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Further Characterization of Reproductive
Abnormalities in mCd59b Knockout Mice: A
Potential New Function of mCd59 in Male
Reproduction
Xuebin Qin, Martin Dobarro, Sylvia J. Bedford, Sean Ferris,
Patricia V. Miranda, Wenping Song, Roderick T. Bronson,
Pablo E. Visconti and Jose A. Halperin
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:6294-6302; ;
doi: 10.4049/jimmunol.175.10.6294
http://www.jimmunol.org/content/175/10/6294
The Journal of Immunology
Further Characterization of Reproductive Abnormalities in
mCd59b Knockout Mice: A Potential New Function of mCd59
in Male Reproduction1
Xuebin Qin,2,3*† Martin Dobarro,2* Sylvia J. Bedford,‡ Sean Ferris,† Patricia V. Miranda,‡
Wenping Song,† Roderick T. Bronson,§ Pablo E. Visconti,‡ and Jose A. Halperin3*†
C
omplement is an effector of the immune system that mediates both acquired and innate responses to defend
against foreign Ags such as those present in bacteria or
heterologous erythrocytes (1). The complement system consists of
⬎12 soluble plasma proteins that interact with one another in three
distinct enzymatic activation cascades known as the classical, alternative, and lectin pathways. The three pathways converge at the
level of C3, a central complement component that is cleaved by
pathway-specific convertases. Thereafter, the common terminal
complement cascade leads to formation of the membrane attack
complex (MAC),4 a macromolecular pore that inserts itself into the
cell membrane causing colloidosmotic lysis of the targeted cell.
*Department of Medicine, Division of Hematology and Oncology, Brigham and
Women’s Hospital, Boston, MA 02115; †Laboratory for Translational Research, Harvard University Medical School, Cambridge, MA 02139; ‡Department of Veterinary
and Animal Sciences, University of Massachusetts, Amherst, MA 01003; and §Rodent
Histopathology Core, Dana Farber/Harvard University Medical School, Boston, MA
02115
Received for publication February 25, 2005. Accepted for publication September
1, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants R01 DK052855 (to
J.A.H.), R01 DK060979 (to J.A.H.), and R01 AI061174 (to X.Q.). P.E.V. and P.V.M.
were supported by National Institutes of Health Grants HD38082 and HD44044 (to
P.E.V.).
2
X.Q. and M.D. contributed equally to this paper.
3
Address correspondence and reprint requests to Dr. Xuebin Qin, Harvard University
Medical School, 1 Kendall Square, Building 600, 3rd Floor, Cambridge, MA 02139;
E-mail address: [email protected] or Dr. Jose A. Halperin, Harvard University Medical School, 1 Kendall Square, Building 600, 3rd Floor, Cambridge, MA
02139; E-mail address: [email protected]
4
Abbreviations used in this paper: MAC, membrane attack complex; LH, luteinizing
hormone; FSH, follicle-stimulating hormone; Crry, complement-related receptor protein; DAF, decay accelerating factor; MCP, membrane cofactor protein.
Copyright © 2005 by The American Association of Immunologists, Inc.
To protect “self” cells from the catastrophic effect of autologous
complement damage, ⬎10 plasma- and membrane-bound inhibitory proteins have evolved to restrict complement activation at
different stages of the activation pathways. In particular, CD59 is
a GPI-linked membrane protein that inhibits formation of the
MAC. Absence of CD59 and other GPI-linked proteins in circulating cells, as the consequence of an acquired somatic mutation in
a clone of hemopoietic-cell precursors, renders the circulating cells
highly susceptible to complement damage and causes the complement-mediated hemolytic anemia known as paroxysmal nocturnal
hemoglobinuria (2). This paradigmatic disease illustrates the protective effect of complement regulatory proteins against autologous complement activation; in their absence, even the basal tickover complement activity is sufficient to lyse erythrocytes and
cause hemolytic anemia.
The reproductive system represents a unique situation in which
cells expressing new Ags are either constantly produced (spermatogenesis) or periodically introduced (insemination and fetal development) in the reproductive tract (3). To avoid constant immune
challenge to a successful pregnancy, several mechanisms of immune tolerance have evolved, including expression of complement
regulators in the female reproductive tissues and the placenta,
where CD59 is highly expressed throughout pregnancy (4 – 6). In
mice, the targeted deletion of CD59 does not result in fetal loss (7).
In contrast, the deletion of mouse complement-related receptor
protein (Crry), a complement regulatory protein only expressed in
mice and rats, results in fetal damage and interruption of pregnancy at or before day 9 (8). The infertility of the Crry⫺/⫺ mother
is the consequence of a complement-mediated attack on the fetus
rather than a reproduction-specific function of Crry⫺/⫺, because
the fetuses survive if the Crry⫺/⫺ mother is also deficient in C3 (8,
9). Another example that highlights the importance of complement
in the pathophysiology of reproduction was provided by a recent
study of a mouse model of the anti-phospholipid Abs syndrome in
0022-1767/05/$02.00
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CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported
recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59bⴚ/ⴚ)
results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities
in mCd59bⴚ/ⴚ mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells
within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a
significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were
similar in mCd59bⴚ/ⴚ and mCd59bⴙ/ⴙ. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the
abnormal reproductive phenotype of mCd59bⴚ/ⴚ, although it was efficient in rescuing their hemolytic anemia. These results
indicate that the male reproductive abnormalities in mCd59bⴚ/ⴚ are complement-independent, and that mCd59 may have a novel
function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex
formation. The Journal of Immunology, 2005, 175: 6294 – 6302.
The Journal of Immunology
Materials and Methods
Mice
The following animal studies have all been reviewed and approved by the
Harvard Medical School Institutional Animal Care and Use Committee
(Animal Welfare Assurance no. A3431-01; International Animal Care and
Use Committee approval date: January 10, 2005).
mCd59b⫺/⫺. Both mCd59b⫺/⫺ and mCd59b⫹/⫹ littermate mice used for
these experiments were offspring derived from intercrossing mCd59b⫹/⫺ in
a B6/129 mixed genetic background, all characterized by PCR genotyping
(7). To avoid the influence of multiple matings on sperm analysis, the
experimental littermates were not mated with females.
mCd59b⫺/⫺C3⫺/⫺. To obtain mCd59b⫺/⫺C3⫺/⫺ (mCd59b and C3 double knockout) and mCd59b⫹/⫹C3⫹/⫹ (both mCd59b and C3 wild type)
littermates, we first crossed mCd59b⫺/⫺ (in B6/129 mixed genetic background) and C3⫺/⫺ (in B6 genetic background; purchased from The Jackson Laboratory) mice to generate mCd59b⫹/⫺C3⫹/⫺ (7, 19). Both
mCd59b⫺/⫺C3⫺/⫺ and mCd59b⫹/⫹C3⫹/⫹ used for experiments were littermate offspring produced from intercrossing mCd59b⫹/⫺C3⫹/⫺, selected
by genotyping as described in Refs. 7 and 19.
Collection of blood and sperm
Blood was collected in tubes containing EDTA by cardiac puncture using
precooled needles and syringes to avoid complement activation. Testes and
epididymides were dissected, the cauda epididymis was separated, and
sperm were collected by squeezing one cauda epididymis from each mouse
into 1 ml of prewarmed HEPES-buffered saline solution containing 1%
BSA (10 mM HEPES, 150 mM NaCl, 0.01 g/ml BSA (pH 7.4)). Epididymal tissue was removed and the solution was filtered through a mesh filter
into a conical centrifuge tube.
Serum levels of luteinizing hormone (LH), follicle-stimulating
hormone (FSH), and testosterone
LH and FSH were determined by RIA at the National Institute of Diabetes
and Digestive and Kidney Disease on a fee-for-service basis as described
in Ref. 20. Testosterone was measured by ELISA following the manufacturer’s recommendations (BioCheck).
Histological analysis of testes
Testes were fixed in Bouin’s solution, 5-mm slices were embedded in
paraffin, and 6-␮m sections were stained with H&E using routine procedures (21). Quantitative analyses were performed on histological sections
obtained from fixed, sectioned testes using a Nikon Eclipse TE2000-E
microscope equipped with a Spot digital camera. The number of multinucleated cells in seminiferous tubules was counted blindly by two independent investigators from two sections of each testis at ⫻20 magnification.
Immunohistochemical analysis
Detection of C3 and C9 deposition in testes. Eight-micrometer serial sections from paraffin-embedded tissue samples were stained with: 1) goat
anti-mouse C3 Ab (ICN Pharmaceuticals) (22) or 2) rabbit anti-rat C9 Ab,
which cross-reacts with mouse C9 (kindly provided by Dr. P. Morgan,
University of Wales) (22). Either anti-goat for anti-C3 or goat anti-rabbit
for anti-C9 conjugated to peroxidase were used as secondary Abs following established procedures (23). Two independent investigators blindly
scored the samples based on the intensity differences between stainings by
each specific Ab and nonimmune IgG.
Detection of apoptotic cells in situ. Apoptotic nuclei in tissue sections
were identified by a TUNEL technique using an in situ apoptosis detection
kit (Apoptag; Chemicon International) according to the manufacturer’s instructions (24, 25). The sections were counterstained with toluidine blue,
dehydrated with ethanol and xylene, and coverslipped. The number of
TUNEL-positive nuclei in 40 ⫻20 microscope fields per sample was recorded blindly by different investigators.
Sperm analysis
Progressive motility. The percentage of progressively motile sperm was
immediately evaluated by placing a 10-␮l drop of the diluted sperm suspension between a prewarmed glass slide and a coverslip, and visually
counted under a phase contrast microscope at ⫻200.
Morphologically normal sperm and sperm count. A total of 10 ␮l of the
sperm suspension was diluted 1/10 into saline-buffered formaldehyde in a
microcentrifuge tube. The relative distribution of morphologically normal
and abnormal sperm was evaluated in a wet slide under a phase contrast
microscope by counting a total of 200 sperm per sample at ⫻1000. The
sperm abnormalities recorded included detached heads, abnormal head
shapes, bent midpieces, droplets, coiled tails, other abnormalities, and immature sperm forms (round cells) (26, 27). We defined abnormal heads as
the sum of detached heads and abnormal sperm heads (type IIa-IIb-IIc,
IIIa-IIIb, IV, and V) (27).
A 10-␮l drop of the above suspension was used to count sperm cells on
a hemocytometer as described in Ref. 28.
Viability. To differentiate live from dead sperm cells we used the live/dead
sperm viability kit (SYBR 14 ⫹ propidium iodide; Molecular Probes) following the manufacturer’s recommendations. Briefly, 5 ␮l of a 10-fold
DMSO dilution of the SYBR 14 dye was mixed with the sperm suspension
and incubated for 5–10 min at 37°C in the dark. Then, 5 ␮l of the propidium iodide solution was added and incubated for an additional 5–10
min. A total of 200 cells were counted for viability evaluation in wet
samples under a fluorescence microscope where propidium-iodide stained
dead cells a fluorescent red.
Sperm function analysis
Capacitation. Sperm capacitation was evaluated by the protein tyrosine
phosphorylation assay (29, 30). Cauda epididymis were dissected and
placed in a petri dish containing bicarbonate-free Wittens medium to allow
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which these Abs produced decidual focal necrosis with C3 deposition and neutrophil infiltration in pregnant mice (10).
In the male reproductive system, spermatogenesis begins at puberty, long after the immune system has developed the ability to
distinguish self from “nonself”, and sperm-surface Ags can trigger
an immune/complement response that would compromise the potential of the sperms for successful fertilization. Thus, it is not
surprising that, in addition to a protective blood-testis barrier, a
variety of nonspecific and specific complement inhibitors, including CD59, are present at high concentrations in the seminal fluid,
and that sperm cells express multiple complement regulators that
prevent complement activation and functional MAC formation
(3– 6, 11, 12). In the seminal plasma, CD59 is present in a GPIanchored, prostasome-associated form that can “deliver” CD59
molecules to the sperm cell (13), and is also highly expressed on
sperm plasma membranes (6). Incubation of sperm with neutralizing anti-CD59 Abs and human serum results in sperm immobilization and lysis, whereas sperm with functional CD59 are protected (14).
Recently, we and others have demonstrated that mice have two
different CD59 genes (mCd59a and mCd59b) (15–17), whereas
most other rodents and humans studied have only one. mCd59b is
highly expressed in testes at the level of mRNA and protein (18),
a finding that suggested a possible testis/reproduction-specific
function of mCd59b. To study the function of CD59 and complement in mouse models of human diseases, particularly the function
of mCd59b, we generated mCd59b⫺/⫺ mice by homologous recombination. These mice express a hemolytic anemia phenotype
comparable to human paroxysmal nocturnal hemoglobinuria, and
progressive loss of male fertility that becomes evident at 5 mo of
age, as we previously reported (7). Furthermore, we demonstrated
that mCd59a protein is significantly down-regulated in
mCd59b⫺/⫺ RBC, which is consistent with the reduced tissue expression of mCd59a mRNA we have reported in mCd59b⫺/⫺ (see
Fig. 6 in Ref. 7). This indicates that the mCd59b⫺/⫺ mouse resembles a mCd59a and mCd59b partial double knockout, explaining the hemolytic anemia phenotype of these mice (X. Qin, M.
Dobarro, S. Bedford, S. Ferris, P. Miranda, W. Song, R. Bronson,
P. Visconti, and J. Halperin, manuscript in preparation). In this
study, we report additional studies on mCd59b⫺/⫺ that indicate
that their reproductive but not their hematological abnormalities
are complement independent, and that mCd59 may have a novel
role in testicular function that is independent of its activity as an
inhibitor of MAC formation.
6295
6296
REPRODUCTIVE ABNORMALITIES IN mCd59b⫺/⫺ MICE
sperm to swim out. Total sperm recovered per mouse was recorded. Sperm
were capacitated for 1.5 h in Wittens medium supplemented with 20 mM
NaHCO3 and 5 mg/ml fatty-acid-free BSA (31). Control sperm were kept
in medium without any supplement. Sperm extracts were obtained by boiling for 5 min with 1% SDS and subjected to SDS-PAGE followed by
immunoblot with anti-phosphotyrosine mAb (4G10; Upstate).
Acrosome reaction. Acrosome reaction was induced by addition of 2 ␮M
calcium ionophore (A23187) during the last 10 min of incubation. Acrosomal staining was performed using Alexa 488-conjugated peanut agglutinin (32) (Molecular Probes).
with abnormal heads between experimental groups were compared by Student’s t test.
We compared the median levels using the Wilcoxon rank sum test and
we compared the proportions in the extremes of the distributions using
Fisher’s exact test. Specifically, we compared proportions with hemoglobin
levels ⬍13 g/100 ml of blood (2 SD in mCd59b⫹/⫹C3⫹/⫹) and proportions
with reticulocyte counts greater than 4.3% (2 SD in mCd59b⫹/⫹C3⫹/⫹).
Assessment of the hematologic profile
Testis histological comparison between mCd59b⫹/⫹ and
mCd59b⫺/⫺ littermate mice at 6 and 10 mo of age revealed that
33% of mCd59b⫺/⫺ mice had abnormal multinucleated cells
within the walls of seminiferous tubules (mean number of abnormal multinucleated cells in mCd59b⫺/⫺ ⫽ 10 ⫾ 8 (n ⫽ 6 per age
group)); these cells were absent in all mCd59b⫹/⫹ mice analyzed
(n ⫽ 6 per age group) (Fig. 1). The TUNEL assay performed on
testicular paraffin sections from 6-mo-old mCd59b⫺/⫺ and
mCd59b⫹/⫹ mice revealed the presence of a significantly greater
number of apoptotic, TUNEL-positive germ cells in mCd59b⫺/⫺
mice (Fig. 1, D, F, and G) than in their age-matched mCd59b⫹/⫹
control mice (Fig. 1, D and E). The comparison of mCd59b⫹/⫹ and
mCd59b⫺/⫺ littermates of different ages (4, 6, and 10 mo old)
showed that there was no difference in the testes to total body
weight ratio, and no evidence of testicular atrophy or of inflammatory cells in the seminiferous tubules (data not shown). Also,
immunostaining of testicular sections with either anti-C3 or
An aliquot of mouse blood was analyzed on an Advia 120 autoanalyzer
(Abbott Laboratories) as previously described in Ref. 7.
Measurement of C activity with hemolytic assay
Statistical analysis
Mean difference of multinucleated cells, apoptotic cells, total sperm, and
viable sperm, as well as motility, viability, and the percentage of sperm
Testes histology, apoptotic cells, and complement deposition
FIGURE 1. Presence of abnormal multinucleated cells and increased apoptotic cells in mCd59b⫺/⫺ testis. A–C, Presence of abnormal multinucleated
cells (red arrow) within the wall of the seminiferous tubules of mCd59b⫺/⫺. A, ⫻10 magnification; B and C, ⫻20 magnification. D–G, There are
significantly more apoptotic cells in mCd59b⫺/⫺ seminiferous tubules (D, F, and G) compared with mCd59b⫹/⫹ (E). The cells were blindly counted by
two different investigators.
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Rabbit erythrocytes were washed in PBS, and a 2% suspension was incubated with an equal volume of mouse anti-rabbit erythrocyte antiserum
(1/250 dilution in PBS; 15 min at 37°C). The Ab-sensitized rabbit erythrocytes were washed with veronal buffered saline (VBS⫹⫹) and resuspended at 1% hematocrit. Fifty microliters of the cell suspension plus 50 ␮l
of mouse serum (10% or 20% in VBS⫹⫹) were added to 96-well plates in
triplicate, and the plates were incubated for 30 min at 37°C. Nonlysed cells
were removed by centrifugation, and hemoglobin in the supernatant was
measured as OD414. Percent hemolysis in each well was calculated as previously published (33).
Results
The Journal of Immunology
6297
anti-C9 Abs did not show an increased complement deposition in
mCd59b⫺/⫺ testes (data not shown). Thus, the presence of abnormal multinucleated cells and of an increased number of apoptotic
germ cells, the main histological findings in the testes of
mCd59b⫺/⫺, do not seem to be associated with an increased complement/MAC deposition, as one would expect from the functional
deletion of CD59.
percentage of motile and viable sperm were all significantly decreased, whereas the percentage of abnormal sperm heads was
significantly higher in mCd59b⫺/⫺ mice (Fig. 2D). Fig. 2, E–G,
show examples of abnormal sperm head morphologies frequently observed in mCd59b⫺/⫺ mice. These results together with the abnormal
histology findings indicate that the progressive loss of male fertility in
mCd59b⫺/⫺ mice may be due to abnormal spermatogenesis.
Sperm abnormalities in mCd59b⫺/⫺
Normal hormonal levels and capacitation-associated tyrosine
phosphorylation in mCd59b⫺/⫺
Comparative analysis of sperm at 3 mo of age showed that relative
to mCd59b⫹/⫹, mCd59b⫺/⫺ mice have a significant decrease in
the total number of live sperm (Fig. 2A), and in the percentage of
viable sperm (Fig. 2C). This decrease in critical sperm parameters
in mCd59b⫺/⫺ was associated with a significant increase in the
percentage of abnormal sperm heads (Fig. 2C).
Sperm quality parameters progressively worsened; at 6 mo of
age, both the number of total and live sperm (Fig. 2B), and the
Fig. 3 shows that there were no significant differences in the serum
levels of the critical reproduction-related hormones FSH (Fig. 3A)
and LH (Fig. 3B); levels of testosterone were also similar in
mCd59b⫺/⫺ and mCd59b⫹/⫹ males (data not shown). Thus, the
progressive loss of male fertility and testicular abnormalities in
mCd59b⫺/⫺ mice do not seem to be caused by low levels of hormones that affect spermatogenesis.
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FIGURE 2. Comparison of the sperm parameters between mCd59b⫺/⫺ and mCd59b⫹/⫹ mice. A and B, Comparing total and live sperm of mCd59b⫺/⫺
with that of mCd59b⫹/⫹ at the age of 3 (A) and 6 mo (B). C and D, Comparison of the mobility, viability, and the percentage of abnormal sperm heads
from mCd59b⫺/⫺ with that of sperm from mCd59b⫹/⫹ at the age of 3 (C) and 6 mo (D). ⴱ, p ⬍ 0.05. Each group contained six males. f, mCd59b⫺/⫺;
䡺, mCd59b⫹/⫹. E–G, Increase in the number of abnormal heads of sperm in mCd59b⫺/⫺ sperm morphologic abnormalities: detached head (arrow) and
class III head (E), class IIIa head (F), and class IIIb head (G).
6298
REPRODUCTIVE ABNORMALITIES IN mCd59b⫺/⫺ MICE
Both sperm capacitation and the acrosome reaction are physiological processes required for successful fertilization. Capacitation is associated with an increase in protein tyrosine phosphorylation. Indeed, Western blot analysis with specific Abs
against phosphotyrosine has become an accepted method to assess sperm capacitation (29, 30, 34). In other cell types, CD59
has been found to be physically connected to the membrane
tyrosine phosphorylation machinery; if this were the case in
sperm cells, CD59 could play a role in sperm capacitation (35–
37). To investigate whether the absence of mCd59 could affect
capacitation-dependent tyrosine phosphorylation and ionophore-induced acrosome reaction in mouse sperm, we analyzed
both tyrosine phosphorylation and acrosome reaction in sperm
obtained by the swim-out method from the epididymis of 3-moold mice. mCd59b⫺/⫺ consistently yielded a significantly lower
number of sperm as compared with mCd59b⫹/⫹ (Fig. 3C).
Analysis of the same number of motile mCd59b⫹/⫹ and
mCd59b⫺/⫺ sperm showed a similar amount of tyrosine phosphorylation (Fig. 3D). As mentioned above, the ability of sperm
to undergo the acrosome reaction is also necessary for fertilization. This process is dependent on calcium influx. Therefore,
to analyze whether mCd59b⫺/⫺ are able to undergo the acrosome reaction, sperm from these mice as well as wild-type control were challenged with calcium ionophore A23187, and the
acrosome reaction was assessed using Alexa 488-conjugated
peanut agglutinin (32). In mCd59b⫹/⫹ and mCd59b⫺/⫺, the percentage of acrosome reaction was similar (Fig. 3E). Together,
both sperm capacitation and the acrosome reaction experiments
suggest that absence of mCd59 does not affect sperm capacitation and/or acrosome reaction.
C3 deficiency does not rescue the sperm abnormalities in
mCd59b⫺/⫺
To investigate further whether the role of CD59 in spermatogenesis is dependent on an efficient complement system, we generated
mCd59b⫹/⫹C3⫹/⫹, mCd59b⫺/⫺C3⫹/⫹, mCd59b⫹/⫹C3⫺/⫺, and
mCd59b⫺/⫺C3⫺/⫺ littermates by intercrossing mCd59b⫹/⫺C3⫹/⫺
followed by genotyping, as described in Materials and Methods.
The functional deficiency of C3 in the different combinations involving the C3⫺/⫺ genotype was confirmed by measuring the serum complement activity as described in (33). Sperm analysis at 6
mo of age revealed that in both mCd59b⫺/⫺C3⫹/⫹ and mCd59b⫺/⫺
C3⫺/⫺ there was a similar decrease in the total and viable sperm
number, as well as in sperm motility and viability as compared
with sperm from mCd59b⫹/⫹C3⫹/⫹ (Fig. 4, A and B). In contrast,
the functional deficiency of C3 rescued the hemolytic anemia phenotype that we originally described in mCd59b⫺/⫺ mice (7), as
shown in Table I. Moreover, 17% of mCd59b⫺/⫺C3⫹/⫹ mice had
a hemoglobin value below 2 SD below the mean in wild-type mice
(i.e., below 13 g/dl), a fraction significantly higher than that in the
mCd59b⫺/⫺C3⫺/⫺ (4.5%) or in the mCd59b⫹/⫹C3⫹/⫹ (3.9%)
groups (Fig. 5A). Similar results were obtained for reticulocyte
counts: 15.8% of mCd59b⫺/⫺C3⫹/⫹ littermates had a reticulocyte
count 2 SD above the mean of their mCd59b⫹/⫹C3⫹/⫹ wild-type
counterparts, a fraction that is also significantly higher than that in
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FIGURE 3. Analysis of the hormonal profile, sperm capacitation, and acrosome reaction in mCd59b⫺/⫺. A and B, There are no significant differences
in FSH (A), LH (B), and testosterone (data not shown) levels between mCd59b⫹/⫹ and mCd59b⫺/⫺ at 6 mo of age (n ⫽ 12). The measurements were done
by RIA as described (20). C, There is a 2.5-fold lower number of motile and live sperm obtained by the swim-out method from the epididymis of
mCd59b⫺/⫺ compared with mCd59b⫹/⫹ (n ⫽ 6, p ⬍ 0.01). D, Sperm capacitation was analyzed by Western blot with anti-phosphotyrosine mAb, as
described in Refs. 29 and 30 (n ⫽ 8, p ⬎ 0.05). ⫹, Wittens medium supplemented with 20 mM NaHCO3 and 5 mg/ml fatty-acid-free BSA. ⫺, Medium
without supplements. E, There is no significant difference in the sperm acrosome reaction (AR) between mCd59b⫹/⫹ and mCd59b⫺/⫺ (n ⫽ 8, p ⬎ 0.05).
The Journal of Immunology
6299
the mCd59b⫺/⫺C3⫺/⫺ (4.5%) or in the mCd59b⫹/⫹C3⫹/⫹ (3.9%)
groups (Fig. 5B).
To understand the incomplete penetrance of the hemolytic anemia of mCd59b⫺/⫺, we assessed the complement activity among
mCd59b⫺/⫺ mice. The percent lysis by each mouse serum is dependent on the activity of C in that particular serum sample. Fig.
5C demonstrates that there is a significant variation in the complement activity between mCd59b⫺/⫺, which may explain the incomplete penetrance of the hemolytic anemia in mCd59b⫺/⫺ (a
similar variation occurs in wild-type mice; data not shown). In
summary, the deficiency of C3 in mCd59b⫺/⫺C3⫺/⫺ rescues the
hemolytic anemia but not the sperm abnormalities of mCd59b⫺/⫺,
a result consistent with a complement-independent function of
mCd59 in spermatogenesis. The alternative explanation that C3
could have a role in spermatogenesis that would result in sperm
abnormalities in C3⫺/⫺ mice (independently of the absence of
CD59) was ruled out because all parameters of sperm function and
quantity were similar in 10-mo-old mCd59b⫹/⫹C3⫺/⫺ and
mCd59b⫹/⫹C3⫹/⫹ littermates, a result consistent with the apparently normal fertility of mCd59b⫹/⫹C3⫺/⫺ (Fig. 6, A and B).
FIGURE 5. C3 deficiency rescues the hemolytic anemia of mCd59b⫺/⫺.
A, Seventeen percent of mCd59b⫺/⫺C3⫹/⫹ mice (n ⫽ 107) had 2 SD below
the mean of their mCd59b⫹/⫹C3⫹/⫹ wild-type counterparts (⬍13 g/dl), a
proportion significantly higher than the 4.5% found in mCd59b⫺/⫺C3⫺/⫺
(n ⫽ 77) or the 3.9% found in mCd59b⫹/⫹C3⫹/⫹ littermates (n ⫽ 75). B,
A total of 15.8% of mCd59b⫺/⫺C3⫹/⫹ littermates had a reticulocyte count
2 SD above the mean of their mCd59b⫹/⫹C3⫹/⫹ wild-type counterparts
(⬎4.3%), a proportion significantly higher than the 4.5% present in mCd59b⫺/
⫺/⫺
⫺C3
or the 3.9% detected in mCd59b⫹/⫹C3⫹/⫹. ⴱ, Fisher’s exact test:
mCd59b⫺/⫺C3⫹/⫹ with mCd59b⫹/⫹C3⫹/⫹ (p ⬍ 0.05); #, Fisher’s exact test:
mCd59b⫺/⫺C3⫺/⫺ with mCd59b⫹/⫹C3⫹/⫹ (p ⬎ 0.05). C, Complement activity in mCd59b⫺/⫺ (B6/129 genetic background) was assessed by the fractional lysis of Ab-sensitized rabbit RBC mediated by each mouse serum (MS)
assayed at different concentrations (10% and 20%). M1, M2, M3, M4, M5,
M6, M7, and M8, individual mCd59b⫺/⫺ mice; P, commercially available
mouse serum; and HI, heat-inactivated mouse serum.
Discussion
A critical role of complement in reproduction has been experimentally characterized by two recent studies showing the deleterious
effect on the fetus of unrestricted or increased complement activation, as seen respectively in pregnant mice after either functional
deletion of Crry (8, 9) or injection of anti-phospholipid Abs (10).
Table I. C3 deficiency rescues the hemolytic anemia in mCd59b⫺/⫺a
Mouse Genotype
Hemoglobin (g/dl)
mCd59b⫹/⫹C3⫹/⫹ (n ⫽ 75)
mCd59b⫺/⫺C3⫹/⫹ (n ⫽ 107)
mCd59⫺/⫺C3⫺/⫺ (n ⫽ 77)
14.95 ⫾ 1.23
14.2 ⫾ 2.21b
14.82 ⫾ 0.89c
a
Mice are from 3– 6 mo old.
Comparison between mCd59b⫺/⫺C3⫹/⫹ and mCd59b⫹/⫹C3⫹/⫹, p ⬍ 0.01 (Student’s t test).
c
Comparison between mCd59⫺/⫺C3⫺/⫺ and mCd59b⫹/⫹C3⫹/⫹, NS, p ⬎ 0.1.
b
FIGURE 6. No significant changes in the sperm quality and quantity of
C3-deficient mice. A, The number of viable sperm from mCd59b⫹/⫹C3⫺/⫺
is not significantly different from mCd59b⫹/⫹C3⫹/⫹, but higher than that
from mCd59b⫺/⫺C3⫹/⫹ at 6 mo of age (n ⫽ 8). B, Neither motility nor
viability from mCd59b⫹/⫹C3⫺/⫺ is significantly different from
mCd59b⫹/⫹C3⫹/⫹, but is higher than that from mCd59b⫺/⫺C3⫹/⫹ at 6 mo
of age (n ⫽ 8). ⴱ, p ⬍ 0.05.
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FIGURE 4. The C3 deficiency does not rescue the sperm abnormalities
in mCd59b⫺/⫺. A, Significant decrease in the number of total or live sperm
from either mCd59b⫺/⫺C3⫹/⫹ or mCd59b⫺/⫺C3⫺/⫺ compared with
mCd59b⫹/⫹C3⫹/⫹ at 6 mo of age (n ⫽ 8). Live sperm number equals total
number of sperm times the percentage of live sperm. B, Significant decrease in sperm motility or viability of either mCd59b⫺/⫺C3⫹/⫹ or
mCd59b⫺/⫺C3⫺/⫺ compared with mCd59b⫹/⫹C3⫹/⫹ at 6 mo of age (n ⫽
8). ⴱ, Comparing mCd59b⫺/⫺C3⫹/⫹ with mCd59b⫹/⫹C3⫹/⫹ (p ⬍ 0.05). #,
Comparing mCd59b⫺/⫺C3⫺/⫺ with mCd59b⫹/⫹C3⫹/⫹ (p ⬍ 0.05).
6300
mCd59b⫺/⫺ mice, indicating that the later is most likely complement independent.
At present, we cannot completely rule out the alternative possibility of a local production of the C3 complement component that
could take place in the testes of C3 knockout mice via an alternative splicing product of C3. However, this alternative explanation
seems unlikely because 1) MAC deposits were absent in the testes
of mCd59b⫺/⫺ mice; 2) a study designed to investigate the role of
local complement production in renal transplantation using the
same C3 knockout mice clearly indicates that there is no local
production of C3 in the kidneys of these mice (46); and 3) the C3
knockout mice are also deficient in acylation-stimulation protein, a
cleavage product of adipocyte C3 that contributes to fat tissue
metabolism (47).
The successful reproduction of young mCd59b⫺/⫺ males, and
the demonstration of normal capacitation-associated tyrosine phosphorylation and ionophore-induced acrosome reaction in
mCd59b⫺/⫺ sperm, strongly support the interpretation that mCd59
may play a complement-independent role in spermatogenesis
rather than in fertilization. This is consistent with the evidence that
expression of mCd59b in spermatozoa progressively increases during their development and maturation (17, 18). Our in vivo data
showing decreased motility of sperm in mCd59b⫺/⫺ is consistent
with the previous demonstration by two different groups that spermatozoa ejaculated from healthy human donors lost their motility
after exposure to anti-human CD59 Abs (48, 49).
Although this study does not define the complement-independent role of CD59 in reproduction, it is tempting to speculate that
CD59 might protect sperm and/or testicular germ cells from attack
by a yet not characterized mechanism independent of complement.
One recent example of an alternative function in reproduction for
a well-known protein is the finding that angiotensin converting
enzyme, a well-characterized zinc peptidase, plays an unexpected
biological role in reproduction cleaving GPI-anchored proteins
from the plasma membrane (50). Alternatively, the role of CD59 in
reproduction could be related to the localization of CD59 in the
sperm lipid rafts. Recently, Cross et al. (51) have shown that, in
noncapacitated sperm, CD59 localizes to lipid rafts, and that it
redistributes after disruption of lipid rafts during the capacitation
process. The molecular mechanism of this postulated new role of
CD59 in reproduction is currently under investigation in our
laboratory.
The number of multinucleate and apoptotic cells was significantly increased in mCd59b⫺/⫺ as compared with mCd59b⫹/⫹
mice (Fig. 1, A–D). The underlying mechanism of increased
multinucleate and apoptotic cells in mCd59b⫺/⫺ needs further investigation. Of note, the number of apoptotic cells was increased in
some mCd59b⫺/⫺ (four of six) but not all mCd59b⫺/⫺ (Fig. 1D).
This heterogeneous phenotype may be due to a different proportion
of B6 and 129 genetic background in our experimental mice. It is
well accepted that the genetic background largely influences phenotypic expression. To minimize the impact of difference genetic
backgrounds, we used littermates (derived from crossing heterozygous pairs); however, this does not totally eliminate differences in
genetic background that may affect phenotypic expression.
The progressive nature of the loss of male fertility in
mCd59b⫺/⫺ is intriguing. It indicates that mCd59 does not play a
role that is indispensable for sperm development/survival, in which
case one would expect the male infertility to be early and absolute
from the beginning of the mouse male fertile cycle, rather than
progressive. Even though we observed a significant reduction in
the total number of normal, motile sperm at the early age of 5 mo
in mCd59b⫺/⫺ (Figs. 2 and 3C), the density of normally motile
sperm at that age seems to be above the minimal threshold required
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A potential role of complement in sperm development, maturation,
and fertilization (38) has long been suspected, but its formal demonstration has remained elusive. Because complement regulatory
proteins, including CD59, decay accelerating factor (DAF), membrane cofactor protein (MCP), and S-protein, are highly expressed
in spermatozoa and present in seminal plasma (5, 6, 39), it has
been postulated that they could be critical in protecting sperm
against complement attack in the female reproductive tract, and/or
play physiological roles in spermatogenesis/sperm maturation.
However, several molecularly engineered mice already developed
to study the function of complement regulatory proteins, including
two mDAF knockout mice (40), and mCd59a- (33) and S-proteindeficient mice (41) did not show any major fertility problem. Several in vitro studies suggested a role for MCP in sperm-egg interaction (42), but the targeted deletion of mouse MCP did not affect
their reproductive efficacy (43). Together, these studies illustrate
that in mice, DAF, mCd59a, MCP, and S-protein are not individually essential for fetal development/survival or for protecting
sperm from complement activation in the male or the female reproductive system. In contrast, the targeted deletion of the mCd59b
gene, resulting in the down-regulation of mCd59a and functional
deficiency of mCd59b, leads to a progressive loss of male fertility,
as we have reported in (7).
The results of the work presented in this study show that
mCd59b⫺/⫺ mice have a significantly lower number and compromised quality of sperm, associated with the presence of abnormal
multinucleated cells, and an increased number of apoptotic cells
within the seminiferous tubules. The multinucleated cells appear to
be arrested spermatogonia that have divided without cytokinesis,
an indication that spermatogenesis does not progress normally inside the seminiferous tubules. These results are consistent with a
defect in spermatogenesis and would explain the intriguing loss of
male fertility that progressively occurs in mCd59b⫺/⫺ mice. Interestingly, this spermatogenesis defect seems to result from a complement-independent function of CD59 because 1) it is not associated with an increased deposition of MAC in the seminiferous
tubules and 2) the functional deficiency of C3, which for all practical purposes makes mice complement insufficient, does not rescue the abnormal testicular phenotype, although it does rescue the
complement-dependent hemolytic anemia (Figs. 4 – 6 and Table I).
Regarding the rescue of the hemolytic anemia of mCd59b⫺/⫺ by
C3 deficiency, it is worth noting that this phenotype does not have
a 100% penetrance in vivo, although 100% of the animals tested
exhibited a significantly increased sensitivity to complement-mediated lysis assayed against a pool of complement-efficient mouse
serum (7). This lower penetrance of the phenotype in vivo is not
unexpected because the hemolytic anemia in mCd59b⫺/⫺ is the net
result of the complement activity in the plasma of the individual
mouse and the level of complement restriction due to the functional absence of Cd59. Assuming that all other complement regulatory factors are similar among individual mCd59b⫺/⫺ mice, the
main remaining variable that might influence the penetrance of the
hemolytic anemia phenotype is the complement activity in each
mouse. It has been established that there are significant differences
in the levels of complement activity in different strains of mice,
between males and females and also among same gender within
the same genetic background (Fig. 5C) (33, 44, 45). The known
difference in complement activity among individual mice is the
most likely explanation for the observation that mCd59b⫺/⫺C3⫹/⫹
mice as a group exhibit anemia, i.e., a significantly lower blood
hemoglobin value than normal littermate controls, but in only 17%
of them, hemolysis is sufficient to bring this value below 2 SD of
the mean in wild-type mice. In summary, C3 deficiency rescues the
hemolytic anemia but not the abnormal reproductive phenotype of
REPRODUCTIVE ABNORMALITIES IN mCd59b⫺/⫺ MICE
The Journal of Immunology
Acknowledgments
We are grateful to Dr. D. C. Tosteson and Dr. M. T. Tosteson for continuous support and critical comments. We are grateful to Dr. Kenney for
testes histological analysis, Dr. H. Aktas for lending his expertise in sperm
functional analysis, and to Dr. L. Grubissich for assistance in the animal
handling. We are also grateful to K. Hazard for careful editing of the
manuscript.
Disclosures
The authors have no financial conflict of interest.
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REPRODUCTIVE ABNORMALITIES IN mCd59b⫺/⫺ MICE