Peritubular cells may modulate Leydig cell–mediated testosterone production through a nonclassic pathway  Jibanananda Mishra, Ph.D., Mukesh Gautam, M.Sc., Rajesh Dadhich, Ph.D., Bhavani S. Kowtharapu, Ph.D., Subeer S. Majumdar, Ph.D.  Fertility and Sterility  Volume 98, Issue 5, Pages 1308-1317.e1 (November 2012) DOI: 10.1016/j.fertnstert.2012.07.1124 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A–E) Computer-generated models of seminiferous tubules. (A, B) Outer view of a seminiferous tubule (A) and sagittal section of a seminiferous tubule showing the arrangements of Lc, Sc, and PTc (B). (C, D) Dissociation of different cell types from seminiferous tubule after the first and second steps of enzymatic digestion. (E) Seminiferous tubular skeleton with Sc and Gc devoid of PTc and Lc. Fertility and Sterility 2012 98, 1308-1317.e1DOI: (10.1016/j.fertnstert.2012.07.1124) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A–L) Cell purity and morphology. (A) Sertoli cells cultured from 18-day-old rats. More than 95% of the cells stained positive for lipid droplets (Oil Red “O” staining), confirming that at least 95% of the cells are Sc. (B) Less than 2% PTc in the Sc culture were alkaline phosphatase positive (arrow). (C) 3β-HSD activity of Percoll-purified Lc. (D) Cross-section of 18-day-old rat testis. (E) Immunohistochemistry of testicular section showing α-smooth muscle actin–positive PTc at the periphery of seminiferous tubules. (F) Immunocytochemistry of purified PTc showing presence of α-smooth muscle actin in the majority of cultured cells. (G) Image in F merged with image of nuclear staining for the same field. (H) Phase contrast and (I) α-smooth muscle actin staining image of cultured PTc displaying ring-like formation. (J) Phase contrast image of nodule-like structures (arrow) generated by Sc and (K) ultraviolet image of such nodules surrounded by α-smooth muscle actin–positive PTc in Sc-PTc coculture. (L) Merged image of K with nuclear (Hoechst) staining. Note that PTc formed ring-like structures in vitro. Fertility and Sterility 2012 98, 1308-1317.e1DOI: (10.1016/j.fertnstert.2012.07.1124) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A–C) Effect of PTc, Sc, and Sc-PTc coculture on T production by Lc. (A) Effect of PTc on Lc (n = 3). (B) Effect of Sc on Lc (n = 3). (C) Effect of Sc-PTc on Lc (n = 5). *P<.05, **P<.01 as compared with T production by Lc alone. Fertility and Sterility 2012 98, 1308-1317.e1DOI: (10.1016/j.fertnstert.2012.07.1124) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A–D) Effect of 10× PTc media on Sc and Lc. (A) Effect of 10× PTc media on lactate production by Sc (n = 5). (B) Effect of 10× PTc media on T production by Lc. C = 50, 100, or 200 μL of 10× PTc media, FT = 50, 100, or 200 μL of 10× PTc media after FSH and T treatment (n = 9). (C) Quantitative real-time PCR analysis of StAR mRNA expression by Lc in response to hCG (n = 4) or (D) 10× PTc-treated media (n = 4). *P<.05, **P<.01. Fertility and Sterility 2012 98, 1308-1317.e1DOI: (10.1016/j.fertnstert.2012.07.1124) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 1 Semiquantitative RT-PCR analysis of StAR gene expression in response to hCG or 10× PTc media treatment on Lc. C = 50, 100, 200 μL of 10× PTc media, FT = 50, 100, 200 μL of 10× PTc media after FT treatment. Lower panel represents densitometric analysis from three repeats. Upper panel is a representative gel picture from one of three such repeats. *P<.05. Fertility and Sterility 2012 98, 1308-1317.e1DOI: (10.1016/j.fertnstert.2012.07.1124) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions