Stefan Schlatt

Sperm from neonatal mammalian testes grafted in mice

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.

Progeny from Sperm Obtained after Ectopic Grafting of Neonatal Mouse Testes

Abstract 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.

GDNF Family Receptor alpha1 Phenotype of Spermatogonial Stem Cells in Immature Mouse Testes

Abstract Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor α1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.

Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo.

While it is well known that serious illness and inflammation reduce male fertility, the mechanisms involved are poorly understood. In adult male rats, a single injection of lipopolysaccharide at doses that induced either mild or severe inflammation, caused a biphasic decline in Leydig cell testosterone production and gonadotropin responsiveness. In the high dose group only, serum LH levels also were reduced; however, intratesticular testosterone concentrations remained at a level adequate to support qualitatively normal spermatogenesis in both treatment groups. Testicular interstitial fluid formation also declined in a dose-dependent fashion after lipopolysaccharide treatment. In the high dose group only, these hormonal and vascular changes were accompanied by an increase in endothelial permeability, microhemorrhage, and inflammatory cells in the testis, followed by vacuolization of round spermatid nuclei, disruption of Sertoli-germ cell contacts at stages I–IV of the cycle of the seminiferous epithelium,...

Inducible Nitric Oxide Synthase in the Rat Testis: Evidence for Potential Roles in Both Normal Function and Inflammation-Mediated Infertility

Abstract In vitro data have indicated that nitric oxide (NO) inhibits Leydig cell testosterone production, suggesting that NO may play a role in the suppression of steroidogenesis and spermatogenic function during inflammation. Consequently, we investigated expression of the inflammation-inducible isoform of NO synthase (iNOS) in the inflamed adult rat testis and the ability of a broad-spectrum inhibitor of NO production, l-nitro-l-arginine methyl ester, to prevent Leydig cell dysfunction during inflammation. Unexpectedly, immunohistochemical and mRNA data established that iNOS is expressed constitutively in Leydig cells and in a stage-specific manner in Sertoli, peritubular, and spermatogenic cells in the normal testis. Expression was increased in a dose-dependent manner in all these cell types during lipopolysaccharide (LPS)-induced inflammation. In noninflamed testes, treatment with the NO synthase inhibitor reduced testicular interstitial fluid formation and testosterone production without any effect on serum LH levels. Administration of the inhibitor did not prevent the suppression of testicular interstitial fluid and testosterone production that occurs within 6 h after LPS treatment. Collectively, these data indicate a novel role for iNOS in autocrine or paracrine regulation of the testicular vasculature, Leydig cell steroidogenesis, and spermatogenesis in the normal testis. The data suggest that increased NO is not the major cause of acute Leydig cell dysfunction in the LPS-treated inflammation model, although a role for NO in this process cannot be excluded, particularly at other time points. Moreover, up-regulation of iNOS may contribute to the seminiferous epithelium damage caused by LPS-induced inflammation.

New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in vitro spermatogenesis has resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional (3D) in vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotrophin support. For the first time, we demonstrate that pre-meiotic male germ cells [revealed by the absence of meiotic marker expression (e.g. Boule)] obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotrophins but appeared independent from the matrix in a 3D culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

Testicular function and fertility preservation in male cancer patients

The testis has been shown to be highly susceptible to the toxic effects of cancer therapy at all stages of life. Young cancer survivors are approximately half as likely as their siblings to sire a pregnancy. Radiation therapy to the testes and high cumulative dose of alkylating agents are the major factors decreasing the probability of fertility. This review aims to present an overview of the current state of knowledge in mechanisms how human spermatogonia proliferate and differentiate and how cancer therapy affects germ cells, what are the options for fertility preservation and what are the clinical risks and limitations related to such procedures. This area of research is discussed in the context of the potential future options that may become available for preserving fertility in male cancer patients.

Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting.

Spermatogonial stem cells open novel strategies for preservation of testicular tissue and fertility preservation in boys and men exposed to gonadotoxic therapies. This review provides an update on the physiology of spermatogonial stem cells in rodent and primate testes. Species‐specific differences must be considered when new technologies on testicular stem cells are considered. Germ cell transplantation is presented as one novel and promising strategy. Whereas this technique has become an important research tool in rodents, a clinical application must still be regarded as experimental and many aspects of the procedure need to be optimized prior to a safe and efficient clinical application in men. Testicular grafting opens another exciting strategy for fertility preservation. Autologous and xenologous transfer of immature tissue revealed a high regenerative potential of immature testicular tissue. Grafting was applied in rodents and primates and resulted in the generation of sperm. Further research is needed before an application in humans can be considered safe and efficient. Despite the current limitations in regard to the generation of sperm from cryopreserved male germline cells and tissues, protocols for cryopreservation of testicular tissue are available and reveal a promising outcome. Since future improvements of germ cell transplantation and grafting approaches can be assumed, bioptic retrieval and cryopreservation of testicular tissue fragments should be performed in oncological patients at high risk of fertility loss since this is their only option to maintain their fertility potential. Pediatr Blood Cancer 2009;53:274–280.

A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys

STUDY QUESTION What clinical practices, patient management strategies and experimental methods are currently being used to preserve and restore the fertility of prepubertal boys and adolescent males? SUMMARY ANSWER Based on a review of the clinical literature and research evidence for sperm freezing and testicular tissue cryopreservation, and after consideration of the relevant ethical and legal challenges, an algorithm for the cryopreservation of sperm and testicular tissue is proposed for prepubertal boys and adolescent males at high risk of fertility loss. WHAT IS KNOWN ALREADY A known late effect of the chemotherapy agents and radiation exposure regimes used to treat childhood cancers and other non-malignant conditions in males is the damage and/or loss of the proliferating spermatogonial stem cells in the testis. Cryopreservation of spermatozoa is the first line treatment for fertility preservation in adolescent males. Where sperm retrieval is impossible, such as in prepubertal boys, or it is unfeasible in adolescents prior to the onset of ablative therapies, alternative experimental treatments such as testicular tissue cryopreservation and the harvesting and banking of isolated spermatogonial stem cells can now be proposed as viable means of preserving fertility. STUDY DESIGN, SIZE, DURATION Advances in clinical treatments, patient management strategies and the research methods used to preserve sperm and testicular tissue for prepubertal boys and adolescents were reviewed. A snapshot of the up-take of testis cryopreservation as a means to preserve the fertility of young males prior to December 2012 was provided using a questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS A comprehensive literature review was conducted. In addition, survey results of testis freezing practices in young patients were collated from 24 European centres and Israeli University Hospitals. MAIN RESULTS AND THE ROLE OF CHANCE There is increasing evidence of the use of testicular tissue cryopreservation as a means to preserve the fertility of pre- and peri-pubertal boys of up to 16 year-old. The survey results indicate that of the 14 respondents, half of the centres were actively offering testis tissue cryobanking as a means of safeguarding the future fertility of boys and adolescents as more than 260 young patients (age range less than 1 year old to 16 years of age), had already undergone testicular tissue retrieval and storage for fertility preservation. The remaining centres were considering the implementation of a tissue-based fertility preservation programme for boys undergoing oncological treatments. LIMITATIONS, REASONS FOR CAUTION The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. WIDER IMPLICATIONS OF THE FINDINGS The clinical and research questions identified and the ethical and legal issues raised are highly relevant to the multi-disciplinary teams developing treatment strategies to preserve the fertility of prepubertal and adolescent boys who have a high risk of fertility loss due to ablative interventions, trauma or genetic pre-disposition.

Germ cell transplantation.

Transplantation of germ cells leads to restoration of spermatogenesis from spermatogonial stem cells. The original description of germ cell transplantation in 1994 has led to new approaches to explore many basic aspects of spermatogonial physiology. Combining germ cell transplantation with culture and cryopreservation of spermatogonia opens new pathways for genetic engineering and conservation of livestock. In addition, autologous transfer of spermatogonia might be used as an approach for fertility preservation in oncological patients. This review summarises the history of germ cell transplantation and presents an outlook on future research on spermatogonial stem cells.