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 In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional (3D) in vitro culture systems.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.

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-AgarCulture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfra-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.

Our results demonstrate that LPS increases the expression levels of the IL-18 family in mouse testis and spleen, but the time of expression differs between the two organs. The presence of IL-18 in the testes might be involved in the regulation of physiological and infection/inflammatory processes, and may be part of the autocrine/paracrine factors that control spermatogenesis. Further studies should be performed to confirm this possibility.

Our results demonstrate for the first time the distant effect of acute localized inflammation on testicular IL-1 levels. Thus, transient inflammatory response to infectious/inflammatory agents at non-testicular sites that elicit systemic IL-1 response should be considered during clinical treatment as a possible factor of male infertility.