PDE2A Is Indispensable for Mouse Liver Development and Hematopoiesis.

Phosphodiesterase 2A (PDE2A) is a cAMP-cGMP hydrolyzing enzyme essential for mouse development and the PDE2A knockout model (PDE2A-/-) is embryonic lethal. Notably, livers of PDE2A-/- embryos at embryonic day 14.5 (E14.5) have extremely reduced size. Morphological, cellular and molecular analyses revealed loss of integrity in the PDE2A-/- liver niche that compromises the hematopoietic function and maturation. Hematopoietic cells isolated from PDE2A-/- livers are instead able to differentiate in in vitro assays, suggesting the absence of blood cell-autonomous defects. Apoptosis was revealed in hepatoblasts and at the endothelial and stromal compartments in livers of PDE2A-/- embryos. The increase of the intracellular cAMP level and of the inducible cAMP early repressor (ICER) in liver of PDE2A-/- embryos might explain the impairment of liver development by downregulating the expression of the anti-apoptotic gene Bcl2. In summary, we propose PDE2A as an essential gene for integrity maintenance of liver niche and the accomplishment of hematopoiesis.

A Three-Dimensional Culture Model of Reversibly Quiescent Myogenic Cells.

Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.

Atrophy, oxidative switching and ultrastructural defects in skeletal muscle of the ataxia telangiectasia mouse model.

Ataxia telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm-knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects, but not cerebellar neurodegeneration and ataxia. We explored whether Atm loss is responsible for skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions. Atm-knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and a switch from glycolytic to oxidative fibers were detected, along with an increase of in expression of slow and fast myosin types (Myh7, and Myh2 and Myh4, respectively) in tibialis anterior and solei muscles isolated from Atm-knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of Z-lines and sarcomeres and mitochondria abnormalities that were associated with an increase in reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex than those in Atm wild-type mice, but with preserved presynaptic terminals. In conclusion, we report for the first time that Atm-knockout mice have clear morphological skeletal muscle defects that will be relevant for the investigation of the oxidative stress response, motor alteration and the interplay with peripheral nervous system in ataxia telangiectasia.

Effects of the hydrostatic pressure in in vitro beating cardiac syncytia in terms of kinematics (kinetic energy and beat frequency) and syncytia geometrical-functional classification.

Many important observations and discoveries in heart physiology have been made possible using the isolated heart method of Langendorff, e.g. the discovery of the very famous Frank-Starling law of the heart. Nevertheless, the Langendorff's method has some limitations and disadvantages such as the probability of preconditioning and a high oxidative stress, leading to the deterioration of the contractile function. To avoid the preceding drawbacks associated to the use of a whole heart, we have alternatively used beating mouse cardiac syncytia cultured in vitro in order to assess the ergotropic and chronotropic effects of both increasing and decreasing hydrostatic pressures. To achieve the preceding aim, we have developed a method based on image processing analysis to evaluate the kinematics of that pressure-loaded beating syncytia starting from the video registration of their contraction movement. We have verified the Frank-Starling law of the heart in in vitro beating cardiac syncytia and we have obtained their geometrical-functional classification. The present method could be used in in vitro studies of beating cardiac patches, as alternative to the Langendorff's heart in biochemical, pharmacological, and physiology studies, and, especially, when the Langendorff's technique is inapplicable. Furthermore, the method could help, in heart tissue engineering and bioartificial heart researches, to "engineer the heart piece by piece".

Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels.

Spontaneous germ cell death by apoptosis occurs during normal spermatogenesis in mammals and is thought to play a role in the physiological mechanism limiting the clonal expansion of such cell population in the male gonad. In the prepubertal rat testis, the most conspicuous dying cells are pachytene spermatocytes, which are also the primary target of the apoptosis experimentally induced by the methoxyacetic acid (MAA). Since we have recently reported that Sertoli cells, the somatic component of the seminiferous epithelium, regulate not only germ cell viability and differentiation but also their death, we have further investigated the mechanism involved in such a control. In this paper we have used the protein clusterin, produced by Sertoli cells and associated with tissue damage or injury, as indicator of germ cell apoptosis in rat seminiferous tubules treated with MAA in the presence or in the absence of omega-agatoxin, a specific inhibitor of P/Q type voltage-operated calcium channels (VOCC's). We performed both a qualitative analysis of clusterin content and germ cell apoptosis by immunofluorescence experiments and a quantitative analysis by in situ end labelling of apoptotic germ cells followed by flow cytometry. The results obtained demonstrate that Sertoli cells modulate germ cell apoptosis induced by methoxyacetic acid also throughout the P/Q-type VOCC's.

Sertoli cell modulates MAA-induced apoptosis of germ cells throughout voltage-operated calcium channels.

Spontaneous cell death by apoptosis--occurring during normal spermatogenesis in mammals--is a prominent event, which results in the loss of up to 75% of the potential number of mature spermatozoa. In the rat testis, the most conspicuous dying cells are pachytene spermatocytes, which are also the primary target of the apoptosis experimentally induced by methoxyacetic acid (MAA). In this paper, we have used clusterin expression as an indicator of germ cell apoptosis in rat seminiferous tubules treated with MAA in the presence or in the absence of voltage-operated calcium channels (VOCCs) inhibitors. We performed both a qualitative analysis of clusterin expression by immunofluorescence experiments and a quantitative analysis of apoptosis by in situ end labeling of apoptotic germ cells followed by flow cytometry. The results obtained demonstrate that Sertoli cells, the somatic component of the seminiferous epithelium, which control male germ cell differentiation, also modulate MAA-induced apoptosis of germ cells throughout voltage-operated calcium channels.