title: Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.
abstract: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation?
  
  
  
  JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro.
  
  
  
  JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation.
  
  
  
  The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro.
  
  
  
  Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF.
  
  
  
  Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones.
  
  
  
  Not applicable.
  
  
  
  First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate.
  
  
  
  Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART.
  
  
  
  The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.
authors.affiliation: Department of Biological Sciences, University of Delaware, 219 Mckinly Lab, Newark, DE 19716, USA.
authors.forename: Kathie Z
authors.initials: KZ
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences, University of Delaware, 219 Mckinly Lab, Newark, DE 19716, USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, University of Delaware, 219 Mckinly Lab, Newark, DE 19716, USA.
authors.forename: Deni S
authors.initials: DS
authors.lastname: Galileo
authors.affiliation: Department of Biological Sciences, University of Delaware, 219 Mckinly Lab, Newark, DE 19716, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Martin-DeLeon
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D004822:Epididymis / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D062745:Junctional Adhesion Molecule A / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D011467:Prostate / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D021381:Protein Transport; D012669:Seminal Vesicles / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D013079:Sperm Maturation / Q000235:genetics*; D013081:Sperm Motility / Q000235:genetics*; D013094:Spermatozoa / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*
chemical_list: D002118:Calcium; D000252:Calcium-Transporting ATPases; D051528:Guanylate Kinases; D062745:Junctional Adhesion Molecule A; C000589038:ATP2B4 protein, mouse; C106868:CASK kinases
keywords: epididymal luminal fluid; epididymosomes; infertility; junctional adhesion molecule A; phosphorylation; post-testicular maturation; sperm maturation