Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans

BACKGROUND: In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. RESULTS: Here, we present evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, we show that Galpha(s) signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. CONCLUSIONS: Regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells.     

Eph and NMDA receptors control Ca2+/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in Caenorhabditis elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. Here, we show that MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. Our results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex.     

Meiotic arrest in human oocytes is maintained by a Gs signaling pathway

In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.     

Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans

In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. Although many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. Here we analyze microtubule behavior in Caenorhabditis elegans oocytes at early stages of the meiotic maturation process using confocal microscopy and live-cell imaging. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. We show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. We present evidence that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic G alpha(o/i) and G alpha(s) pathways and gap-junctional communication with somatic cells of the gonad. We propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors

VAP proteins (human VAPB/ALS8, Drosophila VAP33, and C. elegans VPR-1) are homologous proteins with an amino-terminal major sperm protein (MSP) domain and a transmembrane domain. The MSP domain is named for its similarity to the C. elegans MSP protein, a sperm-derived hormone that binds to the Eph receptor and induces oocyte maturation. A point mutation (P56S) in the MSP domain of human VAPB is associated with Amyotrophic lateral sclerosis (ALS), but the mechanisms underlying the pathogenesis are poorly understood. Here we show that the MSP domains of VAP proteins are cleaved and secreted ligands for Eph receptors. The P58S mutation in VAP33 leads to a failure to secrete the MSP domain as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response. We propose that VAP MSP domains are secreted and act as diffusible hormones for Eph receptors. This work provides insight into mechanisms that may impact the pathogenesis of ALS.     

On the Control of Oocyte Meiotic Maturation and Ovulation inCaenorhabditis elegans

Prior to fertilization, oocytes undergo meiotic maturation (cell cycle progression) and ovulation (expulsion from the ovary). To begin the study of these processes inCaenorhabditis elegans,we have defined a time line of germline and somatic events by video microscopy. As the oocyte matures, its nuclear envelope breaks down and its cell cortex rearranges. Immediately thereafter, the oocyte is ovulated by increasing contraction of the myoepithelial gonadal sheath and relaxation of the distal spermatheca. By systematically altering the germ cell contents of the hermaphrodite using mutant strains, we have uncovered evidence of four cell–cell interactions that regulate maturation and ovulation. (1) Both spermatids and spermatozoa induce oocyte maturation. In animals with a feminized germline, maturation is inhibited and oocytes arrest in diakinesis. The introduction of sperm by mating restores maturation. (2) Sperm also directly promote sheath contraction. In animals with a feminized or tumorous germline, contractions are infrequent, whereas in animals with a masculinized germline or with sperm introduced by mating, contractions are frequent. (3 and 4) The maturing oocyte both induces spermathecal dilation and modulates sheath contractions at ovulation; dilation of the distal spermatheca and sharp increases in sheath contraction rates are only observed in the presence of a maturing oocyte.     

MSP hormonal control of the oocyte MAP kinase cascade and reactive oxygen species signaling

The MSP domain is a conserved immunoglobulin-like structure that is important for C. elegans reproduction and human motor neuron survival. C. elegans MSPs are the most abundant proteins in sperm, where they function as intracellular cytoskeletal proteins and secreted hormones. Secreted MSPs bind to multiple receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. MSP binding stimulates oocyte MPK-1 ERK MAP Kinase (MAPK) phosphorylation, but the function and mechanism are not well understood. Here we show that the Shp class protein-tyrosine phosphatase PTP-2 acts in oocytes downstream of sheath/oocyte gap junctions to promote MSP-induced MPK-1 phosphorylation. PTP-2 functions in the oocyte cytoplasm, not at the cell surface to inhibit multiple RasGAPs, resulting in sustained Ras activation. We also provide evidence that MSP promotes production of reactive oxygen species (ROS), which act as second messengers to augment MPK-1 phosphorylation. The Cu/Zn superoxide dismutase SOD-1, an enzyme that catalyzes ROS breakdown in the cytoplasm, inhibits MPK-1 phosphorylation downstream of or in parallel to ptp-2. Our results support the model that MSP triggers PTP-2/Ras activation and ROS production to stimulate MPK-1 activity essential for oocyte maturation. We propose that secreted MSP domains and Cu/Zn superoxide dismutases function antagonistically to control ROS and MAPK signaling.     

Fertilization initiates the transition from anaphase I to metaphase II during female meiosis in C. elegans

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway.     

SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gα_s–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gα_s–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gα_s–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy.     

G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes

In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.     

The divergent C. elegans ephrin EFN-4 functions inembryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor

The C. elegans genome encodes a single Eph receptor tyrosine kinase, VAB-1, which functions in neurons to control epidermal morphogenesis. Four members of the ephrin family of ligands for Eph receptors have been identified in C. elegans. Three ephrins (EFN-1/VAB-2, EFN-2 and EFN-3) have been previously shown to function in VAB-1 signaling. We show that mutations in the gene mab-26 affect the fourth C. elegans ephrin, EFN-4. We show that efn-4 also functions in embryonic morphogenesis, and that it is expressed in the developing nervous system. Interestingly, efn-4 mutations display synergistic interactions with mutations in the VAB-1 receptor and in the EFN-1 ephrin, indicating that EFN-4 may function independently of the VAB-1 Eph receptor in morphogenesis. Mutations in the LAR-like receptor tyrosine phosphatase PTP-3 and in the Semaphorin-2A homolog MAB-20 disrupt embryonic neural morphogenesis. efn-4 mutations synergize with ptp-3 mutations, but not with mab-20 mutations, suggesting that EFN-4 and Semaphorin signaling could function in a common pathway or in opposing pathways in C. elegans embryogenesis.     

Somatic gonad sheath cells and Eph receptor signaling promote germ-cell death in C. elegans

Programmed cell death eliminates unwanted cells during normal development and physiological homeostasis. While cell interactions can influence apoptosis as they do other types of cell fate, outside of the adaptive immune system little is known about the intercellular cues that actively promote cell death in healthy cells. We used the Caenorhabditis elegans germline as a model to investigate the extrinsic regulators of physiological apoptosis. Using genetic and cell biological methods, we show that somatic gonad sheath cells, which also act as phagocytes of dying germ cells, promote death in the C. elegans germline through VAB-1/Eph receptor signaling. We report that the germline apoptosis function of VAB-1 impacts specific cell death pathways, and may act in parallel to extracellular signal-regulated kinase MAPK signaling. This work defines a non-autonomous, pro-apoptotic signaling for efficient physiological cell death, and highlights the dynamic nature of intercellular communication between dying cells and the phagocytes that remove them.     

Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans

The Eph receptor VAB-1 is required in neurons for epidermal morphogenesis during C. elegans embryogenesis. Two models were proposed for the non-autonomous role of VAB-1: neuronal VAB-1 might signal directly to epidermis, or VAB-1 signaling between neurons might be required for epidermal development. We show that the ephrin VAB-2 (also known as EFN-1) is a ligand for VAB-1 and can function in neurons to regulate epidermal morphogenesis. In the absence of VAB-1 signaling, ephrin-expressing neurons are disorganized. vab-2/efn-1 mutations synergize with vab-1 kinase alleles, suggesting that VAB-2/EFN-1 may partly function in a kinase-independent VAB-1 pathway. Our data indicate that ephrin signaling between neurons is required nonautonomously for epidermal morphogenesis in C. elegans.     

Meiotic cell cycle arrest in mammalian oocytes

Meiotic cell cycle in mammalian oocytes is a dynamic process that involves several stop/go channels. The cell cycle arrest in oocyte occurs at various stages such as diplotene, metaphase‐I (M‐I), metaphase‐II (M‐II), and so called metaphase‐like arrest (M‐III). Leutinizing hormone surge induces meiotic resumption from diplotene arrest in follicular microenvironment by overriding several factors responsible for the maintenance of meiotic arrest. The inhibitory factors are synthesized in oocyte or in the associated follicular somatic cells and transferred to the oocyte. The major factors include hypoxanthine, cyclic adenosine 3′, 5′‐monophosphate, cyclic guanosine 3′, 5′‐monophosphate, reactive oxygen species, protein kinase A, and protein kinase C. In the presence of active protein kinases, epidermal‐like growth factors are produced that activate mitogen‐activated protein kinase in cumulus granulosa cells. The maturation promoting factor, cytostatic factors, and spindle assembly checkpoint proteins are also involved in that maintenance of arrest at various stages of meiotic cell cycle in mammalian oocytes. In this review, we briefly summarize the role of these factors in the maintenance of meiotic cell cycle arrest in mammalian oocytes. J. Cell. Physiol. 223:592–600, 2010. © 2010 Wiley‐Liss, Inc.     

The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.     

Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans.

Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation. We present here a genetic characterization of oocyte maturation, using C. elegans as a model system. We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation. Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I. Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes. The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.