The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform.

BACKGROUND: The von Hippel-Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies. METHODS: We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array. RESULTS: We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172. CONCLUSIONS: The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.

Overexpression of the polarity protein PAR-3 in clear cell renal cell carcinoma is associated with poor prognosis.

The partition-defective 3 (PAR-3) protein is implicated in the development and maintenance of cell polarity and is associated with proteins that mediate the changes in cytoskeleton organization required for cell polarity establishment. In this work, we used two original primary cell lines (R-180 and R-305) derived from clear cell Renal Cell Carcinoma (ccRCC) surgical specimens of a patient with unfavorable clinical course (R-180 cells) and a patient with favorable prognosis (R-305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the PARD3 gene was amplified only in the R-180 cell line that was derived from an aggressive ccRCC. PARD3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. Consistently, PARD3 knockdown in R-180 cells restored the cytoskeleton organization and reduced cell migration in comparison to non-transfected cells. Immunohistochemical analysis of ccRCC samples from a cohort of 96 patients with a follow-up of 6 years revealed that PAR-3 overexpression was correlated with poor survival. Our results suggest that PAR-3 has a role in the clinical aggressiveness of ccRCC, possibly by promoting cell migration.

Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis.

In contrast to the classical model describing the synthesis of androgens and estrogens as restricted to somatic cells, a previous study demonstrated that Xenopus laevis oocytes participate in androgen synthesis. The objective of our study was to determine whether Xenopus oocytes are also involved in estrogen synthesis. More precisely, we analyzed aromatase expression by in situ hybridization and RT-QPCR and measured aromatase activity. Aromatase, the enzyme responsible for estrogen synthesis, appears to be expressed and active not only in the follicular cells but also in the vitellogenic oocytes. During late oogenesis, aromatase oocyte expression and activity decreased concomitantly with the trend observed in surrounding follicular layers. In order to investigate the role of estradiol-17ß (E(2)), we studied its effect on oocyte meiotic resumption. It appears that, as in Rana pipiens, E(2) inhibited the follicle-enclosed maturation of Xenopus oocytes, likely through inhibition of LH-induced maturation-inducing steroid synthesis. In addition, E(2) exerted a slight enhancing action on denuded oocyte maturation whose biological significance remains unclear. Together, our results demonstrate that Xenopus oocyte significantly participates in ovarian E(2) synthesis and this may be a common feature of vitellogenic vertebrates.

Effects of confinement on the self-organization of microtubules and motors.

The regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, "Y-branching" organizations, cortical-like bundles, "rackets," and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo.

Effect of sensory education on willingness to taste novel food in children.

As part of 'EduSens', a project aiming to measure the effect of a sensory education program developed in France on the food behaviour of school children, the present paper shows the results regarding neophobia. One hundred and eighty children (8-10 years old) were involved in the study. Half of them (experimental group) were educated during school-time with the 12 sessions of taste lessons "Les classes du goût" by J. Puisais. The others served as a control group. Food neophobia was evaluated before and after the education period of the experimental group and once again 10 months later. An adapted food neophobia scale was used (AFNS) and the willingness to taste novel food (WTNF) was evaluated by the presentation of eight unknown foods. To improve involvement in the expressed willingness to taste new foods, the children were told that they would have to eat one of the not rejected unknown foods afterwards. Results revealed that, at the end of the education period, in the educated group, declarative food neophobia decreased significantly and participants' willingness to taste novel food seemed to increase compared to the control group. Nevertheless, these effects had disappeared 10 months later. Thus, we have shown that sensory education can influence childrens' food neophobia, but does so only temporarily. This is especially true for the WTNF test, which measures the expression of neophobia in concrete situations, whereas neophobia measured as a psychological trait by the AFNS test hardly changes.

Fish growth hormone receptor: molecular characterization of two membrane-anchored forms.

A RT-PCR approach was used to clone and sequence the full-length growth hormone receptor (GHR) of a teleost fish, the turbot (Scophthalmus maximus). Total liver RNA was amplified by RT-PCR with degenerate primers designed in extracellular and cytoplasmic regions, and a single DNA fragment of 1100 bp was obtained. The entire coding region was obtained by 5' and 3' RACE assays, and comprises an open-reading frame of 633 amino acids. This sequence shows the characteristic motifs of the class I cytokine receptor superfamily, and its amino acid identity with mammalian, avian, reptilian and amphibian GHRs is 32-36%. The 3' RACE also revealed the occurrence of an alternate messenger encoding a membrane-anchored truncated receptor, which could facilitate the production of GH-binding protein in fish species. This report represents the first data on fish GHR sequence, and it provides evidence for the conservation of this receptor throughout vertebrate evolution.

Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes.

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 micron < diameter < 1000 micron) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mos synthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively active raf mRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras --> Raf --> MAPKK --> MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the "Mos synthesis machinery." The present results show that incompetence of Xenopus stage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mos synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.

[In vitro oocyte growth and maturation: a mouse example]. / Croissance et maturation ovocytaire in vitro: l'exemple de la souris.

During the last 20 years, numerous studies have been performed to improve culture conditions in order to allow in vitro growth and meiotic maturation of oocytes isolated from primordial to antral follicles. The main objective of these studies is to define optimal culture conditions which will allow to increase the recovery rate of oocytes that can be successfully fertilized. This paper reviews the more recent advances obtained when studying mouse oocyte development in vitro.

Oocyte control of granulosa cell development: how and why.

Interaction between oocytes and granulosa cells is complex and involves both gap junctions and paracrine signalling factors. Oocyte development in antral follicles is highly dependent on communication with cumulus cells, a subset of granulosa cells that is intimately associated with oocytes. Cumulus cells express characteristics distinct from the mural granulosa cells of preovulatory follicles. The thesis of this paper is that, without the influence of oocytes, the pathway of granulosa cell differentiation in antral follicles leads to the establishment of the mural granulosa cell phenotype. Oocytes in antral follicles abrogate that pathway of granulosa cell differentiation and promote the development of the cumulus cell phenotype. Oocytes may do this in order to control their own microenvironment by regulating differentiation of the supporting cells that are in direct communication with them. Possibly, some aspects of the mural granulosa cell phenotype are antagonistic to, or insufficient for, supporting the final stages of oocyte development. We present evidence that oocytes control their environment by suppressing differentiation of the mural granulosa cell phenotype and promoting differentiation of the cumulus cell phenotype. They achieve this suppression via the secretion of labile paracrine signalling factors. Errors in this regulatory mechanism, whether instigated by defects in the production of oocyte-derived ligands or granulosa cell responses to them, may result in the production of oocytes unable to undergo embryo development or that undergo abnormal follicular development.

Induction of precocious germinal vesicle breakdown (GVB) by GVB-incompetent mouse oocytes: possible role of mitogen-activated protein kinases rather than p34cdc2 kinase.

In contrast to fully grown mouse oocytes, growing oocytes released from their preantral follicles are not capable of resuming meiosis spontaneously. However, when these denuded growing oocytes are incubated for 2-3 days in control medium and then treated with okadaic acid, 95% undergo precocious germinal vesicle breakdown (GVB) and chromosome condensation within 24 h. This study shows that both events apparently occur through a p34cdc2 kinase-independent pathway, since activity of this kinase was detected only after the oocytes had undergone okadaic acid-stimulated GVB. In contrast, microtubule-associated or mitogen-activated protein (MAP) kinases 1 and 2 were activated before GVB and their level of activity was correlated with the percentage of oocytes undergoing GVB, suggesting that these kinases may promote GVB and chromosome condensation under these experimental conditions. In addition, it is shown that MAP kinases accumulate during normal oocyte growth in vivo, but not in cultured denuded oocytes in vitro even when these oocytes become competent to undergo okadaic acid-stimulated GVB. Unlike p34cdc2, the accumulation of MAP kinases requires close physical interactions between the oocytes and the granulosa cells but is not necessary for the oocyte to become GVB-competent. Therefore, it MAP kinases are actually involved in the induction of GVB and chromosome condensation during normal oocyte maturation, acquisition of GVB competence requires oocyte-autonomous accumulation of MAP kinase activator(s) rather than the MAP kinases themselves.

Synthesis and accumulation of p34cdc2 and cyclin B in mouse oocytes during acquisition of competence to resume meiosis.

This study tests the hypothesis 033 that growing murine oocytes, which are incompetent to resume meiosis, are deficient in their content of p34cdc2 and/or cyclin B, the two subunits of maturation promoting factor (MPF). Accumulation of the two MPF components occurred in an asynchronous manner in growing oocytes. Cyclin B content reached maximal levels in oocytes that were not yet competent to undergo germinal vesicle breakdown (GVB), the first obvious morphological manifestation of the resumption of meiosis. Thus, the amount of cyclin B is not the limiting factor rendering these growing oocytes incompetent to undergo GVB. In contrast, synthesis and accumulation of p34cdc2 increased during the period of oocyte growth in vivo when they became competent to undergo GVB. A similar increase in the amount of p34cdc2 also occurred in cultured granulosa cell-free oocytes despite the lack of oocyte growth, but these cultured oocytes did not become GVB competent. Thus, the accumulation of p34cdc2 is probably necessary, but not sufficient, for mouse oocytes to become competent to undergo GVB. This accumulation occurs autonomously in oocytes independently of growth or of the participation of follicular somatic cells.

Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes.

This paper focuses on the relationship between the developmental programs that regulate nuclear and cytoplasmic maturation of mouse oocytes. As oocytes near completion of their growth phase, they become competent to resume meiosis when isolated from the small antral follicles and cultured, but the progression of meiosis in many of these oocytes stops at metaphase I (MI). With further growth and development at the germinal vesicle (GV) stage, however, these oocytes become competent to complete nuclear maturation and progress to metaphase II (MII). In this study, about 44% of the oocytes were arrested at MI after isolation from the small antral follicles of 18-day-old mice and culture for 16 to 17 hr. Upon insemination, these MI-arrested oocytes produced the first polar body, formed pronuclei, cleaved, and developed to the blastocyst stage. Seventy five percent of these blastocysts were triploid, the remainder were diploid. Treatment of MI-arrested oocytes with calcium ionophore resulted in the completion of the first meiotic division and parthenogenetic activation. Moreover, while the pattern of proteins synthesized in MI-arrested oocytes was quite different from that of normal MI oocytes as determined by [35S]methionine labeling and two-dimensional gel electrophoresis, it was similar to that of MII-arrested oocytes. It was therefore concluded that some critical aspects of cytoplasmic maturation can occur in oocytes whose nuclear maturation is arrested at MI. In addition, triggers that promote entry of MII-arrested oocytes into anaphase II are sufficient to drive MI-arrested oocytes into anaphase I and to produce the first polar body when the triggers are generated in mature cytoplasm. The developmental capacity of MII oocytes that matured in vitro after isolation from 18- or 26-day-old mice were compared. The frequency of fertilization and cleavage to the 2-cell stage was equal in both groups. In surprising contrast, the ability to complete the 2-cell stage to blastocyst transition occurred much more frequently when the oocytes were from the 26- than the 18-day-old mice (82 and 27%, respectively). Thus, even though both groups of oocytes had completed nuclear maturation by progressing to MII, oocytes from the smaller follicles of the younger mice were deficient in maternal factors essential for development of embryos beyond the 2-cell stage. Further differentiation of these GV-stage oocytes of small antral follicles is therefore required to produce eggs competent of completing preimplantation development.

Acquisition of meiotic competence by denuded mouse oocytes: participation of somatic-cell product(s) and cAMP.

In contrast to fully grown oocytes, growing mouse oocytes are not capable of undergoing germinal vesicle breakdown (GVB) when released from the follicle unless they are first cultured in somatic-cell-conditioned medium. The first objective of this study was to assess the mechanisms by which oocytes in vitro acquire the ability to resume meiosis in conditioned medium. Whereas most of the denuded oocytes that were initially incompetent of undergoing GVD underwent GVB within 4 days of culture in fibroblast-conditioned medium, oocytes cultured in control medium remained in the GV stage although their viability was sustained as judged by morphological appearance and quantitative and qualitative patterns of protein synthesis. This suggested that the effect of somatic-cell-conditioned medium is inductive rather than simply permissive. When GVB-incompetent oocytes were first incubated in control medium for 1-3 days, a larger percentage underwent GVB following exposure to the conditioned medium or okadaic acid. It was therefore concluded that some aspects of the oocytes' developmental program for the acquisition of GVB competence are oocyte-autonomous but external factors provided by the surrounding somatic cells are probably require for oocytes to become fully GVB-competent. The effect of cAMP on acquisition of GVB competence by growing oocytes was also studied. Dibutyryl cAMP dose-dependently promoted the acquisition of GVB competence by initially GVB-incompetent oocytes. Forskolin, an adenylate cyclase activator, acted in similar way and its effect was potentiated by hypoxanthine, a naturally occurring cAMP-phosphodiesterase inhibitor. Thus cAMP, in addition to maintaining meiotic arrest in GVB-competent oocytes, also participates in the acquisition of GVB competence by growing oocytes.

Secretion of cumulus expansion enabling factor by mouse oocytes: relationship to oocyte growth and competence to resume meiosis.

Fully grown mouse oocytes secrete a factor that enables the surrounding cumulus cells to undergo cumulus expansion upon stimulation of the cumulus cells with follicle-stimulating hormone in vitro. The secretion of the enabling factor by the oocytes is developmentally regulated; it is normally secreted in significant amounts only by oocytes that are competent of undergoing spontaneous germinal vesicle breakdown (GVB) and resuming the first meiotic division. The objective of this study was to determine the relationship, if any, among secretion of this factor, oocyte growth, and the competence to undergo GVB. Three experimental systems were used to assess these relationships. First, 20 to 25% of the oocytes isolated from the small antral follicles of mutant hypogonadal mice were GVB-incompetent even though they had achieved a size typical of GVB-competent oocytes. These large GVB-incompetent oocytes secreted almost as much cumulus expansion enabling factor as the GVB-competent oocytes. Second, GVB-incompetent oocytes cultured under conditions that promote the acquisition of GVB competence but not oocyte growth acquired the ability to secrete the cumulus expansion enabling factor. Third, the precocious activation of maturation promoting factor (MPF) in GVB-incompetent oocytes by okadaic acid did not stimulate the secretion of cumulus expansion enabling factor. It is concluded that the secretion of cumulus expansion enabling factor by oocytes is (1) independent of oocyte growth, (2) independent of competence to undergo GVB, and (3) not induced by the precocious activation of MPF. Thus, although the secretion of the enabling factor normally coincides with the time of acquisition of competence to undergo GVB, the ability to secrete the enabling factor is independent of oocyte maturation. Nevertheless, factors that promote the oocyte's program that leads to competence to undergo GVB probably also promote the ability to secrete the enabling factor.

A nonsteroidal follicular factor is involved in maturation process of Xenopus laevis oocytes.

Xenopus laevis postvitellogenic ovarian follicles release into conditioned medium nonsteroidal factor(s) that potentiate progesterone-induced oocyte maturation. The molecular weight of this factor is between 1000 and 10,000 Da. The potentiating activity is suppressed when follicles are incubated with cyanoketone to inhibit steroidogenesis, suggesting that the secretion of the potentiating factor can be modulated in vitro. These results indicate that the steroid-independent pathway in vitro, previously reported in oocytes exposed to insulin or IGF-1 to induce meiotic resumption, may be of physiological importance.