Testicular Cell Transplantation Research Articles

Optimizing Immature Testicular Tissue and Cell Transplantation Results: Comparing Transplantation Sites and Scaffolds.

For patients who had testicular tissue cryopreserved before receiving gonadotoxic therapies, transplantation of testicular tissues and cells has been recommended as a potential therapeutic option. There are no studies that indicate the generation of sperm after human immature testicular tissue (ITT) or spermatogonial stem cells (SSCs) transplantation. The use of releasing scaffolds and localized drug delivery systems as well as the optimizing transplantation site can play an effective role in increasing the efficiency and improving the quality of testicular tissue and cell transplantation in animal models. Current research is focused on optimizing ITT and cell transplantation, the use of releasing scaffolds, and the selection of the right transplantation site that might restore sperm production or male infertility treatment. By searching the PubMed and Google Scholar databases, original and review papers were collected. Search terms were relevant for SSCs and tissue transplantation. In this review, we'll focus on the potential advantages of using scaffolds and choosing the right transplantation site to improve transplantation outcomes.

Cyp11a2 Is Essential for Oocyte Development and Spermatogonial Stem Cell Differentiation in Zebrafish.

Cytochrome P45011A1, encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), the first and rate-limiting step in steroidogenesis. In zebrafish, cyp11a1 is maternally expressed and cyp11a2 is considered the ortholog of Cyp11a1 in mammals. A recent study has shown that depletion of cyp11a2 resulted in steroidogenic deficiencies and the mutants developed into males with feminized secondary sexual characteristics. Here, we independently generated cyp11a2 mutants in zebrafish and showed that the mutants can develop into males and females in the juvenile stage, but finally into infertile males with defective mating behavior in the adult stage. In the developing ovaries, the cyp11a2 mutation led to stage I oocyte apoptosis and final sex reversal, which could be partially rescued by treatment with P5 but not estradiol. In the developing testes, depletion of cyp11a2 resulted in dysfunction of Sertoli cells and lack of functional Leydig cells. Spermatogonial stem cells (SSCs) in the mutant testes underwent active self-renewal but no differentiation, resulting in a high abundance of SSCs in the testis, as revealed by immunofluorescence staining with Nanos2 antibody. The high abundance and differentiation competence of SSCs in the mutant testes were verified by a novel testicular cell transplantation method developed in this study, by transplanting mutant testicular cells into germline-depleted wild-type (WT) fish. The transplanted mutant SSCs efficiently differentiated into functional spermatids in WT hosts. Overall, our study demonstrates the functional importance of cyp11a2 in early oogenesis and differentiation of SSCs.

IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene.

Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.

PD39-08 EX VIVO HUMAN TESTES AS A PRACTICAL MODEL TO SIMULATE ULTRASOUND-GUIDED GERM CELL AUTOTRANSPLANTATION FOR HUMAN FERTILITY RESTORATION

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-08 EX VIVO HUMAN TESTES AS A PRACTICAL MODEL TO SIMULATE ULTRASOUND-GUIDED GERM CELL AUTOTRANSPLANTATION FOR HUMAN FERTILITY RESTORATION Heiko Yang, John Lindsey, Eva Gillis-Buck, Sudarshan Srirangapatanam, Jared Rosen, Ahmed Hussein, and James Smith Heiko YangHeiko Yang More articles by this author , John LindseyJohn Lindsey More articles by this author , Eva Gillis-BuckEva Gillis-Buck More articles by this author , Sudarshan SrirangapatanamSudarshan Srirangapatanam More articles by this author , Jared RosenJared Rosen More articles by this author , Ahmed HusseinAhmed Hussein More articles by this author , and James SmithJames Smith More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Fertility preservation options are limited for prepubertal boys undergoing gonadotoxic chemotherapy. Cryopreservation of testicular cells and subsequent autotransplantation is a promising experimental technique. The goal of this study was to develop an ex vivo model for urologists to practice ultrasound-guided injection of cellular material into human seminiferous tubules to simulate testicular cell transplantation. METHODS: Ultrasound-guided injections were performed in testes obtained from adult patients undergoing simple orchiectomy at our academic surgical center (Fig. 1). We performed injections using sonographic contrast (Optison™), methylene blue, and fluorescent labeled cells to help assess efficacy of simulated testicular cell transplant (TCT) injection. A characteristic streaming pattern of sonographic contrast in the testis was used to define sonographic success, and the presence of methylene blue and fluorescent labeled cells within the seminiferous tubules was used to determine histologic success. RESULTS: We performed simulated TCT injections in 30 testes obtained from 16 patients undergoing orchiectomy. We were able to achieve sonographic success in 57% of injections and confirmed that sonographic success correlates with histologic success. CONCLUSIONS: We demonstrate a practical means to simulate TCT in humans. As there appears to be a learning curve associated with this procedure, developing this infrastructure to practice these skills is critical prior to clinical implementation. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e670-e671 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heiko Yang More articles by this author John Lindsey More articles by this author Eva Gillis-Buck More articles by this author Sudarshan Srirangapatanam More articles by this author Jared Rosen More articles by this author Ahmed Hussein More articles by this author James Smith More articles by this author Expand All Advertisement Loading ...

O-189 Male fertility restoration by direct transplantation of human infant testicular cells into infertile recipient mouse testis

Abstract Study question Is colonization of human gonocytes and spermatogonial stem cells (SSCs) directly transplanted to seminiferous tubules of busulfan sterilised mice testis during an 8-week period feasible? Summary answer Gonocytes and SSCs from infant boys can settle on the basal membrane and form germline stem cell colonies in the seminiferous tubules of recipient mice. What is known already The neonatal or immature animal provides higher populations of gonocytes and/or SSCs than adults, and the number of transplanted donor SSCs directly affects the colonization rate of the recipient testes. Along with SSC transplantation restoring the recipient’s spermatogenesis, donor gonocyte was also reported to be capable of establishing spermatogenesis in rodents. Study design, size, duration Transplantation of human testicular cells including gonocytes and SSCs into seminiferous tubules of infertile recipient mice. We included 10 infant testis biopsies from which single-cell suspension was transplanted individually into the seminiferous tubules of 10 immunodeficient mice. The immunodeficient mouse testes were injected with busulfan to deplete germ cells. Four weeks later, we did the xenotransplantation. Then after eight weeks, we collected all mouse testes to do further analysis. Participants/materials, setting, methods Testis biopsies were obtained from cryptorchid boys undergoing orchidopexy. After enzymatic digestion of the testis biopsies, dissociated single-cell suspensions were pre-labeled with a green fluorescent dye. Then the single-cell suspensions were transplanted into seminiferous tubules of the infertile recipient mice. Eight weeks later, the presence of gonocytes and SSCs was determined by immunohistochemistry and whole-mount immunofluorescence. Main results and the role of chance Without in vitro propagation, naturally enriched human germline stem cells settled on the basal membrane of seminiferous tubules and survived in the mouse testes at least for two months demonstrating that human gonocytes and SSCs were capable of colonizing the recipient mouse seminiferous tubules. Limitations, reasons for caution The study samples were from infant boys with undescended testes that were more likely to contain gonocytes. It was not possible to determine which germ-cell type at transplantation resulted in the detected gonocytes and SSC colonies after xenotransplantation. Transplantation of gonocytes may include the potential risk of stem cell-related malignancy. Wider implications of the findings Without in vitro propagation, male germline stem cell-based transplantation could provide a relatively safe therapeutic treatment for prepubertal boys with cryptorchidism and boys diagnosed with cancer. This method could also facilitate clinical translation. Trial registration number not applicable

Ex vivo human testes as a practical model to simulate ultrasound-guided testicular cell transplantation for human fertility restoration

<h3>Objective</h3> To develop an ex vivo model to practice ultrasound-guided injection of cellular material into human seminiferous tubules to simulate testicular cell transplantation (TCT). <h3>Design</h3> Simulated TCT injections were performed in human testes removed during orchiectomy. The rete testis was the target site of injection. Successful retrograde infiltration of injected material into the lumen of the seminiferous tubules was detected using ultrasound and confirmed with histology. <h3>Setting</h3> Single academic surgical center. <h3>Patient(s)</h3> Adult patients undergoing orchiectomy for nononcologic indications. <h3>Intervention(s)</h3> The testes were injected with sonographic contrast (Optison), methylene blue, and fluorescent-labeled cells. <h3>Main Outcome Measure(s)</h3> A characteristic streaming pattern of sonographic contrast in the testis was used to define sonographic success, and the presence of methylene blue and fluorescent-labeled cells within the seminiferous tubules confirmed histologic success. <h3>Result(s)</h3> We performed simulated TCT injections in 30 testes obtained from 16 patients undergoing orchiectomy. We were able to achieve sonographic success in 57% of injections and confirmed that sonographic success is correlated with histologic success. <h3>Conclusion(s)</h3> Testicular cell transplantation injections can be practiced using human testes. As there appears to be a learning curve associated with this procedure, developing this infrastructure to practice these skills is critical before implementation in patients.

Гистологическое исследование нативных и криоконсервированных неонатальных семенников крыс после интратестикулярной аллотрансплантации

Cryopreservation and transplantation of testicular fragments and cells are utilized in establishing the germplasm bank of endangered species, line-breeding of animals. Moreover, the transplantation of testicular tissue is involved into investigation of the inborn and acquired defects of testicular tissue. Thus the improvement of cryopreservation and transplantation methods is of great interest. Here, we have compared the findings of histological examination of allotransplanted native and cryopreserved neonatal testis. It has been shown that the usage of DMEM/F12 supplemented with 5 mg/ml bovine serum albumin, 0.05 M sucrose, 20 mM Hepes, 1.5 M dimethylsulfoxide as a cryoprotectant, controlled cooling, preservation at -196°C, water bath warming, followed by intratesticular allotransplantation of neonatal rat testis results in the development of testicular interstitium and spermatogenic epithelium of donor organs into recipient body. Probl Cryobiol Cryomed 2020; 30(1): 34-46

Surrogate testes: Allogeneic spermatogonial stem cell transplantation within an encapsulation device may restore male fertility

Toxic insult to the gonads by chemotherapy or radiotherapy can lead to permanent infertility. It’s an important health concern because each year more than 4000 male patients are at risk of azoospermia in the United States due to gonadotoxicity of the regimens used. There are also several benign/genetic diseases whose natural course can result in infertility without gonadotoxic therapy. Considering the fact that most of these people are cured and survive with the advent of modern medicine, infertility is related to serious psychological and relationship implications and parenthood is a significant issue for those patients. Semen cryopreservation option is available for postpubertal adolescent and adult men, while children do not have this storing option since they do not have mature spermatozoa. However, their testes contain spermatogonial stem cells (SSCs), which are initiators of spermatogenesis. Promising findings in animal studies and human cell lines have encouraged scientists that SSCs may be hope for restoring fertility option of patients who cannot produce functional sperm and who have no other choice to preserve their future fertility. For this reason, several centers around the world already began to collect and cryopreserve testicular tissue or cells with anticipation that SSC-based therapies will be available in the near future; however, an optimal transplantation design in humans is yet to be developed. Here we propose an allogeneic testicular stem cell transplantation with an encapsulation device to restore fertility in patients with infertility. We endeavor to discuss the reliability of this method with the current literature and bring the evidence on its feasibility

FERTILITY PRESERVATION: Testicular transplantation for fertility preservation: clinical potential and current challenges.

Transplantation of testicular tissues and cells has been proposed as a future clinical option for patients who have had testicular tissue cryopreserved prior to receiving gonadotoxic therapies. Whilst this approach remains experimental, success using animal models and successful transplantation of ovarian tissue resulting in live births in female patients provides optimism for the development of clinical applications involving transplantation of testicular tissue in males. Careful consideration must be given to patient groups that may benefit from this approach in the future. Current research is focused on optimising patient selection, methods for tissue cryopreservation and development of transplantation techniques that might restore sperm production or future fertility in males. Crucially, attention must be focused on ensuring safety of transplantation, including eliminating the potential for infection or re-introducing malignancy. Furthermore the genetic/epigenetic integrity of any gametes generated must be ensured to allow generation of normal offspring. This review will provide an overview of the current status of transplantation of testicular tissue and cells for fertility preservation in males.

Production and use of triploid zebrafish for surrogate reproduction

We report for the first time, a comparison of two approaches for artificially induced triploidy in zebrafish (Danio rerio) using cold shock and heat shock treatments. Of the two methods, heat shock treatment proved more effective with a triploid production rate of 100% in particular females. Subsequently, triploid zebrafish larvae were used as recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Production of donor-derived sperm was achieved in 23% of testicular cell recipients and 16% of ovarian cell recipients, indicating the suitability of triploids as surrogate hosts for germ cell transplantation. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos with GFP specific primers. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups.

GDNF family receptor alpha 1 is a reliable marker of undifferentiated germ cells in bulls

Undifferentiated germ cells, including spermatogonial stem cells (SSCs), make up only a very small proportion of germ cells within the testis; for example, 0.03% of germ cells in the mouse testis are SSCs. In this study, we investigated the characteristics of bovine undifferentiated germ cells and developed an enrichment procedure for these cells on the basis of fluorescence-activated cell sorting (FACS), using the specific cell surface marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1). FACS analysis showed that only 0.6% of the total testicular cells were GFRα1-positive. These GFRα1-positive cells had a significantly higher expression of UCHL1, ZBTB16, and DDX4 (all markers of undifferentiated spermatogonial and germ cells) than that of fresh testicular cells. Quantitative reverse-transcription PCR analyses also indicated that the gene expression of BCL6B and NANOS2 was significantly higher in GFRα1-positive cells. Furthermore, xenogeneic transplantation of bovine testicular cells into immunodeficient mice resulted in 4.4-fold more colonies of GFRα1-positive cells than those of fresh testicular cells, indicating that FACS with antibodies to GFRα1 had efficiently enriched putative SSCs from total testicular cells. Collectively, these results demonstrate that GFRα1 could be used as a marker of bovine undifferentiated germ cells, including putative SSCs, and that its expression on SSCs has important implications for the further development of techniques for enriching stem cells from other species.

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments.

Despite their important contribution to the cure of both oncological and benign diseases, gonadotoxic therapies present the risk of a severe impairment of fertility. Sperm cryopreservation is not an option to preserve prepubertal boys’ reproductive potential, as their seminiferous tubules only contain spermatogonial stem cells (as diploid precursors of spermatozoa). Cryobanking of human immature testicular tissue (ITT) prior to gonadotoxic therapies is an accepted practice. Evaluation of cryopreserved ITT using xenotransplantation in nude mice showed the survival of a limited proportion of spermatogonia and their ability to proliferate and initiate differentiation. However, complete spermatogenesis could not be achieved in the mouse model. Loss of germ cells after ITT grafting points to the need to optimize the transplantation technique. Tissue engineering, a new branch of science that aims at improving cellular environment using scaffolds and molecules administration, might be an approach for further progress. In this review, after summarizing the lessons learned from human prepubertal testicular germ cells or tissue xenotransplantation experiments, we will focus on the benefits that might be gathered using bioengineering techniques to enhance transplantation outcomes by optimizing early tissue graft revascularization, protecting cells from toxic insults linked to ischemic injury and exploring strategies to promote cellular differentiation.

Application of dead end-knockout zebrafish as recipients of germ cell transplantation.

Germ cell transplantation is a promising technology for the propagation of endangered or valuable fishes. In this technique, sterile male and female recipient fish are injected with donor germ cells so they can produce viable gametes derived from the donor cells. The dead end (dnd) gene is involved in the migration of primordial germ cells; therefore, dnd-knockout zebrafish are expected to be germ-cell-free, making them suitable recipients for germ cell transplantation. dnd mutants were produced by microinjecting 2 nl of 10 ng/μl cRNAs encoding zinc finger nucleases against dnd into the blastodisc of zebrafish embryos before the cell- cleavage stage. One of the resulting founder males was mated with a wild-type female, and produced heterozygous mutants in the F1 generation. Mating of these F1 mutants produced an F2 generation with approximately 25% of the clutch being homozygous mutant (dnd-knockout) male, and lacking germ cells (as confirmed by expression analyses of vasa). The resulting dnd-knockout zebrafish males were tested for suitability as germ cell transplantation recipients by intraperitoneal transplantation of testicular cells prepared from vasa-gfp zebrafish. GFP-positive germ cells incorporated into the germ-cell-free gonads of the dnd-knockout recipients matured into functional sperm. Progeny tests revealed that the sperm from these dnd-knockout recipients were derived entirely from donor cells. Thus, we demonstrated that homozygous dnd mutants became germ-cell-free males that are able to nurse donor-derived germ cells.

Development of heterotopic transplantation of the testis with the epididymis to evaluate an aspect of testicular immunology in rats.

Transplantation of testicular cells and tissues has been studied for the investigation of immunology of the testis, which is an immunologically privileged organ. However, reports of transplant of the testis at organ level have been extremely limited because of technical difficulties of the orthotopic testis transplantation (OTT) in experimental animals. In the present study, we developed a new and simple model of the heterotopic testis transplantation (HTT), which is donor testis transplantation into the cervical region of recipients, in a syngeneic model in rats [donor Lewis (LEW) graft to LEW recipient]. The duration of HTT was significantly shorter and success rate higher than that of OTT. To histologically evaluate HTT, the local immune responses were compared among the syngeneic model, an acute rejection allogeneic model [donor Augustus Copenhagen Irish (ACI) graft to LEW recipient] and a chronic rejection allogeneic model (donor F344 graft to LEW recipient) at postoperative day 3. We found that allogeneic ACI grafts resulted in mild and not severe orchitic lesions, whereas immune responses of allogeneic F344 grafts seemed intact and were not significantly different from those of syngeneic LEW grafts. These results suggest that our new operative procedure will be useful in future for the investigation of the testicular immunology.

TRANSPLANTATION OF GIANT GOURAMY (Osphronemus gouramy) TESTICULAR CELLS IN EARLY HATCHING TILAPIA (Oreochromis niloticus) LARVAE

Cell transplantation technology has been widely developed in mammals, but less implemented in inland aquaculture fish. Giant gouramy (Osphronemus gouramy) is one of important comercial aquaculture commodities that has a relatively long first reproductive cycle compared to the tilapia (Oreochromis niloticus). Tilapia has a faster reproductive cycle and potential as giant gouramy surrogate broodstock. This study was aimed to applicate testicular cell transplantation technology of giant gouramy in tilapia larvae and to evaluate its success rate trough donor cell colonization. Tilapia larvae of 1-2 dph (days post hatching) and 3-4 dph were injected with ±20.000 giant gouramy testicular cells in the peritoneal cavity, and then groomed for two months. Cell colonization of spermatogonium donor was determined by Polymerase Chain Reaction (PCR), using giant gouramy F1-Growth Hormone and R1-Growth Hormone primers. The result of cell dissociation showed that a bigger giant gouramy (827 g) had lower of spermatogonia composition (4.45%) than a smaller gouramy (608 g) (14.96%). Donor cell colonization in 1-2 dph recipient was higher (100%) than in 3-4 dph recipient (75%). Whereas recipient Survival Rate (SR) was 89.34% for 1-2 dph recipient and 98.96% for 3-4 dph recipient respectively. This study suggests that testicular cell transplantation technology of giant gouramy could be appllied to tilapia (Xenotransplantation) and cell transplantation has a better performance in 1-2 dph than in 3-4 dph recipients. With such success, there is a high potential that the giant gouramy cultivation can be increased through the cell transplantation technology.

Testicular cell transplantation of neon tetra Paracheirodon innesi into common carp fry

&lt;p class="NoParagraphStyle" align="center"&gt;&lt;strong&gt;ABSTRACT&lt;/strong&gt;&lt;/p&gt;&lt;p class="NoParagraphStyle" align="center"&gt;&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt;&lt;p class="NoParagraphStyle"&gt;Neon tetra &lt;em&gt;Paracheirodon innesi&lt;/em&gt; is an ornamental fish that have high export value. However, production is still relatively low due to low fecundity (approximately 180 eggs/female). Technology of testicular cell transplantation of neon tetra as donor to common carp as recipient fish which have high fecundity provides a promising way to overcome problem of neon tetra production. This research was performed to determine the optimum age of common carp fry that is able to receive donor cells and allow high success of transplantation. In this research, the testes of neon tetra fish were dissociated by 0.5% trypsin solution. The testicular cells were labeled with PKH-26 fluorescent dye, and then transplanted into the peritoneal cavity of seven, ten, and 14 days post hatching common carp fry. The results showed that the survival of seven day-old transplanted fry (31.25%) was lower than that of ten day-old (37.75%) and 14 day-old transplanted fry (56.25%). Percentage of fish colonized testicular cells donor at 21 days post-transplantation on seven days old and ten days old fry were similar (80%), while on 14 day-old fry was 60%. Based on the cumulative transplantation success rate (survival and colonization rates), transplantation on 14 days old fry (33.75%) showed higher result compared to transplantation on seven days old fry (25.00%) and ten day-old fry (30.00%). It can be concluded that transplantation of neon tetra testicular cells to common carp fry have been successfully carried out, and the optimum age of common carp fry to transplantation was 14 days after hatching.&lt;/p&gt;&lt;p class="NoParagraphStyle"&gt; &lt;/p&gt;&lt;p class="NoParagraphStyle"&gt;Keywords: transplantation, colonization, testicular cells, common carp, neon tetra&lt;/p&gt;&lt;p class="NoParagraphStyle"&gt; &lt;/p&gt;&lt;p class="NoParagraphStyle"&gt; &lt;/p&gt;&lt;p class="NoParagraphStyle" align="center"&gt;&lt;strong&gt;ABSTRAK&lt;/strong&gt;&lt;/p&gt;&lt;p class="NoParagraphStyle" align="center"&gt;&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt;&lt;p class="NoParagraphStyle"&gt;Ikan neon tetra &lt;em&gt;Paracheirodon innesi&lt;/em&gt; merupakan ikan hias yang memiliki nilai ekspor yang tinggi. Namun demikian, tingkat produksinya masih relatif rendah karena fekunditas ikan neon tetra yang sedikit (sekitar 180 telur/induk). Teknologi transplantasi sel testikular ikan neon tetra (ikan donor) ke ikan mas yang memiliki fekunditas telur yang banyak dan diharapkan mampu mengatasi ketersediaan benih ikan neon tetra. Penelitian ini bertujuan untuk menentukan umur optimum benih ikan mas (calon ikan semang) yang mampu menerima sel donor dengan baik dan memiliki keberhasilan kolonisasi yang tinggi. Testis ikan neon tetra didisosiasi menggunakan larutan tripsin 0,5%. Sel testikular diwarnai dengan PKH-26, kemudian ditransplantasikan ke rongga peritoneal benih ikan mas umur tujuh, sepuluh, dan 14 hari setelah menetas. Hasil penelitian menunjukkan bahwa tingkat kelangsungan hidup ikan mas perlakuan transplantasi umur tujuh hari (31,25%) lebih rendah dibandingkan dengan perlakuan transplantasi umur sepuluh hari (37,50%) dan 14 hari (56,25%). Persentase ikan terkolonisasi sel donor pada hari ke-21 pascatransplantasi pada benih umur tujuh dan sepuluh hari adalah sama (80%), sedangkan transplantasi benih umur 14 hari sebesar 60%. Berdasarkan keberhasilan transplantasi secara kumulatif (tingkat kelangsungan hidup dan kolonisasi), transplantasi pada benih umur 14 hari (33,75%) menunjukkan hasil lebih tinggi dibandingkan dengan perlakuan transplantasi pada benih umur tujuh hari (25,00%) dan benih umur sepuluh hari (30,00%). Transplantasi sel testikular ikan neon tetra pada benih ikan mas telah berhasil dilakukan, dan umur optimum benih ikan mas adalah 14 hari setelah menetas.&lt;/p&gt;&lt;p class="NoParagraphStyle"&gt; &lt;/p&gt;&lt;p class="NoParagraphStyle"&gt;Kata kunci: transplantasi, kolonisasi, sel testikular, ikan mas, ikan neon tetra&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Busulfan pretreatment for transplantation of rat spermatogonia differentially affects immune and reproductive systems in male recipient mice.

Testicular cell transplantation has generally been performed by using immune-deficient recipient mice to investigate the biology of spermatogonial stem cells (SSCs), the production of transgenic animals, and restoration of fertility. Recently, we demonstrated that rat spermatogenesis can occur in the seminiferous tubules of immune-competent recipient mice via pretreatment with busulfan (Myleran, 1, 4-butanediol methanesulfonate, 40mg/kg) after transplantation of rat SSCs. However, considering the immunosuppressive effect of busulfan, there is a possibility that busulfan itself causes immune suppression in immune-competent recipient mice. The aim of this study was to determine the effects of busulfan on the immune system and spermatogenesis in immune-competent recipient mice. The results showed that at 60days after busulfan treatment, just the same time as the transplantation, the recovery could be seen in the immune system including cell counts and functions of T and B lymphocytes in the spleen, but the spermatogenesis was more compromised. This study demonstrated that after busulfan pretreatment the immune system in immune-competent recipient mice had recovered by the time that rat spermatogenesis could occur in the murine testis. It became clear that xenogenic spermatogenesis can be tolerated in seminiferous tubules in the testes of immune-competent mice.

Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis.

Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection.

Restoration of spermatogenesis in infertile male chickens after transplantation of cryopreserved testicular cells

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze).2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks.3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype.4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue.5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.

Testicular cell transplantation into the human testes

To translate spermatogonial stem cell (SSC) transplantation towards a clinical application. Mouse green fluorescent protein (GFP)-positive testicular cells were labeled with (99m)technetium and microbubbles. These labeled cells were injected into the rete testis of isolated human testes under ultrasound guidance. Three different conditions were tested: 1) 800 μL of a 20 million cells/mL suspension; 2) 800 μL of a 10 million cells/mL suspension; and 3) 1,400 μL of a 10 million cells/mL suspension. After injection, the human cadaver testes were analyzed with the use of single-photon-emission computerized tomography (SPECT) imaging and histology. Laboratory research environment. Cadaver testes, obtained from autopsies at the pathology department. Ultrasound-guided injection of mouse GFP-positive testicular cells. Presence of radioactive-labeled cells in the human cadaver testes and GFP-positive cells in the seminiferous tubules. In all of the experimental groups, GFP-positive cells were observed in the seminiferous tubules, near and far from the rete testis, but also in the interstitium. On SPECT, significant difference was seen between the group injected with 800 μL of a 20 million cells/mL suspension (1,654.6 ± 907.6 mm³) and the group injected with 1,400 μL of a 10 million cells/mL suspension (3,614.9 ± 723.1 mm³). No significant difference was reached in the group injected with 800 μL of a 10 million cells/mL suspension. Injecting cells in the human cadaver testis is feasible, but further optimization is required.