Heterogeneous cell population derived from human ovarian follicular liquid: morphological studies and molecular screening

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute [1]. The components of new primary follicles, primitive granulosa and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cells residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The multipotency of a subset of granulosa cells was also established by in vitro differentiation into other cell types [2]. Up to now, luteinizing GCs were considered to be terminally differentiated, unavoidably becoming apoptotic a few days after ovulation. Previously, we have provided evidence for the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitro fertilization techniques [3]. These cells grow in minimal medium condition, without any growth factor (i.e. LIF), that is considered essential according to other procedures [4]. Using immunocytochemistry and flow cytometry we showed that these cells are positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105. However, morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial- like and neural-like shapes. These observations are also supported by the identification of cells expressing specific neural markers, such as neurofilaments and PGP9.5, in addition to vimentin and cytocheratin positive cells. All these data are suggestive of the presence of different cell populations in follicular fluids. To verify this hypothesis we select a panel of markers specific for the different cell populations previously identified and we plan a molecular screening to follow their expression in the follicular fluid derived cells at different times of minimal culture conditions in vitro. Bone marrow derived MSCs were used as a control. For each sample we performed semiquantitative RT-PCR experiments normalizing the cDNAs used as templates on the basis of the number of pseudo-mesenchymal cells morphologically identified in the sample. For this purpose OCT-4 was selected as a stem marker to follow the mesenchymal stem cell population, while FSH-R was used to identify granulosa derived cells; CNTF and beta-3-tubuline were used to discriminate between neural and neuronal cells populations; epithelial and hematopoietic cells were followed using cytokeratin (CK8 and CK10) and CD45 markers, respectively. GAPDH and β-actin specific primers were used on all samples for normalization. Here we compare the results of this molecular screening with the previously obtained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persistence, loss or amplification at different times of in vitro minimal culture conditions

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

Background: Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel. Results: RT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding. Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein. The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells. Conclusion: The results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi

Activation of the CREB/c-Fos pathway during long-term synaptic plasticity in the cerebellum granular layer

The induction of long-term potentiation and depression (LTP and LTD) is thought to trigger gene expression and protein synthesis, leading to consolidation of synaptic and neuronal changes. However, while LTP and LTD have been proposed to play important roles for sensori-motor learning in the cerebellum granular layer, their association with these mechanisms remained unclear. Here, we have investigated phosphorylation of the cAMP-responsive element binding protein (CREB) and activation of the immediate early gene c-Fos pathway following the induction of synaptic plasticity by thetaburst stimulation (TBS) in acute cerebellar slices. LTP and LTD were localized using voltage-sensitive dye imaging (VSDi). At two time points following TBS (15 min and 120 min), corresponding to the early and late phases of plasticity, slices were fixed and processed to evaluate CREB phosphorylation (P-CREB) and c-FOS protein levels, as well as Creb and c-Fos mRNA expression. High levels of P-CREB and Creb/c-Fos were detected before those of c-FOS, as expected if CREB phosphorylation triggered gene expression followed by protein synthesis. No differences between control slices and slices stimulated with TBS were observed in the presence of an N-methyl-Daspartate receptor (NMDAR) antagonist. Interestingly, activation of the CREB/c-Fos system showed a relevant degree of colocalization with long-term synaptic plasticity. These results show that NMDAR-dependent plasticity at the cerebellum input stage bears about transcriptional and post-transcriptional processes potentially contributing to cerebellar learning and memory consolidation

Immediate early genes regulation in rat cerebellar cortex during long-term synaptic plasticity induction

The cerebellum is one of the brain areas involved in learning and memory formation. Long-term synaptic plasticity is thought to play a pivotal role in supporting these functions. Moreover Immediate Early Genes (IEGs) expression and de novo protein synthesis and/or modification have been strictly associated with maintenance of Long-Term Potentiation (LTP) as well as memory consolidation and storage. Two highly conserved signalling cascades, PKA and MAPK, seem to be involved in early- to late-LTP conversion; both pathway can activate CREB transcription factor through phosphorylation and P-CREB has been suggested to initiate the protein synthesis leading to late-LTP induction. The transcription factor c-fos is known to be rapidly and transiently induced in the Nervous System by a variety of stimuli and is thought to be directly involved in processes of neuronal plasticity including LTP. We used rat parasagittal cerebellar slices as a model system in which specific patterns of stimulation delivered to the mossy fibers can induce both Long-Term Potentiation and Long-Term Depression (LTD), depending on local inhibition and other regulating factors. Using Voltage Sensitive Dye (VSD) imaging we obtained high-resolution maps of the spatial distribution of LTP/LTD induced from a Teta Burst Stimulus (TBS) application. Control and stimulated slices were fixed at different times from the TBS application and processed for in situ hybridization or immunohystochemistry in order to detect IEGs mRNA expression patterns and protein expression/modifications. The expression pattern of c-fos and CREB mRNAs and their protein distribution and/or phosphorylation were then correlated with LTP/LTD maps generated by VSD imaging. Preliminary data indicate a significant increase of P-CREB in the granular layer suggesting that CREB phosphorylation is induced as early as 15 minutes post TBS application. In situ hybridization experiments indicate a good correlation between c-fos and CREB mRNAs up-regulation and LTP distribution at 120 minutes post TBS. At the protein level, the comparison of immunofluorescence signals and VSD immaging data indicate a clear correlation between c-Fos and P-CREB distribution and synaptic plasticity patterns. We are planning further experiments to confirm these data and to test our experimental system in the presence of drugs that could interfere with the transcription, translation or post-translational protein regulation

Immediate early genes expression in the cerebellar cortex correlates with LTP and LTD induction

The consolidation of changes following activity-dependent neural plasticity are believed to involve specific patterns of gene expression. In the hippocampus, immediate early genes are thought to contribute to long-term synaptic plasticity (LTP and LTD); this phenomenon may occur also in the cerebellum, in which the transcription factors c-Fos and P-CREB have been identified. The cerebellum granular layer (GL) can manifest both LTP and LTD following a Theta Burst Stimulus (TBS) delivered to the mossy fibers. We have employed VSD imaging in rat cerebellar slices (P18-24) in order to map the spatial distribution of LTP and LTD in the cerebellum GL. Fluorescence changes were correlated to LTP or LTD in two different post-TBS time ranges (15 and 120 min). Slices were then fixed and processed for immunohistochemistry in order to identify levels of c-Fos and P-CREB expression. The induction of long-term plasticity increased the average level of P-CREB both at 15 min (+39±4.9, p<0.01%) and 120 min (+24±7.2, p<0.05%) after TBS. The level of c-Fos was unaltered at 15 min, while it significantly increased at 120 min (+37±8.9, p<0.05%). By spatially correlating longterm synaptic plasticity with the corresponding variation of P-CREB and c-Fos, we observed that regions showing LTP well correlated (p<0.05) with positive variations of P-CREB and c-Fos. Conversely, areas showing LTD correlated exclusively (p<0.05) with negative variations of P-CREB. Slices were also evaluated by in situ hybridization and a similar analysis was performed. The levels of fos and CREB mRNA expression and their spatial correlation with the sign of long-term synaptic plasticity corresponded with the immunohistochemical results. As a further test, VSD recordings showed that the induction of granular layer LTP and LTD could be prevented by applying 50 mM D-APV, a selective NMDA receptor blocker. Moreover, in situ hybridization and immunohistochemistry analysis evidenced that in these conditions both mRNA and protein expression levels of c-fos and CREB were unchanged, confirming the involvement of these two transcription factors in cerebellar granular layer plasticity

Isolamento e caratterizzazione di nuovi geni Zinc Finger espressi in cellule miogeniche di topo

Tesi di dottorato in scienze morfogenetiche citologiche. 6. ciclo. A.a. 1990-94. Relatore Marina Bouche'Consiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - Piazza Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Expression of proteins involved in calcium homeostasis during crista ampullaris regeneration

Most of the events related to the mechano-transduction process in hair cell are regulated by cytoplasmic Ca++ concentrations. Ca++ influx trough the mechano-transduction channels regulates the adaptation process in the stereocilia, while Ca++ entering into the cell through the voltage gated channels regulates the neurotransmitter release. Moreover Ca++ activates the K+ channels that dominate the inhibitory postsynaptic potential at efferent synapses. Cytoplasmic Ca++ concentration is regulated by a complex system of proteins, including the Na+/Ca++ exchanger, Ca++ pumps (PMCAs, SERCAs) and a copious supply of diffusible proteins that buffers free Ca++ in specific cell compartments. These proteins intercept Ca++ near its sites of entry and restrict the spread of the free ion. Moreover this buffering limits the period during which Ca++ local concentration are high enough to trigger its physiological effects. In frog crista ampullaris PMCAs seems to be the most relevant mechanism of Ca++ extrusion since the lack of a functional Na+/Ca++ exchanger. We investigated the distribution of different subtypes of PMCAs finding that each isoform has its specific expression domain along the crista. We also demonstrate a similar compartmentalization for the IP3R, which is involved in Ca++ release from the IP3-sensitive intracellular stores, and for some buffering proteins. These data and the morphological identification of diverse hair cell types differentially distributed along the crista ampullaris, suggest the presence in the sensory epithelium of distinct functional domains where the mechanisms of Ca++ homeostasis regulation are not completely overlapping. This hypothesis is confirmed by the observation that the different regions of the crista ampullaris show a different sensibility to the ototoxic damage induced by gentamicin treatment: although the mechanism of actions of this drug has not jet completely understood, aminoglycosides are known to interfere with Ca++ homeostasis. Our studies on frog crista ampullaris regeneration showed that the complete crista functional activity is restored well afterwards its morphological recovery. Indeed, while the appearance of a stereociliary apparatus expressing a correct pattern of Ca++ pumps seems to be sufficient to restore hair cell basal activity, the recovery of the evoked sensory discharge seems to need also the expression of Ca++ buffering proteins. This study try to correlate the appearance of the different proteins involved in Ca++ homeostasis with the complete functional recovery of the sensory epithelium

Ecto-ATPase activity sites in vestibular tissue: an ultracytochemical study in frog semicircular canals

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function