclosed metaphase II oocytes were exposed for 1 h to 0.01, 0.1, 0.4, 1 and 10 microg/ml propofol (Diprivan) and subjected to a sperm-oocyte fusion test based on the dye (Hoechst 33342) transfer technique. Oocytes exposed to 0.4, 1 and 10 microg/ml propofol showed a significant reduction in the rate of sperm fusion and underwent pronuclei formation at a rate similar to that of sperm fusion. In a second trial, mouse 1-cell and 2-cell embryos were exposed to varying propofol concentrations for 14h and then checked for subsequent development. Although adverse effects were not observed in 2-cell embryos, treatment of 1-cell embryos with propofol concentrations ranging from 0.01 to 10 microg/ml resulted in the inhibition of cleavage to blastocyst stage. We conclude that propofol can negatively influence fertilization in the mouse by impairing the oocyte's ability to fuse with spermatozoa, without interfering with the sperm-induced activation of the cell cycle. Moreover, we document the peculiar sensitivity to propofol of mouse 1-cell embryos as compared with 2-cell embryos.

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs.

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 microM or 50 microM OAG for 10 min resulted in meiosis II completion in approximately 80% of instances. By contrast, at a lower concentration (25 microM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 microM OAG, the cytosolic Ca2+ concentration ([Ca2+]i) increased transiently in all the eggs examined, whereas after the addition of 50 microM OAG, [Ca2+]i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca2+ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway.

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation.

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 microM external Ca2+ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 microM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur.

Protein kinase C-dependent and independent events in mouse egg activation.

The involvement of calcium- or protein kinase C (PKC)-dependent pathways in cortical granule exocytosis (CGE) and pronucleus formation was examined in mouse eggs using the specific PKC stimulator OAG (1-oleyl-2-acetyl-sn-glycerol) at different external calcium concentrations ([Ca2+]e) ranging from 1.7 mM to 0.1 microM. A 10 min exposure of eggs to 150 microM OAG in the presence of 1.7 mM [Ca2+]e caused a large calcium influx, cortical granule release and 82% activation. The increased permeability of the egg membrane to Ca2+ ions after OAG treatment lasted 20 min. At [Ca2+]e lower than 1.7 mM, both OAG-induced calcium influx and CGE decreased, reaching a non-detectable level at 0.1 microM and 100 microM [Ca2+]e, respectively. Resumption of meiosis was not affected by [Ca2+]e above 200 microM but it was reduced at any lower [Ca2+]e, with a minimum activation frequency of 46% at 0.1 microM [Ca2+]e. Loading of eggs with > or = 3 microM of the calcium chelator BAPTA AM (1,2-bis(o-aminophenoxy)ethane-N',N',N',N'-tetraacetic acid-acetoxymethyl ester) prior to OAG treatment caused a reduction in meiosis resumption with 50% of eggs forming pronuclei. Potent inhibitors of PKC, such as acridine orange and sphingosine, did not interfere with OAG-induced CGE. Conversely, these compounds prevented OAG-induced pronucleus formation in a dose-dependent manner with an IC50 (inhibiting concentration, 50%) of 5 microM and 30 microM for acridine orange and sphingosine, respectively. Microinjection of inositol 1,4,5-trisphosphate into eggs at 0.1 microM elicited Ca2+ release from intracellular stores and the cortical reaction, but failed to stimulate pronucleus formation. These results indicate that, in mouse eggs, CGE is a PKC-independent event, and that the transition from M-phase to interphase may require PKC activity for stimulation.

Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells.

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation.

Protein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogenetically by specific PKC stimulators such as 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of 1) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited parthenogenones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence.

Correlates of life satisfaction among military wives.

Military life includes constant change. This study explored the relationship of life satisfaction among military wives with the individual attitudinal and personality variables of perceived social support, locus of control, and temperament. Sixty wives of noncommissioned military personnel were selected as participants. Life satisfaction was found to be related to high levels of perceived social support from family and from friends, to an internal locus of control, and to low levels of emotionality-stress and emotionality-fear. The results supported the role of individual resources for mediating adjustment and enhancing life satisfaction during the changes inherent in military life. Implications for identifying and helping high-risk women emerged.