Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations.

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73 h in fresh IVM, 17.33 h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47 µm vs 11.23 µm in IVM and 11.40 µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37 µm vs 8.12 µm in IVM and 8.16 µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

Polo-like kinase 4 (Plk4) potentiates anoikis-resistance of p53KO mammary epithelial cells by inducing a hybrid EMT phenotype.

Polo-like kinase 4 (Plk4), the major regulator of centriole biogenesis, has emerged as a putative therapeutic target in cancer due to its abnormal expression in human carcinomas, leading to centrosome number deregulation, mitotic defects and chromosomal instability. Moreover, Plk4 deregulation promotes tumor growth and metastasis in mouse models and is significantly associated with poor patient prognosis. Here, we further investigate the role of Plk4 in carcinogenesis and show that its overexpression significantly potentiates resistance to cell death by anoikis of nontumorigenic p53 knock-out (p53KO) mammary epithelial cells. Importantly, this effect is independent of Plk4's role in centrosome biogenesis, suggesting that this kinase has additional cellular functions. Interestingly, the Plk4-induced anoikis resistance is associated with the induction of a stable hybrid epithelial-mesenchymal phenotype and is partially dependent on P-cadherin upregulation. Furthermore, we found that the conditioned media of Plk4-induced p53KO mammary epithelial cells also induces anoikis resistance of breast cancer cells in a paracrine way, being also partially dependent on soluble P-cadherin secretion. Our work shows, for the first time, that high expression levels of Plk4 induce anoikis resistance of both mammary epithelial cells with p53KO background, as well as of breast cancer cells exposed to their secretome, which is partially mediated through P-cadherin upregulation. These results reinforce the idea that Plk4, independently of its role in centrosome biogenesis, functions as an oncogene, by impacting the tumor microenvironment to promote malignancy.

Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation.

Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities.

Centrosome Assembly: Reconstructing the Core Cartwheel Structure In Vitro.

Centrioles are microtubule-based cylinders essential for the formation of centrosomes and cilia. A recent study provides a new cell-free assay that reconstitutes the initial structure formed during centriole assembly - the cartwheel - and proposes a new model for its formation and growth.

Investigating the in Vitro Thermal Stability and Conformational Flexibility of Estrogen Receptors as Potential Key Factors of Their in Vivo Activity.

Among hormone-inducible transcription factors, estrogen receptors (ERs) play important roles in tissue growth and differentiation, via either direct or indirect binding, in the nucleus, to specific DNA targets called estrogen responsive elements (EREs), or through nongenomic pathways. In humans, two estrogen receptor isoforms (hERs), designated hERα and hERß, have been identified. These two hERs, encoded by genes located on distinct chromosomes, exhibit divergent tissue-specific functions and different subcellular distributions depending on their binding status, free or complexed to their cognate ligands. Because it is hypothesized that such distinct behaviors may arise from various conformational stabilities and flexibilities, the effect of salt concentration and temperature was studied on the free and estrogen-activated hERα and hERß. Our results show that the conformational stability of hERß is weakly modulated by salt concentration as opposed to hERα. In addition, we show that the estrogen-bound hERs exhibit a more constrained structure than the unliganded ones and that their conformational flexibility is more affected by diethylstilbestrol binding than that of estradiol, 4-hydroxytamoxifen, or raloxifen. In line with these results, conformational analysis and computational docking were performed on hERα and hERß, which confer molecular support of a diethylstilbestrol-induced restrained flexibility as compared to other ligands. We found that Trp383 in hERα and Trp335 in hERß can closely interact with the NR-box motif of the H12 helix and act as a gatekeeper of the agonist-bound versus antagonist-bound conformations. Altogether, our study contributes to an improved knowledge of the diverse physicochemical properties of full-length hERs, which will help in our understanding of their distinct cellular roles in various cellular contexts.

Mapping molecules to structure: unveiling secrets of centriole and cilia assembly with near-atomic resolution.

Centrioles are microtubule (MT)-based cylinders that form centrosomes and can be modified into basal bodies that template the axoneme, the ciliary MT skeleton. These MT-based structures are present in all branches of the eukaryotic tree of life, where they have important sensing, motility and cellular architecture-organizing functions. Moreover, they are altered in several human conditions and diseases, including sterility, ciliopathies and cancer. Although the ultrastructure of centrioles and derived organelles has been known for over 50 years, the molecular basis of their remarkably conserved properties, such as their 9-fold symmetry, has only now started to be unveiled. Recent advances in imaging, proteomics and crystallography, allowed the building of 3D models of centrioles and derived structures with unprecedented molecular details, leading to a much better understanding of their assembly and function. Here, we cover progress in this field, focusing on the mechanisms of centriole and cilia assembly.

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition.

Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes.

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5'monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-L-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.

EP45 accumulates in growing Xenopus laevis oocytes and has oocyte-maturation-enhancing activity involved in oocyte quality.

The capacity of oocytes to fully support meiotic maturation develops gradually during oocyte growth. Growing oocytes accumulate proteins and mRNAs required for this process. However, little is known about the identity of these factors. We performed a differential proteomic screen comparing the proteomes of growing stage-IV oocytes, which do not undergo meiotic maturation in response to progesterone, with fully grown stage-VI ones, which do. In 2D gels of stage-VI oocytes, we identified a group of four protein spots as EP45 (estrogen-regulated protein 45 kDa), which belongs to the family of serine protease inhibitors and is also known as Seryp or pNiXa. Western blot analysis after mono- and bi-dimensional electrophoreses confirmed the accumulation of certain forms of this protein in oocytes between stages IV and VI. EP45 mRNA was not detectable in oocytes or ovaries, but was expressed in the liver. A low-mobility isoform of EP45 was detected in liver and blood, whereas two (occasionally three or four) higher-mobility isoforms were found exclusively in oocytes, suggesting that liver-synthesized protein is taken up by oocytes from the blood and rapidly modified. Alone, overexpression of RNA encoding either full-length or N-terminally truncated protein had no effect on meiotic resumption in stage-IV or -VI oocytes. However, in oocytes moderately reacting to low doses of progesterone, it significantly enhanced germinal-vesicle breakdown, showing a novel and unsuspected activity of this protein. Thus, EP45 accumulates in growing oocytes through uptake from the blood and has the capacity to act as an 'oocyte-maturation enhancer' ('Omen').

Role of oocyte quality in meiotic maturation and embryonic development.

The quality of oocytes plays a key role in a proper embryo development. In humans, oocytes of poor quality may be the cause of women infertility and an important obstacle in successful in vitro fertilization (IVF). The competence of oocytes depends on numerous processes taking place during the whole oogenesis, but its final steps such as oocyte maturation, seem to be of key importance. In this paper, we overview factors involved in the development of a fully functional female gamete with Xenopus laevis as a major experimental model. Modern approaches, e.g. proteomic analysis, enable the identification of novel proteins involved in oocyte development. EP45, called also Seryp or pNiXa, which belongs to the serpin (serine protease inhibitors) super-family is one of such recently analyzed proteins. This protein seems to be involved in the stimulation of meiotic maturation and embryo development. EP45 is potentially a key factor in correct oocyte development and determining the quality of oocytes.

N-Myristoylation targets dihydroceramide Delta4-desaturase 1 to mitochondria: partial involvement in the apoptotic effect of myristic acid.

This study was designed to analyze the effect of myristic acid on ceramide synthesis and its related lipoapoptosis pathway. It was previously observed that myristic acid binds dihydroceramide Delta4-desaturase 1 (DES1) through N-myristoylation and activates this enzyme involved in the final de novo ceramide biosynthesis step. In the present study, we show first by immunofluorescence microscopy and subcellular fractionation that DES1 myristoylation targets part of the recombinant protein to the mitochondria in COS-7 cells. In addition, native dihydroceramide Delta4-desaturase activity was found in both the endoplasmic reticulum and mitochondria in rat hepatocytes. Dihydroceramide conversion to ceramide was increased in COS-7 cells expressing DES1 and incubated with myristic acid. The expression of the wild-type myristoylable DES1-Gly alone, but not the expression of the unmyristoylable mutant DES1-Ala, induced apoptosis of COS-7 cells. Finally, myristic acid alone also increased the production of cellular ceramide and had an apoptotic effect. This effect was potentiated on caspase activity when the myristoylable form of DES1 was expressed. Therefore, these results suggest that the myristoylation of DES1 can target the enzyme to the mitochondria leading to an increase in ceramide levels which in turn contributes to partially explain the apoptosis effect of myristic acid in COS-7 cells.