Search

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. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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 (4×105 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).>+ cells in comparison with POF rats that did not receive these cells (P < 0.05).> Angpt2 and KDR , showed increased and decreased expressions in POF ovaries, respectively (P < 0.05).>+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [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]

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. [ABSTRACT FROM AUTHOR]

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 in vivo 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 in vivo 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. We have previously shown that implantation of dissociated neonatal pig testis cells under the back skin of immunodeficient mice can lead to regeneration of functional testis tissue, although the results were inconsistent and inefficient. Here, by examining several factors, we dramatically improved the consistency, efficiency and predictability of the model. The testis cell implantation model can be used as an accessible system to study and manipulate testis tissue development and regeneration, and as a unique model for fertility preservation of prepubertal boys undergoing gonadotoxic cancer treatments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 x 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 x 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 8min 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. [ABSTRACT FROM AUTHOR]

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°C with high cell survival rate, and tissue morphology can be preserved for at least 6 days in HTS. [ABSTRACT FROM AUTHOR]

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 × 108 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. [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]

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 crypt-orchid 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. [ABSTRACT FROM AUTHOR]

The article discusses on the development of germ cell fate and seminiferous tubule in bovine testis xenografts. An initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting. Spermatogenesis was observed at the pachytene spermatocyte stage beyond four months of post-grafting. The data shows that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells.

Discusses the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. Full differentiation of the mouse testis allografts; Isolation of mature and viable sperm from the grafts in high concentrations; Intracytoplasmic injection of sperm into mouse oocytes.

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]