The LUCIA beamline at SOLEIL.

Commissioned in May 2004 on the SLS machine, the LUCIA beamline was moved to the synchrotron SOLEIL during the summer of 2008. To take advantage of this new setting several changes to its design were introduced. Here, a review of the various improvements of the mechanics and, mostly, of the optics is given. Described in detail are the results of a new multilayer grating monochromator implemented on the Kohzu vessel already holding the two-crystal set-up. It consists of a grating grooved onto a multilayer (replacing the first crystal) associated to a multilayer (as a second crystal). It allows a shift of the low-energy limit of the beamline to around 500 eV with an energy resolution and a photon flux comparable with those of the previous couples of crystals (KTP and beryl).

Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

cAMP effect on extracellular matrix synthesis in human breast cancer cells.

The effect of a cAMP derivative (N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate: dBcAMP) on the cell cycle and on the synthesis of typical extracellular matrix (ECM) components, i.e. collagen and glycosaminoglycans (GAG), was studied in two hormone-responsive human breast cancer cell lines VHB-1 and MCF-7. The data showed that dBcAMP induced a decrease in the proportion of cells in S + G2 + M phases due to an increase of the non-cycling (G0 phase) cell number as revealed by the Ki-67 antigen immunocytochemical study. The collagen synthesis, estimated by [3H] proline incorporation into the cellular proteins followed by an enzymatic digestion with highly purified bacterial collagenase, was not modified by dBcAMP. In contrast, the GAG synthesis, analysed by [3H] glucosamine incorporation, was increased two-fold in the dBcAMP treated cells. As a comparison we also tested 4-hydroxy-Tamoxifen (4-OH-Tam) since it induces similar cell cycle perturbations as dBcAMP. However, we did not observe a stimulation of the GAG synthesis following 4-OH-Tam treatment. These data demonstrated that the increased GAG synthesis is due to cAMP and is not a consequence of perturbations in the cell cycle. We can therefore assume that the ECM modifications induced by dBcAMP may contribute to the growth inhibition of the hormone-responsive human breast cancer cells.

Intracellular calcium and breast-cancer cell-growth and differentiation.

The growth of normal human breast epithelial cells in vitro, as well as those of other cell types is strongly influenced by the concentration of calcium in the culture medium [Ca++]e. The aim of this study was to ascertain if calcium also affects breast tumor cell growth in vitro. To address this question, the metastatic breast cancer cells MCF-7 were grown at low (0.04 mM, L-Ca) and high (2.5 mM, H-Ca) [Ca++]e. In each culture condition, we estimated intracellular calcium levels (Ca++]i from Indo-1 fluorescence by the ratio method. We showed that [Ca++]i increased with [Ca++]e, the [Ca++]i values ranging from approximately 50 to 250 nM. Changes of [Ca++]i ware accompanied by changes of cell shape and cell kinetic parameters. In H-Ca, cells were flat and 3 times larger than in L-Ca and the percentage of cells in the S+G2+M phases as well as the percentage of Ki-67 positive cells rapidly dropped on days 3-4 of culture in contrast to cells grown in L-Ca. In H-Ca, the cell growth arrest corresponded to maximal [Ca++]i which was stable during the stationary phase; at that time, a switch from H-Ca to L-Ca resulted in a drop of [Ca++]i and a resumption of cell growth.. In H-Ca, modifications in cell differentiation parameters such as diminution of ER expression and increases of lipid content and EMA expression were observed as compared to cells grown m L-Ca. Our results suggest that MCF-7 cells have retained some calcium dependency and that agents that can increase [Ca++]i in breast tumor cells may limit their proliferation and trigger at least a partial differentiation.

Stimulation of axon growth from the spinal cord by a regenerating limb blastema in newts.

The effects of limb blastemas of Pleurodeles waltl on axon growth from fragments of spinal cord were studied in vitro. Cultured in a defined medium, spinal cord fragments regenerated sparse, short axons. The culture of spinal fragments in the presence of blastemas greatly enhanced the length, number and survival of axons. Testing separately each of the two components of the blastema showed that only the mesenchyme exerts a neurotropic effect on the spinal fragments. Other tissues such as muscle or skin had a limited neurotrophic effect. Additionally, the neurotrophic activity of blastemas seems to be dependent of its proliferation status. Compared with blastemas of regenerating limbs from young animals, irradiated blastemas (devoid of mitotic activity) and blastemas of regenerating limbs from old animals or differentiated blastemas (both characterized by a low mitotic activity), exhibited a weaker neurotrophic influence. The blastema neurotrophic factor is not an attachment molecule but a soluble one and cannot be nerve growth factor (NGF) or fibroblast growth factor (FGF). It has a relatively low molecular weight (less than 15 kDa) and its protein nature was ascertained by its sensitivity to heating and proteases. As the production of this mesenchyme-derived neurotrophic factor depends upon mesenchymal cell proliferation of the blastema, we suggest that there is loop of positive regulation between spinal nerves and blastema. Blastema tissues may stimulate nerve regeneration allowing the stimulation of proliferation of blastema cells by regenerating nerve fibers. Alternatively, blastema cells may produce a neurotrophic factor whose secretion might be dependent on cell proliferation.

Apparatus for the in vitro fertilization and culture of human oocytes.

PIP: An apparatus for in vitro fertilization and culture of mammalian eggs that permits close control over variations in temperature, lighting, and pH has been developed to imitate natural conditions as far as possible. All handling of the oocyte and zygote are performed inside a baby incubator which has a system of air saturation using water vapor. A binocular dissecting microscope and an inverted compound microscope are built into the enclosure to permit simultaneous observation of the egg at widely different magnifications. Lighting is restricted to the longer wavelengths, which do not harm mammalian eggs. The microscope is equipped with a camera system. A gas mixture of 5% CO2, 5% O2, and 90% N2 is delivered at a pressure of 1 bar by a double reduction valve arrangement. The gas passes into the incubator through a filter and is humidified and warmed inside the incubator by bubbling through a bottle of sterile distilled water. The gas is finally distributed by a series of culture tubes. The culture tubes are usually kept in darkness; their position is changed as the development of the egg progresses to take advantage of the slight temperature differentials within the apparatus. Less than 1 minute after puncture of the follicle, the fluid is transferred to the incubator and is examined in a petri dish using the stereomicroscope. The oocyte is immediately transferred to a culture tube containing medium and placed at 36.8 degrees Celsius. 16-20 hours after insemination the egg is transferred under the stereomicroscope by pipetting directly into a new tube, where the medium is supplemented by a small quantity of maternal serum. It is kept at 37.2 degrees Celsius. After 2-4 days of culture in vitro, if normal development is observed, the egg is transferred to the patient's uterus. The variation in temperature at any given point in the apparatus is less than .2 degrees Celsius. 114 human oocytes have been collected, of which 58% were fertilized. 82% of the fertilized eggs underwent normal cleavage, and 38 embryos were replaced in the uteri of 34 patients. 8 early pregnancies resulted, of which 2 lasted more than 3 months. The fertilization rate was closely related to the quality of male and female gametes; it was 94% when both were favorable.^ieng

Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.

Eighty-five human embryos fertilized in vitro were frozen and thawed with 1,2 propanediol as a cryoprotective agent. The effects of viability of in vitro culture duration, stage, and morphologic appearance of embryos were examined after thawing. Survival was higher for 2-day-old embryos than for 3-day-old embryos (56% versus 18%) and for 2-, 4-, and 8-cell embryos than for intermediate-cleavage-stage embryos (67% versus 22%). Among 19 regular-cell-size embryos at the 2-, 4-, and 8-cell stage, 15 (79%) kept 50% or more of their initial number of blastomeres after thawing and 9 were intact. The average viability of all 2-day-old frozen-thawed embryos can be estimated at 19%.

High pregnancy rate after early human embryo freezing.

Human embryos produced by in vitro fertilization (IVF) were frozen with 1,2-propanediol as a cryoprotectant. Embryo survival after thawing was related to the presence of a nucleus in frozen cells and decreased with the increasing number of cells in the frozen embryo. None of five embryos frozen 3 or 4 days after IVF survived when thawed. Of 48 early embryos (35 patients) frozen 1 or 2 days after IVF, 42 (87.5%) were transferred in 32 patients. Ten pregnancies were initiated after frozen embryo transfer (ET). If we exclude the three infertile patients who had sexual intercourse in the fertile period, the pregnancy rate for each patient who had 1- or 2-day frozen embryo(s) was 22% (7 of 32). One of the pregnancies was obtained after ET of a 1-cell pronucleated frozen and thawed embryo. The rate of ongoing pregnancies after triple fresh ET was 23%. In patients having four embryos obtained in a single IVF cycle, the expected overall liveborn rate in an IVF-ET program including embryo cryopreservation could theoretically equal that of natural human fertility.

Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program.

Certain factors influencing the success of embryo cryopreservation were analyzed from 124 cycles of in vitro fertilization and embryo transfer (IVF-ET) program in which 193 1- or 2-day embryos were frozen and had already been thawed. There were 100 transfers of one or two surviving embryos from which 26 pregnancies were initiated. Several factors significantly influenced embryo survival after thawing. They were: the developmental stage of frozen embryos; the appearance of the embryo at the time of freezing; and the mode of ovarian stimulation in the IVF cycle. The pregnancy rate after frozen-thawed embryo transfer was higher with 4-cell frozen embryos than with embryos at all other stages combined. There were also tendencies for the pregnancy rate to be higher if a spontaneous luteinizing hormone surge occurred in the transfer cycle or if the duration of embryo storage did not exceed 1 to 2 months. The results obtained support a new policy in IVF-ET programs: it should be advantageous for the sterile couple if the immediate fresh embryo transfer is only performed with the categories of embryos that demonstrate a poor aptitude for survival following cryopreservation procedures.

Cell kinetics (SAMBA 200) of estradiol stimulated long-term phenol red withdrawn cultured breast cancer cells.

When estradiol stimulation was performed on hormone-responsive MCF-7 human breast cancer cells maintained in phenol red-containing medium until 24h before experiments, the cell number and the cell surface transferrin receptor level, an early marker of estradiol stimulation, were strongly increased. In contrast, similar treatment performed on MCF-7 cells grown in phenol red-free medium up to 12 months before experiments induced no stimulating effect on the cell number, although transferrin receptors were still enhanced. Since the appearance of transferrin receptors occurs before the cells begin DNA synthesis in late G1 phase, we assumed that the discrepancy between cell counts and cell surface transferrin receptors might involve cell kinetic perturbations. The proportion of cells in the (S + G2) phases and the duration of the cell cycle phases were analysed using the SAMBA 200 cell image processor. The data presented in this study failed to indicate any block in the cell cycle and the duration of the S and G2 phases were found to be unchanged. The lack of effect of estradiol stimulation on the growth of MCF-7 cells maintained several months without phenol red is not thus a consequence of cell cycle perturbations.

[In vitro evaluation of cutaneous potentials in the triton, Pleurodeles waltlii Michah., by measuring transepithelial potentials]. / Evaluation in vitro des potentiels de surface cutanés chez le triton, Pleurodeles waltlii Michah., par la mesure des différences de potentiel transépithéliales.

Surface potentials previously measured in vivo have been approximately evaluated in vitro by means of an experimental device allowing a simultaneous record of transepithelial potential differences in 2 areas of the same skin piece cut out along the proximo-distal direction of the anterior limb of Pleurodeles waltlii. These potentials are thus explained by unequal transepithelial potential differences in the proximal and distal areas of the same skin sample.

[Effect of osmotic stress on the surface potential of the extremities of a triton, Pleurodeles waltlii Michah]. / Influence des chocs osmotiques sur les potentiels de surface du membre de Triton, Pleurodeles waltii Michah

Surface potentials of Pleurodeles limb have been studied under the action of NaCl solution made hyperosmotic with various chemicals (choline, propionate, sucrose, urea). These potentials were suppressed or lowered to a variable extent according to the solution used. The interpretation of the mechanism of action of these chemicals has led us to propose an hypothesis on the origin of the electrical potentials of the epidermis.

Surface potentials and the control of amphibian limb regeneration.

The relationship between surface potentials and amphibian limb regeneration is examined. The wound surface becomes increasingly positive for several days after amputation but then decreases again, often becoming negative for a variable period during blastemal growth. The same changes of surface potentials are observed during wound healing alone, in the absence of amputation and following amputation of irradiated or denervated limbs. Similar changes occur in non-regnerating frog arms. These observations and other cited reasons lead to the conclusion that surface potentials do not control regeneration.

Sequential transformations of human sperm nucleus in human egg.

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).

Human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes: effect of sperm pretreatment by calcium-ionophore A23187 and freezing-thawing on the penetration rate and polyspermy.

Calcium-ionophore A23187 and freezing-thawing were used as sperm treatments before human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes. The penetration rate (PR) was higher when SI-PVS was performed with calcium-ionophore-treated (28%) or frozen-thawed (51%) sperm than with untreated sperm (8%). Optimal PR occurred when five calcium-ionophore-treated (57%) or frozen-thawed (71%) sperm were injected under the zona pellucida. When the sperm:egg ratio was 1:1, PR was higher for calcium-ionophore-treated (18.5%) or frozen-thawed (27.8%) sperm than for untreated sperm (0.0%). Calcium-ionophore sperm treatment had no effect on the polyspermic oocyte rate (POR) or the mean number of swollen sperm nuclei per penetrated oocyte (Pd) or per injected sperm (SR). This may result from premature oocyte activation induced by Ca-ionophore. However, POR was higher with frozen-thawed (74%) than with untreated (50%) or Ca-ionophore-treated (50%) sperm. Whatever the sperm treatment, there was a trend toward a lower SR as the number of injected sperm increased. Cytoplasmic regulation of polyspermy in the hamster oocyte is discussed.