Figure 1: Testicular cells form normal mammary outgrowths in vivo when transplanted with mouse mECM.

7.5 × 10⁴ testicular cells derived from WC/R26-LacZ mice were inoculated in suspensions of mECM, omental fat ECM, lung ECM, or vehicle (DMEM) into cleared mammary fat-pads of female nude mice. Following full-term pregnancy and 3 weeks of involution to activate the WC/R26-LacZ reporter in the testicular-derived cells, glands were isolated for analysis. (A–C) Examples of X-gal+whole mounts from inoculations of testicular cells and mECM. X-gal+outgrowths were not seen in any of the testicular cells + control (omental fat ECM, lung ECM, and DMEM) groups. (D) Cross-section of a an X-gal stained gland derived from testicular cells. X-gal stain is blue, nuclei are counterstained with nuclear fast red. (E) FISH analysis of testicular derived outgrowths with probes to the X-chromosome (magenta; left panel) and Y chromosome (green, right panel). (F) PCR with primers specific for the Y chromosome. Lane 1: Molecular weight marker; Lane 2, 4, and 5: Testicular cells + mECM outgrowth; Lane 3: Testicular cells + mECM inoculated fat pad with no outgrowth; Lane 6: water; Lane 7&8: MEC + mECM outgrowths; Lane 9&11: outgrowth derived from MEC + testicular cells; Lane 10: MEC + testicular cell inoculated fat pad with no outgrowth; Lane 12: MEC cells; Lane 13: Testicular cells. (G–J) IHC staining for alpha-lactalbumin (G), caseins (H), smooth muscle actin (I), and ERα (J) in outgrowth of testicular cells and mECM in 14 day pregnant host (nuclei counterstained with haematoxylin). Scale bars: A–C = 2 mm; D = 200 μM; E = 100 μM; G-I = 100 μM; J = 200 μM.