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Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique invivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive invivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100×106 cells were larger than those with 50×106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants.

Gonocytes are the only type of germ cells present in the postnatal testis and give rise to spermatogonial stem cells. Purification of gonocytes has important implications for the study and manipulation of these cells and may provide insights for the ongoing investigation of the male germline stem cells. To obtain a pure population of gonocytes from piglet testis cells, a wide range of Nycodenz concentrations were investigated for density gradient centrifugation. We also examined differential plating of testis cells for various culture durations with different extracellular matrix (ECM) components (fibronectin, poly-d-lysine, poly-L-lysine, laminin and collagen Types I and IV). Gonocytes were highly enriched in pellets of testis cells after using 17% Nycodenz centrifugation to a purity of 81±9%. After culturing testis cells on plates precoated with different ECM components for 120 min, the proportion of gonocytes increased among non-adherent cells (suspended in the medium), with fibronectin or poly-D-lysine resulting in the greatest (up to 85%) and laminin in the lowest (54%) gonocyte proportion. Combining the most promising ECM coatings (fibronectin and poly-d-lysine) and further extension of their culture duration to 240 min did not improve final gonocyte purity. However, centrifugation with 17% Nycodenz followed by differential plating with fibronectin and poly-d-lysine coating further purified gonocytes among the collected cells to >90%. These results provide a simple, quick and efficient approach for obtaining highly enriched populations of piglet gonocytes for use in the study and manipulation of these germline stem cells.

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 + or - 1.7%) and DPBS in the lowest (60.3 + or - 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 + or - 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.

Spermatogenesis from many mammalian species occurs in fragments of normal testis tissue xenografted to mice. Here we apply xenografting to the study of testicular pathology. Using the horse model, we investigated whether exposure to a permissive extratesticular environment in the mouse host would rescue spermatogenesis in cryptorchid testicular tissue or in tissue affected by idiopathic testicular degeneration (ITD). In cryptorchid tissue, where the extratesticular environment is abnormal, xenografting induced spermatogenesis up to meiosis in a subpopulation of seminiferous tubules. Thus, spermatogonia survive and partially retain their potential to differentiate in cryptorchid horse testes. In contrast, the primary defect in equine ITD is hypothesised to be tissue autologous. In support of this, xenografting did not restore spermatogenesis to tissue affected by ITD, thus confirming that the testis itself is primarily diseased. This outcome was not affected by supplementation of exogenous gonadotropins to the mouse host or by reconstitution of a normal reproductive regulatory axis supplied by functional porcine testicular xenografts. These studies demonstrate the usefulness of xenografting for the study of testicular pathology.

Gonocytes have germline stem cell potential and are present in the neonatal testis, comprising 5-10% of freshly isolated testis cells. Maximising the number and proportion of gonocytes among freshly isolated testis cells will greatly facilitate their subsequent purification and in vitro study and manipulation. Seven experiments were conducted to evaluate the effects of multiple factors on the efficiency of testis cell isolation from neonatal pigs. We found that the use of a lysis buffer led to elimination of erythrocytes without adversely affecting testis cell isolation. Approximately ninefold as many live cells could be harvested by enzymatic digestion of testis tissues compared with mechanical methods. Digestion with collagenase-hyaluronidase-DNase followed by trypsin resulted in the highest recovery of live cells. However, the proportion of gonocytes ( approximately 7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. Pretreatment of the tissue with cold enzymes increased the recovery of live testis cells. New strategies of combining a gentle enzymatic digestion with two rounds of vortexing resulted in the isolation of testis cells with very high gonocyte proportion. The efficiency of these novel methods could be further optimised to collect testis cells with a gonocyte proportion of approximately 40%. This novel three-step testis cell isolation strategy can be completed within 1 h and can harvest approximately 17 x 10(6) live gonocytes per g testis tissue. Therefore, in addition to elucidating the effects of several factors on testis cell isolation, we developed a novel strategy for the isolation of testis cells that yielded approximately 40% gonocytes in the freshly isolated cells (i.e. four- to eight-fold higher than the proportions obtained using current strategies). This strategy has instant applications in the purification of gonocytes.

The aim of the present study was to examine factors that may affect the outcome of testis tissue xenografting. Recipient factors were examined by grafting small fragments of testis tissue from newborn piglets under the back skin of immunodeficient mice of different strains (severe combined immunodeficiency (SCID) v. nude), sex (male v. female) and gonadal status (intact v. gonadectomised) using a factorial design (eight groups; n = 7 mice per group). Recipient mice were killed after 8 months to compare the gross and histological attributes of the recovered grafts. Overall, approximately 94% of grafts were recovered. Gonadectomy of male or female recipients did not affect any of the measured outcomes of testis tissue xenografting, therefore data were pooled. Overall, in terms of sex, male mice and, in terms of strain, SCID mice tended to show higher gross and histological development of grafts. The group of female nude mice had the lowest graft recovery rate (75%) compared with the other groups (95-100%; P < 0.05). The grafts from male SCID mice were, on average the largest and had the highest percentage of spermatozoa-containing seminiferous tubules among all the groups (P < 0.05). These results suggest that male SCID mice provide a suitable recipient model for testis tissue xenografting and that the mice do not need to be castrated for optimal results.

Embryo development after homologous intracytoplasmic sperm injection (ICSI) with sperm from testis tissue xenografts from pigs or any other farm animal species has not been evaluated critically. Here, we report development of porcine embryos in vitro following ICSI with sperm retrieved from xenografted neonatal pig testis. Small pieces of testis tissue from newborn piglets were grafted under the back skin of castrated immunodeficient mice (n = 4) and the xenografts were collected 8 months after grafting. Spermatozoa were recovered by mincing of the grafted tissue. For comparison, testicular, epididymal and ejaculated spermatozoa were also collected from mature boars. Oocytes injected with xenogeneic spermatozoa were either fixed to determine fertilisation processes (n = 89 in five replicates) or allowed to develop in vitro (n = 143 in four replicates). Xenogeneic porcine spermatozoa were fertilisation competent (24% v. 58%, 68%, 62% or 0% for xenogeneic v. control testicular, epididymal and ejaculated spermatozoa or no spermatozoa, respectively) and embryos developed to the blastocyst stage (8% v. 22%, 27%, 25% or 0%, respectively). These results demonstrate that porcine spermatozoa derived from immature testis tissue xenografted into mice are fertilisation competent, albeit at a lower rate than testicular, epididymal or ejaculated spermatozoa from control boars, and support embryo development after ICSI.