The normal structure and function of sperm are prerequisites for successful fertilization and embryonic development, but little is known about how defective sperm are eliminated during mammalian spermatogenesis. Here, we describe a ubiquitin-dependent, sperm quality control mechanism that resides in the mammalian epididymis, the site of sperm maturation and storage. We used immunofluorescence, electron microscopy, western blotting and pulse-chase experiments to show that ubiquitin is secreted by the epididymal epithelium and binds to the surface of defective sperm. Most of the ubiquitinated sperm are subsequently phagocytosed by the epididymal epithelial cells. A portion of defective sperm escapes phagocytosis and can be found in the ejaculate. Cultured epididymal cells maintain their ability to produce ubiquitin and phagocytose the defective sperm, as well as the ubiquitin-coated microspheres, in vitro. The surprising phenomenon of cell-surface ubiquitination in defective sperm provides a possible mechanism for sperm quality control in mammals and a new marker of semen abnormalities in men and animals.
REFERENCES
Agrawal
Y.
, Vanha-Perttula
T.
(1988
). Electron microscopic study of the secretion process in bovine reproductive organs.
J. Androl
9
, 307
–316
Andonian
S.
, Hermo
L.
(1999
). Cell-and region-specific localization of lysosomal and secretory proteins and endocytic receptors in epithelial cells of the cauda epididymis and vas deferens of the adult rat.
J. Androl
20
, 415
–429
Baarends
W. M.
, Hoogerbrugge
J. W.
, Roest
H. P.
, Ooms
M.
, Vreeburg
J.
, Hoeijmakers
J. H.
, Grootegoed
J. A.
(1999
). Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.
Dev. Biol
207
, 322
–333
Baarends
W. M.
, Roest
H. P.
, Grootegoed
J. A.
(1999
). The ubiquitin system in gametogenesis.
Mol. Cell Endocrinol
151
, 5
–16
Beagley
K. W.
, Wu
Z. L.
, Pomering
M.
, Jones
R. C.
(1998
). Immune responses in the epididymis: implications for immunocontraception.
J. Reprod. Fert. Suppl
53
, 235
–245
Blom
E.
(1966
). A new sterilizing and hereditary defect (the ‘Dag defect’) located in the bull sperm tail.
Nature
209
, 739
–740
Bloom
G.
, Nicander
L.
(1961
). On the ultrastructure and development of the protoplasmic droplet of spermatozoa.
Z. Zellforsch
55
, 833
–844
Chen
H. Y.
, Sun
J. M.
, Zhang
Y.
, Davie
J. R.
, Meistrich
M. L.
(1998
). Ubiquitination of histone H3 in elongating spermatids of rat testes.
J. Biol. Chem
273
, 13165
–13269
Cooper
T. G.
, Yeung
C.-H.
, Meyer
R.
(1989
). Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. I. Vectorial secretion.
Cell Tissue Res
256
, 567
–572
Cooper
T. G.
, Yeung
C.-H.
, Nashan
D.
, Nieschlag
E.
(1988
). Epididymal markers in human infertility.
J. Androl
9
, 91
–101
de Kretser
D. M.
, Huidobro
C.
, Southwick
G. J.
, Temple-Smith
P. D.
(1998
). The role of epididymis in human infertility.
J. Reprod. Fert. Suppl
53
, 271
–275
Flickinger
C. J.
(1982
). The fate of sperm after vasectomy in the hamster.
Anat. Rec
202
, 231
–239
Fouchecourt
S.
, Metayer
S.
, Locatelli
A.
, Dacheux
F.
, Dacheux
J. L.
(2000
). Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins.
Biol. Reprod
62
, 1790
–1803
Fraile
B.
, Martin
R.
, De Miguel
M. P.
, Arenas
M. I.
, Bethencourt
F. R.
, Peinado
F.
, Paniagua
R.
, Santamaria
L.
(1996
). Light and electron microscopic immunohistochemical localization of protein gene product 9.5 and ubiquitin immunoreactivities in the human epididymis and vas deferens.
Biol. Reprod
55
, 291
–297
Hermo
L.
, Dworkin
J.
, Oko
R.
(1988
). Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa.
Am. J. Anat
183
, 107
–124
Hermo
L.
, Oko
R.
, Robaire
B.
(1992
). Epithelial cells of the epididymis show regional variations with respect to the secretion of endocytosis of immobilin as revealed by light and electron microscope immunocytochemistry.
Anat. Rec
232
, 202
–220
Hershko
A.
, Ciechanover
A.
(1998
). The ubiquitin system.
Annu. Rev. Biochem
67
, 425
–479
Igdoura
S. A.
, Hermo
L.
, Morales
C. R.
(1994
). Sulfated glycoprotein-2 synthesized by nonciliated cells of the efferent ducts is targeted to the lysosomal compartment.
Microsc. Res. Tech
29
, 468
–480
Jones
A. R.
(1998
). Chemical interference with sperm metabolic pathways.
J. Reprod. Fert
53
, 227
–234
Laney
J. D.
, Hochstrasser
M.
(1999
). Substrate targeting in the ubiquitin system.
Cell
97
, 427
–430
Lippert
T. H.
, Seeger
H.
, Schieferstein
G.
, Voelter
W.
(1993
). Immunoreactive ubiquitin in human seminal plasma.
J. Androl
14
, 130
–131
Manin
M.
, Lecher
P.
, Martinez
A.
, Tournadre
S.
, Jean
C.
(1995
). Exportation of mouse vas deferens protein, a protein without a signal peptide, from mouse vas deferens epithelium: a model of apocrine secretion.
Biol. Reprod
52
, 50
–62
Martinou
I.
, Desagher
S.
, Eskes
R.
, Antonsson
B.
, Andre
E.
, Fakan
S.
, Martinou
J. C.
(1999
). The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event.
J. Cell Biol
144
, 883
–889
Moore
H. D.
, Curry
M. R.
, Penfold
L. M.
, Pryor
J. P.
(1992
). The culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa.
Fertil. Steril
58
, 776
–783
Morita
Y.
, Tilly
J. L.
(1999
). Oocyte apoptosis: like sand through an hourglass.
Dev. Biol
213
, 1
–17
Parks
J. E.
, Hammerstedt
R. H.
(1985
). Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane.
Biol. Reprod
32
, 653
–668
Parrish
J. J.
, Susko-Parrish
J. L.
, Leibfreid-Rutledge
M. L.
, Critser
E. S.
, Eyestone
W. H.
, First
N. L.
(1986
). Bovine in vitro fertilization with frozen-thawed semen.
Theriogenology
25
, 591
–600
Rajapurohitam
V.
, Morales
C. R.
, El-Alfy
M.
, Lefrancois
S.
, Bedard
N.
, Wing
S. S.
(1999
). Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.
Dev. Biol
212
, 217
–228
Ramalho-Santos
J.
, Moreno
R. D.
, Sutovsky
P.
, Chan
A. W.-S.
, Hewitson
L. C.
, Wessel
G. M.
, Simerly
C. R.
, Schatten
G.
(2000
). SNAREs in mammalian sperm: Possible implications for fertilization.
Dev. Biol
223
, 54
–69
Ramamohana-Rao
A.
, Bane
A.
, Gustafsson
B. K.
(1980
). Changes in the morphology of spermatozoa during their passage through the genital tract in dairy bulls with normal and impaired spermatogenesis.
Theriogenology
14
, 1
–12
Roussel
J. D.
, Stallcup
O. T.
, Austin
C. R.
(1967
). Selective phagocytosis of spermatozoa in the epididymis of bulls, rabbits and monkeys.
Fertil. Steril
18
, 509
–516
Sakkas
D.
, Mariethoz
E.
, St John
J. C.
(1999
). Abnormal spermparameters in humans are indicative of an abortive apoptotic mechanism linked to the fas-mediated pathway.
Exp. Cell Res
251
, 350
–355
Santamaria
L.
, Martin
R.
, Paniagua
R.
, Fraile
B.
, Nistal
M.
, Terenghi
G.
, Polak
J. M.
(1993
). Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium.
Histochemistry
100
, 131
–138
Strous
G. J.
, Govers
R.
(1999
). The ubiquitin-proteasome system and endocytosis.
J. Cell Sci
112
, 1417
–1423
Sutovsky
P.
, Moreno
R.
, Ramalho-Santos
J.
, Dominko
T.
, Simerly
C.
, Schatten
G.
(1999
). Ubiquitin tag for sperm mitochondria.
Nature
402
, 371
–372
Sutovsky
P.
, Moreno
R.
, Ramalho-Santos
J.
, Dominko
T.
, Simerly
C.
, Schatten
G.
(2000
). Ubiquitinated sperm mitochondria, selective proteolysis and the regulation of mitochondrial inheritance in mammalian embryos.
Biol. Reprod
63
, 582
–590
Sutovsky
P.
, Terada
Y.
, Schatten
G.
(2001
). Ubiquitin-based spermassay for the diagnosis of male factor infertility.
Human Reprod
16
, 250
–258
Weil
M.
, Jacobson
M. D.
, Raff
M. C.
(1998
). Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes.
J. Cell Sci
111
, 2707
–2715
Yeung
C. H.
, Sonnenberg-Riethmacher
E.
, Cooper
T. G.
(1998
). Receptory tyrosine kinase c-ros knockout mice as a model for the study of epididymal regulation of sperm function.
J. Reprod. Fert. Suppl
53
, 137
–147
Yeung
C. H.
, Sonnenberg-Riethmacher
E.
, Cooper
T. G.
(1999
). Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms.
Biol. Reprod
61
, 1062
–1069
Yin
Y.
, Stahl
B. C.
, DeWolf
W. C.
, Morgentaler
A.
(1998
). p53-mediated germ cell quality control in spermatogenesis.
Dev. Biol
204
, 165
–171
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