Indian Journal of Ex perimental Bi ology
Vol. 4 1, July 2003, pp. 764-772
Epididymis as a target for contraception
Vrinda Kh ole
Gamete Immunobiology Department. National Institute for Researeh in Reprod uctive Hea lth.
Mumbai. 400 0 12, India
Advantage of using a vacc ine ba sed on sperm anti gens is that it can be used both in males and femal es as individual s
who have anti spenn anti bod ies are llsuall y infertil e bu t oth erw ise hea lthy. Several sperm spec ifi c anti gens ident ifi ed as
prospec ti ve ca ndidates for immunocontracepti on are of tes ticular ori gin . For th e purpose of immunoco ntracep ti on i t may be
desirable not to disrupt spermatogenes is and tes ti cul ar fun cti on. Concept o f post testi cul ar maturation o f sperillatozoa has
been ve ry we ll establi shed. During pos t testi cular voyage sperm atozoa undergo a seri es o f co mpl ex and sequential event s
w hich transforms thc immature immotil e sperm atozoa into mature sperm. A cqu isi ti on o f functi onal maturity is necessary for
progressi ve motilit y, zona pellucida recogniti on culmin at ing in sperm egg binding. Importan ce o f epidid y mal matura ti on is
hi ghli ghted by the fact th at hi gh percentage of male in fe rtilit y in human ori gi ates from the mal func ti on of th e epid idy mi s.
The epidid ymi s has also shown to be in volved in sperm storage and provides an adequate environment for fina l mat uration
of the sperm. It provides a co nd ucive microenvironmeJ1l by virtue of whic h the spermatozoa arc protec ted during th e
storage. In view of thi s it is imperative th at more all enti on needs to be foc used on epididymal anti gens. Th e informati on
obtained will enable us to ide J1l ify epidid y mal anti gens releva J1l 10 fertilit y and also help in in fe rtilit y diagnos is.
Key words : Contrace pti on, Epididy mis. Epidid y mal antigens, Infertilit y di agnosis, Sperm malUrati on
There is an urgent need to prov ide safe and sustained
effecti vc ferti lity co ntro l for th e wo rld popul ati on t.
More and impro ved co ntrace pti ve opti ons is th e need
of th e hour. Thi s is espec ially relevant to males who
currentl y have onl y four limited options: abstin ence,
withdrawa l, co nd oms and vasec tomy. Of th ese
vasectomy is esse nti ally irreversibl e and is not the
preferred method for men who wi sh to father a chi ld
at a later date. Although th e res ults from studi es
in vo lving hormonal method s for male contraception
are convincin g2 it has not yet yielded a marketabl e
product. Vaccine approach for co ntraception would
definitcly be a va luab le addition to th e ex isting
armamentarium of different approaches used for
fa mil y planning. Vaccine based on sperm antigen is
very promising and, therefore, in spite of no real
breakthrough , they are being actively pursued . The
targets have been sperm antigens comi ng either from
the testi s or the epididymis. Reali zing the feasibility
of this approach and th e need for additional methods
for contracepti on Nation al Institute for Child Health
and Human Development (N ICHD) conve ned a
work shop to identify novel strategies involving
testi cul ar and epididymal antigens for developing a
male contraceptive in the 2 1s l centurl.
*For co rrespondence :
E-ma il : vrind ak hole@ hotmai1. co lll
Fax: 9 1-22-241 394 12
Testis as a target for contraception
Tes ti s is th e primary orga n whi ch produces
spermatozoa and, th erefore, has been considered to be
a good target for contracepti on. It is indi cated th at thi s
co uld be done by direct interference at various stages
of sperm atogenesis such as I) interference wi th
meios is and particularly th e cell cycle chec k points 2)
di srupti on of RNA protein interacti on (post
transcription::ll co ntrol) 3) di srupti on of juncti onal
co mpl exes betwee n sertoli ce ll s and ge rm cell s and 4)
attacki ng differen t testicular proteins2 . Number of
testis/sperm specifi c proteins h::l ve been idellli fied
using di fferent approaches and several of th em have
also been cloned and sequenced . The current statu s,
applicati on , relati ve merits and immunoge nicity of
these antigens has been extensively revi ewed 3 .
Epididymis as a target for contraception
Post tes ticular contraception cou ld be achi eved by
interfering pharmacologica ll y or immunologicall y
with the process of sperm maturation in the
epididymis . Maturational changes whi ch the sperm
undergoes during epididymal transit have been show n
to be prerequisites for successful fertili zation. It is
suggested that th eoretically th ese may be interrupted
at different sites. It is poss ible that modifying the
pattern of epididymally secreted protein s may change
the optimal environment for maturation and storage of
spenn 4 .
KH OLE: EPIDIDYMI S AS A TA RGET FO R CONTRACE PTI ON
Advantages of epididymaL antigells for contraception
Vacc in ati on of males for co ntracepti on has not
been possible beyond pre clinica l levels for a va ri ety
of co ncern s. Testi s is an immunologicall y pri vileged
site because of the blood testi s barri er. It may not be
advi sable to immuni ze men with tes ti cular sperm
anti gens as it may lead to irreversible testi cul ar
damage. It is sugges ted th at anti bodi es raised aga inst
anti gens present exclusive ly post testi cul arl y may be
less likely to induce autoimmune orchiti s5 . There are
defi nite ad va ntages in targetin g the epididymi s fo r
co ntraception. Firstl y the onset of in fertility and also
its reversa l has been show n to be far qui cker6 th an any
agent attac king th e testi cul ar producti on of
spermatozoa and seco ndly, as maturin g cell s are
targeted, damage to th e geneti c materi al, a possibl e
sequ elae of effect on di viding germ cells is avo ided 7 .
It wo uld also avoid endoc rin e impa irment of li bid02 .
In order to pursue th e epidid ymi s as a target for
co ntraception there is a need to understand the
di ffe rent rol es pl ayed by epididymal protein s in sperm
mat urati on, and sperm protecti on during storage. The
new tec hn olog ies such as cD A arrays and
proteo mi cs. will help us identi fy th e va ri ous proteins
in vo lved in sperm maturati on immunolog ica l
protec ti on as well as th ose possess ing antimi crobi al
ac ti vity whi ch help in sperm protec ti on. Once such
molec ul es are identi fied we will be ab le to understand
th e importance and co mpos ition of th e epidid ymal
mil eu whi ch pl ays a major role in acqui siti on of
fe rtility, motility.
RoLe of epididymis in sperm maturation
Epididymi s was earlier th ought to be just a pass ive
chan nel through whi ch sperm atozoa travel to be
stored before being ejacul ated. The concept of post
testi cul ar maturati on in mammali an spermatozoa
evolved as a result of th e pioneering studi es of
Benoit8 and Youn g9. But it was Bedfo rd 1o and
Orgebin Cri st ll wh o initi ated in depth inves ti gati ons
and demonstrated th at, sperm whi ch were prevented
from passing along th e epididymal du ct were viable
but did not acquire full fe rtili zing capac ity . Large
amount of informati on has beco me ava il abl e after
1960, about th e stru cture, biochemi cal properti es and
fun cti ons of thi s organ. ]t has been shown th at in the
epididymi s there is co mplex ity in th e cellul ar
propert ies, heteroge nei ty, region specific ex pression
and . pati al and temporal organi zati on of proteins. All
these complexities make this organ very dyn ami c. It is
765
now clea r th at th e maturati on of spermatozoa and the
acqui siti on of motility and fe rtili zing abilit y do not
res ult fro m a pass ive journ ey of spermatozoa but
rath er as a res ult of expos ure to and acti ve interac ti on
with the luminal co ntents of di fferent epidid ymal
.
I?
regions Membrane remodeLLillg
During its epididymal sojo urn the sperm plas ma
membrane undergoes intense changes both in protein
co mposition and loca li zati on on the ga mete. The
sperm plas ma membrane is ori ginall y deri ved from
spermatogo ni a/s permalocy tes . in the testi s but
undergoes
ex tensive
remodeling
du ring
spermi oge nesis in th e testi s, maturati on in the
epididymi s and capac itati on in th e female genital
tract. The epidid ymal maturati onal events take pl ace
du e to the microe nviro nment fo rmed by the lumin al
co ntents of th e epidid ymal duct l3 . Remodeling of the
membrane co uld be bro ught about either by uptake of
sec reted epidid ymal proteins, or processing of
ex isting or acq uired protein s l4 . Process ing of existing
or acquired proteins occ urs as a res ult of some glyca n
modi fy ing enzy mes such as glycos idases and
glycosy ltransferasesl 5 or protein modifying agents
such as endo-proteases and protease inhi bitors 16 that
are found freely in th e luminal fluid or are associa ted
with sperm plas ma membrane. A deficiency of th ese
enzy mes has bee n show n to be res ponsibl e for male
in fe rtilit/ 7 . These remodeling mec hani sms lead to
acqui siti on of spec ifi c fun cti ons by different domains
of the spermatozoa during epididymal transit.
Repositi onin g of protein co mponents to membra ne
domains occ urs as ~ee n in fa mil y of protein s such as
AD AM, (protein s
with
a di sinteg rin
and
metall oproteinase
like domain ) fo r exa mple
fertilin (PH20)l s.IYor membrane proteins such as MDC
(proteins with a Metall oproteinase, a di sintegrin -like
and a cysteine-rich domain ) for exa mpl e 2B ( 0.21 .
Relocati on of proteins can be see n ac ross di ffe rent
domains in proteins whi ch belong LO immunoglobulin
super famil y fo r exa mpl e, CE9 where th e shift is from
principal piece to midpiece 22 .
Every domain of th e sperm is endowed with a
spec ific fun cti on. Head of sperm compri ses of
acroso me and post acroso mal region. Ac roso me is
in vo lved in primary binding to egg pl as ma membrane.
Equ atori al/post ac roso mal region is in vo lved in
seco ndary binding leadin g to fu sion23 . The fl agel lu m
is di vided into midpi ece, principal pi ece and end
piece. Midpiece is associated with genera ti on of
766
INDIAN JEX P BIOL. JULY 200]
energy ( ATP). prin cipal piece w hi ch contain s th e
axoneme and fibrous shea th is responsibl e for
flage ll ar fl exibility and Il1 otilit/~ Protein s of th e
fibrous shea th are impli cated in signaling pathway in
~' .
I'
I
I'
I
1(, 17 Pro teln
.
s permalozo~c · ane In g yco y tl c pa tl\vay- '- .
DE w hich has been loca li zed in th e acrosOIl1e region
A
is shown to have a role in spe rm egg fusio n
26h:Da protein localized in the flage ll a has been
9
shown to have a role in mo tilit /
2x.
Protective role oJflie epididymis
Epid idym is plays a ve ry importan t role In
protect ion of spe rmat ozoa but i s the least studi ed
aspect. Spermatozoa spe lld Il1any da ys tra ve rsin g th e
long epid idyma l duct and are faced w ith a co nstant ly
changing microenvironment. Ep id idy mi s prov ides
co nduci ve mi croe nvi ronment by rapid Iy el i mi na ti ng
th e harmful metabo l ic by products·'o. I t also protec ts
spe rmat ozoa by blood ep idi dym is barri er pro vided by
tight junc ti ons between th e principal ce lls. T hi s
barrier not onl y pro tec ts th e spe rmat ozoa from
ex ternal non conduci ve en vi ron ment but also preve nts
the access to th e immune sys tem 'l . Thi s barrier is
se lec tive and does not all ow passage of hi gh
molec ul ar we ight compo unds lih:e L -gl ucose. in su lin ,
BSA bu t readi ly allows th e passage of water. 0 glucose an d amin o acids showin g th at the hl ood
ep id idy mi s harri er helps in th e Il1ain tenance of
co ndu ci ve mi croenvironment for th e spermatozoa.
Epi did y mi s has also been shO'vv n to playa role in
protect ion of spe rm atozoa from free radi ca ls and
recognition and eli mination of defective spe rm atozoa .
Epid id y mal spe rm ato7.0a are ex tremely vulnerabl e to
ox id ati ve stress. T o ove rcome thi s probl em
epid idym is has a ri ch so urce o f an anti ox id ant
enzyme that scavenges any excess reac ti ve oxygen
metabo lite released by the sperm atozoa durin g
epidid yma l tran sit I!, . Th e- ca put epidid y mi s also
sec retes glutathi one perox ida se w hi ch gets intimately
assoc iated w ith the spe rm su rface whe re it se rves to
remove any H 20 ] genereted by these ce ll s as a
co nsequence of SOD ac ti on . M o lecu les such as
glutathi one
perox id ase,
ca talase,
superox ide
dislllu tase have been show n to protect sperm in th e
epididym is rronl damage due to reac ti ve oxygen
J2
spec ies . Studi es on ex pression o f Il1R A for 13 Defensin - I and 2 in th e initial segment and caput
ep ididym is suggest a ro le for th e epididy mi s in
1
an timi cro bial prot ecti onJ · . Quality con trol of male
gametes is ensured by an acti ve apoptotic pathway
present in s pe rll1 ato~enic lin eage'~ and in mature
.
1, 16
~
17
sperm o f m ice·· ·· and mell" .
Th ough ubiqui tin was prev iously detec ted in
'xW and sem .ll1 al plasma ~o , .Its
Iluman epl'd'd
I yma I ce II s···
import ance in qu alit y control of fertili ty was not
indi cated. A lthough both normal and defec tive sperm
ca rry co nstitut ively ubiquitinated substrates it is onl y
the defec ti ve ones th at get surface ubiquitinated
du rin g epididy mal passage. Suto vsky and cowo rk er. ~I
ha ve shown surface ubiqu i tin ation of sperm and
subsequent phagocy tos is by epidid y ma l epith eli al
ce lls.
Role oj epididymal proteills ill reproductioll
Th ere are several direc t and indirect ev idences
whi ch underlin e th e importan ce of ep ididymal
umber of cli nical
protein s in reproducti on.
ev idcnces show th e correlati on between abnormaliti cs
or disturbances in the epididymal secreti ons and
in re rt ilit / ]·-~~· . In fact hi gh perce ntage of mal e
in fert ilit y in human is beli eved to ori gin ate from th e
malfuncti on of th e e pididymi s~ 5 . I ncomp lete spe rm
maturati on w ithi n th e ep ididy mi s has been sugges ted
to be th e ca use of tota l fa ilure of sperm binding to
zO lla in un success l"ul hum an ill I'il ro fe rtili zation
cases~6 . V asectomy also has show n to ca use
irreversible damage to epidid y mi s and is th ought to be
one of the ca uses of infertilit y even after
vasovasos tomy"7.4X. Human P34 H prote i n wh ieh is
in vo l ved in sperm -zo na pe llucida interact ion is found
to be reduced in certain cases o f idi opa thi c
in fe rtilit y"9. P2Sb and P2Ib protein s (bull homolog ues
o f human P34 H) are associated w ith sub-fertility and
5o
th eir exp ress ion is marh:edly low in sub fen il e bul1 . It
is specu lated th at epididyma l anti gens play a
predominant role in seve ral cases of hu man
in krti lit /I . A ll these ev idences point to th e import ant
ro le of th e epididy mis in bes tow in g on th e spe rm
motility and fertili zin g abi lit y. In view 01" these data it
is clear th at epididymal protein s have a vital role in
maturatioll of sperm atozoa. lead in g to success ful
reproducti on and, therefore, more att entio n needs to
be foc used on identi fica ti on o f epidid y ma l antigens.
Several epidid y mal protei ns have been ident i fi ed
and studi ed for their co ntributi on towa rd s sperm
maturati on. Howeve r. man y more still remain to be
identifi ed and their fun cti ons need to be ascert ained.
Identi fica ti on of newer epidi dyma l protein s and
annonatin g th eir fun cti on w ill help in understandin g
th e mechani sm o f sperm maturati on and th e sequence
of events therein. Thi s will further hel p in se lectin g
epididymal targets ror co ntracepti on w hich w ill
spec i fica ll y alter th e ability of th e sperm to fertili ze
KH OLE: EPIDIDYMI S AS A T A RG ET FOR CONTRACEPTI ON
without any side effec ts. Hence understandin g th e
epididymi s in more detail s will help in treatin g certain
types of male infertility and also to develop male
contracepti ve agents.
Different approaches have been exploited for
identificati on of epididymal proteins such as use of
lectin s52 , . ubtracti ve sc reenin g of th e epididymal
cDNA librar/ 3 , use of the ex pressed sequence tag 54 ,
. 'is and neo nata I to Ien' zatJ.on56'57 Neo nata I
proteomlcs'
tolerizati on is a powerful tool for raising monoclonal
antibodi es to rare or weakly immunogeni c anti gens.
Thi s approac h has bee n used by several inves ti gators
for generatin g monoc lonal antibodies to rare or less
immunogen ic anti gens58 . 64 In thi s approach once a
state of tolera nce to an anti gen is establ ished , the
tolerized animals could be subsequently immuni zed
with a crude preparation of the potential anti ge n
(i mmunoge n). By inducing th e immune toleran ce to
the toleroge n, the immune sys tem will generate an
immune response to onl y th ose epitopes not included
in the tolerogen preparati on. Thi s approach increases
the probability of obtaining antibodies to functionally
significant components that may be weak immunogens.
Identificat ion of ep ididymal proteins by conventi onal imm un ization followed by hybridoma technology has not been very successful , probably beca use
th e testicul ar proteins are more immunoge ni c than the
epididymal proteins. Therefore, the altern ate meth od
of, "Neonatal toleri zati on" also called as subtracti ve
immuni zati on first reported in 199 156 was ex pl oited
by us with slight modifications. Us ing thi s method
we, were ab le to ge nerate an immune respon se to
epididymal anti ge ns in BALB c mi ce 57 . In our protocol
(Fig la) animals were toleri zed to testi cul ar protein
(tolerogen) at birth and were then immuni zed wi th
epididymal sperm protein (imm un oge n) for raising
specific immune response. These studi es demonstrated
that neonata l tolerizati on with testicular proteins
fo llowed by immunizati on with epididymal sperm
proteins enh anced the production of antibodies to
epididymal proteins. Serum from th ese animals
locali zed proteins onl y in co rpu s epithelium and
sperm from corp us and ca uda ep idid ymi s but showed
no reactivit y with tes ti s (Fig. I b) . Us ing se ra from
th ese neo nata ll y tolerized and immuni zed mi ce, we
identified a dominant epididymis specific protein of
molecular we ight approx imately 27 kDa whi ch was
used to raise polyc lonal antibodies in rabbit. The
polyclonal ant ibody was very spec ific to epidid ymis
as see n by ELIS A, Western blot and IHe. The
antibody iden tifi ed a 27 kDa protein fro m corpus
767
epididymis as well as sperm from corpus and cauda
region . The protein identified was present on th e
midpiece as seen by indirec t immunoflu orescence and
was androgen regulated and was also developmenta ll y
reg ul ated65 . These results indica ted that thi s protein is
sec reted mainl y in th e corpu s and acq uired by th e
spermatozoa during passage through these regions.
The prese nce of th e protei n in co rpu s and cauda may
be due to its secretion by the principal cell s of th e
corpus. The principal cell s of the corpus have a well
developed endopl asm ic reticulum and golgi
apparatus, which indi cates th at they are in volved in
active protein sy nthesis. There are so me reports where
principal cells of epididymis have geen shown to
incorporate labeled Hmino ac ids and transport
radiolabelled molecules through th e ce Il 66 .li7. Principal
cell s have been show n to be act ive ly in vo lved in th e
ph ys iologica l fu nctions of the epidid ym is, involv ing
endocytosis 68 and sec reti onli9. It is repo rted that
proteins sec reted by principal cell s may interact with
sperm atozoa in th e lumen of epid idyma l duct and
enabl e spermatozoa to develop motilit/ o and
fertilit/ I . It has been show n that co rpu s/caud a
juncti on pl ays a major role in sperm maturat ion72-7.J.
Fertilization rate is show n to be hi gher in sperma tozoa
from corpus th an from caput as seen in in -v it ro
fertilization 75.76. Sperm motility and preg nan cy rates
were also fo und to be significantly improved when
the vas was surgicall y joined to the corpu s in those
pati ents who received specifi c tubul e vasoep idid ymo. 77·7~ . S'II1ce
stomy f or 0 bstru ctJ· ve azoospermia
spe rm atozoa beco me motile and fe rtil e in th e corpus
and ca ud a ep ididymis, the protein identifi ed in thi s
study is probab ly a sperm maturat ion protein.
Monoc lonal an tibod ies have been ex tensively used
for identifi cati ons of sperm antigens (Fig. 2).
Monoclonal antibodi es provide a powerful analytical
tool, allowing recognition of individual determinants
in a co mplex antigeni c structure and have becn
applied to studi es in reproducti ve biolog/ lJ . Sera from
infertile male and femal e or vasec tomized male have
also provcd to bc a good source of an ti sperm ant ibody
for characteri zati on of sperm antigen in vo lved in
fertility. Using sera from in fe rti le male Poulton el 01. 51
identified a 18 KDa sperm protein of epidi dymal
origin and suggested that auto immune infertility
mi ght rep rese nt a respo nse to the epididymal rather
than tes ti cular sperm. He furth er suggested that
monoclonal anti bodi es ra ised to such unique and
immunologica ll y access ible spe rm coa ting an ti gens in
IND IAN J EX P BIOL. JUL Y 2003
768
using polycl onal serum from the T I mouse used for
fus ion loca li zed anti gens in different reg ions of the
sperm such as acrosome. post acrosome, equator,
midpiece and tail. Th is indi cated th at epidid ymal
proteins are located on different reg ions of the sperm
and are li kely to pl ay doma in speci fic ro les such as
sperm-egg interaction , acrosome react ion and motility
w hich are essen ti al for fert ili za tion . It was interestin g
to note th at th e pattern of II F locali zati on was seen to
be identi cal in both glu tera ldehyde fix ed spermatozoa
smcared on glass sli de as well as spermatozoa in
th e epidid ymis rath er than in the testi s would seem to
preseIll a th eoreti ca l soluti on to male inferti lity.
We generated large number of hybridomas, using
Balbc mice which were toleri zed to testi cular proteins
57
and immuni zed w ith epididymal sperm prote ins .
Maj ority of clones showed hi gh reactivity w ith
epididymal sperm prote in while a small number were
si
fou nd to react with tes ti cular sperm protein . Thi s
ind icated that neonates were succes sfull y tol eri zed to
testi cular anti gen and mounted immune response to
epididymal protein s. Im munonuoresccnt loca li za ti on
Neonatal Tolerization-Immunization Protocol
Mi ce injected with toleroge n (testicular protein ) Day 0
Mice injected with tolerogen (testicular protei n) Day 5
~
Mice bled retro orbitally to check reactivity with testicular proteins Day 21
Immunized with epididymal sperm protein
Two boosters at 2 weeks interval
Bled retro orbitally to check titer against epididymal and testicular sperm proteins
Fig. I (a) -- Fl ow chan showing protocol
Testis
rur lIeo natal to leriza ti oll ami illlilluni za ti on
Corpus
Cauda
Fig. 1(b) - llllllluliohi stoc hellli ca l locJ li zat ion of amige ns usin g serum from neo nat all y to leri zed imilluni zed mouse. Test icul ar sect ion
shows no stai ning. The co rpus sccti o n shows locali zation in the supranu clea r region of th e e pitheliulll and o n sperill atozoa. The cauda
epid idyillal section shows no sta ining in the epithe liulll bu t strong sta ini ng on the spcrmalOzoa in the lumen
KHOLE: EPIDIDYMI S AS A TARGET FOR CONTRACEPT ION
769
Fi g. 2 - lrn rnun ochemical and funct ional characteri zat ion of antigen using monoclonal alllibod ies.
al-;)4 Representa ti ve pictu rc of immunohi stochemi ca l loca li zation in differclll regions of cpidid y mi s using a monoc lonal antibod y. (al )
Proximal cap ut shows no staining. (a2) Distal cap ut shows sorne loca li za ti on. (a3) Corpus epi th elium shows strong localizati on.
Spermatozoa in thc lumen arc also st ained. (a4) Cauda cpid idymal rcg ion shows localizat ion on ci li ary lining and spermatozoa.
b J -b 3: lmmunolluorcscen t locali zat ion of antigcns on sperm atozoa using diffcrent monoclonal ant ibodies. (b I ) M ab V2C4E2 stains
acrosoma l regi on. (b2) M ab V3C8 , V3F4F4 stain post acrosoma l and eq uatori al region. (b3) M ab V I B8E 10. V3C I 0 stain rnid piece
rcgion
c l -c 2: Agg lutin at ion pallern using differclll mAbs. (c I ) Radial pallcrn of agg lutinati on sccn wi th mAb V2C4E2 , V3C8. V3F4F4. (c2)
Cornct shape pallcrn of agglutination secn with mAb V I B8E I O. V 3C I O.
suspenSion. Thi s obse rvation along with th e
agglutination pattern indicated that th e proteins
identified by the mon oclonal antibodies are on th e
surface of sperm . Surface locali zation of sperm
antigens is one of th e criteria for ideal co ntraceptive
targets.
Earlier studi es from our laborator/ 2 have sho wn
th at pass ive immuni zation with antibodies to 26 kDa
epididymal specific protein were very effective in
bringing about antifertility effect in both female and
male mice. In case of the femal e mi ce, th e antibody
showed a dose dependent reducti on in th e antifertility
effec t and was found to be effective on ly before
fertilization . Whereas, in case of male mi ce, th e
antibody was able to enter th e epidid ymi s within 24 hI'
and inhibit sperm maturation and sperm fun ctio n.
Fertility was reduced to 100% after about 5 days of
antibody administration. Histologically, the effect was
partially reversed after a week and co mpl etely
reversed after two weeks. These studies clearl y
indicated that where spermatoge nesis is un affected ,
the recovery of fertility was faster.
Advantage of using a vaccine based on sperm
ant igen is th at it can be successfully used both in
males and females, as indi vid uals who have antisperm
antibodies are usuall y infertile but otherwise
health l 3 . Of th ese ant igens the epid idyma l anti ge ns
make better ca ndidates for imIl1unocontraception as
they inhibit onl y post testi cular maturati on of
sperm atozoa, mak ing th em infertil e wit hout affecting
770
INDIAN J EXP BIOL, JULY 2003
testi cul ar function8~. It is suggested that antigens th at
do not cause testicular pathology but can access th e
epididymal lumen and eliminate the spermatozoa or
impede their function , with potenti al reversible
antifertility effect would be worth y of co nsiderati on
for immunocontraception.
Pos t testicul ar approaches to male con tracept ion
are yet in its infancy and have not reached the clinical
phase. It would be necessary to investigate the
molecular physiology of sperm maturation and
epididymal fun ction. Thi s would be poss ible on ly if
we identify target molecules . Once thi s is ac hieved
these molecules could th en be blocked by spec ific
pharmacological agents with a rapid onset of act ion.
Acknowledgement
J wi sh to thank my students Saurabh ' Joshi ,
Sandeep Ranpura and Shagufta Khan for co ntributing
to so me of the studi es described in this revi ew.
References
2
3
4
5
6
7
8
9
10
II
Aitk n J R, l mmunocontraceptive vaccines for human use.
J Reprod 111111111110 1. 57 (2002) 273 ,
DePuo lo L V , Hinton B T & Braun R E. Male Contraception:
View, to the 2 1" Century , Beth esda. MD. USA. 9- 10
September 1999, TEM . II (2000) 66.
Naz R K , Fertilization related sperm antigen s and their
immunocontraceptive potenti al, AII1 J Reprod 1111111 UII 01, 44
(2000) 4 1,
Cooper T G. The epididymi s as a site of co ntracep ti ve attack.
in Spermatogenes is, Fe rtilizatioll .Colltraceptioll, Molecular.
Cellu/a r and Endoc rine events in Male Reproductioll, Edited
by E N iesc hl ag E and U-F Huberni cht (S prin ger- Verl ag,
Berlin) 1992,4 19.
Jones W R. Gamete Immunology. HUIII Reprod, 9 ( 1994) ·
828,
Hegde U C. Gopa lkri shnan K. Kh ole V & Shartiya 0,
Ant ibody di rec ted to a 26 kDa epi didymal sperm protein
inhibits sperm maturation,function and fertility signi f ica ntl y
in mouse, in Reproducti ve IlIIlIIull ology. Ed ited by S K Gupta
(Narosa Publishing House, New Delhi , India) 1999,3 16,
I-linton ST, The epididy mal mi croenv iro nment. A site of
attack for a male con tracept i ve?, Ill vest Urol, 18 ( 1980) I .
Benoit J, Sur la signifi ca ti on fonc hi onelle des sec reti ons
epididy maire et deferentielle, COlliI' Relld Soc Bioi. 84
(192 1)951.
Young WC ( 193 1) , Functional changes undergone by
spermatozoa during their passage through the epid idy mis and
vas deferens in guinea pi g, Fertilizing abilit y and embryo nic
mortalit y in docs insem inated with epididymal spermatozoa .
AIIII Bioi Allilll Bioc" elll Birl/I" vs. 7: 373-389 . .J Exp Bioi , 8:
151.
Bedford J M , Efreet of duc t li gati on on the fertili zing ab il ity
of spermat ozoa in th e epid idy mi s of rabbit. J Erp Zool. 166
(1967) 27 1.
Orgebin-Crist M C. Maturation of spermatozoa in the rabbit
epididym is, Fcrtili zing abilit y and cmbryoni c mortali ty in
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
does inseminated wi th epidid y mal sperm atozoa. A IIII Bioi
Allill1 Biochem Biophys, 7 ( 1967) 373.
Jervis K M & Roba ire B, Dynamic changes in gene expression
al ong th e rat epididy mi s, Bioi Rep rod , 65 (2(l0 I ) 696,
Hinton B T & Palladino M A , Epidid ymal epithelium : its
contribution to th e formation of a luminal Ouid
microenv ironmcnt. . Mic rosc Res Tech, 30 ( 1995) 67,
Jones R, Plasma membrane stru cture and remode lling during
sperm maturati on in the epididym is, J Refirod Fe rti!, Suppl
53 ( 1998) 73,
Tuls iani
R P, O rgebin-Cri st M C & Skudl arek M 0 , Role
of luminal Ouid glycosy ltransferases and glycos idases in th e
mod ifi ca ti on of rat sperm pl asma membrane glycop rotei ns
du ring epididymal maturation , J Reprod Ferril, Suppl 53
( 1998) 85.
Dacheux J L, Gatt i J L & Dacheu x F. Contributi on of
epididym al secretory prote ins for sperm atozoa maturati on.
M icroscopy Res Tech. 61 (2003) 7.
Corrales J J, Bu rgo R M , Miralles J M & Villar E,
Abn ormaliti es in sperm ac id glycosidases from infertil e men
wit h idi opathi c oligoasthenoteratozoospermia, Ferti/ Stel'il
73 (2000) 470.
Primakoff P & My les 0 G, The ADAM gene famil y: surface
proteins with adhes ion and protease activity. Trend s Genet
16 (2000) 83.
Blobel CP, Functional process ing of fertilin : ev idence for a
criti cal ro le of proteolysis in sperm maturati on and
ac tivati on. Rev Reprod. 5 (2000) 75.
Frayne J, Jury J A , Barker H L & Hall L , Th e MDC family
of protein. and their processin g during epid idyma l transit.
JRF Supplem elll . 53 ( 1998) 149.
Jones R, M a A. Hou S T , Shalgi R & Hall L, T esti cu lar
biosynthesis and epididymal endoproteoly ti c processing of
rat sperm surface antigen 2B I , J Cell Sci, 109 ( 1996) 256 1,
Cesari o M M & Bartles J R. Compartmenta lizati on,
processing and red istribution of th e pia, ma membrane
protein CE9 on rodent sperm atozoa, Relationshi p of the
annulu s to domain boundarie s in th e plasma membrane of the
tail , J Cell Sci, 107 ( 1994) 561.
Saxena 0 K. Oh-oka T . Kadomatsu K , M uramatsu T &
Toshimori K, Behav iou r of a sperm surface transmembrane
glycoprotein basigin during ep ididymal matu ra tion and its
role in fertili zation. Rep rodll ct ioll , 123 (2002) 435,
Si Y & Okuno M . The sl iding of the fibrous shea th throu gh
the axoneme proximally together with mi crotu bul e ex tru sion,
Ex/' Cell Res. 208 ( 1993) 170,
M ei X , Si ngh I S, Erli chman J & Orr G A. Clonin g and
characteri za ti on of a test is-spec ific, developmelltall y
regulated A-kinase- anchorin g protein (TAKAP-80) present
on th e fi brous sheath of rat sperm, E/l I' J Biochelll. 246
( 1997) 425.
Westh off 0 & Kamp G.Glyceraldehyde 3-ph osphate
dehydrogenase is bou lld to the fi brous sheath of mammalian
sperm atozoa. J Cell Sci, 110 (1997) 182 1,
M ori C, Nakamura N. Welch J E. Gotoh II, Goulding E H.
Fujioka M & Eddy EM, M ouse sperm atogenic ce ll -spec ific
ty pe I hexokinase (/IlHk I -s) transcript s arc ex pressed by
alt ernati ve spli cing from th e mHk I gene and th e H K I -S
protein is loca li zed main ly in the sperm tail, Mol Reprod
Dev, 49 ( 1998) 374.
M art inez P S. Conesa 0 & Cuasn icu P S. Pote ntial
co ntracep ti ve usc of epidid y mal protei ns: Ev ide nce for the
KHOLE: EPIDIDYMIS AS A TARGET FOR CONTRACEPT!ON
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
part icipat ion of spec ific antibody that is against rat
cpididy mal protein DE in malc and femal e fert ility
inhi bition. J Rep rod 111I1/1U1lol. 29 ( 1995) 3 1.
Olson G E. Lifsics M R, Winfrey V P & Rifkin J M ,
M od ificati on of the rat sperm nagellar Illeillbrane during
Illaturati on in the epididymis. J Alldmlog.\', 8 ( 198-t ) 129.
Hinton B T. Pallad ino M A, Rud olph D, Z i Ji an L & Labu s J
C, The role of the epidid y mi s in the protecti on o f
sperm atoLOa, Cll rr Topics Dev lliol. 33 ( 1996) (i I .
Pollanen P & Cooper T G, Illllllullology of te sti cu lar
excurrent duct, J Reprud 11111/1111/01.26 ( 1994) 207.
Gi lle G & Sigler K, Oxidat i ve st ress and li ving ce ll s. Folia
Micmbiol. 40 ( 1995) IJ I.
Palladin o M A , Mallonga T A & Mishra M S. M essenger
RNA (m l< A) expression for th e antimi crob ial peptides
beta -defen sin- I ;Jnd beta-dcfcnsin-2 in th e III a Ie rat
reproductivc tract: be ta-defensi n- I mR ' A in initial segment
and caput epididymid is is regulated by androgens and not
bacterial lipopol ysacc harides . Bioi Rep rod, 68 (2003) 509.
Sinha H AP & Swerdlof R S. Horillonal and geneti c con trol
of gerlll ce ll apoptosi s in th e testis. ReI' Rep rod. 4 ( 1999) 38.
Weil M , Jacobson M D & Rail M C. Arc cas pa ses involved
in the death of ce ll s with a tran scripti ona lly inac ti ve nucleus'!
Sperm and chi cken ery throcy tes. J Cell Sci. I II ( 1998) 2707.
Yin Y, Stah l B C, DeWol f W C & Morgent aler A. p53 mediated ge rm cell qual ity contro l in spermatogenesis, Del'
Hiol, 204 ( 1998) 165.
Sakkas D. Mariethoz E & St John J C. Ab norma l sperm
parameters in human arc indica ti ve of an abortive apop toti c
Illec hani slll linked to the fas- Illcdiated pathway. E.rp Cell
Res, 25 1 ( 1999) 350.
Fraile B, Martin R, DcMig uel M P. Arcnas M I. Bcthcn cou rt
F R. Pei nado F. Pani agua R & Santa illaria L , Li ght and
electron microscopic immunohistochemica l loca li zation of
prote in gene product 9.5 and Ub iquitin illlmunoreac ti vities in
th e human epididym is and vas defe ren s. Bioi Reprod, 55
( 1996) 29 1.
Santaillaria L. Martin R. Pani agua R, Fraile B. istal M.
Tercn ghi G & Polak J M , Protei n gene product 9.5 and
ubiquitin immunoreactivit ies in rat epididy mi s epitheliu m.
H istochelllis/ry . 100 ( 1993) 13 1.
Lippert T H. Seeger H. Schieferstein G & Voel ter W .
Immunoreactive Ubiqu itin in human seminal plasma.
J Alldrol. ( 1993) 14, 130.
Sutovsky p. Moreno R, Ramalh o-Sa ntos J. Dom in ko T,
Th oillpson W E, & Schallen G A putati ve. ubiquitindependent mechanism for th e recogn itioll alld elimin ation of
defeeli ve Spenl1;.tLOzoa. J Cell Sci. I 14 (200 I ) 1665.
Fichorova R N & Nakov L S. The usc of ELI SA to cvaluate
human antibody binding lO epidid y mal sperm from di fferent
spec ies, Alii J Reprod 1111111111101.29 ( 1993) 109 .
Fichorova R N. Dimitrova E, akov L. Tzvetkov D, Penkov R
& Taskov H. Detecti on of an tibodies towards epididymal sperm
antigens: A n obligatory step in evaluati on of human
immunologic infert ility? AII1 J Reprod 1111111111101, 33 ( 1995) 34 1.
Bl aquier J A, Cameo M S, Stephan y D. Piazza A , Te zon J, &
Sherin s R J. Abnorma l distribution of epidid y mal anti gens on
spermalozoa frolll infert ile men, Fer/if S/eril. 47 ( 1987) 302.
Lunde T . Chri sl ian sen E & Purvi s E. Epididymi s. AnatolllY
fun cti on and pathology, Tidssk r Nor Laegej(JI"CrI. 11 0 ( 1990)
3 1 \ 9.
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
771
l3edford J M & Kim H H. Sperm/egg bi nding pallern s and
oocy te cyto logy in retro~pect i ve analy sis of fertili zation
failure ill vi/ro. HIIIII Rep rod. 8 ( 1993) 453.
Guill emelle C. Thabet M , Dompierre L. & Su lli va n R, Some
v;Jsectom ized men are characterized by low leve ls of P34H.
an epididymal sperlll protein. J Alldrol. 20 1(99) 214 .
Turn er T T. Reley T A, Vagnell i M . Fli ck inger C J. Caldwe ll
J A & Hunt D F. Post vasectomy alterat ions in protein
sy nthesis and sec reti on in the rat caput ep i d i dymidi~ are not
repaired afte r vasectomy. J Alldrol . 2 1 (2000) 276.
Boue F & Su lli van R. Cases of human in fert il ily arc
associated wi th th e absence of P3-t H an epididy mal sperm
anti gen. Bioi Reprod. 54 ( 1996) 101 8.
Parent S, Lelievre L , Brindle Y & Sull i van R. Bul l subfertility is assoc iated with low levels of sperm membrane
anti gen. Mol Reprod Del', 52 ( 1999) 57 .
Poulton T A, Everard D. Baxby K. & Parslow J M.
Characte ri za tion of a sperm Loating alltoantigen react ing
with antisperm antibodies of infertil e males using
monoc lonal an tibodies. Br J Obs/e/ Gyl/ecol . 103 ( 1996) 463.
Sri astav A. Matu ration -dependent gl ycopro tein s conta ining
bo th N-and O-linked oli gosacc haridcs in epididYlllal sperm
pl as ma membrane of rhesus monkeys (Macaw IIIl1lalla) . .I
Reprod Fer/ii, 119 (2000) 24 1.
Hol land M K & Ni xso n B. The spec ifi cit y of epididy mal
secreLOry proteins, 1111 J Reprod Fertil (S uppI 53). ( 1998 ) 197 .
Kirchhoff C. Molecular characterizat ion of epididymal
protein s. ReI' Rl'prod. 3 ( 1998) 86.
Synt in P, Dacheux F, Druat X. Galli J L. Okaillura N &:
Dacheux J L. Characteri zation and identifi cat ion of proteins
secreted in the varioll s regions of the adult boar epididymi~ ,
Bioi Reprod, 55 ( 1996) 956.
Ensrud K M & Hamilton D W .Use of neonatal tolerizalion
and chemica l immunosupression for producti on of
monoc lonal antibod ies to Illaturation specific sperm surf;Jce
molecule. J AI/drol, 12 ( 199 1) 305 .
Khole V , Joshi S A & Singh S, Identil'icati on of epididymis
specific an ti gen by neonatal LOlerizat ion. Alii J /?eprod
11//11/1/1 /01, 44 (2000) 350.
Go lumbski G S Jr & D iamond R L, The use of toicriz;Jtion in
the producti o:l of monoc lonal antibodies against minor
alll igeni c determinant. Anal IJiochelll, 154 ( 1986) 373.
Hllekri eld S. A monoclonal antibody to a uni que ccrcbeller
neuron generaled by
im munosupn.:ss ion and rap id
imll1unization. Sciel/ ce, 237 ( 1987) 67.
Ou S k, M cDonald C & Patt erson P H. Co mpari son of two
techniques for targetin g the produelion of monoc lonal
antibodi es aga inst parti cular antigens, J 111111/1/1101 MeillUds.
145 ( 199 1) I I I.
William s C V , Stech man C L , & M cLoon S C, Subtracti ve
imm unization tec hniq ue for the production of 1110noclonal
antibod ies to rare anti gens. BiOiechl/iqlles. 12 ( 1992) 842.
Imam S K. Esteban E F, & Young L L , Generation ofa murine
monoc lonal antibod ies to normal mam mary epitheli um usi ng
mice rendered iml11unotolerant to mali gnant l11al11mary
epitheliul11. J HislOchelll Cytochelll . 42 ( 1994) 585.
L ebron J A. Shen H, Bj orkman P & Ous J, Tolcri zation of
ael ult mice to iml11unodominanl protein before 1110noe lonal
anti body prod uction, J 111111111/101 Me/hods . 222 ( 1999) 59 .
Sieister H M & Rao G A.S trategies to generate anti bodies
capab le of di stingui shing between protein s with > 90%
am ino acid identit y . .Illlllllll/w l M e/hods. 252 (2Q(1I ) 12 1.
INDI AN J EXP SIOL. J ULY 2003
772
65
66
f,7
68
f,9
7()
71
72
73
Jos hi S A. Shaikh S. Ranpura S & Kh o le V V. Postnatal
deve lop ment and testos tero ne de pe nde nce or an e pididymal
pro tein identifi ed by nco natalt olc ri za ti on. Repmdllcl ioll. 125
(2003a) 495.
Fli ck inge r C J. SYlllhcs is. transport and secrcti on or protc in
in thc initi a l seg ment or thc mousc epi d id ymis as stu d ied hy
electron microsco pc radioa ut og raph y. Bioi Reflrod. 20 ( 11)79)
1015.
Fli ck ingc l' C J. Rcg io nal difTc rences In sy nth c, is.
int racel lul ar transport and scc reti on of protc in in mouse
epiuidym is. Bioi Reflrod. 25 ( 198 I) R71 .
I-I enll ll L. Barin K & Ok o R. And rogc n bind ing protein
secrct io n and end ocy tos is by princ ipal cc ll s in th c auul t ra t
cpididy mi s and duri ng pos tn atal dc:vcloplllcnl. J /llltlml. 19
( 199X ) 527.
Legarc C. Gaudrea ult C. St-JaC(IU CS S & Sulli va n R. P341-1
' perm pro tein is preferentiall y ex pressc u by thc hum an
corpu: epi di dy mi dis . Elillouill (}log\'. 1-10 ( 1999 ) 33 18.
Jaiswal B S & liaju mdc r G C. Biochcmical para mctcrs
rcg ul atin g rorward motil ity initi ~lti o n ill I'il ro in go t ill1lllature
cpi d id ymal spennat ozoa . Neflm d Ferri l /) e l '. 10 ( 1998) 299 .
Smit hwick E 13 & Youn g L G. Immuno his toc hcmical
local izat ion of epi didy mal sccrctory g lycop rotc in EP I in th c
adu lt malc chimpanzec. Tiss lle Cell Res. 3 ( 1999) 154.
Phel ps 13 M & Myles D G. The g uin c~ 1 pi g sperm pl as ma
me lllhrane protein. P1-I 20. rcaches th e su rface via two
tran.,port pa th ways and hecomcs localized to a do mai n afte r
an in itia l uniform di ,t ri bllli on. /)cl ' Bioi. 12:1 ( 1987) 63.
AnaKawc 0 O. Sharm a S. I-I o ll 1-1 13. Haru v D & Ge rt on G I.
Matura ti on of gui nea pig spcrm in the ep idi dy mi s in vo lves
the nll)d ificat ion of pruaeros in o li gosacc hari dc side chai ns.
Mol Rt'f1ro D CI'. 29 ( 199 1) 294.
n
Setc hdl 13 p. Maddocks S & Brooks D E. Anato lll Y.
vasculatu re. innervat ion. and riuids or thc male rep rodu cti ve
75
76
77
71\
tract. in The Phvsio logy o/ Repmdllclioll Edited by E Kn ob il
& JD cill (RJve n Press. Ne w York ) 1994. 1063 .
Mahadeva n M M & T ro unso n A O. Remova l o r th e cumu lus
oo phorus rrom th e human oocy tc 1'0 1' in vi tro fertili za tio n.
Ferril Sleril. 43( 19)15) 263 .
Pryor J P. Surgica l re trievJ I of cpi didymal spcnna to,wa
Lallcel. 2 ( 1987) 134 1.
Fogdes talll I. Fall M & Nil sso n S. Microsurgical
epididYlllovasos to lll Y in th c treatlllcnt of occ lusive
al.Oos pcrlni a. Fen il Sl eril. 46 ( 19X6) 925.
Schoys illall R J & l3edrord J M. The role or th e hU Ill:.tn
epididYllli s in sperm maturati on and sperlll storage as
rc lkcted in th e co nseque nces of epidi dy movasostomy. Fen il
Sll'I'il. 46 ( 1986 ) 2"J3.
79
Si lbc r S J. Role or epi didY llli , in sperm Illaturation. UroloK.I'.
:13 ( 19X9) 47 .
80
XI
Be llve A R & Moss S B. Mon()clo na l :lI1ti bodi es as probc or
re product ivc Mecha ni, m. Bioi Reprod. 28 ( 19X3) I.
Jos hi S A. Ranp ura S. Shaikh S & Khole \' V. Monoc lonal
anti bodi es to epidi dy mi s , pcc ifi c prote ins using Illice
re nd ered immune lO il:rant to tes ti cular pro tei n, . J AI/dml. 24
(20()3 b).
82
Hcgde U C. Premchand ran S. Fern andcs J [ & Kh ole V.
Imillunoc hc mica l c haracteri zati on and antifertilit y cfrcct or
lllatu l',lt io n anti gc ns or sperillat ozoa. in ClIrrt'1lI COl/ celilS ill
Fer/ ili lv l? e~ lI la l i(J/l (/ l/rI /?l'l' rodllcl iol/. edited by C P Puri c'
P F A Van Lllllk (Wil ey Easte rn Li mitcu. NelV Delh i. India )
1994.237.R2.
1\3
Bronson R. Illl lll uno log ic inrcrtilit y. in 114({I/({gelll (,111 of
illlpOlel/ce al/d il{/(' n ililv. edit ed by Whit chead E D and
ogler 1-1 M. (Harper and Row). 1994. 390.
X4 Coope r T G. Waites G M 1-1 & Niesc hla g E. T he epi did ym is
and male fertil ity. A SY lllpos ium report. 111 1 J ;I mimi, l)
( 1986) XI .