Expression of ciliated bronchial epithelium 1 during human spermatogenesis  Christiane Pleuger, M.Sc., Daniela Fietz, Dr.Med.Vet., Katja Hartmann, Dr.Med.Vet., Hans-Christian Schuppe, Dr.Med., Wolfgang Weidner, Dr.Med., Sabine Kliesch, Dr.Med., Mark Baker, Ph.D., Moira K. O'Bryan, Ph.D., Martin Bergmann, Dr.Rer.Medic.  Fertility and Sterility  Volume 108, Issue 1, Pages 47-54 (July 2017) DOI: 10.1016/j.fertnstert.2017.05.019 Copyright © 2017 American Society for Reproductive Medicine Terms and Conditions

Figure 1 mRNA expression and protein localization of CBE1 in normal spermatogenesis. (A) CBE1 mRNA is clearly localized in cytoplasm of primary pachytene spermatocytes, showing by in situ hybridization. Insets show the lower magnification and the matching negative control using the sense probe. (B) Staging according to Bergmann and Kliesch (17). (C) Western blot analysis revealed that CBE1 protein (29.9 kDa) was only localized in testicular biopsy samples but not in ejaculate samples (α-tubulin, 50 kDa, was used as loading control). (D) Immunohistochemical detection showed a clear stage-specific localization of CBE1 within the flagellum of spermatids from stage V (black arrow) to the sperm release in stage II. In the flagellum of spermatids in stage IV, CBE1 was missing (white arrow). (E) CBE1 was not detected in the flagellum of mature sperm, in contrast to the control α-tubulin. (F–H) Immunoelectron microscopy showed CBE1 (black arrows) clearly associated with the microtubules of the manchette (F), the head-tail coupling area (G), and the flagellum (H) of elongating spermatids, indicating strongly an influence not only in the intramanchette but also in the intraflagellar transport. Fertility and Sterility 2017 108, 47-54DOI: (10.1016/j.fertnstert.2017.05.019) Copyright © 2017 American Society for Reproductive Medicine Terms and Conditions

Figure 2 CBE1 expression is altered in impaired spermatogenesis. (A) In contrast to the constant mRNA expression in normal spermatogenesis (NSP), CBE1 was not expressed in specimens showing a Sertoli cell only syndrome (SCO), indicating that CBE1 is only expressed in germ cells and not in somatic Sertoli cells. (B) Although the mRNA was localized in primary pachytene spermatocytes in normal spermatogenesis (n = 8), CBE1 was significantly reduced in maturation arrests at the level of early round spermatids (SDA, n = 8) and primary spermatocytes (STA, n = 8). The CBE1 expression was normalized with β-actin and KASH5 (specific spermatocyte marker) to account for variations in the amount of spermatocytes in the samples. (C) Western blot shows that the protein was missing in SDA samples in comparison to normal spermatogenesis (α-tubulin was used as loading control). (D and E) Immunohistochemical analysis of selected SDA samples, which contain (D) a rare amount of elongating spermatids, showed (E) an absence of CBE1 in the flagellum. Fertility and Sterility 2017 108, 47-54DOI: (10.1016/j.fertnstert.2017.05.019) Copyright © 2017 American Society for Reproductive Medicine Terms and Conditions