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Male reproductive capacity has fallen considerably in recent decades; in addition, the incidence of testicular cancer has increased in many developed countries. The cause of this phenomenon is unknown, but environmental toxicants are considered a major contributing factor. To study potential reproductive toxicants, robust in vitro testis models are needed. We have recently established a porcine testis organoid system with a high resemblance to the architectures of innate testis tissue. Here, we further investigated the testis morphogenesis, cell maturation, and endocrine function of the testis organoids. We also challenged this system with abiraterone, a steroidogenic inhibitor, to validate its suitability as an in vitro platform for endocrine toxicology tests. Our results showed that the testis cells in the organoids reorganize into testis cordal structures, and the cordal relative areas increase in the organoids over time of culture. Moreover, the diameters and cell numbers per cross-section of the cordal structures increased over time. Interestingly, Sertoli cells in the organoids gradually underwent maturational changes by showing increased expression of androgen receptors, decreased expression of the anti-müllerian hormone, and formation of the blood-testis barrier. Next, we confirmed that the organoids respond to hormonal stimulation and release multiple sex hormones, including testosterone, estradiol, and progesterone. Finally, we showed that the production of testosterone and estradiol in this system can be inhibited in response to the steroidogenic inhibitor. Taken together, our organoid system provides a promising in vitro platform for male reproductive toxicology studies on testis morphogenesis, somatic cell maturation, and endocrine production., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Ali Honaramooz reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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.

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.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P < 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P < 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P < 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Introduction: Cell-based therapies with certain cell types are touted as novel and hopeful therapeutic intervention in the clinical setting. Here, we aimed to assess the regenerative potential of c-Kit + cells in the rejuvenation of ovarian tissue and fertility rate in rat model of premature ovarian failure (POF). Methods: Rats were treated with 160 mg/kg/BW of 4-vinylcyclohexene dioxide for 15 days. Freshly enriched rat bone marrow-derived c-Kit + (MACS) and c-Kit - cells (4×10 5 cells/10 µL) were transplanted into the ovaries of treatment and control animals. Prior to transplantation as well as 2, 4, 6, and 8 weeks post-transplantation, randomly-selected rats were euthanized and ovarian tissues were subjected to pathophysiological examinations and real-time PCR analyses. Results: POF status was confirmed by the presence of pathological features and a decreased number of immature and mature follicles compared with the control group ( P  < 0.05). Histological examination revealed a substantial reduction of atretic follicles in POF rats receiving c-Kit + cells in comparison with POF rats that did not receive these cells ( P  < 0.05). Compared with the control samples, angiogenesis-related genes, Angpt2 and KDR , showed increased and decreased expressions in POF ovaries, respectively ( P  < 0.05). c-Kit + cells had potential to restore angiogenesis in the ovarian tissue within normal ranges. Systemic levels of FSH did not significantly change in pre- or post-transplantation time points for any group ( P  > 0.05). Notable reduction of collagen deposition was found in c-Kit-treated rats. Transplantation of c-Kit + cells also restored the reduced fertility rate ( P  < 0.05). Conclusion: The administration of c-Kit + cells can modulate angiogenesis and pathological changes, leading to the rejuvenation of ovarian function of a rat model of premature menopause., (© 2022 The Author(s).)

Limb wounds are common in horses and often develop complications. Intravenous multipotent mesenchymal stromal cell (MSC) therapy is promising but has risks associated with intravenous administration and unknown potential to improve cutaneous wound healing. The objectives were to determine the clinical safety of administering large numbers of allogeneic cord blood-derived MSCs intravenously, and if therapy causes clinically adverse reactions, accelerates wound closure, improves histologic healing, and alters mRNA expression of common wound cytokines. Wounds were created on the metacarpus of 12 horses. Treatment horses were administered 1.51-2.46 × 10 8 cells suspended in 50% HypoThermosol FRS, and control horses were administered 50% HypoThermosol FRS alone. Epithelialization, contraction, and wound closure rates were determined using planimetric analysis. Wounds were biopsied and evaluated for histologic healing characteristics and cytokine mRNA expression. Days until wound closure was also determined. The results indicate that 3/6 of treatment horses and 1/6 of control horses experienced minor transient reactions. Treatment did not accelerate wound closure or improve histologic healing. Treatment decreased wound size and decreased all measured cytokines except transforming growth factor-β3. MSC intravenous therapy has the potential to decrease limb wound size; however, further work is needed to understand the clinical relevance of adverse reactions.

The normal developmental anatomy and histology of the reproductive tract of the honey bee drone, Apis mellifera (Linnaeus, 1758), has been well documented. The post-emergence maturation changes of the accessory glands are likewise well understood, but the normal histological changes of the testicle undergoing physiologic atrophy are not well characterized. To address this knowledge gap, herein we describe the anatomy and sequential histological stages of normal testicular atrophy of drones sampled daily from emergence to sexual maturity in the spring (June) and early summer (July). Testicular histological changes during maturation are characterized by the following stages: I) conclusion of spermiogenesis; II) evacuation of spermatodesms from tubular lumens; III) progressive follicular cell atrophy, and IV) complete atrophy and collapse of testicular parenchyma. Tubular changes occur in a basilar to apical direction where segments closer to the vas deferens are histologically more mature than corresponding apical segments. In addition, the rate of testicular maturation was found to change with seasonal progression. This description of physiologic testicular atrophy should be useful for future studies investigating potential pathological effects of stressors on drone testes during sexual maturation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

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.

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.

Spermatogenic failure is believed to be a major cause of male infertility. The establishment of a testis organoid model would facilitate the study of such pathological mechanisms and open the possibility of male fertility preservation. Because of the complex structures and cellular events occurring within the testis, the establishment of a compartmentalized testis organoid with a complete spermatogenic cycle remains a challenge in all species. Since the late 20th century, a great variety of scaffold-based and scaffold-free testis cell culture systems have been established to recapitulate de novo testis organogenesis and in vitro spermatogenesis. The utilization of the hydrogel scaffolds provides a 3D microenvironment for testis cell growth and development, facilitating the reconstruction of de novo testis tissue-like structures and spermatogenic differentiation. Using a combination of different strategies, including the use of various scaffolding biomaterials, the incorporation of the living cells with high self-assembling capacity, and the integration of the advanced fabrication techniques, a scaffold-based testis organoid with a compartmentalized structure that supports in vitro spermatogenesis may be achieved. This article briefly reviews the current progress in the development of scaffold-based testis organoids while focusing on the scaffolding biomaterials (hydrogels), cell sources, and scaffolding approaches. Key challenges in current organoid studies are also discussed along with recommendations for future research., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interest disclosure The authors certify that there are no conflicts of interest., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

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 mm 3 , each) and cell aggregates (100 × 10 6 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., (© 2020 American Society of Andrology and European Academy of Andrology.)

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.

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 × 10 8 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 × 10 8 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.

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.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.

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.

Horses often sustain cutaneous wounds and healing can be prolonged and difficult to treat. Compared to body wounds, limb wounds heal slower and are more likely to develop exuberant granulation tissue. Differences in healing rates and exuberant granulation tissue formation is attributed to abnormal cytokine profiles. CXCL8 and its receptor CXCR2 are involved in acute inflammation whereas CXCL10 and its receptor CXCR3 are involved in inflammation resolution. β- arrestin-2 regulates inflammation through internalization of G-protein coupled receptors (GPCRs) including CXCR2 and CXCR3. Gene expression of these five inflammation associated proteins have not been previously identified in equine cutaneous tissue and may play a role in dysregulation of inflammation in equine limb wounds. The mRNA expression levels were measured using QuantiGene Plex Assay from cutaneous biopsies collected from surgically created wounds on the limb and thorax on days 0, 1, 2, 7, 14, and 33 from two horses. The mRNA expression levels were measured in mean fluorescent intensity and graphed. We were successful in identifying all five proteins for the first time in equine cutaneous tissue. Preliminary results suggest that there are different expression patterns for CXCL8, CXCR2 and β-arrestin-2 between the limb and thorax but not for CXCL10 and CXCR3. Differential regulation of CXCL8, CXCR2 and β-arrestin-2 may further explain why limb wounds heal differently than body wounds and warrants further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 × 10 4 testis cells/cm 2 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 × 10 4 testis cells/cm 2 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., Competing Interests: All experimental procedures involving animals were approved by the University of Saskatchewan’s Institutional Animal Care and Use Committee (Protocol# 20080042).Not applicable. Portions of this study were presented in the form of an abstract at the 41st American Society of Andrology annual conference.The authors declare that they have no competing interest.

Three-dimensional (3D)-printed constructs made of polycaprolactone and chondrocyte-impregnated alginate hydrogel (hybrid cartilage constructs) can mimic the biphasic nature of articular cartilage, thus offering promise for cartilage tissue engineering applications. Notably, the regulatory pathway for medical device development requires validation of such constructs through in vitro bench tests and in vivo preclinical examinations for premarket approval. For this, noninvasive imaging techniques are required for effective evaluation of the progress of these cartilage constructs, especially when implanted in animal models or human subjects. However, characterization of the individual components of the hybrid cartilage constructs and their associated time-dependent structural changes by currently available noninvasive techniques is challenging as these constructs contain a combination of hydrophobic and hydrophilic biomaterials with different refractive indices. In this study, we report the use of a novel synchrotron radiation inline phase contrast imaging computed tomography (SR-inline-PCI-CT) approach for noninvasive (in situ) characterization of 3D-printed hybrid cartilage constructs that has been implanted subcutaneously in mice over a 21-day period. In parallel, traditional invasive assays were used to evaluate the in vivo performance of the implanted hybrid cartilage constructs with respect to their cell viability and secretion of cartilage-specific extracellular matrix over the 21-day period postimplantation in mice. SR-inline-PCI-CT allowed striking visualization of the individual components within the 3D-printed hybrid cartilage constructs, as well as characterization of the time-dependent structural changes after implantation. In addition, the relationship between the implanted constructs and the surrounding tissues was delineated. Furthermore, traditional assays showed that cell viability within the cartilage constructs was at least 70% at all three time points, and secretion of alcian blue- and collagen type 2-positive matrices increased progressively over the 21-day period postimplantation. Overall, these results demonstrate that the 3D-printed hybrid cartilage constructs have good in vivo performance and validate their potential for regeneration of articular cartilage in vivo. In addition, SR-inline-PCI-CT has demonstrated potential for longitudinal and noninvasive monitoring of the functionality of 3D-printed hybrid cartilage constructs in a way that is translatable to other soft tissue engineering applications.

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed., (© 2016 S. Karger AG, Basel.)

In tissue engineering, non-invasive imaging of biomaterial scaffolds and tissues in living systems is essential to longitudinal animal studies for assessments without interrupting the repair process. Conventional X-ray imaging is inadequate for use in soft tissue engineering due to the limited absorption difference between the soft tissue and biomaterial scaffolds. X-ray phase-based imaging techniques that derive contrast from refraction or phase effects rather than absorption can provide the necessary contrast to see low-density biomaterial scaffolds and tissues in large living systems. This paper explores and compares three synchrotron phase-based X-ray imaging techniques-computed tomography (CT)-diffraction enhanced imaging (DEI), -analyzer based imaging (ABI), and -phase contrast imaging (PCI)-for visualization and characterization of low-density biomaterial scaffolds and tissues in situ for non-invasive soft tissue engineering assessments. Intact pig joints implanted with polycaprolactone scaffolds were used as the model to assess and compare the imaging techniques in terms of different qualitative and quantitative criteria. For long-term in vivo live animal imaging, different strategies for reducing the imaging radiation dose and scan time-reduced number of CT projections, region of interest, and low resolution imaging-were examined with the presented phase-based imaging techniques. The results demonstrated promising capabilities of the phase-based techniques for visualization of biomaterial scaffolds and soft tissues in situ. The low-dose imaging strategies were illustrated effective for reducing the radiation dose to levels appropriate for live animal imaging. The comparison among the imaging techniques suggested that CT-DEI has the highest efficiency in retaining image contrast at considerably low radiation doses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This article presents the data of using three phase-based X-ray imaging techniques to characterize biomaterial scaffold and soft tissues in situ, as reported in our study "Low-dose phase-based X-ray imaging techniques for in situ soft tissue engineering assessments" [1]. The examined parameters include the radiation dose, scan time, and image quality, which are all critical to longitudinal in situ live animal assessments. The data presented were obtained from three dimensional imaging of scaffolds in situ cartilage by means of synchrotron-based computed tomography-diffraction enhanced imaging (CT-DEI), analyzer based imaging (CT-ABI), and in-line phase contrast imaging (CT-PCI) at standard and low dose imaging modalities.

Elk velvet antler (EVA) has been used in traditional Oriental medicine for centuries to promote general health; however, little evidence for its effect on bone development is available. We investigated the effects of lifelong exposure of Wistar rats to a diet containing 10% EVA on physical growth and bone development. Measurements included weekly body weights, blood chemistry and kidney and testis/ovary indices (sacrificed at 5, 9, or 16 weeks of age), and bone traits of the femur bones by peripheral quantitative computed tomography (pQCT). Mean body weights were higher in the EVA group at 4-8 weeks in males and at 5 weeks of age in females. The kidney indices were greater in EVA dietary supplemented male rats at 5 and 16 weeks of age, in females at 16 weeks of age, and testis/ovary indices at 5 weeks of age. The femoral length was increased in both males and females at 5 weeks, and several pQCT-measured parameters had increased in EVA males and females. The activity of alkaline phosphatase (ALP) increased in EVA group while the content of calcium and phosphorus did not differ among groups. Our results seem to support a role for dietary supplementation of EVA on growth and bone development in this model.

Brominated flame retardants are chemicals with fire quenching properties which are extensively used in manufacturing. Historically, less regulated use of legacy brominated flame retardants (BFRs) for a number of years has resulted in ubiquitous contamination of the environment. As a result, some of the more persistent BFRs have been phased out and are being replaced by a next generation of brominated compounds for which there is little toxicological data. The study investigated effects of 2-ethylhexyl tetrabromobenzoate (TBB), 1,2,5,6-tetrabromocyclooctane (TBCO), and bis-(2-ethylhexyl) tetrabromophthalate (TBPH)on steroidogenesis in a porcine primary testicular cell model. TBB did not affect sex-steroid production in this cell model; rather the data suggest a flux towards synthesis of aldosterone and cortisol via up-regulation of CYP21A2. At the greatest concentrations of TBCO and TBPH tested greater production of sex hormones testosterone (T) and estradiol (E2) was observed. Effects were mediated by regulation of multiple molecular targets in the steroidogenesis pathway; CYP11A in the case of TBPH and CYP17A1 in the case of TBCO. This investigation is the first of its kind to use a testicular mixed population cell model to investigate mechanism(s) of action of three chemically diverse compounds currently used in commercial fire retardants.

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

The purpose of this study was to evaluate the long-term outcome of testis tissue xenografting from immature deer. Testis tissue was collected post-mortem from a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 7 mice; 8 fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo from grafting for up to 14 mo post-grafting. The retrieved xenografts were evaluated for seminiferous tubular density (per mm(2)) and tubular diameter, as well as for seminiferous tubular morphology and identification of the most advanced germ cell type present in each tubule cross section. Overall, 63% of the grafted testis fragments were recovered as xenografts. Testis tissue xenografts showed a gradual testicular development starting with tubular expansion by 2 mo, presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed sperm by 12 mo post-grafting. The timing of complete spermatogenesis generally corresponded to the reported timing of sexual maturation in white-tailed deer. This study demonstrated, for the first time, that testis tissue xenografting from immature deer donors into recipient mice can successfully result in testicular maturation and development of spermatogenesis in the grafts up to the stage of sperm production. These results may therefore provide a model for salvaging genetic material from immature male white-tailed deer that die before reaching sexual maturity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10-15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.

Elk velvet antler (EVA) has a traditional use for promotion of general health. However, evidence of EVA effects at different lifestages is generally lacking. This paper investigated the effects of long-term maternal dietary EVA supplementation on physical, reflexological and neurological development of rat offspring. Female Wistar rats were fed standard chow or chow containing 10% EVA for 90 days prior to mating and throughout pregnancy and lactation. In each dietary group, 56 male and 56 female pups were assessed for physical, neuromotor, and reflexologic development postnatally. Among the examined physical developmental parameters, incisor eruption occurred one day earlier in pups nursing dams receiving EVA. Among neuromotor developmental parameters, duration of supported and unsupported standing was longer for pups nursing EVA supplemented dams. Acquisition of neurological reflex parameters (righting reflex, negative geotaxis, cliff avoidance acoustic startle) occurred earlier in pups nursing dams receiving EVA. Longterm maternal EVA supplementation prior to and during pregnancy and lactation accelerated certain physical, reflexologic, and neuromotor developmental milestones and caused no discernible adverse effects on developing offspring. The potential benefits of maternal EVA supplementation on postnatal development warrants further investigation to determine whether EVA can be endorsed for the promotion of maternal and child health.

The objective was to evaluate the long-term outcome of testis tissue xenografting from neonatal bison calves as a model for closely related rare or endangered ungulates. Testis tissue was collected postmortem from two newborn bison calves (Bison bison bison) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice; eight fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo after grafting (for up to 16 mo). The retrieved xenografts were evaluated for seminiferous tubular density, tubular diameter, seminiferous tubular morphology, and identification of the most advanced germ cell type. Overall, 69% of the grafted testis fragments were recovered as xenografts. Xenografts weight increased (P < 0.02) approximately four-fold by 2 mo and 10-fold by 16 mo post-grafting. In testis xenografts, gradual maturational changes were evident, manifested as the first detection of the following at the times specified: seminiferous tubule expansion, 2 mo; spermatocytes, 6 mo; round spermatids, 12 mo; and elongated spermatids, 16 mo. Furthermore, there were differences between the two donor calves regarding the efficiency of spermatogenesis in xenografts. The timing of complete spermatogenesis approximately corresponded to the reported timing of sexual maturation in bison. This study demonstrated, apparently for the first time, that testis tissue xenografting from neonatal bison donors into recipient mice resulted in testicular maturation and complete development of spermatogenesis in the grafts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.

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.

Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor-derived sperm production. Therefore, this system represents a major development in the study of spermatogenesis and a unique functional assay to determine the developmental potential and relative abundance of spermatogonial stem cells in a given population of testis cells. The application of this method in farm animals has been the subject of an increasing number of studies, mostly because of its potential as an alternative strategy in producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. This paper highlights the salient recent research on germ cell transplantation in farm animals. The emphasis is placed on the current status of the technique and examination of ways to increase its efficiency through improved preparation of the recipient animals as well as isolation, purification, preservation, and transgenesis of the donor germ cells.

To optimize the procedure for testis tissue xenografting, we grafted 2, 4, 8, or 16 small fragments of immature porcine testis tissue under the back skin of immunodeficient castrated mice (n = 10 mice/group). At 8 months post grafting, the graft recovery rate did not differ between groups; however, not only the total but also the average graft weights were higher (by approximately 12-fold and approximately 2.5-fold, resp.) in mice receiving 16 fragments than those receiving 2 fragments (P < .05). The recipient mice with 16 fragments had the largest vesicular glands (indicators of testosterone release by the grafts) compared with those with 2 fragments (P = .007). The grafts in the group of 16 fragments also had more (P < .05) percentage of tubules with round spermatids than those of the group of mice receiving 2 fragments. Therefore, recipient mice can be grafted with at least 16 testis tissue fragments for optimal results.

The objective of this study was to develop effective strategies for hypothermic preservation of immature porcine testis tissue to maintain structural integrity and cell viability. In Experiment 1, testes from 1-week-old piglets were used to study the effects of tissue sample size (as intact testes or fragments of 100-or 30 mg) and the use of one of 9 different media on hypothermic preservation of the testis tissue for 6 days. The examined media included: Dulbecco's phosphate-buffered saline (DPBS), Dulbecco's modified Eagle's medium (DMEM), Leibovitz L15 (L15), L15 with fetal bovine serum (FBS, at 10%, 20% or 50%), HypoThermosol solution-FRS (HTS), Ham's F12, and Media 199. On days 0, 3, and 6, testis tissues were digested to compare the cell survival rates. Tissue sections were also semi-quantitatively assessed to determine the efficiency of different preservation strategies. There was no effect of testis sample size (P > 0.05), but cell survival rates of testis cells isolated from preserved testis tissues changed depending on the media and day (P < 0.05). Testis tissue within HTS did not show morphological changes after 6 days. In Experiment 2, two of the top performing media (20% FBS-L15 and HTS) were selected for immunocytochemical detection of gonocytes. Proportions of gonocytes (%) in isolated testis cells, however, did not differ between the two media on days 0, 3, or 6. These results show that testis tissue can be maintained for 3 days at 4 degrees C with high cell survival rate, and tissue morphology can be preserved for at least 6 days in HTS.

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.

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.

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.

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%.

Recovery of germ cells could be an option for preservation of the genetic pool of endangered animals. In immature males, xenografting of testis tissue provides the opportunity to recover sperm from these animals. In adult animals, xenografting has been less successful, but de novo morphogenesis of functional testis tissue from dissociated testis cells could be an alternative. To assess the potential use of these techniques in endangered bovid species, the domestic sheep was used as a model. Testes from 2-week-old lambs were grafted as tissue fragments or cell suspensions into nude mice. Grafts were recovered at 4, 8, 12 and 16 weeks post grafting. For isolated cells, two additional time points at 35 and 40 weeks after grafting were added. In addition, to analyse the possible effect of social stress among mice within a group on the development of the grafts, testis tissue grafts were recovered 13 weeks post grafting from mice housed individually and in groups. Complete spermatogenesis occurred in sheep testis xenografts at 12 weeks, similar to the situation in situ. Isolated sheep testis cells were able to reorganize and form functional testicular tissue de novo. Housing mice individually or in groups did not have any effect on the development of xenografts. Xenografting of testis tissue might be useful to obtain sperm from immature endangered ungulates that die prematurely. Testis tissue de novo morphogenesis from isolated cells could open interesting options to recover germ cells from mature males with impaired spermatogenesis.

Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences.

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.

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.

During mammalian development, morphogenesis of the testis requires the coordinated interplay of somatic cells to form seminiferous cords in which the primitive germ cells reside. These cords are the precursor of the functional male gonad and as such form the basis of male fertility. Cell migration during mammalian organogenesis and formation of complex tissues, such as the testis, are difficult to study in situ. Herein, we report extensive rearrangement of cells to regenerate complete functional testis tissue after implantation of isolated neonatal porcine testis cells under the skin of immunodeficient mice. Somatic cells and germ cells reorganized into structures that have remarkable morphologic and physiologic similarity to normal testis tissue, forming the endocrine and spermatogenic compartment of the testis. This unique in vivo system provides an accessible model for the study of testicular morphogenesis that could be especially useful in nonrodent species.

Spermatogenesis can occur in testis tissue from immature bulls ectopically grafted into mouse hosts; however, efficiency of sperm production is lower than in other donor species. To elucidate a possible mechanism for the impaired spermatogenesis in bovine testis xenografts, germ cell fate and xenograft development were investigated at different time points and compared with testis tissue from age-matched calves as controls. Histologically, an initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting without an increase in germ cell apoptosis. From 2 months onward, the number of germ cells increased. In contrast, a continuous increase in germ cell number was seen in control tissue. Pachytene spermatocytes were observed in some grafts before 4 months, whereas in the control tissue they were not present until 5 months of age. Beyond 4 months post-grafting spermatogenesis appeared to be arrested at the pachytene spermatocyte stage in most grafts. Elongated spermatids were observed between 6 and 8 months post-grafting, similar to the controls, albeit in much lower numbers. Lumen formation started earlier in grafts compared with controls and by 6 months post-grafting tubules with extensively dilated lumen were observed. A donor effect on efficiency of spermatogenesis was also observed. These results indicate that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells and impaired meiotic and post-meiotic differentiation. The characterization of spermatogenic efficiency will provide the basis to understand the control of spermatogenesis in testis grafts.