Seminar organized by Marie-Hélène Verlhac, from 22 to 27 juin 2009
John Carroll, Anna Castro (organisateur), Rey-Huei Chen, Manqi Deng, Alexei Evsikov, Gary Gorbsky, Evelyn Houliston, Patricia Hunt, Laurinda Jaffe, Catherine Jessus, Keith Jones, Takeo Kishimoto, Jacek Kubiak, Johne Liu, Thierry Lorca, James Maller, Alex McDougall, Hiro Ohkura, Terry Orr-Weaver, Marie-Hélène Verlhac (organisateur)
Sexual Reproduction is a fascinating area of Developmental Biology, which allows an organism to perpetuate itself in a non-clonal way via the formation of gametes. The key cellular process that distinguishes gamete formation from clonal division is Meiosis. An oocyte accomplishes all the essential functions of mitosis plus other specific ones related to gametogenesis such as: recombination of homologous chromosomes, asymmetric partitioning of its cytoplasm to preserve its maternal stores as well as preparing itself for fertilization.
During the meeting, the speakers covered many essential aspects of the regulation of meiotic divisions in oocytes, such as how oocytes resume meiosis, how they regulate meiotic progression, how evolution has used core-signalling pathways for sexual reproduction in different species, and how our environment can affect fertility. In this short synopsis, we can neither cover all the aspects raised by the participants nor report the intense and stimulating discussions that took place after every presentation.
Oocytes are arrested in the ovary in prophase I of meiosis. Depending on the species, the resumption of meiosis requires either an increase or a decrease in cAMP levels. In mouse oocytes, the LH surge induces a decrease in cAMP levels. Laurinda Jaffe explained how the level of cAMP is regulated in mouse oocytes. In these oocytes, there is a constant production of cAMP due to constitutive activation of an orphan GPR3 receptor. The level of cAMP in the oocyte is decreased through the activation of PDE3A, a phosphodiesterase responsible of cAMP hydrolysis that is inhibited by cGMP. Follicular cells produce cGMP, which diffuses into the oocyte by way of intracellular gap junctions.
Dr Jaffe’s working model is that the LH surge induces closure of the gap junctions between follicle cells and oocytes. This causes a decrease in cGMP levels, a release of PDE3A activity and, therefore, a drop in cAMP level in the oocyte, which triggers meiotic resumption. Although progesterone added in the external medium is a potent inducer of meiotic resumption in Xenopus oocytes, Catherine Jessus suggested that a combination of different steroids, such as testosterone and androsterone, could also activate different receptors both externally and internally in vivo. As in mouse oocytes, activation of these receptors leads to the regulation of PKA activity. Using a mutant of PKA that works as a Shokat kinase, James Malleridentified a new uncharacterized PKA substrate, BCSC-1 (Breast Cancer Suppressor Candidate one), which could potentially negatively regulate the G2/M transition in Xenopus oocytes. Jacek Kubiak proposed that some proteins, as EP45, may act as oocyte maturation enhancers without inducing GVBD by themselves.
The interactions between the various steroids and receptors leads to the synthesis of a small amount of Cyclin B, a major player for the auto-amplification loop triggering massive MPF activation and the G2/M transition. At prophase I arrest and at mitotic entry, MPF is maintained inactive by the inhibitory kinases, Myt1 and Wee1. Upon M-phase entry, Cdc25 dephosphorylates the inhibitory sites and promotes MPF activation. New evidence presented by Gary Gorbsky and Thierry Lorca indicates that the equilibrium between the inhibitory kinases Myt1 and Wee1 and the stimulatory phosphatase Cdc25 are active not only at M-phase entry but also during M phase and at mitotic exit. John Carroll and Keith Jones also addressed the issue of the control of prophase I arrest. They show that mouse oocytes accomplish prophase I-arrest by preventing Cyclin B accumulation through the APCcdh1-dependent degradation of this protein. In particular, John Carroll presented evidence demonstrating that Emi1 regulates entry into M-phase via the modulation of APCcdh1. Keith Jones addressed the issue of spatial control of Cyclin B degradation as being important for its regulated destruction.
Terry Orr-Weaver showed that in Drosophila oocytes, Cyclin A appears at maturation in correlation with the disappearance of Bruno protein, a translational repressor. The mRNA for Cyclin A is poly-adenylated, most certainly by the polyA-polymerase Gld2 at maturation, at the same time as it is translated. Cyclin A thus appears to be an essential player of meiosis resumption in Drosophila.
Three speakers showed evidence of the important role for a recently discovered kinase, Greatwall, in the control of the M-phase state. As shown by Thierry Lorca, Anna Castro and Michael Goldberg, Greatwall is essential in maintaining the phosphorylation status of MPF substrates. They showed that Greatwall interacts with PP2A and regulates its activity negatively, thereby promoting the maintenance of the M-phase state. A role of PP2A in chromosome segregation was also suggested by Alex McDougall. He presented data indicating that chromosome segregation in meiosis might be regulated by an inhibitor of PP2A interacting with Shugoshin on kinetochores.
Terry Orr-Weaver and John Carroll addressed the importance of the control of APC/C activity in the regulation of the meiosis I to anaphase I transition. Terry Orr-Weaver showed that Cortex is an essential meiotic specific regulator of APC/C from the Cdc20 family, which regulates Cyclin A levels and may also regulate other meiotic specific substrates. A genetic suppressor screen for meiotic specific substrates of Cortex is currently being performed in Terry’s lab.
Most meiotic oocyte spindles are assembled in the absence of true centrosomes. Hiro Ohkura explained how the phosphorylation of BAF by NHK1 is essential for karyosome formation, downstream of the DNA double-strand break/meiotic checkpoint repair pathway in drosophila. Errors in karyosome formation in mutant oocytes for NHK1 lead to split chromosome mass, themselves inducing split meiotic spindles. Marie-Hélène Verlhac also explained how chromatin influences meiotic spindle formation, via HURP in mouse oocytes. Indeed oocytes from hurp-/- female present oscillations in spindle length showing that HURP is essential to maintain meiotic spindle bipolarity over time. As frightening as it is, Patricia Hunt explained how exposure to Bisphenol A (BPA), a product coming out from damaged plastic wear, affects fertility by inducing strong meiotic abnormalities, BPA has a strong influence on meiosis I spindle assembly. There was an intense debate on the issue of the presence of a spindle assembly checkpoint (SAC) in meiosis I. Jacek Kubiak produced evidence, using bisected oocytes, that indeed there is an active and relatively robust SAC in meiosis I. Manqi Deng showed that DNA beads with no kinetochores induce a meiotic spindle, which undergoes anaphase progression, cytokinesis at the same time as the oocyte chromosomes and the beads are separated in one spindle pole. Patricia Hunt argued that if there is a spindle checkpoint, it must function slightly differently to the one observed in mitosis, and is not as efficient at least for certain bivalents. Manqi Deng and Johné Liu have analyzed how an asymmetric division occured. By following the first meiosis in live Xenopus oocytes using confocal imaging, Johné Liu showed that mutually exclusive domains of Cdc42 and RhoA activation mediate outpocketing of the oocyte membrane allowing polar body extrusion.
Takeo Kishimoto and Evelyn Houliston, using respectively starfish and jellyfish oocytes, came up with a novel view of a core Mos/MAPK signalling pathway essential for oocyte maturation in spawning organisms. This core CSF module would have different targets depending on the model system: for example in starfish it would have both p90rsk-dependent and independent functions. Alexei Evsikov concluded this meeting by showing that we can learn a lot about the process of oocyte formation by comparing the enrichment of ESTs from oocytes and early embryos coming from different species.
As anticipated, this meeting settled important issues but also generated a high number of key questions. A few examples: how are gap junctions between follicular cells and the mammalian oocyte closed in response to LH treatment? Which physiological steroids are involved in meiosis resumption in Xenopus? How is the synthesis/degradation of Cyclin B locally regulated during the prophase I arrest? Apart from Drosophila Cortex, are there other essential meiosis specific APC/C regulators in oocytes? How is Greatwall activation regulated during meiotic maturation? Which are the key substrates of Greatwall in meiosis and mitosis? How is DNA replication inhibited during the two meiotic transitions? How is the meiotic spindle organized in the absence of canonical centrosomes in most oocytes? Is the spindle assembly checkpoint in meiosis I function as efficient as in mitotic cells? How is the asymmetry of meiotic divisions ensured both in terms of the control of spindle positioning and regulation of polar body extrusion by small GTPases? Which are the effectors of the core Mos/MAPK signalling pathway in different model systems? Can we understand oocyte-specific mechanisms by analyzing data from comparison of different EST collections?