Enhanced Suppression of Fertility Can be Achieved by Priming with FSHR and Eppin and Further Boosting with Their B-cell Epitope Peptides

Abstract

In our previous study on adult male mice, we had identified one immunodominant epitope in hEppin and three epitopes in hFSHR that caused fertility inhibition. But it only demonstrated a moderate inhibitory effect on fertility, and the antifertility effect was unsatisfactory.
Based on the protein prime-peptide boost inoculation modalities, we further investigated whether the antifertility capacity could be enhanced by a combined immunization with the two antigens.
The results displayed a enhanced suppressed fertility (F2EP2C 6.67%) in male mice similar to that seen after four separate administrations of the two proteins (F12E-4 5%). The most likely mechanism by which this antifertility efficacy was achieved was probably through the production of antibodies that led not only to impairment of spermatogenesis but also to inhibition of sperm motility. Moreover, this treatment also induced high concentrations of neutralizing antibodies which were secreted into the lumen of the epididymis.
Thus, a combination immunization with hFSHR and hEppin enhanced the contraceptive effects and may provide a better means of immunocontraception.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Full text

Enhanced Suppression of Fertility Can be Achieved by Priming
with FSHR and Eppin and Further Boosting with Their B-cell
Epitope Peptides
Ping Yan
1,2
, Wei He
1
, Yuzhang Wu
2
, Zhengqiong Chen
1
, Haiyang He
2
, Bing Ni
2
, Ji Zhang
2
, Xia Yang
2
,
Zigang Shen
2
, Xiaolan Fu
2
, Zhiqing Liang
1
, Jintao Li
2
1
Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing, China;
2
Institute of Immunology, Third Military University, Chongqing, China
Keywords
Epididymal protease inhibitor, follicle-
stimulating hormone receptor,
immunocontraception, spermatozoa, testis
Correspondence
Zhiqing Liang, Department of Obstetrics and
Gynecology, Southwest Hospital, Third Military
Medical University, 30 Gaotanyan Street,
Shapingba District, Chongqing 400038, China.
E-mail: zhi.lzliang@gmail.com
and
Jintao Li, Institute of Immunology, Third
Military Medical University, 30 Gaotanyan
Street, Shapingba District, Chongqing 400038,
China.
E-mail: ljtqms@yahoo.com.cn
Submission November 8, 2014;
accepted March 10, 2015.
Citation
Yan P, He W, Wu Y, Chen Z, He H, Ni B,
Zhang J, Yang X., Shen Z, Fu X, Liang Z, Li J.
Enhanced suppression of fertility can be
achieved by priming with FSHR and Eppin and
further boosting with their B-cell epitope
peptides. Am J Reprod Immunol 2015; 74:
156–168
doi:10.1111/aji.12381
Problem
In our previous study on adult male mice, we had identified one immu-
nodominant epitope in hEppin and three epitopes in hFSHR that caused
fertility inhibition. But it only demonstrated a moderate inhibitory effect
on fertility, and the antifertility effect was unsatisfactory.
Method of study
Based on the protein prime–peptide boost inoculation modalities, we
further investigated whether the antifertility capacity could be enhanced
by a combined immunization with the two antigens.
Results
The results displayed a enhanced suppressed fertility (F2EP2C 6.67%) in
male mice similar to that seen after four separate administrations of the
two proteins (F12E-4 5%). The most likely mechanism by which this
antifertility efficacy was achieved was probably through the production
of antibodies that led not only to impairment of spermatogenesis but
also to inhibition of sperm motility. Moreover, this treatment also
induced high concentrations of neutralizing antibodies which were
secreted into the lumen of the epididymis.
Conclusion
Thus, a combination immunization with hFSHR and hEppin enhanced
the contraceptive effects and may provide a better means of immuno-
contraception.
Introduction
As the world’s population continues to soar, it is
affecting economic growth and development in
every country. Presently, if the male partner desires
to assume family planning responsibilities, he is
hampered by the lack of suitable methods acceptable
to the couple. Thus, there is an urgent need for a
better contraceptive method that is acceptable, effec-
tive, reversible and available both in the developed
and in the developing nations. Contraceptive vac-
cines (CVs) may meet the requirements as highly
effective, safe, inexpensive and easily accessible male
contraceptives. Mammalian reproduction begins
with the production of male and female gametes fol-
lowed by their unification within a fertilized egg.
The identified molecular targets for immunocontra-
ception are classified into three main categories: hor-
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
156
ORIGINAL ARTICLE

monal [gonadotropin-releasing hormone (GnRH),
follicle-stimulating hormone (FSH) and luteinizing
hormone (LH)],
1,2
gamete-associated components,
3,4
and embryonic components [human chorionic gona-
dotropin (hCG)].
5
As for hormonal targets, FSH plays a key role in
reproduction. It is essential for normal function of
Sertoli cells in males which is to support spermato-
genesis. It is proven that various discrete, discontinu-
ous regions of the extracellular domain (ECD) play
an important role in interaction between FSH and its
receptor.
6,7
The ECD of FSH receptor (FSHR) has
also been considered a suitable target for a male vac-
cine, as it is expressed exclusively on testicular Ser-
toli cells.
8,9
With respect to gamete-associated
components, research has focused on the relation-
ship between sperm antigens and fertility in past
decades. Among numerous molecular candidates
related to sperm, epididymis protease inhibitor (Ep-
pin) is a promising candidate. Eppin on the surface
of spermatozoa and in seminal plasma is bound to
semenogelin,
10
which is the principle protein com-
ponent of the ejaculate coagulum. The Eppin–seme-
nogelin complex provides antimicrobial protection
for spermatozoa and aids its survival in the female
reproductive tract.
11
It has been reported that male
monkeys immunized with Eppin showed effective
immunocontraceptive effect. Seven of nine males
produced high titers to Eppin, and all these high-
titer monkeys were infertile.
12
In our previous research, we identified an immu-
nodominant B-cell epitope in hEppin.
13
Also, three
B-cell epitopes in hFSHR were identified, and their
antifertility efficiency was explored.
14
Employing a
protein prime/peptide boost vaccination modality,
even the dominant peptide (pep2 of hFSHR and
pepC of hEppin) demonstrated a moderate inhibitory
effect on fertility in immunized adult male mice, but
the antifertility effect was unsatisfactory. There may
be potential for a combination of B-cell epitope pep-
tide from hFSHR with that of hEppin to promote the
contraceptive efficacy. Thus, we explored the advan-
tages of simultaneous immunization with these two
peptides, and this achieved a relatively high fertility
inhibition in male mice.
Materials and methods
Animals
Specific pathogen-free BALB/C male mice (8 weeks
old) were purchased from the Animal Research
Centre of the Academy of Military Medical Science
(Beijing, China). The animals were housed in a tem-
perature- and light cycle-controlled facility, with
food and water supplied at all times. The mice were
randomly divided into six groups (10 mice in each
group) and ear-coded. Protocols for use of animals
were according to Chinese Animal Care for Labora-
tory Animals and followed the protocols approved
by the Animal Care and Use Committee of the Third
Military Medical University.
Synthesis of Peptides and Production of
Recombinant Protein
In our previously study,
13,14
we identified one domi-
nant peptide located in Eppin and three peptides in
hFSHR that led to antifertility effects. The sequences
of the peptides involved in hFSHR and hEppin used
in this study are shown in Table I. Briefly, the three
epitopes of FSHR were synthesized in tandem with a
promiscuous foreign T-cell epitope, PADRE,
15
and
the immunodominant B-cell epitope of Eppin was
conjugated with a spacer residue ‘GGG’ and promis-
cuous helper T-cell epitope by Invitrogen Biotech-
nology Co. (Shanghai, China), purified by reverse-
phase high-performance liquid chromatography
(HPLC) to a purity of >95%, and then validated by
mass spectrometry.
The segment of ECD in hFSHR (including NO. 1-
140aa and NO. 120–349aa) and the entire open
Table I Peptides Used to Immunize Mice
Peptide Helper epitope Epitope peptide sequence Location (amino acids) in protein
2 aKXVAAWTLKAAaZ IELRFVLTKLRVI 32–44 (hFSHR ECD)
3 aKXVAAWTLKAAaZ RNAIELRFVL 29–38 (hFSHR ECD)
4 aKXVAAWTLKAAaZ DYMTQARGQRSSLAED 285–300 (hFSHR ECD)
C LSEIKGVIVHRLEGV MFVYGGCQGNNNN 103–115 (hEppin)
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 157
B-CELL EPITOPE PEPTIDES OF FSHR AND EPPIN

reading frame (ORF) of Eppin were produced and
purified as described previously.
13,14
Concisely, the
essential fragments were amplified by PCR using
Marathon-Ready
TM
cDNA (Clontech Laboratories,
Inc., Mountain View, CA, USA) as template. The
PCR products were ligated into plasmid pET32a after
the sequence accuracy was confirmed. The proteins
were purified and separated from Trx-His after factor
Xa cleavage according to the supplier’s instruction
(New England Biolabs, Beverly, MA, USA). The
purified recombinant hFSHR proteins were desig-
nated as F140 and F240, respectively.
Immunization Schedule
The sexually mature BALB/C male mice were vacci-
nated four times, with the first boost immunization
administered 2 weeks after initial immunity was
achieved, and this was repeated at 1 weekly interval
for 3 weeks. The different vaccination modalities are
presented in greater detail in Table II. The dosage
applied was 60 lg for recombinant protein and
80 lg for peptide. The immunogen was diluted in
100 lL PBS (pH 7.2) and emulsified with an equal
volume of Freund’s complete adjuvant for the first
injection (Sigma, St. Louis., MO, USA) and Freund’s
incomplete adjuvant (Sigma) for subsequent injec-
tions. Each mouse received a total of 200 lL of the
emulsion subcutaneously.
Monitoring Antibody Titer
For the determination of immune response, tail blood
samples were collected on certain days (Fig. 1). The
plasma was then prepared and stored at 70°C until
needed for measurements. Antibody titers were
determined by standard enzyme-linked immunosor-
bent assay (ELISA). Ninety-six-well microtiter plates
(Costar Inc., New York, NY, USA) were coated with
purified Eppin protein (130 ng/well) dissolved in car-
bonate buffer [pH 9.6], and these were used for
ELISA. After removing the excess antigen, the wells
were washed and blocked. Serial dilutions of sera
generated from inoculated mice were added into
wells and incubated at 37°C for 1 h. After washing,
bound antibody was detected by horseradish peroxi-
dase-conjugated goat anti-mouse IgG antibody
(100 lL/well, 1:5000 dilution) followed by the addi-
tion of the TMB substrate (eBioscience, San Diego,
CA, USA). The reaction was terminated with 1M
H
2
SO
4
, and the OD values read at 450 nm in micro-
plate reader (Bio-Rad Laboratories Ltd., Hercules, CA,
USA). The end-point titer of the individual plasma
sample was considered positive if the absorbance was
twofold higher than the background.
Breeding Study
Each immunized male mouse was caged with three
randomly selected females of proven fertility for
1 week. Female mice were examined for vaginal
plugs each morning, and progeny size was recorded.
Two such mating trials were carried out at 2 weeks
and 6 weeks, respectively, after the final injection.
Inhibition of Antisera to Human Sperm Motility
Normal human semen was collected from donors
whose semen was assessed as normal by computer-
assisted sperm analysis (CASA), and the actively
Table II Summary of Vaccination Protocols
Group named Prime vaccination
a
Boost vaccination
b
PBS (control) PBS PBS
F12E-1 Recombinant protein F240 +F140 +Eppin PBS
F12E-4 Recombinant protein F240 +F140 +Eppin Recombinant protein F240 +F140 +Eppin
F2EP2C Recombinant protein F140 +Eppin P2 +PC
F2EP3C Recombinant proteinF140 +Eppin P3 +PC
F1EP4C Recombinant protein F240 +Eppin P4 +PC
F1, designated as F240, the purified recombinant proteins including NO. 120-349aa of the extracellular domain in hFSHR; F2, designated as
F140, the purified recombinant proteins including NO. 1-140aa of the extracellular domain in hFSHR; E, the purified recombinant hEppin pro-
tein; P2, designated as peptide 2 of hFSHR; P3, designated as peptide 3 of hFSHR; P4, designated as peptide 4 of hFSHR; PC, designated as
peptide C of hEppin.
a
Add equal volume of complete Freund’s adjuvant (Sigma).
b
Add equal volume of incomplete Freund’s adjuvant (Sigma).
American Journal of Reproductive Immunology 74 (2015) 156–168
158 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
YAN ET AL.

motile spermatozoa were recruited by the ‘swim-up’
procedure
16
andadjustedto20910
6
cell/mL in F-10
medium at 37°C. The recovered spermatozoa were
reassessed by CASA to evaluate the impact of the proce-
dure on sperm motility. Antisera from immunized mice
wereaddedintosemenandheldat37°C for 30 min
and 3 h, respectively. Then, 4 lL of semen sample was
loaded in a Makler’s chamber, and a range of 5–10
fields were acquired for sperm motility analysis.
17
Evaluation of Epididymal Sperm
Two weeks after the last fertility assay was performed,
five male mice of each group were randomly anaes-
thetized after measuring body weight and collecting
blood through cardiac puncture. Subsequently, the
right cauda epididymidis was excised to collect epidid-
ymal sperm, followed by sperm counting, sperm
motility evaluation, and hypo-osmotic swelling test as
was performed in our previous study.
14
Elongated Spermatid Counts
The right testis of killed male mice was removed
carefully and weighed. After decapsulation, the
testicular parenchyma was homogenized using a Tis-
sue Tearor homogenizer at maximum speed (12,000
g) for 1 min in 2 mL of normal saline containing
0.05% Triton X-100 and 3.8 mMsodium azide.
Homogenization-resistant elongated spermatids were
counted and denoted as total number of cells per
testis.
18
Detection of Serum IgA in Epididymis Lavages
The whole left epididymis was dissected according to
the method delineated earlier.
19
The epididymis was
kept away from fat tissue and placed in 200 lL PBS
supplemented with 0.1% BSA. The epididymis
lumens were flushed three times and discarded
before the remaining fluid in epididymis lumina was
expressed. The supernatant was collected after cen-
trifugation. The specific IgA antibody in epididymis
lavage was determined in duplicates with ELISA.
The specific IgA antibody in serum was assayed
simultaneously to eliminate the possibility of con-
tamination by traces of blood. The isotype of specific
antibodies was detected using HRP-conjugated anti-
IgA (Southern Biotech, Los Angeles, CA, USA) as
secondary antibodies as previously described.
20
0 week
2 weeks
4 weeks
6 weeks
8 weeks
10 weeks
12 weeks
15 weeks
18 weeks
21 weeks
24 weeks
27 weeks
30 weeks
33 weeks
1
10
100
1000
10,000
100,000
1,000,000
Means reciprocal of end-point dilution
Weeks after primary immunization
PBS
F12E-1
F12E-4
F2EP2C
F2EP3C
F1EP4C
Fig. 1 The sera IgG titer against Eppin in vaccinated male BALB/C mice. Specific immune response in vaccinated male BALB/c mice. ↑on the X-
axis represent immunization time and ↓represent the time of cohabited. Group F12E-4 received all four injections with recombinant protein F240,
F140, and Eppin. Group F2EP2C received recombinant protein F140 plus Eppin priming and pep2 plus pepC boosting. Group F2EP3C received
recombinant protein F140 plus Eppin priming and pep3 plus pepC boosting. Group F1EP4C received recombinant protein F240 plus Eppin priming
and pep4 plus pepC boosting. Group F12E-1 inoculated recombinant protein F240, F140, and Eppin only once at primary. As a control, the group
PBS immunized with mixture containing only adjuvant. Protein prime-peptide boost group (group F2EP2C, F2EP3C, and F1EP4C) induced potent
antibodies in males which were similar to the protein boost group (group F12E-4), but were significantly different from that of F12E-1 group and
PBS group as well (P<0.001).
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 159
B-CELL EPITOPE PEPTIDES OF FSHR AND EPPIN

Measurement of Serum Testosterone and LH
Levels
The serum prepared after killing animals was used to
detect serum testosterone (T) and LH level by radio-
immunoassay (RIA) kits obtained from North Biolog-
ical technology Research Institute (Beijing, China).
It had a sensitivity of 0.02 ng/mL, cross-reactivity of
0.011% with 5a-dihydrotestosterone, 1.2 910
5%
with androstenedione, 0.032% with progesterone
and a negligible cross-reactivity with other steroids
related to testosterone. The minimum detectable
concentration of LH was ~1.0 mIU/mL. The intraco-
efficients of variation were <10%. All samples were
analyzed in a single assay to avoid interassay varia-
tion. The results were consistent with intra-assay
variations of 7% for T and 8.5% for LH.
Histological Analysis
After male mice were killed, the left testes were dis-
sected, then immediately fixed, paraffin-embedded,
sectioned and subjected to hematoxylin–eosin stain-
ing according to the standard procedure.
21
Statistical Analysis
All data were presented as means S.E.M. and ana-
lyzed using SPSS version 11.0 (SPSS Inc., Chicago,
IL). Data were analyzed by independent-samples t-
test and between three or more groups by the one-
way ANOVA. Pearson chi-squared test was performed
to assess the significant difference of percentage
among each group. A difference was considered sig-
nificant when P-values were <0.05.
Results
Antibody Titration
It can be seen from the results presented in Fig. 1 that
an Eppin-specific IgG antibody response was detected
in immunized animals. The values demonstrated are
over the titers shown in the males vaccinated with
the immunogen only once (group F12E-1), and this
was the same as the PBS group. The mean reciprocal
of end-point titers showed an increasing trend (shown
in Fig. 1). The PBS group provided the measure for
the background level. After primary immunization, in
all peptide boost groups [group F2EP2C (received
recombinant protein F140 +Eppin prime–peptide
2+C boost), F2EP3C (received recombinant protein
F140 +Eppin prime–peptide 3 +C boost) and
F1EP4C (received recombinant protein F240 +Eppin
prime–peptide 4 +C boost)], the antigen-specific IgG
antibody could be detected and was similar to the pro-
tein boost group [group F12E-4 (received all four
injections with recombinant protein F240, F140, and
Eppin)]. However, in the PBS group, it never
exceeded 500, and in group F12E-1 (received recom-
binant protein F240, F140, and Eppin only once at
primary), it once achieved a peak of about 1:14,400
after 4 weeks of prime immunization but then
dropped sharply. The highest antibody titers were
seen in the protein or peptide boost groups at week
12, and these were significantly different from the
PBS group (P<0.001). This suggested that the pro-
tein prime/peptide boost vaccination schedule could
effectively elicit strong immune responses against
these immunogens.
In vivo Fertility Test
All treated males were caged 1 week with female
mice at two and 6 weeks after the last boost, respec-
tively. All females mated within the expected inter-
val after placement with the immunized male mice,
which suggest the libido of the treated animals was
normal. When male mice were placed with
untreated female mice, the antibody titers among
the groups were significantly different (P<0.001,
Fig. 2a). The result indicates that the protein or pep-
tide boost groups (groups F12E-4, F2EP2C, F2EP3C,
and F1EP4C) exhibited significantly reduced average
litter size compared with the PBS group (Fig. 2b,
P<0.001). The female mice in group F12E-4 deliv-
ered the fewest offsprings (2.00 0.01), followed by
group F2EP2C (2.50 0.19), group F2EP3C
(4.13 0.22) and group F1EP4C (4.21 0.20).
However, group F12E-1 (8.35 0.16) showed no
significant difference compared to the control PBS
group (8.50 0.10, P>0.05). The fertility rate of
female mice mating with treated animals also
decreased remarkably when compared to the PBS
group (Fig. 2c, P<0.05). The females in group
F12EC-4 showed the lowest fertility as 5%, followed
by groups F2EP2C (6.67%), F2EP3C (25.00%), and
F1EP4C (25.76%). Compared with the results of the
previous study,the males vaccinated with FSHR
plus Eppin four times or boosted with peptide 2, 3
or 4 plus C displayed smaller litter sizes and lower
fertility rates than those targeted with FSHR or
American Journal of Reproductive Immunology 74 (2015) 156–168
160 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
YAN ET AL.

Eppin singly. In particular, the F2EP2C group
showed a very low fertility rate that was similar to
the F12EC-4 group, both being lower than 10%.
This suggested that targeting with both FSHR and
Eppin leads to better antifertility effects than using
single target.
Inhibition of Antisera to Human Sperm Motility
As the data show that the fertility rates of mice in
groups, F12E-4 and F2EP2C, were strikingly reduced,
it was necessary to investigate this depressive activity
on spermatozoa. A normal human sperm suspension
8.50 8.35
2.00
2.50
4.13 4.21
0
1
2
3
4
5
6
7
8
9
10
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
Number of the pups
**
**
**
**
89.17%
80.00%
5.00%
6.67%
25.00%
25.76%
0% 20% 40% 60% 80% 100%
PBS
F12E-1
F12E-4
F2EP2C
F2EP3C
F1EP4C
Percentage fertility
**
**
*
*
0
1
2
3
4
5
6
7
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
End-point titer (log10)
6 weeks
10 weeks
****
(a)
(b)
(c)
Fig. 2 In vivo fertility analysis of inoculated
groups. (a) The sera IgG titer (log 10) of male
mice when mated with females. Two fertility
assays were carried out at six and ten weeks
after first injection. *indicated significant
difference from group PBS (P<0.05). (b)
Progeny size of BALB/c mice with different
immunization schedule. **indicated
significant difference from the control PBS
group (P<0.001). (c) The fertility rate of
female mice mated with immunized males.
*indicated significant difference compared
with group PBS (P<0.05). **indicated group
F12E-4 and group F2EP2C significant different
from groups F2EP3C and F1EP4C (P<0.05).
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 161
B-CELL EPITOPE PEPTIDES OF FSHR AND EPPIN

was aliquoted and mixed with sera from immunized
male mice and sera from the PBS group as control.
The progressive motility and viability of sperm as
assessed by CASA in treated groups are presented in
Table III (means S.E.M.). A significant inhibition is
seen when compared with that of the PBS group
(P<0.001). When treated in the same way, sera from
group F12E-1 showed no motility inhibition to
human sperm (P>0.05).
Sperm Analysis of Immunized Mice
To explore the mechanism of suppressed fertility in
immunized mice, sperm analysis was carried out as
follows. Sperm obtained from the cauda epididymis
were analyzed with respect to sperm count, motility,
and sperm membrane functional integrity. The data
show sperm count was significantly lower in groups
F12E-4, F2EP2C, F2EP3C, and F1EP4C where the
corresponding sperm motility was reduced remark-
ably (P<0.001), and this was consistent with a
reduction in fertility (Table IV).
The spermatogenic capacity was dependent on
Sertoli cell for each of these cells can only nurse a
finite number of germ cells. Based on this, the num-
ber of homogenization-resistant elongated spermat-
ids in testis was calculated. Data suggested that in
the fertility-depressed groups (groups F12E-4,
F2EP2C, F2EP3C, and F1EP4C but not F12E-1),
males presented with a small testis (Fig. 3a) and sig-
nificantly fewer homogenization-resistant spermatids
(Fig. 3b) when compared to the control group
(P<0.05), while in animals only immunized with
FSHR plus Eppin once (group F12E-1), no significant
change was observed for testis weight and testis
sperm content.
Antibody Subclasses Induced in Response to
Immunization
The local IgA levels in epididymis lavage fluid were
comparable among the different groups. The data
were presented as means S.E.M. (Fig. 4). With
respect to Eppin-specific IgA antibodies, the male
mice which received four injections of both proteins
(group F12E-4) displayed the highest IgA concentra-
tion (1.23 0.10), followed by group F2EP2C
(1.14 0.12), group F2EP3C (1.04 0.11), and
group F1EP4C (0.94 0.17), which was completely
different from that of the PBS group (0.12 0.01,
P<0.001). The F140-specific IgA and F240-specific
IgA were similar to Eppin. A single inoculation with
recombinant proteins (group F12E-1) failed to
induce a high IgA response (0.24 0.05) in the epi-
didymis, which showed no difference when com-
pared to the control group (P>0.05). The IgA
Table III Inhibition Effects of Antisera Derived from Immunized Mice on Human Sperm
Group named
% Progressive motility % Viability
30 min 3 h 30 min 3 h
PBS (control) 56.59 3.26 50.86 3.41 81.91 2.32 79.22 2.62
F12E-1 50.58 3.22 44.86 1.89 74.42 3.55 69.23 3.41
F12E-4 28.80 3.71** 18.55 1.53** 50.97 3.04** 38.67 1.61**
F2EP2C 30.60 3.69** 24.17 2.58** 55.85 2.73** 49.29 3.29**
F2EP3C 32.67 2.46** 27.56 2.84** 58.53 3.60** 53.22 3.68**
F1EP4C 33.18 3.74** 27.40 3.79** 59.48 2.51** 54.62 2.36**
Data represent the means S.E.M. Values are significantly different at **P<0.001 from controls.
Table IV Effect of FSHR Prime–Peptide Boost on the Seminal
Parameters of Inoculated Mice
Group
named
Sperm count
(910
6
) Motility (%) HOS (%)
PBS (control) 13.30 0.63 62.72 0.93 82.72 1.10
F12E-1 11.82 0.61 58.84 1.75 78.48 1.49
F12E-4 5.22 0.60** 12.56 1.23** 29.30 1.65**
F2EP2C 5.58 0.55** 11.90 1.35** 33.38 2.22**
F2EP3C 6.78 0.38** 12.62 1.22** 36.28 1.95**
F1EP4C 6.66 1.18** 13.28 1.21** 38.56 1.97**
HOS, hypo-osmotic swelling.
Data represent the means S.E.M.
**Values are significantly different at P<0.001 from controls.
American Journal of Reproductive Immunology 74 (2015) 156–168
162 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
YAN ET AL.

concentration in serum analyzed synchronously
revealed fairly low levels of IgA (data not shown)
giving an end-point titer of <100. Therefore, this
suggested that the contamination from serum had a
negligible impact on the IgA concentration in epidid-
ymis lavage.
Determination of Sera Hormone Levels
Compared with control males, mice with reduced fer-
tility showed significantly reduced serum T levels
(Fig. 5, P<0.001); however, males immunized with
FSHR and Eppin once showed similar T levels to con-
trol ones (P>0.05). The T level (ng/mL) in group
F12E-4 was 2.372 0.040, followed by groups
F2EP2C (2.436 0.054), F2EP3C (2.462 0.050),
and F1EP4C (2.472 0.040), and group F12E-1
(2.700 0.042) showed identical results to the PBS
group (2.728 0.032).
In mice presenting with inhibited fertility, the sera
levels of LH were normal to slightly elevated levels
with no significant variations when compared to the
PBS group (P>0.05, data not shown).
Histological Examination
Following killing, the testes and epididymides from
vaccinated and control mice were weighed and
fixed. Paraffin-embedded 5-lm sections were
stained with hematoxylin and eosin. The histologi-
cal examinations of testis and epididymis were
carried out using a 209objective to evaluate differ-
ences from organs obtained from vaccinated and
control animals (Fig. 6). In males with reduced fer-
0
10
20
30
40
50
60
70
80
90
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
Testis weight (mg)
**
**
** **
0
1
2
3
4
5
6
7
8
9
10
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
Homogenization-resistant spermatids (million/testis)
** **
** **
(a)
(b)
Fig. 3 Testis weight and calculation of
homogenization-resistant elongated
spermatids of each group. Testis tissue was
dissected from mice at 2 weeks after the last
fertility assay. (a) Testis weight (mg) of
inoculated mice. (b) The number of
homogenization-resistant elongated
spermatids in testis, which was expressed as
millions per testis. Values are shown as
mean S.E.M., n=5 per group, and
** indicated significant difference against
group PBS (P<0.001).
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 163
B-CELL EPITOPE PEPTIDES OF FSHR AND EPPIN

tility, these animals demonstrated smaller diameter
of testicular seminiferous tubules, broadened inter-
stitial spaces, disorder arrangement, and lower den-
sities of spermatogenic cells, with variable but
reduced levels of sperm in the seminiferous tubules
with differing degrees. This phenomenon was
extraordinarily obvious in group F12E-4, showing
only one layer of spermatogenic cells in some semi-
niferous tubules and sperm being absent in some of
the lumens. No obvious change was seen in all
Antibody levels in epididymal
lavage (A450 nm)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
** **
** **
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
PBS F12E-1 F12E-4 F2EP2C F2EP3C
Antibody levels in epididymal
lavage (A450 nm)
**
**
**
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
PBS F12E-1 F12E-4 F1EP4C
Antibody levels in epididymal
lavage (A450 nm)
** **
(a)
(b)
(c)
Fig. 4 Determination of the specific IgA level
in epididymis lavage fluid. (a) Eppin-specific
IgA antibodies in epididymis lavage fluid. (b)
F140-specific IgA antibodies in epididymis
lavage fluid. (c) F240-specific IgA antibodies in
epididymis lavage fluid. The group F12E-4,
F2EP2C, F2EP3C and F1EP4C immunization
regimen induced a similarly higher IgA
concentrations against group PBS
(** indicates P<0.001) and group F12E-1
failed to induce a high IgA response
(P>0.05) against group PBS.
American Journal of Reproductive Immunology 74 (2015) 156–168
164 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
YAN ET AL.

epididymides and testes from the PBS and F12E-1
groups.
Discussion
Nowadays, the only contraceptive options available to
men are vasectomy, condoms, and early withdrawal.
However, these methods could not meet the needs of
all the different people in their reproductive age.
Thus, it calls for developing male contraceptives that
are more effective, economical, and acceptable. A
contraceptive vaccine (CV) is a promising approach
for male contraception. In order to develop a safe,
effective, and long-acting CV, appropriate targets
Sera testosterone
concentration (ng/mL)
2.10
2.20
2.30
2.40
2.50
2.60
2.70
2.80
PBS F12E-1 F12E-4 F2EP2C F2EP3C F1EP4C
**
** ** **
Fig. 5 Sera testosterone levels in immunized
males. Sera testosterone levels of treated
male mice were assayed by RIA. The serum
was prepared after killing of animals. Data
were presented as means S.E.M. n=5 per
group. **indicated significant difference
against group PBS (P<0.001).
(a) (b)
(c) (d)
(e) (f)
Fig. 6 Histological sections of testis of
immunized adult male mice. Testis tissue
dissected from mice at 2 weeks after the last
fertility assay was sectioned and stained with
hematoxylin–eosin. Slices were photographed
at the same magnification (20 objective). (a)
group PBS; (b) group F12E-1; (c) group F12E-
4; (d) group F2EP2C; (e) group F2EP3C; (f)
group F1EP4C. Mice immunized with F12E-4,
F2EP2C, F2EP3C, and F1EP4C regimen
showed changes of reduced diameter of the
testicular seminiferous tubules, broadened
interstitial spaces, disorder arrangement, and
lower densities of spermatogenic cell (black
arrow), with variable but reduced levels of
sperm in the seminiferous tubules (red arrow)
with differing degrees. While testis from PBS
and F12E-1 groups showed normal
seminiferous tubule diameter and
spermatogenium. Photomicrographs are
representative of histochemistry in tissue
from n=5 mice per group. Bar =50 lm.
American Journal of Reproductive Immunology 74 (2015) 156–168
ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 165
B-CELL EPITOPE PEPTIDES OF FSHR AND EPPIN

within the reproductive process which are obedient
to interference with antibodies should be primarily
considered. Apart from egg and sperm antigens, the
hormone FSH which plays a role in the development
of gametes is potential target for CV. FSH exerts its
function on spermatogenesis through its receptor
(FSHR) which is expressed only on Sertoli cell
22,23
and regulates germ cell viability in synergy with tes-
tosterone.
24
Targeting FSH could provide the basis for
a male antifertility vaccine.
25
Previous research
revealed that blocking FSH signalling leads to fertility
inhibition.
26
Using a combined online prediction facil-
ity and molecular docking, we previously identified
three epitope-based peptides in extracellular domain
(ECD) of hFSHR and observed moderate antifertility
effects in immunized male mice.
14
However, FSH-based vaccine caused oligospermia
rather than azoospermia, which hindered further pro-
gress. Furthermore, spermatozoa have proteins that
are of great importance in functional regulation of
mature sperm, with a potential to be targets for CV.
One promising candidate is epididymis protease
inhibitor (Eppin), a ~133 amino acid protein secreted
by Sertoli cells and epididymal epithelial cells
27,28
in
an androgen-dependent manner and functions as a
serine protease inhibitor. The Eppin–semenogelin
complex modulates prostate-specific antigen (PSA)
activity and provides antimicrobial protection for sper-
matozoa in the female reproductive tract.
10,11,28,29
It
was reported that anti-Eppin antibodies from infertile
monkeys inhibited Eppin from binding to semenog-
elin.
30
In our earlier study, we observed moderate
fertility inhibition in male mice vaccinated with a B-
cell epitope of Eppin and a Th2 epitope derived from
measles.
13
Inoculation with rhFSHR or rhEppin alone only
produced moderated fertility inhibition in male mice,
which is not very satisfactory. Taking advantage of
FSHR and Eppin in males where they are located
only in reproductive system, we investigated the
antifertility capacity of uniting the application of the
two antigens. An effective vaccine needs to elicit a
potent immune response, has a rapid onset and a
long duration of activity. In our previous study,
these properties were fulfilled by employing a pro-
tein prime–peptide boost strategy,
13,14
in addition
avoiding the disadvantages caused by administering
a full-length protein or a short peptide.
31,32
In this
study, we employed the protein prime–peptide boost
regimen as before which we had proven to greatly
enhance the immune responses and produced
specific high IgG antibody response in immunized
male mice, especially in the group that received a
united vaccination.
In our previous study,
13,14
using a protein prime–
peptide boost inoculation modality, male mice
immunized with peptide C from rhEppin showed a
fertility rate of 26.7%, while the fertility rates of
mice immunized with rhFSHR peptide 2, 3, and 4
were 26.67%, 46.67%, and 45.00%, respec-
tively.
13,14
Thus, immunization with a combination
of rhFSHR and rhEppin resulted in enhanced anti-
fertility effect, particularly in group F2EP2C which
demonstrated a fertility rate near to zero as protein
prime/boost group. Meanwhile, boosting with an
immunodominant peptide as opposed to protein did
not aggravate but rather reduce the side-effects such
as serum testosterone reduction and weight loss of
testes. In brief, with respect to antifertility efficacy,
the united administration presented outstanding
effects with slightly lower antifertility rates when
compared with those of proteins homologous boost
but superior to single antigen employment. With
regard to the antifertility mechanism, in our previ-
ous study, it showed inhibition of sperm motility in
mice primed with Eppin and boosted with peptide
C,
13
and decreased sperm count in cauda epididymi-
dis, and reduced homogenization-resistant spermat-
ids in testis of mice received FSHR prime–peptide
boost inoculation.
14
With these confirmed results, it
was reasonable to explain the antifertility effects of
combined vaccination with intercepting FSH signal-
ling led to spermatogenesis disorder and anti-Eppin
antibodies caused inhibition of sperm motility. As an
effective contraceptive vaccine, it would need to pro-
duce effective antibody concentrations in the local
genital tract. In this study, the data suggest that IgA
was higher in the epididymis lumen of protein prim-
ing–peptide-boosted animals, above all in the com-
bined immunization, and the motility of sperm
depressed significantly as well, and this was consis-
tent with inhibition effects of antisera on normal
human sperm. This may be one reason for the better
antifertility efficacy of combined vaccination.
In conclusion, combined immunization against sev-
eral antigens may prove ultimately to be a better way
to ensure complete protection from contraception.
Our results propose that combination priming of
rhFSHR with rhEppin and immunodominant pep-
tides boosting could elicit strong humoral responses
and produce high neutralizing antibodies in the
lumen compartment of the reproductive tract, there-
American Journal of Reproductive Immunology 74 (2015) 156–168
166 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
YAN ET AL.

fore resulting in a fertility inhibition near to zero in
male mice, and this was far superior to a single
administration. Therefore, uniting vaccination
against FSHR and Eppin may provide an ideal target
for male immunocontraception in the future.
Acknowledgements
This research was funded by grants from National
Key Technology R & D Program of China (Grant
2006BAI03B12). We appreciate the help of Dr Dev
Sooranna for kindly editing this manuscript.
Conflict of interest
The authors declare that there is no conflict of inter-
est that would prejudice the impartiality of this sci-
entific work.
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