Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2+/− ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm.

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

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In the glandular stomach, gap junctional intercellular communication (GJIC) plays an important role in the gastric mucosal defense system, and loss of GJIC is associated with ulcer formation. In spite of the high incidence of gastric ulcers in horses, particularly at pars nonglandularis, the presence of gap junctions in the equine stomach has not yet been studied. The objective was to obtain basic data on the distribution of gap junction protein connexin 32 (Cx32) in the different regions of normal equine gastric mucosa. Samples of mucosa were taken from seven horses at cardiac, fundic, and pyloric region and pars nonglandularis. To detect Cx32, immunohistochemical staining and Western blot analysis were performed. Corresponding mRNA was shown by RT-PCR and localised in tissue sections by in situ hybridisation. Cx32 was found in the glandular regions, whereas it was not detectable in squamous mucosa. Within the glandular epithelium, Cx32 was abundant in surface and foveolar cells and decreased towards the proliferative zone of the glands. These results suggest that gap junctions develop during the maturation of surface cells. Whether the lack of Cx32 at pars nonglandularis contributes to its susceptibility for developing ulcers, has to be further elucidated.