Reduced cytochrome oxidase activity and increased protein tyrosine phosphorylation of mitochondria-rich fractions of buffalo (Bubalus bubalis) spermatozoa after a cycle of freezing and thawing.

The motility and fertility of mammalian spermatozoa are compromised when they are cryopreserved. Sperm mitochondrial proteins play a vital role in conferring motility. However, the effects of cryopreservation on mitochondria-specific proteins remain primarily unexplored in domestic animals, including buffaloes, so the present study aimed to evaluate this issue. Mitochondria were isolated from both non-cryopreserved and cryopreserved buffalo spermatozoa by sonication followed by sucrose density gradient ultracentrifugation. The purity of the mitochondrial preparation was assessed by cytochrome oxidase assay and electron microscopy. Mitochondria separated from cryopreserved buffalo spermatozoa were associated with significantly lower (P ≤ 0.05) cytochrome oxidase activity as compared with non-cryopreserved spermatozoa. The intensities of two low-molecular-mass mitochondrial proteins (30.1 kDa and 26.1 kDa) were significantly reduced as compared with the non-cryopreserved group. In addition, in cryopreserved buffalo sperm mitochondria, the intensities of three tyrosine phosphorylated proteins (126.6, 106.7 and 26 kDa) increased significantly compared with the non-cryopreserved group. Of these, tyrosine phosphorylation of the 26-kDa mitochondrial protein of cryopreserved sperm was very intense and unique because it could not be detected in the mitochondria of non-cryopreserved sperm. Thus, the study confirmed that both cytochrome oxidase activity and the proteins of buffalo sperm mitochondria undergo significant cryogenic changes in terms of quantity and quality after a cycle of freezing and thawing and this may be one of the important causes of reduced post-thaw motility and fertility of cryopreserved buffalo spermatozoa.

Expressed sequence tag analyses of three leukocyte subpopulations in ayu Plecoglossus altivelis altivelis, separated by monoclonal antibodies.

Ayu Plecoglossus altivelis altivelis are one of the most economically important fish for freshwater aquaculture in Japan. We conducted expressed sequence tag analyses of three leukocyte subpopulations, thrombocytes, neutrophils, and B lymphocytes in ayu using a next generation sequencer. The sequencing and de novo assembly yielded 22,494, 22,733, and 16,505 contigs from the thrombocyte, neutrophil, and B lymphocyte cDNA libraries, respectively. Pathways involving endocytosis, phagosomes, and lysosomes, were found in all three cDNA libraries using pathway analysis. The thrombocyte cDNA library contained 2894 unique sequences, including CXC chemokine receptor 4 and MHC class II. Cytokine and cytokine receptor genes such as interleukin (IL)-1ß, IL-8, IL-1 receptor (IL-1R), IL-8RA, and IL-8RB were found among the 3056 unique sequences of the neutrophil cDNA library. Typical B lymphocyte related genes such as B cell linker protein, immunoglobulin (Ig) M, IgD and transforming growth factor ß were found in the 1590 unique sequences of the B lymphocyte cDNA library. In summary, a large number of immune-related genes were identified from the three leukocyte cDNA libraries. Our results represent a valuable sequence resource for understanding the immune system function in ayu.

Studies on morphology and cytochemistry in blood cells of ayu Plecoglossus altivelis altivelis.

Peripheral blood cells from ayu, Plecoglossus altivelis altivelis, were separated using a density gradient. Blood cells were then smeared using Shandon Cytospin and subjected to cytochemical staining. Blood cells were categorized based on morphological and cytochemical characteristics, and the density fractionation range and nucleus area/cell area ratio were observed. Lymphocytes are distinguished from neutrophils by their basophilic cytoplasm and Golgi-like field. The features of chromatin in thrombocytes are different from those of lymphocytes or neutrophils, but some small neutrophils have similar chromatin. Therefore, it is necessary to perform peroxidase staining to distinguish small neutrophils from thrombocytes. Basophils have large basophilic granules in cytoplasm. Based on density fractionation of blood cells, thrombocytes in the low-density area were separated from other blood cells. Identification of peripheral blood cells from ayu was possible with these staining methods. Monocytes/macrophages from spleen are specifically positive for esterase staining by α-naphthyl butyrate. As a result, thrombocytes, lymphocytes, neutrophils, basophils and monocytes/macrophages were identified in smears from peripheral blood or spleen tissue. In this paper, we confirmed that the peripheral blood corpuscles of ayu are able to be identified using the present staining methods.

Isolation and characterization of hematopoietic progenitor cells in canine bone marrow.

For ultimate diagnoses of canine leukemia or malignant lymphoma, we sought to isolate hematopoietic progenitor cells (HPCs) from canine bone marrow (BM) using physiological phenotypes. Canine BM cells were separated by equilibrium discontinued density centrifugation, and HPCs, detected by in vitro colony formation, were significantly enriched in the relatively low density (LD) fraction. In flow cytometry, many CD34 or MHC class II expressing cells were detected in the LD fraction, but these were not significantly enriched. When the LD cells were separated, using a cell-sorting method, into cells with high affinity of wheat germ agglutinin (WGAhigh) and cells with WGAlow, almost all multipotent HPCs (MHPCs) and HPCs committed to myeloid lineage were found in the WGAhigh population. When the WGAhigh population was further stained for rhodamin 123, almost all MHPCs were included in the dull population (Rhlow), but not in the bright one (Rhhigh). Morphologically, most Rhlow cells were round, blastic cells containing a large nucleus with nucleoli and narrow cytoplasm. Based on these results, we suggest that all of the MHPCs in canine BM show the Rhlow WGAhigh LD phenotype, and may contain hematopoietic stem cells, which are the primitive HPCs.