Regulated upon activation normal T-cell expressed and secreted originating from the epididymis differentially associates with viable and defective spermatozoa  Zhen Li, M.D., Zhi-jian Sun, M.Sc., Cheng-gong Liao, M.Sc., Li Ma, M.Sc., Bing-fang Ma, B.Sc., Yuan-qiang Zhang, M.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2661-2667 (May 2010) DOI: 10.1016/j.fertnstert.2010.01.053 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in human reproductive tissues. (A) Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of RANTES in human reproductive tissues. The PCR products were found in uterus, epididymis, and prostate, but not in testis and sperm. Negative control consisted of PCR mixture without template complementary DNA (cDNA). (B) Line drawing showing human epididymal regions. (C) Protein extracted from of the epididymis and testis were analyzed by Western blot. RANTES was detected relative strong in the caput (Cap) and cauda (Cau) epididymis, weak in corpus (Cor), none in testis (Te). (D, left) In situ hybridization (ISH) of RANTES messenger RNA (mRNA) in human epididymis and teistis. Positive signal were present along the efferent and epididymal epithelia cells, but not in the testis. The ISH control was obtained by using a sense probe. Right. Immunohistochemistry (IHC) of RANTES in human epididymis and testis. Positive reactions were found restricted in ciliated cell (small arrow) of the efferent duct, narrow (large arrow), apical (arrowhead), and basal cell (fat arrow) in the epididymal duct, but not in the testis. The IHC control was obtained by omitting the primary antibody. (E) The cell localization of human RANTES, F4/80, and clusterin and their colocalization in corpus epididymis by immunofluorescence. The green signal of RANTES (Alexa 488 labeled) coincide with the red signal of F4/80 (Alexa 594 labeled for basal cell, top) but not with the clusterin (Alexa 594 labeled for principle cell, bottom). Bars = 20 μm. Fertility and Sterility 2010 93, 2661-2667DOI: (10.1016/j.fertnstert.2010.01.053) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in mouse epididymis. (A) Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of RANTES in mouse epididymis. The PCR products were found relatively strong in caput (Cap) and corpus (Cor), but not in cauda (Cau). (B) Western blot analysis of RANTES protein along the epididymis. (C) The relative amounts of RANTES messenger RNA (mRNA) (real-time quantitative PCR analysis) and protein (Western blot analysis) level in different regions of epididymides (n = 3). (D) Cellular of RANTES localization in mouse epididymis. Positive reactions were limited in ciliated cells (small arrow) of efferent duct (Ed), apical (arrowhead), and narrow (large arrow) cells of the initial segment (Ins), caput (Cap), and corpus (Cor) epididymis. No positive reaction was shown in basal cell. Control was obtained by omitting the primary antibody. Bar = 20 μm. Fertility and Sterility 2010 93, 2661-2667DOI: (10.1016/j.fertnstert.2010.01.053) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in mouse epididymis during postnatal development. (A) Western blot detected RANTES protein expression during postnatal development epididymis. RANTES expression was first detected in epididymis of 28-day-old mouse and increased with development. (B) The relative amount of RANTES protein in the mouse epididymis at different stages of development (n = 3). Fertility and Sterility 2010 93, 2661-2667DOI: (10.1016/j.fertnstert.2010.01.053) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunolocalization of regulated upon activation normal T-cell expressed and secreted (RANTES) in human and mouse epididymal spermatozoa. (A) Immunohistochemical studies of RANTES on human epididymal spermatozoa, positive reactions were located on midpiece of the spermatozoa. Bar = 10 μm. (B) Indirect immunofluorescence analysis of RANTES protein on mouse epididymal spermatozoa, RANTES positive signals (red) were mainly located in the midpiece of the spermatozoa. (C) RANTES (red signal) was detected on the head and midpiece of abnormal spermatozoa derived from mouse epididymis. Abnormalities shown include lack of tail (a), swollen (enlarged) head (b), twin spermatozoa (c), twin head (d), twin tail (e), folded tail (f), and misshaped sperm heads (g). RANTES binds to the midpiece of normal spermatozoa (h). Bar = 5 μm. Fertility and Sterility 2010 93, 2661-2667DOI: (10.1016/j.fertnstert.2010.01.053) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions