Using a testis regeneration model, FGF9, LIF, and SCF improve testis cord formation while RA enhances gonocyte survival.

Implantation of testis cell aggregates from various donors under the back skin of recipient mice results in de novo formation of testis tissue. We used this implantation model to study the putative in vivo effects of six different growth factors on testis cord development. Recipient mice (n = 7/group) were implanted with eight neonatal porcine testis cell aggregates that were first exposed to a designated growth factor: FGF2 at 1 µg/mL, FGF9 at 5 µg/mL, VEGF at 3.5 µg/mL, LIF at 5 µg/mL, SCF at 3.5 µg/mL, retinoic acid (RA) at 3.5 × 10-5 M, or no growth factors (control). The newly developed seminiferous cords (SC) were classified based on their morphology into regular, irregular, enlarged, or aberrant. Certain treatments enhanced implant weight (LIF), implant cross-sectional area (SCF) or the relative cross-sectional area covered by SC within implants (FGF2). RA promoted the formation of enlarged SC and FGF2 led to the highest ratio of regular SC and the lowest ratio of aberrant SC. Rete testis-like structures appeared earlier in implants treated with FGF2, FGF9, or LIF. These results show that even brief pre-implantation exposure of testis cells to these growth factors can have profound effects on morphogenesis of testis cords using this implantation model.

Brief exposure of neonatal testis cells to EGF or GDNF alters the regenerated tissue.

We have previously shown that implantation of testis cell aggregates under the back skin of immunodeficient mice results in de novo regeneration of testis tissue. We used this unique model to investigate the effects of epidermal growth factor (EGF) and glial cell-derived neurotrophic factor (GDNF) on testis cord development. Neonatal piglet testis cells were briefly (<1 h) exposed to either low (L: 0.02 µg/mL) or high (H: 2 µg/mL) doses of EGF, GDNF, or vehicle (control), before implantation in recipient mice. Randomly selected implants were removed from each mouse at 1, 2, 4, and 8 weeks post-implantation. GDNF-L implants showed increased testis cord development over time, and EGF-L implants had increased cross-sectional area. The ratio of regular cords decreased over time in EGF-H and GDNF-H implants and was replaced by a higher ratio of irregular cords in GDNF-H. EGF-L and GDNF-H implants were quickest to display rete testis-like structures. Overall, the lower dose of each growth factor was more effective than its higher dose in improving the implantation outcomes. This is the first comprehensive assessment of these key growth factors on de novo formation (regeneration) of testis tissue. Lay summary: In recent decades, testicular cancer rates have quadrupled in young men while sperm counts have dropped by half. Both conditions may be related to exposure of fetuses or infants to noxious substances causing disruption of normal testis development. To study the effects of any putative factor on testis development, we established an animal model of testis tissue regeneration. We collected newborn piglet testes after routine castration, used enzymes to completely dissociate testis cells, exposed the cells to two key growth factors (EGF or GDNF), and implanted the cells under the back skin of recipient mice, acting as live incubators. We then examined implant samples after 1, 2, 4, or 8 weeks and assessed testis regeneration. Overall, the high dose of each growth factor had adverse effects on the formation of normal testis. Therefore, this novel implantation model may also be used to study the effects of potentially harmful substances on testis development.

Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy.

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

Homing and Engraftment of Intravenously Administered Equine Cord Blood-Derived Multipotent Mesenchymal Stromal Cells to Surgically Created Cutaneous Wound in Horses: A Pilot Project.

Limb wounds on horses are often slow to heal and are prone to developing exuberant granulation tissue (EGT) and close primarily through epithelialization, which results in a cosmetically inferior and non-durable repair. In contrast, wounds on the body heal rapidly and primarily through contraction and rarely develop EGT. Intravenous (IV) multipotent mesenchymal stromal cells (MSCs) are promising. They home and engraft to cutaneous wounds and promote healing in laboratory animals, but this has not been demonstrated in horses. Furthermore, the clinical safety of administering >1.00 × 108 allogeneic MSCs IV to a horse has not been determined. A proof-of-principle pilot project was performed with two horses that were administered 1.02 × 108 fluorescently labeled allogeneic cord blood-derived MSCs (CB-MSCs) following wound creation on the forelimb and thorax. Wounds and contralateral non-wounded skin were sequentially biopsied on days 0, 1, 2, 7, 14, and 33 and evaluated with confocal microscopy to determine presence of homing and engraftment. Results confirmed preferential homing and engraftment to wounds with persistence of CB-MSCs at 33 days following wound creation, without clinically adverse reactions to the infusion. The absence of overt adverse reactions allows further studies to determine effects of IV CB-MSCs on equine wound healing.

The study and manipulation of spermatogonial stem cells using animal models.

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.

Validation of ultrasound biomicroscopy for the assessment of xenogeneic testis tissue grafts and cell implants in recipient mice.

BACKGROUND: Subcutaneous grafting/implantation of neonatal testis tissue/cells from diverse donor species into recipient mice can be used as an in vivo model to study testis development, spermatogenesis, and steroidogenesis. Ultrasound biomicroscopy (UBM) allows obtaining high definition cross-sectional images of tissues at microscopic resolutions. OBJECTIVES: The present study was designed to (a) validate the use of UBM for non-invasive monitoring of grafts/implants overtime and to (b) correlate UBM findings with the morphological attributes of recovered grafts/implants. MATERIALS AND METHODS: Testis tissue fragments (~14 mm3 , each) and cell aggregates (100 × 106 cells, each) obtained from 1-week-old donor piglets (n = 30) were grafted/implanted under the back skin of immunodeficient mice (n = 6) in eight analogous sites per mouse. Three-dimensional transcutaneous Doppler UBM was performed, and a randomly selected graft and its corresponding implant were recovered at 2, 4, 6, and 8 weeks. RESULTS: Graft/implant weight (P = .04) and physical height (P = .03) increased overtime. The dynamics of physical length and volume increases over time differed between tissue grafts and cell implants (P = .02 and 0.01 for sample type*time interactions, respectively). UBM-estimated volume was correlated with the post-recovery weight and volume of the grafts/implants (r = 0.98 and r = 0.99, respectively; P < .001). Pre- and post-recovery length and height of the grafts/implants were positively and strongly correlated (r = 0.50, P = .01; r = 0.70, P = .001) and so were the areas covered by cordal, non-cordal, or fluid-filled cavities between UBM and histology (r = 0.87, P < .001). DISCUSSION AND CONCLUSION: UBM findings correlated with physical attributes of the grafts/implants, validating its use as a non-invasive high-fidelity tool to quantify the developmental changes in ectopic testis tissue grafts and cell implants, potentially leading to a reduction in the number of recipient mice needed for similar experiments.

Regeneration of testis tissue after ectopic implantation of porcine testis cell aggregates in mice: improved consistency of outcomes and in situ monitoring.

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.

Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes.

BACKGROUND: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens. RESULTS: Confluency of cells was optimal (>90% by ~6 d) when 3.0 × 104 testis cells/cm2 containing ~40% gonocytes were used. Incubating the cells at 35 °C or 37 °C resulted in similar cell number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradually settled over the next 5 d. Consequently, not changing the medium for 7 d (as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes". CONCLUSION: We found that gonocytes require the presence of a critical minimum number of somatic cells for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cells/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.