R. Davey

Irradiation Enhances the Efficiency of Testicular Germ Cell Transplantation in Sheep

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species.

A comparison of methods for preparing enriched populations of bovine spermatogonia

The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 x 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 microg mL(-1) laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 microg mL(-1) DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.

Expression of promyelocytic leukaemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential plating

Identification and enrichment of spermatogonial stem cells (SSCs) are critical steps in testis germ cell transplantation. The present study shows that expression of the protein promyelocytic leukaemia zinc-finger (PLZF) does not occur in all cells, only in gonocytes in neonatal testis (Stage 1) and a subpopulation of Type A spermatogonia in peripubertal (Stage 2), prepubertal (Stage 3) and post-pubertal (Stage 4) ovine testes. Dolichos biflorus agglutinin (DBA) lectin binding does not occur at any stage of testis development. The numbers of putative undifferentiated spermatogonia, germ cells and Sertoli cells were assessed by PLZF, VASA and vimentin staining, respectively. In paraffin sections, the percentage of PLZF-positive cells per tubule in samples derived from Stage 2 testis (12.2 + or - 2.8%) was twofold higher than that from Stage 1 testis (6.4 + or - 0.4%), but the percentages decreased in Stage 3 and Stage 4 testes (4.6 + or - 0.7% and 3.1 + or - 0.6%, respectively). Single cell suspensions from Stage 1 and Stage 2 testis were generated by two-step enzymatic digestion. The spermatogonia were enriched by 2 h and 2 + 16 h (overnight) differential plating on 0.2% gelatin-coated coated flasks. For Stage 1 testes, a sixfold increase in PLZF-positive cells was observed in 2 h differential plating and an almost 10-fold increase was produced following 2 + 16 h enrichment. There was less than a twofold increase in PLZF-positive cells between the 2 h and 2 + 16 h differential plating. A similar level of enrichment efficiency was also obtained for Stage 2 testis, but the percentage of PLZF-positive cells in the final enrichment was approximately one-third of that Stage 1. The efficiency of isolation and/or enrichment of PLZF-positive cells appears to depend on the maturity of the testis and the neonatal testis is better suited for isolation of gonocytes and/or putative SSCs.

Detection of tubule boundaries based on circular shortest path and polar‐transformation of arbitrary shapes

In studies of germ cell transplantation, counting cells and measuring tubule diameters from different populations using labelled antibodies are important measurement processes. However, it is slow and sanity grinding to do these tasks manually. This paper proposes a way to accelerate these processes using a new image analysis framework based on several novel algorithms: centre points detection of tubules, tubule shape classification, skeleton‐based polar‐transformation, boundary weighting of polar‐transformed image, and circular shortest path smoothing. The framework has been tested on a dataset consisting of 27 images which contain a total of 989 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually and the novel approach can achieve a better performance than two existing methods.

Microsatellite detection of donor-derived sperm DNA following germ cell transplantation in cattle

Although autologous and heterologous transplantation has resulted in colonisation of recipient testes in cattle, the ability of the transplanted spermatogonial stem cells to complete spermatogenesis has not yet been determined. The objective of the present study was to identify and validate microsatellite markers that can distinguish the genotype of different individuals and therefore can be used to detect the presence of donor DNA in recipient semen samples. In a previous study by this group, successful colonisation of recipient testes by heterologous transfer using a fluorescent dye was shown. In the present work, some of the same recipient animals were investigated further to monitor donor-derived sperm production. The bovine microsatellite detection method was developed specifically to test the ejaculates of the recipients and can also be used to pre-match individuals before germ cell transplantation. Semen was collected from the recipients 52-98 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In one of the recipients, all collected semen samples were shown to be positive for donor-derived cells; however, the percentage of donor spermatozoa in the recipient ejaculate declined with time. The donor DNA was also detected in both single cell suspensions and testis tissue from this recipient. These results demonstrate for the first time that testicular germ cell transplantation between different breeds of cattle is feasible and the recipients thereof are able to produce spermatozoa of donor origin. This technology has potential applications in livestock breeding systems and may provide an alternative to artificial insemination.

Cell Microarrays for the Screening of Factors That Allow the Enrichment of Bovine Testicular Cells

Cell microarrays can serve as high‐throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface‐engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue‐derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker™ staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell‐based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 x 10(6), 9 x 10(6), or 18 x 10(6)cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7+/-1.2%, P<0.05) compared to those cells in 20 ml freezing bags (46.7+/-0.1%) or 1.5 ml cryovials (46.3+/-2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2+/-0.6%) than a 10 min cooling (56.0+/-2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 x 10(6) and 18 x 10(6)cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.

Claudin-8 expression in Sertoli cells and putative spermatogonial stem cells in the bovine testis

Adhesion molecules are expressed by both adult and embryonic stem cells, with different classes of adhesion molecules involved in cell-membrane and intercellular contacts. In this study the expression of the adhesion molecule claudin-8 (CLDN8), a tight-junction protein, was investigated as a potential marker for undifferentiated spermatogonia in the bovine testis. We found that CLDN8 was expressed by both spermatogonia and a subset of Sertoli cells in the bovine testis. We also showed co-expression of GFRα1 in testis cells with CLDN8 and with Dolichos biflorus agglutinin-fluorescein isothiocyanate (DBA-FITC) staining. We observed co-enrichment of spermatogonia and CLDN8-expressing Sertoli cells in DBA-FITC-assisted magnetic-activated cell sorting (MACS), an observation supported by results from fluorescence-activated cell sorting analysis, which showed CLDN8-expressing cells were over-represented in the MACS-positive cell fraction, leading to the hypothesis that CLDN8 may play a role in the spermatogonial stem-cell niche.

A shorter interval between irradiation of recipient testis and germ cell transplantation is detrimental to recovery of fertility in rams

The objective of the current study was to identify an optimal time period for donor cell transplantation after irradiation in sheep. The testes of recipient rams were treated with a single dose of 15 Gray (Gy) irradiation followed by germ cell transplantation either 3 or 6 weeks later. Transplantation of donor cells at 6 weeks after irradiation resulted in production of donor sperm by all five recipient rams compared with 4 of 11 rams transplanted at 3 weeks. Rams transplanted 3 weeks post-irradiation appeared to show reduced libido and fertility. Two rams produced sperm with low motility (< 20%) and two other rams were azoospermic. More than 1 year after cell transfer, there were heavy infiltrates of CD45-positive cells and more fibrous tissue in 9 of 14 recipient testes (seven rams) that received cells 3 weeks after irradiation. Taken together, these results suggest that the interval between irradiation of recipients and germ cell transplantation affects the success rate of the procedure, with a 6-week interval preferable. The elevated inflammatory/immune reaction may be responsible, at least in part, for the reduced fertility and low libido observed in the rams that received cells 3 weeks post-irradiation.

Transplanted germ cells persist long-term in irradiated ram testes

Testicular germ cell transplantation provides a tool to study transgenesis, spermatogenesis and to increase production efficiency in livestock industries. Isolated testicular germ cells can be transplanted into testes of livestock breeds to generate sperm of donor origin. In sheep, methods have been developed previously to isolate cell populations from ram testes and transplant these into irradiated testes of recipient rams. This has resulted in rams producing sperm derived from the donor cells and a number of the recipient animals have produced donor-derived offspring from the introduced spermatogonial cells. Microsatellite genotyping data presented here demonstrates that these rams continue to produce sperm of donor origin for at least 5 years post-transplantation. This research provides new evidence of the stability of transplanted germ cells in a commercially important species, and with further refinements to cell isolation, transplantation and recipient preparation, this technology should find use in breeding systems to increase livestock production efficiency.