Dalia Galiani

Reactive oxygen species are indispensable in ovulation

Ovulation is stimulated by the preovulatory surge of the pituitary luteinizing hormone (LH). Because the ovulatory response is commonly identified with inflammation, we explored the involvement of reactive oxygen species (ROS) in this process. Our experiments show that administration of broad-range scavengers of oxidative species into the ovarian bursa of mice, hormonally induced to ovulate, significantly reduced the rate of ovulation. LH-induced cumulus mucification/expansion, a necessary requirement for ovulation, was prevented by antioxidants both in vivo and in an ex vivo system of isolated intact ovarian follicles. Along this line, H2O2 fully mimicked the effect of LH, bringing about an extensive mucification/expansion of the follicle-enclosed cumulus–oocyte complexes. Impaired progesterone production was observed in isolated follicles incubated with LH in the presence of the antioxidant agents. Furthermore, LH-stimulated up-regulation of genes, the expression of which is crucial for ovulation, was substantially attenuated upon ROS ablation. This system was also used for demonstrating the role of ROS in phosphorylation and activation of the EGF receptor as well as its downstream effector, p42/44 MAPK. Together, our results provide evidence that ovarian production of ROS is an essential preovulatory signaling event, most probably transiently triggered by LH.

The Proteasome Is Involved in the First Metaphase-to-Anaphase Transition of Meiosis in Rat Oocytes

Abstract The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.

Dissociation between the inhibitory and the stimulatory action of cAMP on maturation of rat oocytes.

The possible mediatory role of cAMP in the induction of oocyte maturation by luteinizing hormone (LH) is not yet clear since evidence for both inhibitory and stimulatory actions of the nucleotide on the oocyte has been provided. To elucidate the role of cAMP in regulation of oocyte meiosis we tried in the present study to dissociate between the inhibitory and stimulatory action of this nucleotide on oocyte maturation. To induce maturation, oocytes enclosed by their follicles were transiently exposed to either dibutyryl cAMP (dbcAMP) or to the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Inhibition of maturation was obtained by the addition of the above agents to either follicle-enclosed oocytes incubated in the presence of LH or isolated cumulus-free oocytes that mature spontaneously in vitro. We found that inhibition of oocyte maturation is obtained by a relatively low dose of either dbcAMP or MIX while higher concentrations of these agents are required to induce oocyte maturation. Coupling of the oocyte to the cumulus cells, as expressed by the fraction of labeled uridine transferred from the cumulus cells to the oocyte following exposure of the follicle-enclosed cumulus-oocyte complex to MIX, was also determined. We found that uncoupling of the oocyte from the cumulus cells corresponded with the induction, but not inhibition of oocyte maturation, both by its concentration dependence and time-course. We suggest that cAMP has a dual role in regulation of oocyte maturation. Lower levels of the nucleotide act to maintain meiotic arrest, while elevated levels of cAMP mediate LH action to induce meiosis resumption.

Inhibition of Rat Oocyte Maturation and Ovulation by Nitric Oxide: Mechanism of Action

Established gap junctional communication (GJC) in the ovarian follicle is essential for maintaining the oocytes in meiotic arrest. Alternatively, LH-induced reinitiation of meiosis is subsequent to breakdown of GJC. It was recently reported that nitric oxide (NO) inhibits maturation in rat follicle-enclosed oocytes and elevates GJC in cultured mesangial cells. Taking these observations into account, we hypothesized that NO prevents reinitiation of meiosis by antagonizing the effect of LH on GJC in the ovarian follicle. Indeed, we found that NO interferes with LH-induced disruption of GJC as well as with the decrease of the expression of the gap junction protein GJA1 (previously known as CONNEXIN43). We also demonstrated that NO prevents activation of LH-induced mitogen-activated protein kinases (MAPKs) 1 and 2 and inhibits cumulus expansion. Along this line, incubation of ovarian follicles with an inhibitor of soluble guanylate cyclase, which is a downstream NO effector, induced on its own oocyte maturation as well as cumulus expansion. Unlike previous studies, we show here that elevation of NO resulted in inhibition of ovulation. We conclude that the mechanism by which NO inhibits LH-induced oocyte maturation possibly involves a negative effect on MAPK activation and, in turn, interference with interruption of GJC. This action of NO in the ovarian follicle is apparently mediated by cGMP. In addition, the negative effect of NO on ovulation may be subsequent to its inhibitory effect on cumulus expansion. Together, this study suggests that the preovulatory decrease in NO concentrations is a prerequisite for the ovarian response to LH.

Inactivation of M-Phase Promoting Factor at Exit from First Embryonic Mitosis in the Rat Is Independent of Cyclin B1 Degradation

Abstract Exit from M-phase and completion of cell division requires inactivation of M-phase promoting factor (MPF), a heterodimer composed of the regulatory cyclin B1 and the catalytic p34cdc2 kinase. Inactivation of MPF is associated with cyclin B1 degradation that is brought about by the ubiquitin-proteasome pathway. Our study examined the role of the proteasome in the first mitosis of rat embryos and its participation in the regulation of cyclin B1 degradation and MPF inactivation. We show that in the early zygote the proteasome is evenly distributed in the ooplasm and the nucleus, whereas during mitosis it accumulates on the spindle apparatus. We further demonstrate that inhibition of proteasomal catalytic activity prevents 1-cell embryos from undergoing mitosis. This mitotic arrest is associated with the presence of relatively high amounts of cyclin B1, which unexpectedly does not result in elevated MPF activity. Our findings strongly imply that completion of the first embryonic division depends on proteasomal degradation and that cyclin B1 is included among its target proteins. They also provide the first evidence that MPF inactivation at this stage of development is not solely dependent upon cyclin B1 degradation and is insufficient to allow the formation of the 2-cell embryo.

Maturation-Promoting Factor Governs Mitogen-Activated Protein Kinase Activation and Interphase Suppression During Meiosis of Rat Oocytes

Abstract Meiosis is a particular example of a cell cycle, characterized by two successive divisions without an intervening interphase. Resumption of meiosis in oocytes is associated with activation of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). The activity of MPF declines during the transition between the two meiotic divisions, whereas the activity of MAPK is sustained. Attempts to disclose the interplay between these key regulators of meiosis in both amphibian and mammalian oocytes generated contradictory results. Furthermore, the enzyme that governs the suppression of interphase in mammals is still unidentified. To our knowledge, we provide herein the first demonstration in a mammalian system that inhibition of MPF at reinitiation of meiosis abrogated Mos expression and MAPK activation. We also show that oocytes, in which reactivation of MPF at completion of the first telophase was prevented, exhibited an interphase nucleus with decondensed chromosomes. Inhibition of MAPK did not interfere with the progression to the second meiotic metaphase but, rather, resulted in parthenogenic activation. We conclude that in rat oocytes, MPF regulates MAPK activation and its timely reactivation prevents the oocytes from entering interphase.

Expression and modification of PKA and AKAPs during meiosis in rat oocytes.

Meiosis in oocytes is initiated during fetal life, arrested around birth and resumed after puberty. Meiotic arrest is controlled by a cAMP-dependent protein kinase (PKA)-mediated cAMP action. We examined oocytes for the presence and modulation of the regulatory (R) subunits of PKA and the A-kinase anchoring proteins (AKAPs) that target PKA to specific subcellular locations. We found that rat oocytes express the two regulatory subunit isoforms, RI and RII of PKA. Immunocytochemistry revealed that the regulatory subunits underwent cellular translocation upon resumption of meiosis. We also demonstrated the presence of a novel 140 kDa AKAP, AKAP140 that exhibited a retarded electrophoretic motility at reinitiation of meiosis. The mobility shift of AKAP140 was susceptible to alkaline phosphatase and prevented by inhibition of p34cdc2 kinase. We conclude that rat oocytes express AKAP140 that is phosphorylated during meiosis. AKAP140 phosphorylation is sensitive to p34cdc2 kinase inhibitors. We hypothesize that AKAP140 and its phosphorylation state may influence the translocation of the R subunits of PKA throughout resumption of meiosis.

Rat oocytes induced to mature by epidermal growth factor are successfully fertilized

Epidermal growth factor (EGF), which is a known mitogen, can also induce resumption of meiosis in the rat oocyte. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by EGF in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. For further clarification of the effect of EGF on steroidogenesis in the ovarian follicle, progesterone concentrations were determined by radioimmunoassay. We found that oocytes matured by EGF (100 ng/ml) were successfully fertilized. Even though their rate of fertilization was relatively lower as compared to that of oocytes stimulated by luteinizing hormone (LH) both in vitro and in vivo (61%, 79%, and 83% respectively), once fertilized they exhibit an equal potential for further development (EGF: 48%, LH: 45%). On the other hand, EGF-induced progesterone production was very poor. These findings strongly support the idea that both mitogenesis and meiogenesis can be mediated by common signals. The results further suggest that progesterone production and oocyte maturation, in the rat, are independent events.

Epithelial cell transforming protein 2 (ECT2) depletion blocks polar body extrusion and generates mouse oocytes containing two metaphase II spindles.

Completion of the first meiosis in oocytes is achieved by the extrusion of the first polar body (PBI), a particular example of cell division. In mitosis, the small GTPase RhoA, which is activated by epithelial cell transforming protein 2 (ECT2), orchestrates contractile ring constriction, thus enabling cytokinesis. However, the involvement of this pathway in mammalian oocytes has not been established. To characterize the role of ECT2 in PBI emission in mouse oocytes, the small interfering RNA approach was employed. We found that ECT2 depletion significantly reduces PBI emission, induces first metaphase arrest, and generates oocytes containing two properly formed spindles of the second metaphase. Moreover, we describe, for the first time, that before PBI emission, RhoA forms a ring that is preceded by a dome-like accumulation at the oocyte cortex, next to the spindle. This unique mode of RhoA translocation failed to occur in the absence of ECT2. We further found that the Rho-dependent kinase, a main RhoA effector, is essential for PBI emission. In addition, we demonstrate herein that ECT2 is subjected to phosphorylation/dephosphorylation throughout meiosis in oocytes and further reveal that PBI emission is temporally associated with ECT2 dephosphorylation. Our data provide the first demonstration that an active cyclin-dependent kinase 1, the catalytic subunit of the maturation-promoting factor, phosphorylates ECT2 during the first meiotic metaphase and that cyclin-dependent kinase 1 inactivation at anaphase allows ECT2 dephosphorylation. In conclusion, our study demonstrates the indispensable role of the maturation-promoting factor/ECT2/RhoA pathway in PBI extrusion in mouse oocytes.

Excess cholesterol induces mouse egg activation and may cause female infertility

Significance Production of functional sperm and eggs requires a complex process called meiosis. Meiosis in mouse and human eggs pauses at a stage called metaphase II (MII) arrest until fertilization by sperm. After fertilization, eggs released from MII arrest complete meiosis and develop into new individuals. In analyzing the female infertility of genetically altered mice, we discovered that excess cholesterol can trick mouse eggs into behaving as though they were fertilized (released from arrest), thus disrupting the normal synchrony between fertilization and completion of meiosis and rendering them dysfunctional. These findings suggest that abnormal cholesterol metabolism may contribute to some forms of human female infertility. The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.