PSV-12 Fertility following germline transplantation in sterile NANOS2 knockout surrogate bulls.

Although the prognosis of childhood cancer survivors has increased dramatically during recent years, chemotherapy and radiation treatments for cancer and other conditions may lead to permanent infertility in prepubertal boys. Recent developments have shown that spermatogonial stem cell (SSC) transplantation may be a hope for restoring fertility in adult survivors of childhood cancers. For this reason, several centres around the world are collecting and cryopreserving testicular tissue or cells anticipating that, in the near future, some patients will return for SSC transplantation. This review summarizes the current knowledge and utility of SSC transplantation techniques. The aim of this narrative review is to provide an overview of the currently used experimental injection techniques for SSC transplantation in animal and human testes. This is crucial in understanding and determining the role of the different techniques necessary for successful transplantation. A comprehensive review of peer-reviewed publications on this topic was performed using the PubMed and Google Scholar databases. The search was limited to English language work and studies between 1994 (from the first study on SSC transplantation) and April 2019. Key search terms included mouse, rat, boar, ram, dog, sheep, goat, cattle, monkey, human, cadaver, testes, SSC transplantation, injection and technique. This review provides an extensive clinical overview of the current research in the field of human SSC transplantation. Rete testis injection with ultrasonography guidance currently seems the most promising injection technique thus far; however, the ability to draw clear conclusions is limited due to long ischemia time of cadaver testis, the relatively decreased volume of the testis, the diminishing size of seminiferous tubules, a lack of intratesticular pressure and leakage into the interstitium during the injection on human cadaver testis. Current evidence does not support improved outcomes from multiple infusions through the rete testes. Overall, further optimization is required to increase the efficiency and safety of the infusion method. Identifying a favourable injection method for SSC transplantation will provide insight into the mechanisms of successful assisted human reproduction. Future research could focus on reducing leakage and establishing the optimal infusion cell concentrations and pressure.

BackgroundSpermatogonial stem cell (SSC) transplantation technology as a promising option for male fertility preservation has received increasing attention, along with efficient SSC purification technology as a necessary technical support; however, the safety of such application in patients with tumors remains controversial.MethodsIn this study, we used a green fluorescent protein mouse xenograft model of B cell acute lymphocytic leukemia. We isolated and purified SSCs from the testicular tissue of model mice using density gradient centrifugation, immune cell magnetic bead separation, and flow cytometry. The purified SSCs were transplanted into convoluted seminiferous tubules of the nude mice and C57BL/6 male mice subjected to busulfan. The development and proliferation of SSCs in the recipient testis were periodically tested, along with whether B cell acute lymphocytic leukemia was induced following SSC implantation. The genetic characteristics of the offspring obtained from natural mating were also observed.ResultsIn testicular leukemia model mice, a large number of BALL cells infiltrated into the seminiferous tubule, spermatogenic cells, and sperm cells in the testis tissue decreased. After spermatogonial stem cell transplantation, the transplanted SSCs purified by immunomagnetic beads and flow cytometry methods colonized and proliferated extensively in the basement of the seminiferous tubules of mice; a large number of spermatogenic cells and sperm were found in recipient testicular tissue after 12 weeks of SSC transplantation. In leukemia detection in nude mice after transplantation in the three SSC purification groups, a large number of BALL cells could be detected in the blood of recipient mice 2–3 weeks after transplantation in the density gradient centrifugation group, but not in the blood of the flow cytometry sorting group and the immunomagnetic bead group after 16 weeks of observation.ConclusionsIn this study, we confirmed that immunomagnetic beads and flow cytometry methods of purifying SSCs from the testicular tissue of the testicular leukemia mouse model could be safely applied to the SSC transplantation technology without concomitant tumor implantation. The results thus provide a theoretical basis for the application of tumor SSC cryopreservation for fertility preservation in patients with tumors.

The dog is recognized as a highly predictive model for preclinical research. Its size, life span, physiology, and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model has not been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for preclinical models of genetic diseases. Specifically, we i) established markers for identification and tracking canine spermatogonial cells; ii) established methods for enrichment and genetic manipulation of these cells; iii) described their behavior in culture; and iv) demonstrated engraftment of genetically manipulated SSC and production of transgenic sperm. These findings help to set the stage for generation of transgenic canine models via SSC transplantation.

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.

Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility. Key words: Spermatogonia; Stem cells; Cell culture techniques; Transplantation; Mice

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

Using the male gamete for assisted reproduction: past, present, and future.

With the exception of the domestic cat, all members of the family Felidae are considered either endangered or threatened. Although not yet used for this purpose, spermatogonial stem cell (SSC) transplantation has a high potential to preserve the genetic stock of endangered species. However, this technique has not previously been established in felids. Therefore, we developed the necessary procedures to perform syngeneic and xenogeneic SSC transplants (eg, germ cell [GC] depletion in the recipient domestic cats, enrichment and labeling of donor cell suspension, and the transplantation method) in order to investigate the feasibility of the domestic cat as a recipient for the preservation and propagation of male germ plasm from wild felids. In comparison with busulfan treatment, local x-ray fractionated radiation was a more effective approach to depleting endogenous spermatogenesis. The results of both syngeneic and xenogeneic transplants revealed that SSCs were able to successfully colonize and differentiate in the recipient testis, generating elongated spermatids several weeks posttransplantation. Specifically, ocelot spermatozoa were observed in the cat epididymis 13 weeks following transplantation. As donor GCs from domestic cats and ocelots were able to develop and form mature GCs in the recipient environment seminiferous tubules, these findings indicate that the domestic cat is a suitable recipient for SSC transplantation. Moreover, as modern cats descended from a medium-size cat that existed approximately 10 to 11 million years ago, these results strongly suggest that the domestic cat could be potentially used as a recipient for generating and propagating the genome of wild felids.

Sustained spermatogenesis in adult males relies on the activity of spermatogonial stem cells (SSCs). In general, tissue-specific stem cell populations such as SSCs are influenced by contributions of support cells that form niche microenvironments. Previous studies have provided indirect evidence that several somatic cell populations and the interstitial vasculature influence SSC functions, but an individual orchestrator of niches has not been described. In this study, functional transplantation of SSCs, in combination with experimental alteration of Sertoli cell content by polythiouracil (PTU)-induced transient hypothyroidism, was used to explore the relationship of Sertoli cells with SSCs in testes of adult mice. Transplantation of SSCs from PTU-treated donor mice into seminiferous tubules of normal recipient mice revealed a greater than 3-fold increase in SSCs compared to those from testes of non-PTU-treated donors. In addition, use of PTU-treated mice as recipients for transplantation of SSCs from normal donors revealed a greater than 3-fold increase of accessible niches compared to those of testes of non-PTU treated recipient mice with normal numbers of Sertoli cells. Importantly, the area of seminiferous tubules bordered by interstitial tissue and percentage of seminiferous tubules associated with blood vessels was found to be no different in testes of PTU-treated mice compared to controls, indicating that neither the vasculature nor interstitial support cell populations influenced the alteration of niche number. Collectively, these results provide direct evidence that Sertoli cells are the key somatic cell population dictating the number of SSCs and niches in mammalian testes.

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.

The most promising procedure to restore fertility in male childhood cancer patients is spermatogonial stem cell transplantation (SSCT). Although the efficiency of SSCT has been proven in the mouse model, its safety needs to be investigated too before considering any implementation in the clinic. To examine the incidence of genetic abnormalities after SSCT, the karyotypes of donor-derived spermatozoa and offspring were analyzed. Donor cells were obtained from prepubertal mice and introduced in the seminiferous tubules of genetically sterile W/W(v) mice. Five to 10 months after SSCT, DNA was extracted from epididymal sperm to perform array comparative genomic hybridization (aCGH) analysis. In addition, spermatozoa, liver and kidney from the offspring were subjected to aCGH analysis. Numerical chromosomal aberrations could not be detected in spermatozoa from transplanted males, nor in their offspring. The few genetic deviations (deletions, amplifications) observed were all polymorphisms. No major genetic alterations could be detected after SSCT. These data are supportive for further development of SSCT as a strategy for fertility restoration.

Transplantation of spermatogonial stem cells from fertile, transgenic donor mice to the testes of infertile recipients provides a unique system to study the biology of spermatogonial stem cells. To facilitate the investigation of treatment effects on colonization efficiency an analysis system was needed to quantify colonization of recipient mouse seminiferous tubules by donor stem cell-derived spermatogenesis. In this study, a computer-assisted morphometry system was developed and validated to analyze large numbers of samples. Donor spermatogenesis in recipient testes is identified by blue staining of donor-derived spermatogenic cells expressing the E. coli lacZ structural gene. Images of seminiferous tubules from recipient testes collected three months after spermatogonial transplantation are captured, and stained seminiferous tubules containing donor-derived spermatogenesis are selected for measurement based on their color by color thresholding. Colonization is measured as number, area, and length of stained tubules. Interactive, operator-controlled color selection and sample preparation accounted for less than 10% variability for all collected parameters. Using this system, the relationship between number of transplanted cells and colonization efficiency was investigated. Transplantation of 10(4) cells per testis only rarely resulted in colonization, whereas after transplantation of 10(5) and 10(6) cells per testis the extent of donor-derived spermatogenesis was directly related to the number of transplanted donor cells. It appears that about 10% of transplanted spermatogonial stem cells result in colony formation in the recipient testis. The present study establishes a rapid, repeatable, semi-interactive morphometry system to investigate treatment effects on colonization efficiency after spermatogonial transplantation in the mouse.

Induction of spermatogenesis under 3-dimentional tissue culture conditions by in vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue

The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production systems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of spermatogonial stem cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production systems in which AI is difficult to implement.

In search of an efficient injection technique for future clinical application of spermatogonial stem cell transplantation: infusion of contrast dyes in isolated cadaveric human testes