Tetraspanins and Mouse Oocyte Microvilli Related to Fertilizing Ability.

Our electron microscopy observations demonstrate for the first time that the number of microvilli on the mice oocyte membrane decreases when meiosis progresses from prophase I to metaphase II (MII) stage, and the morphology of the microvilli also changes. Microvilli are significantly shorter and larger on the ovulated oocyte membrane than at the previous stages. Although clathrin vesicles clearly disappear during oocyte maturation, exosome-like vesicles begin to be secreted at the metaphase I stage, more strongly at the MII stage. Multivesicular bodies are visible only at the MII stage. Since several oocyte tetraspanins are involved in the gamete interaction, Cd9 being congregated on the MII oocyte microvilli, we analyzed the effect of tetraspanin deletion on oocyte membrane morphology. The Cd9-/- and Cd9-/- Cd81-/- deletions are associated with a decreased microvilli density on the MII oocyte surface. Microvilli thickness is significantly increased whatever the deleted tetraspanin gene be. Only Cd9 deletion clearly disturbs the vesicular traffic, increasing the number of clathrin and exosome vesicles. Additional investigations are necessary to elucidate how tetraspanins modulate the microvilli morphology, likely in relation with cytoskeleton. The role of oocyte exosomes in gamete adhesion/fusion remains to be further studied.

Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-ß-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes.

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Review: Lamin A/C, caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.

After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.

Sperm-egg interaction: is there a link between tetraspanin(s) and GPI-anchored protein(s)?

Both female mice deficient in CD9 tetraspanin- and oocyte-specific glycosyl-phosphatidylinositol-anchored family proteins showed severely reduced fertility due to the failure of sperm-egg fusion. This raises the question of a link between these two groups of proteins at the oocyte membrane. We propose two hypotheses to explain why the absence of one of these proteins from the oocyte membrane results in the same phenotype. The first hypothesis envisages different levels of control by these molecules of the common induced signaling cascade. The second relies on the known involvement of these molecules in the overall organization of the plasma membrane. Their disappearance could thus prevent sperm-egg fusion either by disruption of the signaling cascade and/or by an important disorganization of the oocyte membrane. In this review, describing their structural and functional characteristics, and using published results on the oocyte, we try to analyze how these two protein families could interact.

Whole-body or isolated ovary (60)Co irradiation: effects on in vivo and in vitro folliculogenesis and oocyte maturation.

For the first time, the effects of low doses of gamma-radiation were studied on folliculogenesis and on isolated oocytes. After irradiation of adult mice, even at the lowest dose, a drastic loss of primordial follicles was observed in serial sections of ovaries, with, in opposite, no effect on the other follicle stages. Moreover, oocytes freshly recovered from the largest antral follicles of irradiated adult ovaries exhibited significantly less regular Ca(2+) oscillations than controls. Finally, in vitro folliculogenesis demonstrated a smaller diameter of preantral follicles recovered from irradiated juvenile ovaries compared to control, and an increase in follicle atresia. Further on, PLC-beta1 localization was not affected in the enclosed oocytes whereas chromatin configuration revealed that a quarter of them had prematurely resumed meiosis or was fragmented. These results raise the question of the risk of genetic and teratogenic effects on women submitted to chronic exposure even of very low radiation.

Caspase-2(L), caspase-9, and caspase-3 during in vitro maturation and fragmentation of the mouse oocyte.

Several studies have shown that apoptotic pathways control fragmentation of unfertilized ovulated oocyte, induced by doxorubicin. But very few have investigated the basis of this process, from prophase I to later stages. Our results revealed the presence of caspase-2(L), caspase-9, and caspase-3 in their zymogen and cleaved forms in the oocyte before meiosis resumption. Caspase-2(L) and caspase-9 were detected in the nucleus of GV-oocytes in a distribution related to chromatin configuration. The inhibition of caspase activity by Z-VAD-fmk accelerated the transition from metaphase I to metaphase II, and caspase-9 and caspase-3 were detected along the meiotic spindle. Surprisingly, Western blot analysis revealed that the three cleaved caspases were present in similar amounts in healthy and fragmented oocytes and caspase inhibition did not prevent doxorubicin-induced apoptosis. Our results suggest that, if cleaved, caspases may be dispensable for final oocyte death and they could be involved in regulating the maturation process.

The role of PLC beta 1 in the control of oocyte meiosis during folliculogenesis.

The dynamics of the subcellular distribution of PLCbeta1 was investigated during meiosis competence acquisition and meiosis resumption in relation to oocyte diameter and to nonsurrounded-nucleolus or surrounded-nucleolus chromatin configurations. Oocytes collected after both in vivo and in vitro folliculogenesis were studied. In both conditions, at the beginning of the process, most oocytes exhibited a nuclear PLCbeta 1 associated with a nonsurrounded-nucleolus chromatin configuration. Then at the final stage of the process, the factors shifted mainly toward a cytoplasmic PLCbeta1 and a surrounded-nucleolus chromatin configuration, typical of a preovulatory fertilizable oocyte. Additionally, only germinal vesicle oocytes with a diameter > 75 microm, and exhibiting cytoplasmic PLC beta1 distribution and surrounded-nucleolus chromatin configuration, resumed meiosis. Our findings demonstrate a strong correlation between oocyte diameter, chromatin configuration, and PLCbeta1 localization. Thus, PLCbeta1 localization appears to be a key factor determining the progressive acquisition of meiotic competence and final resumption of meiosis.

The nucleolus of the maternal gamete is essential for life.

The mammalian oocyte is a round cell arrested at prophase I of meiosis. It is characterized by the presence of a large nucleus, called the germinal vesicle, in the middle of which is the nucleolus. Before it can be fertilized, the oocyte must resume meiosis, enter metaphase II and be ovulated. The nucleolus is dissolved during this process. However, the nucleoli of the male and female pronuclei in the zygote are both of maternal origin. A recent paper1 demonstrates that the maternal nucleolus, together with other nucleoplasmic elements, is essential for early embryonic development. These nucleolar and nucleoplasmic factors remain undetermined.

Natural uranium disturbs mouse folliculogenesis in vivo and oocyte meiosis in vitro.

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.

The phosphoinositide-phospholipase C (PI-PLC) pathway in the mouse oocyte.

As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.

The PKC pathway and in particular its beta1 isoform is clearly involved in meiotic arrest maintenance but poorly in FSH-induced meiosis resumption of the mouse cumulus cell enclosed oocyte.

PKC modulators were used to investigate the role of the PKC pathway either on the maintenance of meiotic arrest or on FSH-induced maturation of mouse cumulus cell enclosed oocytes (CEOs). (1) Whereas PKC activation (PMA 8 microM) overcomed clearly the HX-maintained meiotic arrest (83.7 +/- 3.6% vs. 16.1 +/- 10.6% GVBD oocytes), PKC inhibition (Calphostin C 100 nM) did not. On the contrary, it better maintained the meiotic arrest than HX alone. (2) No significant effect of PKC activation or inhibition was observed. (3) HX alone maintained PKCbeta1 in the cytoplasm, whereas FSH and PKC activation induced partly its translocation into the nucleus. The results show that whereas the PKC pathway is clearly involved in maintenance of the meiotic arrest through PKCbeta1, it is not involved in FSH-induced meiosis of CEOs.

Regulation of spontaneous meiosis resumption in mouse oocytes by various conventional PKC isozymes depends on cellular compartmentalization.

In this study, we investigated the spatio-temporal distribution of conventional protein kinases C (cPKC) isoforms PKC-alpha, PKC-betaI, PKC-betaII and PKC-gamma in mouse oocytes. The cPKCs were present in the cytoplasm at the start of the process and migrated to the nucleus (or germinal vesicle) before germinal vesicle breakdown, except for PKC-gamma which remained cytoplasmic. In both compartments, the fully phosphorylated form corresponding to the 'mature' enzyme was revealed for PKC-alpha, PKC-betaI and PKC-betaII. Microinjection of specific antibodies against each isozyme in one or the other cell compartment at different times of the meiotic process, permitted us to observe the following: (1) When located in the cytoplasm at the beginning of the process, PKC-alpha is not implicated in germinal vesicle breakdown, PKC-betaI and PKC-gamma are involved in maintaining the meiotic arrest, and PKC-betaII plays a role in meiosis reinitiation. Furthermore, just before germinal vesicle breakdown, these cytoplasmic cPKCs were no longer implicated. (2) When located in the germinal vesicle, PKC-alpha, PKC-betaI and PKC-betaII are involved in meiosis reinitiation. Our data highlight not only the importance of the nuclear pathways in the cell cycle progression, but also their independence of the cytoplasmic ones. Further investigations are however necessary to discover the molecular targets of these cPKCs to better understand the links with the cell cycle progression.

Meiosis resumption, calcium-sensitive period, and PLC-beta1 relocation into the nucleus in the mouse oocyte.

We aimed to determine whether the breakdown of the germinal vesicle of the mouse oocyte and the nuclear import of phospholipase C-beta1 were calcium-dependent. We chelated Ca2+ ions with BAPTA-dextran at different times after the release of the oocyte from the ovarian follicle, i.e. after meiosis resumption has started, and we studied the effects on the kinetics of germinal vesicle breakdown, and on the migration of phospholipase C-beta1. We discriminate between two key-periods of calcium-sensitivity during the process of meiosis resumption. During the first hour, changes in the cytosolic Ca2+ especially promoted the migration of phospholipase C-beta1 into the nucleus, whereas changes in the nuclear concentration of Ca2+ were not implicated. Moreover, at this time, the cytosolic calcium pathway is PLC-beta1-dependent. By contrast, during the second hour following the onset of meiosis resumption, and thus just previous GVBD, the PLC-beta1-dependent Ca2+ signals in both cellular compartments were equally necessary for the resumption of meiosis. This particular period of the meiotic process corresponds to the moment when the phospholipase C-beta1 has strongly migrated into the nucleus. Our results highlight also the role played by the nucleus during the second key-period in the control of the GVBD via a Ca2+-dependent pathway.

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation.

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.