Cell proliferation : a potential compensatory mechanism for tissue damage during hibernation

Cell proliferation is usually depressed during hibernation due to its highly energy-consuming nature. However, this process is not depressed all the time, with previous studies finding an increase in cell proliferation in the brain, testis and skeletal muscle tissues of some hibernators during hibernation. In our study, we investigated the relative mRNA expression levels of ZBED1, an important transcription factor associated with cell proliferation, in five tissues of the greater horseshoe bat across pre-hibernation, deep hibernation and post-hibernation. Increased expression of ZBED1 during deep hibernation was only observed in the brain and skeletal muscle tissues, but not in the other tissues studied, suggesting a tissue-heterogeneity of the cell proliferation. Increased cell proliferation may compensate for tissue damages under stressful conditions during hibernation.

Hibernation is an energy-conservation strategy used by animals to survive in the stressful conditions.Many functions of animals are depressed during hibernation [1] .Cell proliferation, involving the synthesis of DNA and protein, cell cycle, and cell division, is an energetically demanding process, which is expected to be depressed during torpor [1,2] .Previous studies have focused on the highly proliferative intestinal epithelial cells to study the rates of cell division during hibernation [3][4][5] , and such processes important for cell proliferation, including DNA synthesis and mitosis, have been found to be greatly reduced in intestinal epithelial cells during deep torpor [3,4] .The attenuation of mitosis in intestinal epithelial cells is interpreted as the blockage of cells in the G2 phase of cell cycle during torpor, which is assumed to minimize errors in cell replication under hypothermia [4,6] .However, cell proliferation is not always depressed during hibernation, at least not in all tissues.Cerri et al. (2009)  reported that the degree of cell death was higher in the parenchyma of the cerebral hemispheres and optic tectum of hibernating frogs compared with active ones, meanwhile, higher cell proliferation occurred in the corresponding ventricular zones.It seems to be a neuroprotective strategy that cell proliferation was increased in the brain, which was utilized by hibernating frogs to avoid neurological damages RESEARCH HIGHLIGHT [7]   .Recently another study found that the numbers of muscle satellite cells involved in muscle growth and repair also increased during hibernation, which may protect the skeletal muscle of hibernators from atrophy and dysfunction after extended periods of disuse [8] .In addition, some hibernators come into full sexual activity during hibernation, and this is associated with the rise of cellular proliferation in the testes at this period [9] .Spermatogenic recrudescence in the involuted testes usually begins shortly after the mid-hibernal interval and continues at a gradually accelerating pace through the remaining weeks of dormancy [9] .These studies suggest cell proliferation may be of importance during hibernation.
ZBED1 (zinc finger, BED-type containing 1), previously called DREF (DNA replication-related element-binding factor), a transcription regulatory factor controlling many cell proliferation-related genes, plays a key role in the regulation of cell proliferation [10] .Given the important role of ZBED1 in cell proliferation, an increased or decreased expression of ZBED1 gene during hibernation may reflect the change of cell proliferation in the tissues of hibernators.In order to determine the change of cell proliferation in different tissues during hibernation, we investigated the relative mRNA expression levels of ZBED1 gene in five tissues of the greater horseshoe bat (Rhinolophus ferrumequinum) (Figure 1), including heart, kidney, brain, liver and skeletal muscle, across pre-hibernation (September), deep hibernation (December and April) and post-hibernation (May) [11] .We found that the expression of ZBED1 gene only increased in the brain and skeletal muscle tissue during deep hibernation compared with pre-hibernation and post-hibernation.However, when compared with pre-hibernation and post-hibernation, the expressions of ZBED1 gene in the heart, kidney and liver were all decreased during deep hibernation, though these changes were not statistically significant.Our results therefore suggest that the rate of cell proliferation is variable in different tissues during hibernation.The tissue-heterogeneity of the potential cell proliferation during deep hibernation might imply different intensities of stresses confronted by those different tissues.
Cell proliferation in the brain and skeletal muscle tissues of R. ferrumequinum was inferred to be increased during deep hibernation based on the increased expression of ZBED1 gene.Previous studies also found increases of cell proliferation in the brain, testis, and skeletal muscle [7][8][9] .This is somewhat surprising, because cell proliferation is usually expected to be depressed during hibernation due to its energetically-demanding nature, as has been found in intestinal epithelial cells, lymphocyte, and hippocampus [3,6,12,13] .The potential increase of cell proliferation in those specific tissues may suggest an existence of specific pressures in those tissues during torpor.
During onset and maintenance of torpor, the brain suffers diverse stresses, such as ischemia-induced oxidative stress and thermal stress [1,14] .These stresses are harmful or even lethal to non-hibernators, and an increased cell proliferation may help to compensate for cell death and the onset of neurological damages.It is reported that the arctic ground squirrels, an obligate hibernator, can even tolerate at least 10 min of global ischemia without detectable neuronal injury in the active state [15,16] .In addition to the brain, the skeletal muscle tissue may suffer stresses during hibernation.For instance, inactivity in humans (e.g., limb immobilization) is well known to lead to atrophy of skeletal muscle, loss of muscle tone and impaired strength [17] .However, in hibernating animals, such as ground squirrels and bats, though the reduction of their skeletal muscle mass is observed, neither atrophy nor dysfunction is found in their skeletal muscle after hibernation [18,19] .The possible cell proliferation may help to maintain the function of skeletal muscle during hibernation, thereby partly accounting for the lack of muscle atrophy in hibernators after extended periods of disuse during hibernation.
If ZBED1 is involved in the regulation of cell proliferation and is of importance for compensating for possible tissue damage during torpor, we may expect positive selection underlying the evolution of ZBED1 in the hibernating taxon compared with its non-hibernating counterparts.To test this, we further conducted a comparative evolution analysis of ZBED1 between hibernating subspecies (Central-East and Northeast mtDNA clades) and non-hibernating subspecies (Southwest mtDNA clade) of the greater horseshoe bat in China.Consistent with our expectations, positive selection signals were only detected in the hibernating subspecies [11] , suggesting an adaptive evolution of ZBED1 under the pressures during hibernation.
In summary, we investigated relative mRNA expression level of ZBED1, an important transcript factor controlling cell proliferation in five tissues of R. ferrumequinum across pre-hibernation, deep hibernation and post-hibernation, and an increased expression of ZBED1 was only observed in the brain and skeletal muscle tissues during deep hibernation, suggesting a potential increase of cell proliferation in the two tissues.The potential increase of cell proliferation in the two tissues is suggested to compensate for their possible tissue damages during hibernation.Further genetic analyses of ZBED1 show evidence of positive selection unique to the hibernating subspecies of the greater horseshoe bat.Our results suggest that ZBED1 may be a potential candidate gene for hibernation.