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In vitro spermatogenesis (IVS) has important applications including fertility preservation of prepubertal cancer patients; however, thus far, IVS has only been achieved using mouse models. To study the effects of growth factors on the maintenance of testicular tissue integrity, germ cell numbers, and potential induction of IVS using a porcine model, we cultured small testicular fragments (~2 mg) from 1-wk-old piglets under six different media conditions (DMEM + 10%KSR alone or supplemented with GDNF, bFGF, SCF, EGF, or a combination of all) for 8 weeks. Overall, tissues supplemented with GDNF and bFGF had the greatest seminiferous tubule integrity and least number of apoptotic cells. GDNF-supplemented tissues had the greatest number of gonocytes per tubule, followed by bFGF-supplemented tissues. There was evidence of gradual Sertoli cell maturation in all groups. Moreover, histological examination and the expression of c-KIT (a marker of differentiating spermatogonia and spermatocytes) and STRA8 (a marker of the pre/meiotic stage germ cells) confirmed the induction of IVS in all groups. However, GDNF- and bFGF-supplemented tissue cultures had greater numbers of seminiferous tubules with spermatocytes compared to other groups. In conclusion, overall, GDNF and bFGF supplementation better maintained the tissue integrity and gonocyte numbers and induced IVS in cultured testicular tissues. [ABSTRACT FROM AUTHOR]

Simple Summary: One out of every 650 children is affected by cancer, and about half of survivors become permanently infertile due to the side effects of cancer treatments. Pre-treatment testicular biopsies can be collected and cryopreserved for future use to uphold biological fatherhood. Currently, the only safe approach to produce haploid germ cells is through in vitro spermatogenesis (IVS), which has only been fully achieved in mice. Pigs are a more suitable animal model for the study of testicular development and IVS, not only because of their anatomical and physiological similarities to humans but also their long prepubertal period. This study explored optimal conditions for the long-term culture of neonatal porcine testicular tissues as a first step in achieving IVS from pigs, as a model. In this study, we investigated the effects of testicular tissue size, tissue preparation method, and serum source in media on long-term culture of neonatal porcine testicular tissues. We showed that small intact testicular tissue fragments (~2 mg) cultured in knockout serum replacement could be effectively maintained in vitro for up to 4 weeks of culture. Moreover, the presence of viable germ cells after 4 weeks of culture indicates that this approach may be applicable for IVS in pigs. Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) intervene in the COX (cyclooxygenase) pathways which generate two important inflammation mediators, prostaglandins (PGs) and leukotriene (LTs). Contradictory claims regarding the effect of NSAIDs in asthmatic patients continues to be an issue. The present study investigated the effects of COX inhibitors on the responsiveness of the tracheal tract and on the levels of LTC4 and PGE2 in cells of the bronchoalveolar lavage fluid in an allergic guinea pig model. Materials and Methods: Adult male Dunkin-Hartley guinea pigs (250 − 300 g) were divided into seven groups of six animals each. Four COX inhibitors, aspirin (200 mg/kg and 20 mg/kg), indomethacin (10 mg/kg), ketoprofen (10 mg/kg), and celecoxib (25 mg/kg), were given orally on day 17 to allergy induced guinea pigs at 0, 12, and 24 h before ovalbumin challenge on day 18. PGF2 and LT4 were measured in the bronchoalveolar lavage fluid as well as inflammatory cell count and total protein. Tracheal responsiveness to acetylcholine (Ach) and histamine (His) also was evaluated. Results: An augment in the response of the trachea to Ach and His, as well as overt allergenic signs including short breath, wheezing and sneezing, was observed. The most significant increase in tracheal hyper-responsiveness was observed in the ketoprofen-treated group with similar but less pronounced changes observed in the indomethacin-treated group. Although some variables increased with the aspirin and celecoxib treatments, overall the tracheal sensitivity was reduced. Inflammatory cells including eosinophils and neutrophils corresponded to the changes observed for each treatment group. Conclusion: Ketoprofen and indomethacin increased the tracheal sensibility to Ach and His; therefore, their administration is not recommended in patients susceptible to allergy. [ABSTRACT FROM AUTHOR]

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications. [ABSTRACT FROM AUTHOR]

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air–liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications. [ABSTRACT FROM AUTHOR]

Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks.

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., (© The authors.)