**1. Introduction**

Satellite cells (SCs) represent a population of postnatal mononucleated stem cells [1] that are located between the basal lamina and the sarcolemma of skeletal muscle fibers, and are clearly detectable by means of electron microscopy [2]. SCs are able to occasionally fuse with muscle fibers in order to compensate for the muscle turnover caused by daily wear and tear, or support muscle hypertrophy, thereby underpinning skeletal muscle plasticity [3,4]. SCs exert their physiological role in close interplay with their local environment (the SC niche; [5]). While several non-SC cell populations are involved in muscle plasticity [6], SCs represent a key factor in muscle growth and regeneration, and research thereupon is steadily increasing over time. SCs are typically in a quiescent state, showing a minimum amount of cytoplasm and organelles therein [7]. Upon stimulation (e.g., physical exercise or muscle damage), SCs activate and re-enter the cell cycle, leading to proliferation and/or differentiation. Activation of SCs implies that a transition takes place in the cell nucleus from a low/absent to a high transcriptional activity [8]. The most obvious morphological counterpart of such a transition is a reduction in the amount of heterochromatin, and it was shown that the activation and differentiation of SCs are characterized by an important shift from condensed to lightly packed chromatin [9].

Hibernation is an adaptation to adverse winter conditions adopted by several mammals [10], which is characterized by greatly reduced metabolic activity and lowered body temperature while maintaining homeostasis. Upon arousal, bodily functions are resumed in full. Accordingly, cells in the organs of hibernating animals undergo periodical cycles of deactivation and reactivation. Intriguingly, skeletal muscle mass and strength are preserved during hibernation as well as the fiber size despite long periods of immobilization, contrary to what happens in non-hibernating mammals [11–13]. Protective mechanisms apparently take place in skeletal muscle during hibernation, which most likely involve, among others, inhibition of proteolysis, a decrease in autophagy and increased oxidative capacity [14,15]. The preservation of muscle mass during hibernation could also involve SC activation; however, the role of SCs in the prevention of atrophy has barely been investigated [16,17]. In particular, to the best of our knowledge, the structural and functional characteristics of the cell nucleus in SCs during hibernation have not been investigated so far.

Over the last several years, the morphology of the cell nucleus during the hibernation/arousal cycle has been studied in several tissue types of different species in our laboratory [18–27]. In the skeletal muscle, we found that the fine structure of the muscle fiber is well preserved during hibernation in the edible dormouse (*Glis glis*, Gliridae), with myonuclei showing morphological evidence of transcriptional activity [28]. In the present work, we investigated whether hibernation affects the structural and functional features of the SC nucleus. Both the ultrastructural and immunocytochemical characteristics of the SC nuclei were analyzed in active and hibernating edible dormice, with a focus on the key nuclear constituents involved in RNA transcription and processing. The results show that the SC nuclei are similar in hibernating and euthermic dormice, suggesting that factors other than SC activation are operating during hibernation in the immobilized skeletal muscle to prevent atrophy.

### **2. Materials and Methods**

This is a retrospective study conducted on specimens obtained from six male edible dormice captured in 1998/1999 for the purpose of multiple investigations upon permission from the Regione del Veneto (Decreto n. 76 del 20 Gennaio, 1998). Adult (approximately 1–2-year-old) wild-living animals were trapped and maintained in an outdoor animal house supplied with food and bedding material. Under such conditions, they spontaneously began to hibernate in November and awoke in March. Three animals were killed during the euthermic period (June–July), and three during deep hibernation (January, after at least three days of continuous torpor). Euthermic animals were decapitated under deep anesthesia; hibernating animals were taken from the cage and immediately decapitated.

Samples of the right quadriceps muscle were processed for transmission electron microscopy for either morphology or immunocytochemistry.

For morphological analysis, muscle samples were fixed by immersion in 2.5% (*v*/*v*) glutaraldehyde and 2% (*v*/*v*) paraformaldehyde in 0.1 M Sørensen phosphate buffer pH 7.4 at 4 ◦C for 2 h, post-fixed with 1% (*v*/*v*) OsO4 at 4 ◦C for 1 h, dehydrated through graded acetone and embedded in Epon 812. Ultrathin sections (70–80 nm in thickness) were placed on copper grids coated with a Formvar layer and stained with Reynold's lead citrate prior to observation.

For immunocytochemistry, muscle samples were fixed by immersion in 4% (*v*/*v*) paraformaldehyde in 0.1 M Sørensen phosphate buffer at 4 ◦C for 2 h, washed, treated with 0.5 M NH4Cl in PBS 0.1 M pH 7.4 to block free aldehydes, dehydrated with ethanol and embedded in LR White resin polymerized under UV light. Ultrathin sections (70–80 nm in thickness) were placed on nickel grids coated with a Formvar-carbon layer and treated with the following probes: mouse monoclonal antibodies directed against the active phosphorylated form of RNA polymerase II (Abcam, Cambridge, MA, USA; ab24759) and the DNA/RNA hybrid molecules [29], both occurring at pre-mRNA transcription sites [28,30]; the (Sm)snRNP (small nuclear RiboNucleoProtein) core protein (Abcam; ab3138), involved in the co-transcriptional splicing of pre-mRNA [31]; the myogenic differentiation transcription factor D (MyoD; [32] (Abcam; ab16148)); and rabbit polyclonal antibody directed against the SC-specific paired box protein 7 (Pax7) transcription factor [33] (Abcam; ab34360). In detail, the sections were

floated for 3 min on normal goat serum (NGS) diluted 1:100 in PBS, incubated for 17 h at 4 ◦C with the primary antibody diluted in PBS containing 0.1% (*w*/*v*) bovine serum albumin (Fluka, Buchs, Switzerland) and 0.05% (*v*/*v*) Tween 20. After rinsing, sections were floated on NGS, and then reacted for 30 min at room temperature with the specific secondary 12 or 6 nm gold-conjugated antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) diluted 1:10 in PBS. Finally, the sections were rinsed and air-dried. The control grids were treated as above, but the primary antibody was omitted from the incubation mixture, and then processed as described. After the immunocytochemical procedure, the sections were treated with Uranyl Acetate Replacement Stain (Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min and lead citrate for 45 s in order to weakly stain the heterochromatin and visualize the structural constituents in the interchromatin space. All grids were observed in a Philips Morgagni TEM operating at 80 kV and equipped with a Megaview III camera for digital image acquisition.

Quantitative assessment of immunolabelling was carried out by estimating the gold particle density over the interchromatin space (i.e., the nucleoplasmic region devoid of heterocromatin clumps) in sections treated in the same run. Briefly, the surface areas of the nucleoplasmic region and the heterochromatin were measured in 10 randomly selected electron micrographs (×22,000) of SC nuclei from each animal using a computerized image analysis system (AnalySIS Image processing, Soft Imaging System GmbH, Münster, Germany). The interchromatin space area was calculated, the gold particles present over the interchromatin space were counted and their density was expressed as number/μm2. Background evaluation was carried out on the resin (in the areas devoid of tissue) of the immunolabelled samples as well as on the tissue of control samples. The same procedure was used to assess the density of perichromatin granules (PG; representing sites of storage and/or transport of spliced pre-mRNA [34]) over the interchromatin space.

To estimate the amount of heterochromatin, the percentage of the heterochromatin area within the total nucleoplasm area was calculated.

For each analyzed variable, the Kolmogorov–Smirnov two-sample test was performed in order to verify the hypothesis of identical distribution among animals in each group, and then the mean ± standard error of the mean (SEM) was calculated. Comparisons of variables in the two groups (euthermic and hibernating) were performed with one-way ANOVA (significance set at *p* ≤ 0.05).
