166 Use of ECM-based three-dimensional scaffolds for stimulating bovine spermatogonia differentiation and meiotic division in vitro

Accelerating genetic selection to obtain animals that are more resilient to climate change and have a lower environmental impact would greatly benefit by a substantial shortening of the generation interval. One way to achieve this goal is to generate male gametes directly from embryos. However, spermatogenesis is a complex biological process that can be partially reproduced in vitro only in mice, whose pluripotent stem cells-derived spermatogonia-like cells can be differentiated into spermatids and used to obtain offspring. In cattle, it is possible only to propagate spermatogonia without inducing an effective meiotic division. A possible strategy to obtain haploid gametes is to generate a three-dimensional (3D) niche that favors the functional interaction between Sertoli cells and spermatogonia. A decellularized 3D scaffold is a possible option because it accurately replicates the in vivo topography, and its extracellular matrix provides essential biomechanical cues for cellular interactions and differentiation. As a first step, we developed a protocol for the creation of a testicular 3D bio-scaffold. Three adult bovine testes were decapsulated. Testicular parenchyma fragments of 0.5 cm³ were frozen at −80°C for at least 24 h, thawed at 37°C for at least 30 min, and immersed in 0.3% SDS and then in 1% of TRITON X-100. This step was followed by a 24-h PBS wash on a shaker at 150 rpm. Decellularization efficiency was evaluated through histological analyses; the functionality of scaffolds was assessed by repopulating them with 0.8 × 10⁶ bovine Sertoli cells, obtained from 1-mm³ parenchyma fragments cultured in DMEM/F12 supplemented with 20% FBS, 2 mM glutamine, and 2% antibiotic/antimycotic solution in an incubator at 37°C with 5% CO₂ for 2 weeks. The experiments were performed in triplicate, and Student’s t-test or ANOVA with Tukey’s post-hoc were used for statistical analysis. Hematoxylin/eosin (HE) and DAPI staining demonstrated that the 3D scaffolds were devoid of cells, while preserving an intact ECM microarchitecture. Crossmon’s trichrome, alcian blue, and orcein staining showed the preservation of the main ECM components. The estimation of volume density (vol/vol) for collagen, elastin, and glycosaminoglycan by stereological quantifications using the Delesse principle demonstrated no significant differences of ECM composition between decellularized bio-scaffolds and native tissue. Repopulation experiments showed rapid cell adhesion and migration during 1 week of culture. Cell density analysis using the image analysis software ImageJ and the specific Cell Counter plugin, indicated a 3.5-fold increase over the culture period and confirmed the ability of bio-scaffolds to encourage cell homing and proliferation. This protocol generates decellularized scaffolds with no cytotoxic effects that provide a suitable support for the ex vivo culture of testicular cells, offering a solution for recapitulating spermatogenesis in vitro. Coupled with the effective differentiation of Sertoli-spermatogonia complexes from embryonic cells, this approach may be an effective way to recapitulate male meiosis in vitro.

This research was supported by the Agritech National Research Center funded from the European Union Next-Generation EU (Piano Nazionale di Ripresa e Resilienza; PNRR).