Development and Disease-Dependent Dynamics of Spermatogonial Subpopulations in Human Testicular Tissues.

Cancer therapy and conditioning treatments of non-malignant diseases affect spermatogonial function and may lead to male infertility. Data on the molecular properties of spermatogonia and the influence of disease and/or treatment on spermatogonial subpopulations remain limited. Here, we assessed if the density and percentage of spermatogonial subpopulation changes during development (n = 13) and due to disease and/or treatment (n = 18) in tissues stored in fertility preservation programs, using markers for spermatogonia (MAGEA4), undifferentiated spermatogonia (UTF1), proliferation (PCNA), and global DNA methylation (5mC). Throughout normal prepubertal testicular development, only the density of 5mC-positive spermatogonia significantly increased with age. In comparison, patients affected by disease and/or treatment showed a reduced density of UTF1-, PCNA- and 5mC-positive spermatogonia, whereas the percentage of spermatogonial subpopulations remained unchanged. As an exception, sickle cell disease patients treated with hydroxyurea displayed a reduction in both density and percentage of 5mC- positive spermatogonia. Our results demonstrate that, in general, a reduction in spermatogonial density does not alter the percentages of undifferentiated and proliferating spermatogonia, nor the establishment of global methylation. However, in sickle cell disease patients', establishment of spermatogonial DNA methylation is impaired, which may be of importance for the potential use of this tissues in fertility preservation programs.

Strains matter: Success of murine in vitro spermatogenesis is dependent on genetic background.

The current strategy to preserve fertility of male prepubertal cancer patients consists of cryopreservation of a testicular tissue biopsy containing spermatogonial stem cells (SSCs). While in humans, fertility restoration strategies from prepubertal testicular tissues are still under investigation and have not yet resulted in complete germ cell differentiation, in mice various studies have described production of sperm and offspring through testicular organ culture and transplantation of in vitro propagated SSCs. Organ culture has shown to be successful in generating mature spermatozoa when using testicular fragments from various mouse strains, including CD1 and C57BL/6 J. Conversely, in vitro proliferation of SSCs from C57BL/6 J mice is highly inefficient when compared to other strains such as DBA2 or hybrid mice of C57BL/6 J and DBA2 with 75% C57BL/6 J background (B6D2F2). In this study, we investigated in vitro spermatogenesis by organ culture using testicular tissue from C57BL/6 J and B6D2F2 mice. Whereas spermatogenesis was initiated and completed in C57BL/6 J fragments, it could not be effectively supported in B6D2F2 testicular tissue. While maturation of Sertoli cells and Leydig cells functionality appeared to be identical between the two strains, in B6D2F2 tissue spermatogenesis did not proceed past the spermatocyte step, followed by a rapid decline of the number of all germ cells in the fragments. This suggests that the spermatogenic potential in vitro is dependent on specialized sites in the genome and therefore the organ culture conditions suboptimal for some strains of mice.

High glucose levels affect spermatogenesis: an in vitro approach.

Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas-liquid interface with either 5, 25 or 50mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increased Sertoli cell number (P<0.05) and decreased tubular luminal area (P<0.01), suggesting an impairment of this somatic cell type. Moreover, higher proliferative activity in a TM4 Sertoli cell line exposed to high D-glucose was found (P<0.05) without compromising cell viability (P>0.05), further suggesting altered Sertoli cell maturation. Overall, high D-glucose concentrations may lead to impairment of Sertoli cell function, which, given their significant role in spermatogenic control, may compromise male fertility.

Male germline stem cells in non-human primates.

Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs). These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys) are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs) are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28) during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of human fertility preservation strategies.