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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.

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 vitrobench tests and in vivopreclinical 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 vivoperformance 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 vivoperformance 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.

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 Hellsheterozygote 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.

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.

Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys (Macaca mulatta) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.

Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technqiue can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.

Spermatogonial stem cells form the foundation of spermatogenesis, and their transplantation provides a unique opportunity to study spermatogenesis and may offer an alternative approach for animal transgenesis. This study was designed to extend the technique of spermatogonial transplantation to an economically important, large-animal model. Isolated immature pig testes were used to develop the intratesticular injection technique. Best results of intratubular germ cell transfer were obtained when a catheter was inserted into the rete testis under ultrasound guidance. The presence of infused dye or labeled cells was confirmed in the seminiferous tubules from 70 of 89 injected isolated testes. Infusion of 3–6 ml of dye solution or cell suspension could fill the rete and up to 50% of seminiferous tubules. The technique was subsequently applied in vivo. Donor cells included testis cells from 1- or 10-wk-old boars (from the recipients’ contralateral testis or unrelated donors) and those from mice carrying a marker gene. Porcine testis cells were labeled with a fluorescent marker before transplantation. Testes were examined for the presence and localization of labeled donor cells immediately after transplantation or every week for 4 wk. Labeled porcine donor cells were found in numerous seminiferous tubules from 10 of 11 testes receiving pig cells. These results indicate that germ cell transplantation is feasible in immature pigs, and that porcine transplanted cells are retained in the recipient testis for at least 1 mo. This study represents a first step toward successful spermatogonial transplantation in a farm animal species.

The mechanisms controlling the pulsatile release of gonadotropins in prepubertal heifers are not completely understood. We examined the role of excitatory amino acid neurotransmitters, via activation of N-methyl-d-aspartate (NMDA) receptors, in the control of pulsatile LH and FSH release during prepubertal development in heifers. Hereford heifer calves received 4.7 mg/kg of N-methyl-d,l-aspartic acid (NMA), a potent NMDA receptor agonist (n = 5, i.v.), or saline (n = 5, i.v.), as single doses, at 4, 8, 12, 24, 36, and 48 wk of age. Blood samples were collected every 15 min, for 1 h before and 9 h after injection, on the days of treatment. Injection of NMA resulted in an acute release of LH (p< 0.001) in 0, 3, 3, 4, 5, and 5 calves (p< 0.01) and of FSH (p< 0.001) in 0, 1, 2, 4, 3, and 2 calves at 4, 8, 12, 24, 36, and 48 wk of age, respectively. The peak response of LH and FSH release to NMA was at 15 min posttreatment, and these peak responses were highest at 36 wk of age (p< 0.05). We suggest that neuroexcitatory amino acids, through NMDA receptors, are involved in prepubertal development of LH and FSH secretion in heifer calves.

The study and manipulation of testis cells require their preservation for various length of time. There are situations where it is necessary to maintain cells in suspension for up to several days (e.g., shipment of cells between collaborating labs). In such cases, culturing the cells at 37°C may not be suitable and resorting to cryopreservation will compromise the overall cell viability. This study was designed to develop strategies for maintaining the viability of isolated testis cells at room or refrigeration temperatures. Testes were collected by castration of one-week-old piglets and transferred to the lab within 1 hour in ice-cold PBS. After removal of the tunica albuginea and connective tissue, testis parenchyma was minced using fine scissors and cells were dissociated following a two-step enzymatic digestion procedure (with 0.2% collagenase-IV + 0.1% hyaluronidase + 0.01% DNase-I at 37°C for 15 min, followed by 0.25% trypsin with 2.21 mM EDTA for an additional 5 min). Cells were filtered (40μm pore size) and depleted of erythrocytes with lysis buffer (0.156 M NH4Cl, 0.01 M Na2EDTA, 0.1 M KHCO3, Ph=7.4). The resultant cells were then kept at either room or refrigeration temperatures in polypropylene tubes containing one of the following media: PBS, DMEM, DMEM with FBS (10%, 20%, or 50%), FBS, or Leibovitz (L-15) (n≥8 replicates each). Cell viability was assessed daily for 6 consecutive days (day of cell isolation as Day 0) using trypan blue exclusion (0.4% solution). By Day 3, storage at refrigeration temperature maintained significantly more live cells than at room temperature in all preservation media, except in PBS. At this time, while under room temperature conditions no significant differences in cell viability were observed among different media, cells stored at refrigeration temperature in DMEM (76.4±2.6% of live cell compared to Day 0, Mean±SEM), DMEM+50% FBS (76.7±2.7), FBS (77.6±3), or L-15 (81±5.7) had significantly better viability than those in PBS (60.3±1.9). Similarly on Day 6, all media preserved significantly more live cells at refrigeration temperature than those at room temperature, with the exception of L-15 in which cells at room temperature had maintained a surprisingly high viability similar to those at refrigeration temperature. On Day 6 under room temperature, cells maintained in DMEM+50% FBS (51.3±2.6), FBS (40.7±6.2), or L-15 (53.7±9.1) had maintained significantly more viable cells than those in PBS (0±0), or DMEM+10% FBS (14.6±3.1). At refrigeration temperature, cells kept in DMEM+50% FBS (64±4.5), FBS (74.4±3.8), or L-15 (72.2±5.6) also survived significantly better than those in PBS (42.9±3.5). Based on these results, for refrigeration of porcine testis cells, DMEM+50% FBS, FBS, or L-15 are equally suitable media. This research was supported by NSERC and SHRF grants (AH).

Recovery of germ cells is an important tool to preserve the genetic resources of endangered animals. In adult males, sperm cannot always be collected and cryopreserved and testis tissue xenografting has shown little success. Formation of functional testis tissue after xenografting of dissociated testis cells has been reported for pigs and rodents and could be an alternative to the conservation of germ cells from adults. To develop this approach with bovid testis tissue, de novomorphogenesis of testis tissue was characterised in sheep testes. Preliminary work showed that isolated sheep testis cells are able to form testicular tissue de novobut germ cell differentiation had not occurred at 16 weeks postgrafting. Therefore, the objective of this study was to investigate if germ cell differentiation could be supported in testis tissue formed de novoby increasing time to tissue recovery or by increasing testosterone levels by co-grafting with intact testis tissue. Testes from 14 day old Suffolk lambs were processed in two ways: Part of every testis was cut into small tissue fragments and from the remaining testis tissue a single cell suspension was prepared by two-step enzymatic digestion. Eight pellets of 50x106cells were transplanted under the back skin of 4 NCR-nude mice and 4 cell pellets and 4 pieces of testis tissue were grafted into another 4 NCR-nude mice. At the time of transplantation the cell pellets contained 1.27 ± 0.50 % germ cells, 55.47 ± 5.19 % Sertoli cells, 6.13 ± 0.92 % myoid cells and 4.60 ± 1.39 % Leydig cells. Histological examination was performed at 35 and 40 weeks after grafting (n=4 mice per time point). At 35 weeks postgrafting, no differentiated germ cells were found in the isolated cells grafted alone but single cell aggregates co-grafted with tissue contained pachytene spermatocytes. The weight of the seminal vesicles, an indicator of circulating levels of testosterone, was significantly higher (291.7 ± 81.7 mg) in mice carrying cell and tissue co-grafts, than in mice carrying only cell grafts (83.2 ± 69.9 mg). At 40 weeks after grafting, the seminal vesicle weights had increased 2 fold in both groups. Elongated spermatids were present in grafts from cells transplanted alone while germ cells in grafts of cells co-grafted with tissue showed no differentiation. These results show that isolated sheep testis cells are able to reorganize and form functional testis tissue. More time is required to complete germ cell differentiation than in intact animals and the efficiency of spermatogenesis is low, possibly due to the low number of spermatogonia harvested after enzymatic digestion. Increased levels of testosterone accelerated the onset of spermatogenesis in single-cell grafts. De novoformation of testis tissue might provide a novel alternative for germ line preservation in bovid species.Supported by NIH/NCRR RR17359-05, USDA/CSREES/NRICGP 03-35203-13486 and Spanish Ministry of Education and Science (BES-2004-4112).

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.

Gonocytes can be identified in situ and in histological cross-sections by their unique morphological attributes and distinctive topography within the seminiferous cords/tubules; however, specific biomarkers are required for their accurate quantification. In search for such a specific biomarker for piglet gonocytes, we encountered an intense auto-fluorescence in both neonatal testis tissue and disassociated testis cells. This intrinsic fluorescence was not previously described in neonatal pig testis, and could blur the distinction between specific and non-specific immune-fluorescence signals, interfering with characterization of piglet gonocytes. Therefore, the aim of the present study was to examine this intrinsic fluorescence in both the piglet testis tissue and cells, followed by developing an effective method to block the auto-fluorescence. We found that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescent microscope, confocal laser scanning microscope, or flow cytometry. The emission wavelength of the auto-fluorescent substance ranged from 425 to 700 nm, a range that could potentially interfere with the commonly used fluorophores. Following treatment of the testis tissue sections with Sudan Black B for 10-15 min or testis cells for 8 min, the intrinsic fluorescence was completely masked, allowing specific staining of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin within Leydig cell granules was mainly responsible for the observed intrinsic fluorescence in piglet testes. The method developed in the present study will facilitate the identification and characterization of piglet gonocytes using immuno-fluorescence techniques.(poster)

The purpose of this study was to evaluate the long-term outcome of testis tissue xenografting from bison and deer as models for closely-related rare or endangered ungulates. Testis tissue was collected post-mortem from 2 newborn bison calves (Bison bison bison) and a 2-mo-old white-tailed deer fawn (Odocoileus virginianus). Small fragments of donor testis tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice for bison xenografts and n = 7 mice for deer xenografts; 8 fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo from grafting for up to 16 mo for bison xenografts, and 14 mo post-grafting for deer xenografts. The retrieved xenografts were evaluated for seminiferous tubular density (per mm2) 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, 69% of the grafted testis fragments from donor bison and 63% of those from donor deer were recovered as xenografts. The weight of bison testis tissue xenografts increased (P < 0.02) ~4-fold by 2 mo and ~10-fold by 16 mo post-grafting. In bison xenografts, gradual maturational changes were evident in the form of seminiferous tubule expansion starting at 2 mo, first appearance of spermatocytes at 6 mo, round spermatids at 12 mo, and elongated spermatids at 16 mo post-grafting. For bison grafts, a donor effect on efficiency of spermatogenesis was also observed. Testis tissue xenografts from donor white-tailed deer also showed a gradual testicular development starting with tubular expansion also by 2 mo and presence of spermatocytes by 6 mo post-grafting, but round and elongated spermatids by 8 mo, followed by fully-formed spermatozoa by 12 mo post-grafting. The timing of complete spermatogenesis roughly corresponds to the reported timing of sexual maturation in these species. This study demonstrated, for the first time, that testis tissue xenografting from neonatal/immature bison or deer donors into recipient mice can successfully result in testicular maturation and development of spermatogenesis in the grafts up to the stage of spermatozoa production. These results may therefore provide a model for salvaging genetic material from immature males of rare or endangered ungulates that die prematurely.(platform)

Preparation of the recipient testes can enhance the outcome of germ cell transplantation. Whether neonatal pig gonocytes have stem cell potential has not been shown. Therefore, the objectives of the present study were 1) to examine the effects of neonatal piglet testis irradiation on testis development and subsequent endogenous spermatogenesis, and 2) to investigate the potential of donor neonatal piglet gonocytes in establishing spermatogenesis after homologous transplantation into irradiated recipient testes. Nine-day-old piglets underwent daily local irradiation of testes using fractionated gamma-rays at doses of 0 (control), 1, 2 or 3 Gray (n = 6 piglets/group) for 3 consecutive days. Two months after irradiation, half of the pigs in each group were sacrificed and the testes examined. At the same time, the remaining pigs in each group were used as recipients for donor neonatal piglet testis cells. The donor cells were injected, through the rete testis under ultrasound guidance, into one testis of each irradiated and non-irradiated recipient animal, and the contra-lateral testis was injected with saline as an internal control. Two months after transplantation (i.e., 4 months after irradiation), pigs were sacrificed and the testes collected for examination. Compared with the control (0 Gray), at both 2- and 4 months post-irradiation, the testis weight indices from the group of pigs undergoing daily irradiation doses of 3 Gray were smaller, seminiferous tubule density from 2- and 3-Gray groups were lower, and tubule diameter in all irradiated testes were also lower (P < 0.05). Two months after irradiation with doses of 2 or 3 Gray, the relative number of germ cells per 1,000 Sertoli decreased (P < 0.05). At 4 months post-irradiation, spermatogenic differentiation up to elongating spermatids was observed in all non-irradiated testes, and while no spermatogenic development was found in any of the irradiated testes injected with only saline; after gonocyte transplantation, a small number of tubules in the groups of 1- and 2-Gray contained spermatogenesis up to elongating and round spermatids. In conclusion, local irradiation of recipient piglet testes with as low as 1 Gray (for 3 days) could completely suppress the endogenous spermatogenesis, and gonocyte transplantation into irradiated recipients led to initiation of spermatogenesis.(poster)

Gonocytes are the first identifiable germ cells in the testis and progenitors of spermatogonial stem cells. The objective of this study was to maximize gonocyte isolation yield to facilitate the in vitro study and manipulation of these male germline stem cells. Fresh testis tissues from 3-day-old piglets were subjected to mechanical dissociation (Protocols 1: Teasing, 2: Mincing, 3: Sieving, or 4: Grinding), enzymatic digestion (Protocol 5: Collagenase IV (Col)-Dnase1, 6: Col-Dnase1+Col-Dnase1, 7: Col-Dnase1+ Trypsin-EDTA, or 8: Col-Dnase1-Hyaluronidase+Trypsin-EDTA) and enzymatic digestion under cold temperature or pressurized conditions (Protocols 9: Col-Dnase1 for 5-hrs at 4°C followed by 37°C digestion, 10: Col-Hyaluronidase-Dnase1 for 5-hrs at 4°C + Trypsin-EDTA, 11: Pressurized-Col-Dnase1 for 5-hrs at 4°C + 37°C digestion, or 12: Pressurized-Col-Hyaluronidase-Dnase1 for 5-hrs at 4°C +Trypsin-EDTA) (n = 7 replicates each). Additionally, intact testes and testis tissue fragments (30 mg and 100 mg) were maintained at 4°C with PBS, DMEM, Liebovitzs L-15, L-15 with FBS (10%, 20% or 50%), Ham-F10, M199 or HypoThermosol-FRS for 6 days. On preservation Day 0, Day 3 and Day 6, testis tissues were digested (n = 6), and fixed for semi-quantitative morphological analyses (n = 4). Enzymatic methods resulted in the recovery of significantly more testis cells with significantly higher viability than mechanical dissociation methods, while Protocol 10 resulted in the highest cell yield (mean ± SEM, 27.14 ± 0.83×106per 100-mg testis tissue) and viability (95.91% ± 2.56), Protocol 4 had the lowest viability (36.17% ± 2.29) and Protocol 3 had the lowest cell yield (3.25 ± 0.84×106). Of the enzymatic digestion protocols, Protocol 8 yielded the highest total live cell number (22.72 ± 2.44×106) and its efficiency was significantly increased by cold enzyme treatment (26.02 ± 1.27×106). Compared to fresh tissue dissociation, total live cell number collected on Day 3 and Day 6 dropped significantly, with HTS-FRS (50.43% ± 16.65, 26.19% ± 17.94) and 20% FBS L-15 (45.94% ± 17.52, 21.43% ± 11.19) consistently resulting in the highest numbers. These two media also resulted in the best preservation of morphology. In conclusion, digestion of testis tissue with Col-Dnase1-Hyaluronidase followed by Trypsin-EDTA appears superior to other methods and could be enhanced by adding the enzymes during the cold temperature storage, prior to digestion at 37°C. Cooling of testis tissue may decrease the total live cell recovery by approximately 50% by Day 3 and 75% by Day 6.(poster)

Testicular cell isolation is a starting step for culture, enrichment, modification or transplantation of spermatogonial stem cells. Therefore, the efficiency of enzymatic dissociation of the testis tissue is a potential limiting factor for such stem cell applications. Protocols used for testis cell isolation vary depending on the species from which the testes are collected and among laboratories, and little research has compared the virtue of different protocols. Therefore in search of an optimized protocol, we started by comparing the dissociation efficiency of collagenase type I and IV, as either type has been used in similar protocols. The testes of oneweek old piglets were surgically removed, decapsulated, trimmed, minced and exposed to collagenase (either type I or IV, 0.2% in DMEM) for 30 min to be digested, followed by an additional 20 min digestion with hyaluronidase (0.2% in DMEM). The suspension was then washed (centrifuged at 350X gfor 5–7 min and the supernatant was discarded) and a combination of trypsin (0.25%) with EDTA (2.21 mM) and DNase I (0.2%) in HBSS was added for 15 min for further digestion, before fetal bovine serum (FBS) was supplied to stop the digestion. The suspension was washed again and resuspended in DMEM with 12% FBS and filtered through a 40 micrometer nylon filter. Cell populations were counted and the cell viability was determined using trypan blue exclusion. Total cell number harvested per gram testicular parenchyma were 332 ± 42 X 106(mean ± SEM, n = 13) and 335 ± 26 X 106 (mean ± SEM, n = 14), for collagenase type I and IV, respectively, displaying no significant difference. Cell viability rates were 78 ± 2% (mean ± SEM, n = 13) and 83 ± 2% (mean ± SEM, n = 14) for collagenase type I and IV, respectively, showing no significant difference (p = 0.1). These results show that in terms of the viability and cellular yield, collagenase type I and IV are equally efficient; however, our observations of the final populations of isolated cells seemed to suggest that collagenase type IV resulted in far fewer cell clusters than collagenase type I. Further research is required to evaluate the stem cell activity of the cell populations obtained from each protocol. (poster)

Lymphoid specific helicase (Lsh) is a member of the SNF2-helicase family of chromatin remodeling proteins. Lsh is ubiquitously expressed and essential in embryonic development. We demonstrated recently that Lsh plays a critical role in female meiosis. Because Lsh null mice die at birth, a potential phenotype in male meiosis cannot be studied in these animals. Grafting of tissue from mutant mice to immuno-tolerant wild type recipients allows the study of postnatal tissue differentiation even in animals with a neonatal lethal phenotype. Therefore, we employed allografting of testis tissue from Lsh -/- mice to immuno-deficient wild type host mice to enable the study of postnatal germ cell development and differentiation. Testes were harvested at day 18.5 of gestation from Lsh -/-, +/- and +/+ mice of 24 litters and grafted ectopically under the back skin of 30 immuno-deficient, castrated male recipient mice (3-6 grafts/mouse). Each mouse received at least one graft from each genotype. To compare proliferative activity of germ cells between Lsh +/+ and -/- testis tissue, 3 recipient mice were treated with BrdU for 1 h before grafts were harvested at 1 wk post grafting. Double immunocytochemistry for BrdU incorporation and a germ cell marker (UCHL1) revealed significantly fewer dividing germ cells in tissue grafts from Lsh -/- mice than in grafts from Lsh +/+ animals (22.4 + 3.8 % vs 39.2 + 11.8 %, respectively, P<0.05). Grafted tissue from the remaining mice was harvested at 4, 6 and 8 weeks after grafting (n= 6, 10 and 11 mice, respectively) and analyzed histologically for spermatogenic differentiation. While spermatogenesis proceeded through meiosis with the appearance of round and elongated spermatids in the seminiferous tubules in grafts from Lsh heterozygote and wild type donor testes, pachytene spermatocytes were the most advanced germ cell type present in grafts from Lsh -/- mice at all analysis time points. These results indicate that proliferation of spermatogonia is reduced and that germ cell differentiation is arrested in the absence of Lsh, indicating an essential role for Lsh also during male meiosis. This study also highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity. Meiotic germ cells harvested from testis tissue allografts can now be studied to define the mechanism underlying the observed arrest of spermatogenesis and to further characterize the role of Lsh in meiotic chromatin remodeling. Supported by RR17359 (NCRR/NIH) and by NCI contract # N01-C0-12400. (platform)