Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

Abstract

Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cellcell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

Fundamentals of rapid injection molding for microfluidic cell-based assays

“…The device is based on a design previously published for primary testis cell culture studies. 32 Maintaining a planar surface to allow reliable solvent bonding presents design challenges in the context of injection molding. Areas of varying thickness result in regions of the device cooling and solidifying at different rates and thus variable shrinking arises.…”

Micromilling: a method for ultra-rapid prototyping of plastic microfluidic devices

“…21 Carney and co-workers cultured primary fetal testis cells in mixed and compartmentalized co-culture to study microenvironmental factors regulating steroid production and organ co-culture assays. 22 However, the method remains underutilized as a microfabrication method compared to other methods. This is largely because of presumed high start-up costs, the need for large equipment and lab space, and the need for unique technical expertise.…”

Experimental and theoretical investigation into simultaneous deburring of microchannel and cleaning of the cutting tool in micromilling

“…Researchers have used micromilling extensively for fabrication of microfluidic devices including Kit-On-A-Lid-Assays (KOALA) in which multiple slides fabricated by micromilling can be combined for fluid delivery, 15 cell capture devices with oil and aqueous interfaces, 16 and devices for microenvironmental studies on primary foetal testis cells. 17 The machinability studies on polymer materials revealed that the elastomers like PDMS (polydimethylsiloxane) are difficult to process using micromilling. 12 PMMA has higher machinability compared to PDMS, and PMMA microfluidic devices have the advantage of having least hydrophobic nature 18 compared to other polymers.…”