Cell and Tissue Research

, Volume 372, Issue 1, pp 135–147 | Cite as

Pattern of protein expression in the epididymis of Oligoryzomys nigripes (Cricetidae, Sigmodontinae)

  • Tatiana Prata Menezes
  • Eric Hill
  • Arlindo de Alencar Moura
  • Marina D. P. Lobo
  • Ana Cristina O. Monteiro-Moreira
  • Sylvie Breton
  • Mariana Machado-Neves
Regular Article


In the epididymis, epithelial cells work in a concerted manner to create a luminal environment for sperm maturation, transport, and storage. However, the cell functions may be affected by anthropogenic factors, causing negative impacts on male fertility. In our study, we describe the pattern of protein expression in the epithelium and luminal fluid from epididymis of Oligoryzomys nigripes, a South American sigmodontine rodent whose reproductive biology has been little studied. Nine animals were captured from a preserved area of Atlantic Forest, where the exposure to anthropogenic influences is minimal. Epididymides were processed for histological analysis under light and epifluorescence microscopy, in which we used cell-specific markers aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Other samples were assessed for protein expression using shotgun proteomics. Similar to laboratory rodents, principal cells expressed AQP9 in their stereocilia. Basal cells, identified by KRT5 labeling, presented lateral body projections and a few axiopodia going toward the lumen. Clear cells expressed V-ATPase in their sub-apical vesicles and microplicae, and showed different shapes along the duct. Shotgun proteomics detected 51 proteins from epididymal supernatant. Most of them have been previously described in other species, indicating that they are well conserved. Twenty-three proteins detected in O. nigripes have not been described in epididymis from other South American sigmodontine rodents, confirming that the secretion pattern is species-specific. Our findings in O. nigripes from a protected area may help to create a baseline for studies investigating the effects of anthropogenic factors on functionality of the epididymal epithelium.


Epithelial cells Epididymal fluid Proteome Morphometry Reproductive biology 



The authors thank Ana Carolina Torre Morais, Maytê Koch Balarini, Kyvia Lugate, Nayara Magalhães, Rafael Reis and Mário José de Oliveira for their support during the collection of the specimens, and Mariana M Castro for revising the manuscript. This research was supported by Dr. Arlindo A A Moura and Dr. Sylvie Breton along with their research teams. Tatiana P Menezes has been granted a scholarship of Brazilian Federal Agency for Support and Evaluation of Graduate School (CAPES). Mariana Machado-Neves participated as a visiting professor in the Program of Membrane Biology at Massachusetts General Hospital (MGH) with a fellowship from Fundação de Amparo a Pesquisa de Minas Gerais (FAPEMIG; process number ECT00054-1).

Compliance with ethical standards

Conflict of interest

None of the authors have any conflict of interest to declare.

Supplementary material

441_2017_2714_MOESM2_ESM.docx (76 kb)
Table S1 (DOCX 76 kb)


  1. Aitken RJ (2006) Sperm function tests and fertility. Int J Androl 29:69–75CrossRefPubMedGoogle Scholar
  2. Amann RP (1962) Reproductive capacity of dairy bulls. III. The effect of ejaculation frequency, unilateral vasectomy, and age on spermatogenesis. Am J Anat 110:49–67CrossRefPubMedGoogle Scholar
  3. Badran HH, Hermo LS (2002) Expression and regulation of aquaporins 1, 8, and 9 in the testis, efferent ducts, and epididymis of adult rats and during postnatal development. J Androl 23:358–373PubMedGoogle Scholar
  4. Bhatia VN, Perlman DH, Costello CE, McComb ME (2009) Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem 81:9819–9823CrossRefPubMedPubMedCentralGoogle Scholar
  5. Belleannée C, Da Silva N, Shum WW, Marsolais M, Laprade R, Brown D, Breton S (2009) Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9. Biol Reprod 80:134–143CrossRefPubMedPubMedCentralGoogle Scholar
  6. Belleannée C, Belghazi M, Labas V, Teixeira-Gomes AP, Gatti JL, Dacheux JL, Dacheux F (2011) Purification and identification of sperm surface proteins and changes during epididymal maturation. Proteomics 11:1952–1964CrossRefPubMedGoogle Scholar
  7. Beu CC, Orsi AM, Domeniconi RF (2009) Structure of the lining epithelium of the cauda epididymis of the golden hamster. Anat Histol Embryol 38:49–57CrossRefPubMedGoogle Scholar
  8. Breton S, Smith PJ, Lui B, Brown D (1996) Acidification of the male reproductive tract by a proton pumping (H+)-ATPase. Nat Med 2:470–472CrossRefPubMedGoogle Scholar
  9. Breton S, Tyszkowski R, Sabolic I, Brown D (1999) Postnatal development of H+ ATPase (proton-pump)-rich cells in rat epididymis. Histochem Cell Biol 111:97–105CrossRefPubMedGoogle Scholar
  10. Castro MM, Kim B, Hill E, Fialho MC, Puga LC, Freitas MB, Breton S, Machado-Neves M (2017) The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat. Histochem Cell Biol 147:39–48CrossRefPubMedGoogle Scholar
  11. Cheung KH, Leung GP, Leung MC, Shum WW, Zhou WL, Wong PY (2005) Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat. J Gen Physiol 125:443–454CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cornwall GA (2009) New insights into epididymal biology and function. Hum Reprod Update 15:213–227CrossRefPubMedPubMedCentralGoogle Scholar
  13. Cornwall GA, von Horsten HH, Swartz D, Johnson S, Chau K, Whelly S (2007) Extracellular quality control in the epididymis. Asian J Androl 9:500–507CrossRefPubMedGoogle Scholar
  14. Da Silva N, Silberstein C, Beaulieu V, Piétrement C, Van Hoek AN, Brown D, Breton S (2006a) Postnatal expression of Aquaporins in epithelial cells of the rat Epididymis. Biol Reprod 74:427–438CrossRefPubMedGoogle Scholar
  15. Da Silva N, Piétrement C, Brown D, Breton S (2006b) Segmental and cellular expression of aquaporins in the male excurrent duct. Biochim Biophys Acta 1758:1025–1033CrossRefPubMedGoogle Scholar
  16. Dacheux JL, Dacheux F, Labas V, Ecroyd H, Nixon B, Jones RC (2009) New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus). Reprod Fertil Dev 21:1002–1007CrossRefPubMedGoogle Scholar
  17. Dacheux JL, Belleannée C, Guyonnet B, Labas V, Teixeira-Gomes AP, Ecroyd H, Druart X, Gatti JL, Dacheux F (2012) The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa. Syst Biol Reprod Med 58:197–210CrossRefPubMedGoogle Scholar
  18. Dacheux JL, Dacheux F (2014) New insights into epididymal function in relation to sperm maturation. Reproduction 147:R27–R42CrossRefPubMedGoogle Scholar
  19. Di-Nizo CB, Ventura K, Ferguson-Smith MA, O’Brien PCM, Yonenaga-Yassuda Y, Silva MJJ (2015) Comparative chromosome painting in six species of Oligoryzomys (Rodentia, Sigmodontinae) and the Karyotype evolution of the genus. PLoS ONE 10:e0117579CrossRefPubMedPubMedCentralGoogle Scholar
  20. Domeniconi RF, Orsi AM, Beu CCL, Felisbino SL (2007) Morphological features of the epididymal epithelium of gerbil, Meriones unguiculatus. Tissue Cell 39:47–57CrossRefPubMedGoogle Scholar
  21. Domeniconi RF, Souza AC, Xu B, Washington AM, Hinton BT (2016) Is the Epididymis a series of organs placed side by side? Biol Reprod 95:1–8CrossRefGoogle Scholar
  22. Fernandez CDB, Porto EM, Arena AC, Kempinas WG (2007) Effects of altered epididymal sperm transit time on sperm quality. Int J Androl 31:427–437CrossRefGoogle Scholar
  23. Gannon WL, Sikes RS (2007) Guidelines of the American Society of Mammalogists for the use of wild mammals in research. J Mammal 88:809–823CrossRefGoogle Scholar
  24. Gatti JL, Castella S, Dacheux F, Ecroyd H, Metayer S, Thimon V, Dacheux JL (2004) Post-testicular sperm environment and fertility. Anim Reprod Sci 82-83:321–339CrossRefPubMedGoogle Scholar
  25. Guyonnet B, Dacheux F, Dacheux JL, Gatti JL (2011) The epididymal transcriptome and proteome provide some insights into new epididymal regulations. J Androl 32:651–664CrossRefPubMedGoogle Scholar
  26. Griffiths GS, Galileo DS, Aravindan RG, Martin-DeLeon PA (2009) Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes. Biol Reprod 81:562–570CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hermo L, Schellenberg M, Liu LY, Dayanandan B, Zhang T, Mandato CA, Smith CE (2008) Membrane domain specificity in the spatial distribution of aquaporins 5, 7, 9 and 11 in efferent ducts and epididymis of rats. J Histochem Cytochem 56:11221–11135CrossRefGoogle Scholar
  28. Huang Q, Luo L, Alamdar A, Zhang J, Liu L, Tian M, Eqani SAMAS, Shen H (2016) Integrated proteomics and metabolomics analysis of rat testis: mechanism of arsenic-induced male reproductive toxicity. Sci Rep 6:32518CrossRefPubMedPubMedCentralGoogle Scholar
  29. Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137–138AGoogle Scholar
  30. Kim B, Roy J, Shum WW, da Silva N, Breton S (2015) Role of testicular luminal factors on basal cell elongation and proliferation in the mouse epididymis. Biol Reprod 92:1–11CrossRefGoogle Scholar
  31. Leung GP, Cheung KH, Leung CT, Tsang MW, Wong PY (2004) Regulation of epididymal principal cell functions by basal cells: role of transient receptor potential (Trp) proteins and cyclooxygenase-1 (COX-1). Mol Cell Endocrinol 216:5–13CrossRefPubMedGoogle Scholar
  32. Majewska AM, Kordan W, Koziorowska-Gilun M, Wysocki P (2017) Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides. Reprod Domest Anim 52:107–114CrossRefPubMedGoogle Scholar
  33. Martins JAM, Souza CEA, Silva FDA, Cadavid VG, Nogueira FC, Domont GB, de Oliveira JTA, Moura AA (2013) Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Rumin Res 113:115–127CrossRefGoogle Scholar
  34. Menezes TP, Castro MM, Vale JA, Moura AAA, Lessa G, Machado-Neves M (2017) Proteomes and morphological features of Calomys tener and Necromys lasiurus (Cricetidae, Sigmodontinae) epididymides. J Mammal 98:579–590CrossRefGoogle Scholar
  35. Moura AA, Souza CE, Stanley BA, Chapman DA, Killian GJ (2010) Proteomics of cauda epididymal fluid from mature Holstein bulls. J Proteome 73:2006–2020CrossRefGoogle Scholar
  36. Oliveira RL, Campolina-Silva GH, Nogueira JC, Mahecha GA, Oliveira CA (2013) Differential expression and seasonal variation on aquaporins 1 and 9 in the male genital system of big fruit-eating bat Artibeus lituratus. Gen Comp Endocrinol 186:116–125CrossRefPubMedGoogle Scholar
  37. Ohkawa H, Inui H (2015) Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants. Pest Manag Sci 71:824–828CrossRefPubMedGoogle Scholar
  38. Park Y-L, Battistone MA, Kim B, Breton S (2017) Relative contribution of clear cells and principal cells to luminal pH in the mouse epididymis. Biol Reprod 96:366–375CrossRefPubMedGoogle Scholar
  39. Pastor-Soler N, Bagnis C, Sabolic I, Tyszkowski R, McKee M, Van Hoek A, Breton S, Brown D (2001) Aquaporin 9 expression along the male reproductive tract. Biol Reprod 65:384–393CrossRefPubMedGoogle Scholar
  40. Pietrement C, Sun-Wada G-H, da Silva N, Mckee M, Marshansky V, Brown D, Futai M, Breton S (2006) Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis. Biol Reprod 74:185–194CrossRefPubMedGoogle Scholar
  41. Pietrement C, da Silva N, Silberstein C, James M, Marsolais M, Van Hoek A, Brown D, Pastor-Soler N, Ameen N, Laprade R, Ramesh V, Breton S (2008) Role of NHERF1, cystic fibrosis transmembrane conductance regulator, and cAMP in the regulation of aquaporin 9. J Biol Chem 283:2986–2996CrossRefPubMedGoogle Scholar
  42. Primiani N, Gregory M, Dufresne J, Smith CE, Liu YL, Bartles JR, Cyr DG, Hermo L (2007) Microvillar size and espin expression in principal cells of the adult rat epididymis are regulated by androgens. J Androl 28:659–669CrossRefPubMedGoogle Scholar
  43. Rego JPA, Crisp JM, Moura AA, Nouwens AS, Li Y, Venus B, Corbet NJ, Corbet DH, Burns BM, Boe-Hansen, McGowan MR (2014) Seminal plasma proteome of ejaculated Bos indicus bulls. Anim Reprod Sci 148:1–17CrossRefPubMedGoogle Scholar
  44. Robaire B, Hermo L (1988) Efferent ducts, epididymis, and vas deferens: structure, functions and their regulation. In: Knobil E, Neill JD (eds) The physiology of reproduction. Ravenress, New York, pp 999–1080Google Scholar
  45. Robaire R, Hinton BT (2015) The epididymis. In: Knobil (4th) Knobil and Neill’s physiology of reproduction. Elsevier, Cambridge, pp 691–771CrossRefGoogle Scholar
  46. Rosalino LM, Martins PS, Gheler-Costa C, Lopes PC, Verdade LM (2013) Allometric relations of Neotropical small rodents (Sigmodontinae) in anthropogenic environments. Zool Sci 30:585–590CrossRefPubMedGoogle Scholar
  47. Roy JW, Hill E, Ruan YC, Vedovelli L, Păunescu TG, Brown D, Breton S (2013) Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis. Am J Physiol Cell Physiol 305:C436–C446CrossRefPubMedPubMedCentralGoogle Scholar
  48. Roy J, Kim B, Hill E, Visconti P, Krapf D, Vinegoni C, Weissleder R, Brown D, Breton S (2016) Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging. Nat Commun 7:10666CrossRefPubMedPubMedCentralGoogle Scholar
  49. Salas PJ, Forteza R, Mashukova A (2016) Multiple roles for keratin intermediate filaments in the regulation of epithelial barrier function and apico-basal polarity. Tissue Barriers 4:e1178368CrossRefPubMedPubMedCentralGoogle Scholar
  50. Salazar-Bravo J, Pardiñas UFJ, D'Elía G (2013) A phylogenetic appraisal of Sigmodontinae (Rodentia, Cricetidae) with emphasis on phyllotine genera: systematics and biogeography. Zool Scr 42:250–261CrossRefGoogle Scholar
  51. Schimming BC, Bauman CAE, Pinheiro PFF, Matteis R, Domeniconi RF (2017) Aquaporin 9 is expressed in the epididymis of immature and mature pigs. Reprod Domest Anim.
  52. Serre V, Robaire B (1998) Segment-specific morphological changes in aging brown norway rat epididymis. Biol Reprod 58:497–513CrossRefPubMedGoogle Scholar
  53. Sidorkiewicz I, Zareba K, Wolczyński S, Czerniecki J (2017) Endocrine-disrupting chemicals-mechanisms of action on male reproductive system. Toxicol Ind Health.
  54. Shin JH, Lee SH, Kim YN, Kim IY, Kim YJ, Kyeong DS, Lim HJ, Cho SY, Choi J, Wi YJ, Choi JH, Yoon YS, Bae YS, Seong JK (2016) AHNAK deficiency promotes browning and lipolysis in mice via increased responsiveness to β-adrenergic signalling. Sci Rep 6:23426CrossRefPubMedPubMedCentralGoogle Scholar
  55. Shum WW, Da Silva N, Mckee M, Smith PJ, Brown D, Breton S (2008) Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia. Cell 135:1108–1117CrossRefPubMedPubMedCentralGoogle Scholar
  56. Shum WWC, da Silva N, Brown D, Breton S (2009) Regulation of luminal acidification in the male reproductive tract via cell-cell crosstalk. J Exp Biol 212:1753–1761CrossRefPubMedPubMedCentralGoogle Scholar
  57. Shum WWC, Ruan YC, da Silva N, Breton S (2011) Establishment of cell-cell cross talk in the epididymis: control of luminal acidification. J Androl 32:576–586CrossRefPubMedPubMedCentralGoogle Scholar
  58. Shum WWC, Hill E, Brown D, Breton S (2013) Plasticity of basal cells during postnatal development in the rat epididymis. Reproduction 146:455–469CrossRefPubMedPubMedCentralGoogle Scholar
  59. Shum WW, Smith TB, Cortez-Retamozo V, Grigoryeva LS, Roy JW, Hill E, Pittet MJ, Breton S, da Silva N (2014) Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis. Biol Reprod 90:1–10CrossRefGoogle Scholar
  60. Sipilä P, Björkgren I (2016) Segment-specific regulation of epididymal gene expression. Reproduction 152:R91–R99CrossRefPubMedGoogle Scholar
  61. Souza ACF, Marchesi SC, Ferraz RP, Lima GDA, Oliveira JA, Machado-Neves M (2016) Effects of sodium arsenate and arsenite on male reproductive functions in rats. J Toxicol Environ Health A 79:274–286CrossRefPubMedGoogle Scholar
  62. Souza CE, Rego JP, Lobo CH, Oliveira JT, Nogueira FC, Domont GB, Fioramonte M, Gozzo FC, Moreno FB, Monteiro-Moreira AC, Figueiredo JR, Moura AA (2012) Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. J Proteome 75:4436–4456CrossRefGoogle Scholar
  63. Turner T (1995) On the epididymis and its role in the development of the fertile ejaculate. J Androl 16:292–298PubMedGoogle Scholar
  64. Turner TT, Bomgardner D, Jacobs JP, Nguyen QAT (2003) Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice. Reproduction 125:871–888CrossRefPubMedGoogle Scholar
  65. Turner TT, Johnston DS, Jelinsky SA, Tomsig JL, Finger JN (2007) Segment boundaries of the adult rat epididymis limit interstitial signaling by potential paracrine factors and segments lose differential gene expression after efferent duct ligation. Asian J Androl 9:565–573CrossRefPubMedGoogle Scholar
  66. van Tilburg MF, Salles MG, Silva MM, Moreira RA, Moreno FB, Monteiro-Moreira AC, Martins JA, Cândido MJ, Araújo AA, Moura AA (2015) Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S). Int J Biometeorol 59:561–573CrossRefPubMedGoogle Scholar
  67. Weksler M, Bonvicino CR (2005) Taxonomy of pigmy rice rats genus Oligoryzomys bangs, 1900 (RODENTIA, SIGMODONTINAE) of the brazilian cerrado, with the description of two new species. Arquivos do Museu Nacional 63:113–130Google Scholar
  68. Zar JH (2010) Biostatistical analysis, 5th edn. Prentice-Hall, Englewood CliffsGoogle Scholar
  69. Zhu S, Chim SM, Cheng T, Ang E, Ng B, Lim B, Chen K, Qiu H, Tickner J, Xu H, Pavlos N, Xu J (2016) Calmodulin interacts with Rab3D and modulates osteoclastic bone resorption. Sci Rep 6:37963CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Tatiana Prata Menezes
    • 1
  • Eric Hill
    • 2
    • 3
  • Arlindo de Alencar Moura
    • 4
  • Marina D. P. Lobo
    • 5
  • Ana Cristina O. Monteiro-Moreira
    • 5
  • Sylvie Breton
    • 2
  • Mariana Machado-Neves
    • 1
  1. 1.Department of General BiologyFederal University of ViçosaViçosaBrazil
  2. 2.Center for Systems Biology/Program in Membrane Biology/Nephrology Division, Massachusetts General HospitalHarvard Medical SchoolBostonUSA
  3. 3.Micro Video Instruments, IncAvonUSA
  4. 4.Department of Animal ScienceFederal University of CearáCearáBrazil
  5. 5.Laboratory of Proteomics, School of PharmacyUniversity of FortalezaCearáBrazil

Personalised recommendations