Abstract. The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro. Key words: Gonocytes, Spermatogonia, Spermatogenesis, Stem cells, Testis (J. Reprod. Dev. 57: [355][356][357][358][359][360][361][362][363][364] 2011) erm cells originate from primordial germ cells (PGCs), which are primary cells on the germline lineage in embryos. After migration to the genital ridge, male germ cells become gonocytes [1]. At a certain period after birth, gonocytes migrate to the basement membrane of the testis and differentiate to type A spermatogonia including spermatogonial stem cells (SSCs). SSCs have the potential to self-renew and generate differentiated germ cells, resulting in the production of large numbers of spermatozoa throughout most or all of adult life. Thus, gonocytes have key roles in producing SSCs and initiating spermatogenesis. In mice, the transition of gonocytes to SSCs begins 3 days after birth [2]. Although some report have shown the postnatal testis development in large domestic species including cattle (Bos indicus [3], Bos taurus [4,5]), little is known about gonocytes during their development in cattle.Specific germ cell markers have been identified in the mouse testis. One such marker is VASA, a DEAD (asparagineglutamine-alanine-asparagine) box protein 4 (DDX4) that is required for male germ cell development in mice [6,7]. Additionally, stem cell characteristics of mouse SSCs were determined by a transplantation a...

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig-human xenotransplantation.

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IntroductionOxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA). Anthocyanin is a plant antioxidant. We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs) and explored possible mechanisms by which AEBS might exert anti-arthritic effects.Material and MethodsCIA was induced in DBA/1J mice. Cytokine levels were measured via enzyme-linked immunosorbent assays. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed by measuring the ratio of phosphorylated IκB to total IκB via Western blotting. Th17 cells were stained with antibodies against CD4, IL-17, and STAT3. Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels.ResultsIn the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS decreased Th17 cell numbers in spleen of CIA mice. Additionally, AEBS repressed differentiation of Th17 cells and expression of Th17-associated genes in vitro, in both splenocytes of naïve DBA/1J mice and human PBMCs. In vitro, the numbers of both human and mouse tartrate-resistant acid phosphatase+ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS.ConclusionsThe anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.

Regulator of calcineurin 3 (RCAN3), an endogenous regulator of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, inhibits the phosphatase activity of calcineurin, the nuclear translocation of NFAT, and the NFAT downstream pathway. To investigate the effects of RCAN3 on T-cell regulatory function and the development and progression of inflammatory arthritis, we studied the effects of RCAN3 transfection on regulation of Th17 cell differentiation in a murine T-lymphoma cell line and primary splenic CD4 T cells. Overexpression of RCAN3 suppressed Th17 cell differentiation through the down-regulation of RAR receptor orphan receptor γT mRNA and up-regulation of forkhead box P3 mRNA. In mice with collagen-induced arthritis, injection of an RCAN3-overexpression vector controlled arthritis development in vivo. Injection of RCAN3 reduced the formation of osteoclasts and expression of inflammatory cytokines in vivo. Antioxidants stimulated the expression of RCAN3 in vitro, and combination therapy with pcDNA-RCAN3 had a synergistic suppressive effect on the development of arthritis. These data suggest that RCAN3 may be an effective treatment for rheumatoid arthritis.