Sally Stockwell

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.

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.

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.

Gene expression profiles of BMP4, FGF10 and cognate inhibitors, in the skin of foetal Merino sheep, at the time of secondary follicle branching

Abstract:  The high concentration of secondary branched follicles is a distinctive feature of the Merino sheep. These follicles initiate from 100 days of gestation. Here, we report a transition in abundance of the BMP4 and FGF10 morphogens occurring at this time. At 103 days of gestation, FGF10 gene expression dropped steadily from maximal levels, in a trend that continued until day 143. Conversely, from day 105, BMP4 transcript levels rapidly increased to maximal levels that were maintained until 131 days, before declining. This profile closely matches reported changes in branched follicle numbers, which peak in density at day 134. SPRY4, a known regulator of FGF10, increased to maximal levels concomitant with the fall in FGF10, suggesting a relationship. Levels of the BMP4 inhibitor NOG matched the initial rise of BMP4, with a fivefold spike at 108 days; but consistent with the rise in BMP4, this high level was not sustained.

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.

Examination of basement membrane components associated with the bovine seminiferous tubule basal lamina

Immunohistology has been used to examine the distribution of certain components of the basement membrane (BM) associated with bovine spermatogonial germ cells that are located within the seminiferous tubules. Histology was performed on testis tissue from Brahman cattle (Bos indicus) of three different age groups: pre-pubescent (4-6 months), juvenile (8-10 months) and adult (18-24 months) animals. There were no major changes in the BM composition apparent between these three age groups, except for certain lectin staining. These data suggest that the predominant collagen type IV component may have an alpha3 and alpha4 composition, although other chains, including the alpha5 and alpha6 chains, were also present. Possibly the main laminin type present was laminin 121 (alpha1beta2gamma1), although other variants were also present. Both nidogen-1 and perlecan, which are normal BM components, were also found as part of the seminiferous tubule BM. Interstitial collagens, such as type I, III and VI collagens, were found in the peritubular space, but were not part of the BM itself, although type VI collagen was most visible in the peritubular zone adjacent to the tubules. Examination of the BM with a range of lectins gave strong staining for (glcNAc)(2) entities, weak positive staining for alpha-l-fuc, but little or no staining for alpha-galNAc and (glcNAc)(3) at all ages, whereas staining for alpha-gal, beta-gal(1-->3)galNAc and alpha-man showed developmental changes.

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.

Depletion of testis cell populations in pre-pubertal Bos indicus cattle by irradiation

Recovery of spermatogenesis following a single dose of irradiation was evaluated in pre-pubertal Brahman bulls, after receiving a single dose of 3, 6, 9 or 12Gray (Gy) irradiation. Biopsy samples of testis tissue were collected and processed for immunohistology at various times following irradiation. Spermatogenic recovery was defined by the changes in tubule diameter, and absolute numbers of undifferentiated spermatogonia (PLZF positive cells) and Sertoli cells (GATA-4 positive cells) per tubule cross section. The effect of irradiation on the depletion of testicular cells was dose-dependent. Immunohistological results from both the 9 and 12Gy group showed degeneration of seminiferous tubules, compared with other doses and controls. From 2 weeks after the treatment, irradiation resulted in a significant and dramatic reduction in tubule diameter (up to 40%), number of undifferentiated spermatogonia (up to 90%) and Sertoli cells (up to 70%), which was sustained for up to 16 weeks post-irradiation in 9 and 12Gy groups (P<0.0001). However, a moderate depletion effect was observed in the 6Gy treatment groups, compared with 9 and 12Gy doses. The 6Gy treatment had significant effects on spermatogonia (up to 79% reduction) and Sertoli cell (30% reduction) numbers following irradiation (P<0.0001). In contrast, the 3Gy dose had no significant effect at either 3 or 5 weeks post-irradiation on tubule diameter, spermatogonia or Sertoli cells. In conclusion, the results from the current study suggest that treatment of recipient testes with a single dose of 6Gy irradiation can temporarily deplete spermatogonial cells in pre-pubertal Brahman bulls, whilst minimising the impact on Sertoli cells and tubule morphology.