In Vitro and In Vivo Culture Systems for Development of Porcine Testis Cells and Tissue

Abstract

The studies presented in this thesis were designed to identify, examine, and manipulate potential factors associated with the development of porcine testis cells and tissue using in vitro and in vivo culture systems. The objective of the first in vitro study was to examine several conditions for short-term culture of testis cells to optimize the maintenance and propagation of neonatal porcine gonocytes. We found that culturing testis cells at 3.0 × 10⁴ cells/cm² containing ~40% gonocytes in DMEM+10% FBS, at 37 °C, and without changing the medium for 7 days, improves in vitro maintenance, proliferation, and formation of gonocyte colonies. The goal of the second in vitro study was to apply these optimized conditions for prolonged culture to study the behavior, colony formation, and ultrastructure of gonocytes. We observed that gonocytes extensively migrate after developing various cytoplasmic projections, rapidly propagate, and form pluripotent embryoid body-like colonies (EBLC). The first in vivo study was designed to establish an efficient method for implantation of neonatal porcine testis cell aggregates under the back skin of recipient mice, and to explore the use of ultrasound biomicroscopy (UBM) for the non-invasive in vivo evaluation of implants. We showed that the subcutaneous injection approach, as compared with the conventional surgical approach, offers a less invasive route for implantation of cell aggregates, and results in a more consistent de novo formation of testis tissue in the implants. UBM proved to be a unique means for the non-invasive monitoring of implants and the prediction of the outcomes. The second in vivo study was aimed at utilizing this improved cell implantation model to examine the effects of pre-implantation exposure of testis cells to various growth factors (i.e., EGF, GDNF, FGF2, FGF9, VEGF, LIF, SCF, and RA) on de novo morphogenesis of porcine testis tissue. We observed that each growth factor had a distinct effect on development of the testis tissue, ranging from enhanced development to improved gonocyte survival, and promotion of certain types of seminiferous cords. In summary, we discovered several critical elements for the in vitro and in vivo culture systems for the development of testis cells and tissue.