The mammalian testis

The mammalian testis is a bi-compartmental organ consisting of seminiferous tubules (approx. 90% of adult mass) and an interstitium (approx. 10% of adult mass) as depicted in Fig. 1. The tubular wall consists of a basement membrane with underlying contractile peritubular cells. The tubules present the sperm generating compartment. The seminiferous epithelium is a unique and exquisitely complex structure with both germ cells and Sertoli cells undergoing major structural and morphological changes during the spermatogenic process. The interstitial space contains the steroidogenically active Leydig cells as a second testis specific somatic cell type and is home to many more cell types including fibroblasts, immune cells and blood vessels. The research in the Schlatt team looks at many histological aspects of testis specific cells and uses a range of tools for detailed histo(patho)logical, cellular and molecular analysis of the testis.

Fig.1. Generalized schematic drawing depicting the mammalian testicular anatomy and cell biology.

Testicular stem cells

Spermatogonia are located at the basal membrane of the seminiferous compartment. A subpopulation of spermatogonia functions as stem cells. In contrast to other adult stem cells in the organism, spermatogonial stem cells show unique features. As progeny from primordial germ cells and constituents of the germ line they are theoretically immortal. Spermatogonia inherit the germline of the species at a critical point when germ cells need to mitotically expand prior to meiosis. A very high number of sperm is needed to maintain fertility of males. Therefore stem cell spermatogonia must maintain DNA-integrity against the accumulation of replication errors and exposure to environmental mutagens. Spermatogonial stem cells have therefore a key position in controlling balanced evolutionary changes of the genome. The research group has developed a number of tools to visualize spermatogonia and their expansion in human and monkey testes (Fig. 2). The group uses these tools to explore basic mechanisms of spermatogonial physiology during normal and pathological stages of prepubertal development and adulthood.

Fig. 2: Visulazation of proliferating spermatogonial stem cells in whole mounts of macaque testes by confocal (a, b) or bright field (c) microscopy. Cells in S-phase of the cell cycle were labeled with BrDU.

In all mammals, both type A and B spermatogonia undergo a series of mitotic divisions to produce germ cell cohorts that enter into meiosis. Thus the size of the spermatogonial population and the extent of premeiotic expansion is a key determinant for the output of mature sperm. The Schlatt group explores features of spermatogonial stem cells with a strong focus on species specific differences in kinetics (Fig. 3). The group has pioneered the description of spermatogonial kinetics in primates and has proposed models for spermatogonial expansion in monkeys and man. Many research aspects are related to explore the details of spermatogonial functions. 

Fig. 3: Scheme of male germ cell differentiation in mouse, rhesus monkey and man. Spermatogonial subtypes: As Asingle; Apr Apair; Aal Aaligned; I Intermediate; B B-spermatogonium. Spc spermatocyte; RS round spermatid; S sperm.

Dysfunction of spermatogonial stem cells or faulty stem cell niches affect stem cell turnover and may be responsible for spermatogenic failure. Many research aspects in the Schlatt team deal with options to better understand and influence spermatogonial stem cell function in the primate testis. In vitro and in vivo approaches are used to explore the morphogenetic and regulatory processes involved in testicular stem cell function which have direct clinical relevance for infertile patients as well as for the development of fertility preservation techniques.

Fertility protection in prepubertal boys

Survival rates of cancer patients improved over the last decades. Therefore, quality of life of these patients after successful treatment has become an important concern. One side effect of the cancer treatment is temporary or permanent subfertility or even infertility due to the depletion of testicular stem cells. Cryopreservation of immature testicular tissues is therefore offered in few European centers to prepubertal and pubertal boys at risk for germ cell loss. In these tissues, germ cells have not yet initiated spermatogenic differentiation, but experimental protocols for the derivation of sperm from the spermatogonia that are present in these tissues are currently being developed

 

Description of a diagnostic scheme for histological evaluation of the fertility potential of immature human testis biopsies

We developed a diagnostic fertility score based on immunohistochemical stainings that will provide information on the status of the testicular tissues with regard to the prospect of using the material for spermatogonia based approaches for the derivation of sperm.

 

Development of novel approaches for generation of sperm from testicular stem cells

This work is integral part of the GrowSperm consortium (http://www.growsperm.eu) funded by a Marie Skłodowska Curie International Training Network.

One project establishes in vitro culture approaches to induce re-assembly of human primary testicular cells. With this, we aim at gaining further insight in molecular and cellular mechanisms, testicular cell interactions and dynamics during human testicular tubulogenesis. It is the goal to obtain tubule-like-structures from testicular single-cell suspensions from human adult tissue samples after enzymatic digestion and culture under conventional and three-dimensional (Matrigel® and collagen scaffolds) conditions.

Other projects in this research area focus on the dynamics involved in germ cell differentiation. Here we use nonhuman primate models employing different age groups (neonatal, prepubertal and adult) of both available monkey species: Marmoset (Callithrix jacchus) and Macaque (Macaca fascicularis). We use ectopic xenografting of monkey tissue onto immune-deficient nude mice hosts and explore the effect of the hosts being male, female, intact or castrated on supporting spermatogenic induction. We also explore long term in vitro culture strategies (from tissue-to-cellular level) for germ cell differentiation and the effect of culture conditions for expansion of monkey spermatogonial stem cells. One of the sub-project focusses on establishing a testicular organoid culture system for primate species. We also explore the use of microfluidic devices.

 

Gonadal damage by X-irradiation

The damaging effects of irradiation are poorly understood. Again we employ our preclinical non-human primate models and xenografting into nude mice to explore the molecular and cellular mechanisms leading to germ cell depletion in boys undergoing irradiation cancer therapy. We have successfully established an approach to identify irradiation-induced changes in transcription and protein expression of somatic cells in prepubertal macaque testicular xenografts. We were able to show that irradiation affects somatic cell function and that gene expression analyses on xenografts is a valid approach in order to provide insight into the regulation of germ cell function by somatic cells. We also employ migration assays in combination with immunohistochemical stainings to investigate the migratory response of primate testicular cells towards somatic cell-secreted niche factors. Providing additional information on the underlying mechanisms of germ cell function might offer beneficial therapy targets to treat male infertility, in particular in adult survivors of childhood cancer.

Novel analytical tools for sperm assessment

Conventional sperm parameters are unable to detect sperm abnormalities at biochemical and molecular level. We aim to explore new approaches to establish tests for the functional status of human sperm. New analytical techniques such as Raman Microspectroscopy open the potential to assess the biochemical fingerprint of different sperm cell compartments in a non-destructive manner. Multiparametric Flow Cytometry is capable to determine simultaneously multiple parameters in individual sperm. Bot tools alone or in combination present opportunities for novel assessment of multiple sperm features.