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.

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey.

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.

Sexing of chicken feather follicle, blastodermal and blood cells.

The production of chicken chimeras using donor and acceptor cells which can be of opposite sex has necessitated the utilization of methods developed to distinguish the sex of chickens. We demonstrate one of these methods, based on the polymerase chain reaction which amplifies the EcoRI repeat unit of the fowl W chromosome, and how this technique may be used to sex various cell types in chickens as well as small numbers of blastodermal cells. Our results demonstrate the ability to sex chickens using EcoRI primers, specific for the W chromosome, from as little as 2 ng of female genomic DNA isolated from blood and feathers--the latter being the result of DNA extraction from only one feather. Also evident in this study is the detection of the W chromosome by PCR from approximately 50 blastodermal cells originating from the developing blastodisc at stage X.

Expression of human erythropoietin gene in the mammary gland of a transgenic mouse.

WAP is being recognized as the principal milk protein expressed in pregnant or lactating females of several mammalian species. Recently, it has been shown that the 6.3-kb 5' untranslated region of the rWAP gene is able to control, and almost completely restrict, the expression of the transgene into the mammary gland of the transgenic animal. We cloned the genomic fragment carrying the rWAP gene locus from the rabbit phage genomic library and used the 8.5-kb long 5' untranslated part of the rWAP gene to target the expression of hEPO, cloned from the human phage genomic library, into the mammary gland of the mouse. The vectors, carrying either the hEPO gene or the rWAP-hEPO hybrid gene, were injected into the mouse ova, and 12 transgenic animals were identified by PCR and Southern blot from the progeny of 168 tested littermates. Transgenic mice were viable, fertile and displayed a normal development. Recombinant human erythropoietin was produced in the milk of a transgenic mouse female at a secretion level of 5.3 mIU/ml, as detected by ELISA. Despite the low production of the transgenic glycoprotein in the milk we demonstrate that the hybrid gene can be expressed in the mammary gland of the host animal. Thus, WAP-based recombinant vectors, with additional optimizing modifications, can be useful for production of therapeutic proteins in the transgenic mammals.

[Achievements and applications in making chicken chimeras using BCs].

The technology of producing chicken chimeras using blastodermal cells is very important not only in the field of transgenic chicken bioreactor but also in searching for efficient ways to conserve avian genetic resource. The basic processes for producing chicken chimeras consist of: (1) Setting up the color model; (2) Separating and dissociating of donor embryos; (3) Compromising of the recipient embryos; (4) Windowing and recovering the recipient eggs; (5) Cells injecting; (6) Method of hatching. The progress, obstacles and prospects of producing chicken chimeras via BCs were discussed in this paper.

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.

Preparation of fowl testes as recipient organs to germ-line chimeras by means of gamma-radiation.

1. The aim of the study was to investigate the possibility of preparing adult fowl testes for the production of exogenous germ-lines by eradication of recipient spermatogenesis using gamma-radiation. 2. A comparison between several radiation therapy treatments (based on 60Co isotope) of male testes was conducted using gamma-rays of 18, 22 and 26 Gy in a single dose or repeated doses of 5 x 8 Gy over a 15-d period. Sperm concentration and motility were determined after each treatment. 3. Altered spermatogenesis was observed after a single treatment dose of 18 Gy, while single doses of 26 Gy were followed by reduced sperm numbers (from 22 x 10(9) to 31 x 10(6) sperm/ml) within 60 to 100 d after treatment. After a single treatment of 26 Gy sperm motility was reduced by 50%. In contrast, a fractionated treatment (5 x 8 Gy) with gamma-rays halted spermatogenesis 39 d after the distribution of the first 8 Gy dose. 4. Observations of the seminiferous tubules by electron microscopy performed 12 months after this treatment confirmed that moderate doses of gamma-rays (8 Gy) distributed repeatedly (5 x) over a limited period (15 d) sterilise adult fowl testes but maintain morphologically normal somatic (Leydig and Sertoli) cell populations.

Association of (3H) DNA with fowl spermatozoa and their in vitro fertilisation of hamster ova.

1. Fowl spermatozoa incubated with DNA were used for the fertilisation of zona-free hamster oocytes. 2. Two categories of spermatozoa were detected: those which were not labelled and those which showed a very high degree of labelling (19 +/- 2% of the whole population). 3. In the hamster oocytes exposed to treated spermatozoa the labelled sperm heads were identified.

Intramagnal insemination of hens can eliminate negative influence of lipofectin on fertilising ability of spermatozoa.

1. After intramagnal insemination egg production decreased drastically during the first two days and was equivalent to egg production of hens inseminated intravaginally for the remaining period of collection. 2. After magnal insemination, the fertility of eggs collected during the first week was 36.2% and only 3.6% during the second week. 3. In the case of intramagnal insemination, egg fertility in the first week was 88.1%, in the second week 81.8% and the third week 52.3%. 4. The eggs laid during the first day after intramagnal insemination were 83.3% fertile, indicating that treated spermatozoa fertilised the newly ovulated egg within 20 minutes of ovulation.

Response of avian nuclei to mammalian maturation promoting factor (MPF).

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.