Restoration of spermatogenesis in infertile male chickens after transplantation of cryopreserved testicular cells.

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze). 2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks. 3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype. 4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue. 5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.

Expression of the chicken GDNF family receptor α-1 as a marker of spermatogonial stem cells.

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

Identification of various testicular cell populations in pubertal and adult cockerels.

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

Particulate 1,3-beta-D-glucan, carboxymethylglucan and sulfoethylglucan--influence of their oral or intraperitoneal administration on immunological respondence of mice.

The effect of orally or intraperitoneally administered particulate 1,3-beta-D-glucan (PBG), carboxymethylglucan (CMG) or sulfoethylglucan (SEG), obtained from the culture filtrate of Saccharomyces cerevisiae, on the functions of murine peritoneal adherent cells (PC) (peroxidase activity, nitric oxide synthesis), on relative organ mass and on proliferation of splenocytes was determined. The modulating activities after parenteral and non-parenteral administration of these polysaccharides were compared. Significant enhancement of NO production was observed only after in vitro cultivation of PC in the presence of lipopolysaccharide (LPS) in groups of mice treated repeatedly orally with CMG, PBG and SEG at a dose of 50 mg/kg body mass. Peroxidase activity increased significantly after repeated oral administration of CMG and PBG at doses 150 and 50 mg/kg, SEG 150 mg/kg body mass. The peroxidase activity and NO synthesis in mice given a single intraperitoneal injection of glucans (15 mg/kg body mass) were slightly higher than those after oral administration. Neither a significant enhancement of relative organ mass nor enhancement of the proliferative response of splenocytes to in vitro added stimuli (LPS, phytohemagglutinin) after repeated oral or single intraperitoneal administration of beta-glucans was observed.

[Serologic detection of antibodies to Helicobacter pylori in children and adolescents using ELISA]. / Sérologický prukaz protilátek proti Helicobacter pylori u deti a dospívajících metodou ELISA.

Serum antibodies anti-Helicobacter pylori (H.p.) which are specific against antigenic determinants of the bacterial body, were determined in 183 non-ulcer dyspeptic children and adolescents, aged 7- to 20 years. 108 patients (59%) of the same group underwent fibroendoscopy for direct detection of H.p. in bioptic specimens of the stomach and duodenum microbiological methods. Discrimination analysis revealed a overall positive correlation between the results of the two methods: P < 0.01. For detection of specific IgG antibodies against H.p., evaluation by the P/N ratio was used, i.e. the absorbance of the examined serum (P) was compared with the mean absorbance of three negative serum controls (N). The specificity of the ELISA test in this examination was 96.1%, the sensitivity of 90.3%. The mentioned ELISA method for detection of specific IgG antibodies against H.p. is comparable with direct microbiological prove of H.p. presence in mucus specimens in our group examined. It is a very important non-invasive method useful for primary detection of H.p. infection.