Indian Journal of Experimental Biology Vol. 4 1, July 2003, pp. 764-772

Epididymis as a target for contraception

Vrinda Khole

Gamete Immunobiology Department. National Institute for Researeh in Reproductive Hea lth. Mumbai. 400 0 12, India

Advantage of using a vacc ine based on sperm antigens is that it can be used both in males and females as individuals who have anti spenn anti bod ies are llsuall y infertile bu t otherw ise hea lthy. Several sperm spec ifi c anti gens ident ified as prospecti ve candidates for immunocontracepti on are of tes ticular ori gin . For the purpose of immunocontracepti on i t may be desirable not to disrupt spermatogenes is and tes ti cular functi on. Concept of post testi cul ar maturation of sperillatozoa has been very we ll establi shed. During post testicular voyage spermatozoa undergo a seri es of complex and sequential events which transforms thc immature immotile spermatozoa into mature sperm. Acqu isi ti on of functional maturity is necessary for progressive motilit y, zona pellucida recogniti on culminat ing in sperm egg binding. Importance of epididymal matura ti on is highlighted by the fact that high percentage of male in fertilit y in human origi ates from the mal func ti on of the epid idymi s. The epididymis has also shown to be involved in sperm storage and provides an adequate environment for fina l maturation of the sperm. It provides a conducive microenvironmeJ1l by virtue of which the spermatozoa arc protected during the storage. In view of thi s it is imperative that more allention needs to be foc used on epididymal antigens. The informati on obtained will enable us to ide J1l ify epididymal antigens releva J1l 10 fertilit y and also help in in fertilit y diagnos is.

Keywords : Contraception, Epididymis. Epididymal antigens, Infertilit y diagnosis, Sperm malUration

There is an urgent need to prov ide safe and sustained effecti vc ferti lity control for the world popul ati on t. More and improved contracepti ve opti ons is the need of the hour. This is espec ially relevant to males who currentl y have onl y four limited options: abstinence, withdrawa l, condoms and vasectomy. Of these vasectomy is essenti ally irreversible and is not the preferred method for men who wi sh to father a chi ld at a later date. Although the results from studies in volving hormonal methods for male contraception are convincing2 it has not yet yielded a marketabl e product. Vaccine approach for contraception would definitcly be a va luab le addition to the ex isting armamentarium of different approaches used for fa mily 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 the need for additional methods for contraception National Institute for Child Health and Human Development (N ICHD) convened a workshop to identify novel strategies involving testicul ar and epididymal antigens for developing a male contraceptive in the 2 1 s l centurl.

* For correspondence : E-mail : vrindakhole@ hotmai1. colll Fax: 9 1-22-241 394 12

Testis as a target for contraception Tes ti s is the primary organ which produces

spermatozoa and, therefore, has been considered to be a good target for contraception. It is indicated that thi s could be done by direct interference at various stages of spermatogenesis such as I) interference wi th meios is and particularly the ce ll cycle check points 2) di sruption of RNA protein interacti on (post transcription::l l control) 3) di sruption of juncti onal complexes between sertoli ce ll s and germ 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 them have also been cloned and sequenced . The current statu s, application , relati ve merits and immunogenicity of these antigens has been extensively reviewed3

.

Epididymis as a target for contraception Post tes ticular contraception cou ld be achieved by

interfering pharmacologica lly or immunologicall y with the process of sperm maturation in the epididymis . Maturational changes which the sperm undergoes during epididymal transit have been shown to be prerequisites for successful fertili zation. It is suggested that theoretically these may be interrupted at different sites. It is poss ible that modifying the pattern of epididymally secreted proteins may change the optimal environment for maturation and storage of spenn4

.

KH OLE: EPIDIDYMI S AS A TA RGET FOR CONTRACEPTION 765

Advantages of epididymaL antigells for contraception Vacc in ati on of males for contraception has not

been possible beyond pre clinica l levels for a va riety of concerns. Testi s is an immunologicall y pri vileged site because of the blood testi s barrier. It may not be advi sable to immunize men with tes ticular sperm anti gens as it may lead to irreversible testi cul ar damage. It is suggested that anti bodies raised against antigens present exclusively post testi cul arly may be less likely to induce autoimmune orchiti s5

. There are defi nite ad vantages in targeting the epididymis fo r contraception. Firstly the onset of in fertility and also its reversa l has been shown to be far qui cker6 than any agent attacking the testi cular producti on of spermatozoa and secondly, as maturing cell s are targeted, damage to the geneti c materi al, a possible sequelae of effect on di viding germ cells is avoided7

.

It would also avoid endocrine impairment of li bid02 .

In order to pursue the epididymis as a target for contraception there is a need to understand the di ffe rent roles pl ayed by epididymal proteins in sperm maturation, and sperm protecti on during storage. The new technolog ies such as cD A arrays and proteomics. will help us identi fy the va rious proteins in vo lved in sperm maturati on immunolog ica l protec ti on as well as those possess ing antimicrobial ac ti vity whi ch help in sperm protec ti on. Once such molecules are identi fied we will be ab le to understand the importance and co mpos ition of the epididymal mil eu which pl ays a major role in acqui siti on of fe rtility, motility.

RoLe of epididymis in sperm maturation Epididymi s was earlier thought to be just a pass ive

chan nel through which spermatozoa travel to be stored before being ejacul ated. The concept of post testicul ar maturation in mammali an spermatozoa evolved as a result of the pioneering studies of Benoit8 and Young9

. But it was Bedfo rd 1o and Orgebin Cri st ll who initi ated in depth inves ti gati ons and demonstrated that, sperm whi ch were prevented from passing along the epididymal duct were viable but did not acquire full fe rtili zing capacity . Large amount of information has become ava il abl e after 1960, about the structure, biochemical properti es and functi ons of thi s organ. ]t has been shown that in the epididymi s there is complex ity in the cellul ar propert ies, heterogenei ty, region speci fic ex pression and . patial and temporal organization of proteins. All these complexities make this organ very dynami c. It is

now clear that the maturati on of spermatozoa and the acqui siti on of motility and fe rtili zing ability do not result fro m a pass ive journey of spermatozoa but rather as a result of exposure to and acti ve interac ti on with the luminal contents of di fferent epididymal

. I ? regions -

Membrane remodeLLillg During its epididymal sojourn the sperm plasma

membrane undergoes intense changes both in protein composition and loca li zati on on the gamete. The sperm plas ma membrane is ori ginall y deri ved from spermatogoni a/spermalocytes . in the testi s but undergoes ex tensi ve remodeling duri ng spermiogenesis in the testi s, maturati on in the epididymis and capacitation in the female genital tract. The epididymal maturational events take pl ace due to the microenvironment fo rmed by the luminal contents of the epididymal duct l3 . Remodeling of the membrane could be brought about either by uptake of sec reted epididymal proteins, or processing of ex isting or acq uired proteins l4 . Process ing of existing or acquired proteins occurs as a result of some glycan modi fy ing enzy mes such as glycosidases and glycosy ltransferasesl 5 or protein modifying agents such as endo-proteases and protease inhi bitors 16 that are found freely in the luminal fluid or are associa ted with sperm plasma membrane. A deficiency of these enzy mes has been shown to be responsibl e for male in fe rtilit / 7

. These remodeling mechani sms lead to acqui siti on of spec ifi c functi ons by different domains of the spermatozoa during epididymal transit.

Repositioning of protein co mponents to membrane domains occurs as ~een in fa mil y of protein s such as ADAM, (proteins with a di sintegrin and metall oproteinase like domain ) fo r example fertilin(PH20)l s.IY or membrane proteins such as MDC (proteins with a Metalloproteinase, a di sintegrin -like and a cysteine-rich domain) for example 2B ( 0.21 .

Relocation of proteins can be seen across di ffe rent domains in proteins which belong LO immunoglobulin super family fo r exampl e, CE9 where the shift is from principal piece to midpiece22

.

Every domain of the sperm is endowed with a spec ific fun ction. Head of sperm compri ses of acroso me and post acrosomal region. Acrosome is in volved in primary binding to egg pl asma membrane. Equatori al/post ac rosomal region is in volved in secondary binding leading to fusion23

. The fl agel lu m is di vided into midpiece, principal pi ece and end piece. Midpiece is associated with genera ti on of

766 INDIAN JEXP BIOL. JULY 200]

energy (ATP). principal piece which contains the axoneme and f ibrous sheath is responsible for flage llar fl exibility and Il1otilit/~ Proteins of the fibrous shea th are impli cated in signaling pathway in

~' . I' I I' I 1(, 17 P . spermalozo~c· ane In g yco ytl c patl\vay- '- . ro teln DE which has been loca li zed in the acrosOIl1e region is shown to have a role in sperm egg fusio n2x. A 26h:Da protein localized in the flage lla has been shown to have a role in motilit / 9

Protective role oJflie epididymis Epid idym is plays a very importan t role In

protect ion of spermat ozoa but is the least studi ed aspect. Spermatozoa spe lld Il1any days tra versing the long epid idymal duct and are faced w ith a constant ly changing microenvironment. Ep id idymi s prov ides conduci ve microenvi ronment by rapid I y el i mi nat i ng the harmful metabo l ic by products·'o. I t also protec ts spermat ozoa by blood ep ididym is barri er pro vided by tight juncti ons between the principal ce l ls. T his barrier not onl y pro tec ts the spermat ozoa from ex ternal non conduci ve en vi ron ment but also prevents the access to the immune system'l . Thi s barrier is se lec tive and does not all ow passage of hi gh molecul ar weight compounds lih:e L -gl ucose. insu lin , BSA bu t readi ly allows the passage of water. 0 -glucose an d amino acids showing th at the hlood ep id idymi s harri er helps in the Il1ain tenance of conducive microenvironment for the spermatozoa.

Epididymi s has also been shO'vv n to playa role in protect ion of spermatozoa from free radi ca ls and recognition and eli mination of defective spermatozoa. Epid idymal spermato7.0a are ex tremely vulnerable to ox idati ve stress. To overcome thi s probl em epid idym is has a ri ch source o f an anti ox idant enzyme that scavenges any excess reacti ve oxygen metabo lite released by the spermatozoa during epididyma l tran sit I!, . Th e- caput epididymi s also secretes glutathione perox idase which gets intimately assoc iated w ith the sperm su rface where it serves to remove any H20 ] genereted by these ce ll s as a consequence of SOD acti on . M olecu les such as glutathione perox idase, ca talase, superox ide dislllu tase have been shown to protect sperm in the epididym is rronl damage due to reacti ve oxygen spec iesJ2

. Studi es on expression o f Il1R A for 13 -Defensin - I and 2 in the initial segment and caput ep ididymis suggest a ro le for the epididymi s in an timi crobial protecti onJ

·1

. Quality con trol of male gametes is ensured by an act i ve apoptotic pathway present in s pe rll1ato~enic lineage'~ and in mature

. 1, 16 ~ 17 sperm o f mice·· ·· and mell" .

Though ubiqui tin was prev iously detec ted in I 'd'd I II 'xW . ~o . luman epl I yma ce s··· and sem ll1 al plasma , Its importance in quality control of fertili ty was not indicated. A lthough both normal and defective sperm carry constitut ively ubiquitinated substrates it is onl y the defec ti ve ones that get surface ubiquitinated du ring epididymal passage. Sutovsky and coworker. ~I have shown surface ubiqu i tinat ion of sperm and subsequent phagocy tos is by epididymal epitheli al ce l ls.

Role oj epididymal proteills ill reproductioll There are several direc t and indirect ev idences

which underline the importance of ep ididymal proteins in reproducti on. umber of cli nical ev idcnces show the correlation between abnormaliti cs or disturbances in the epididymal secreti ons and in re rt ilit/]· -~~· . In fact high percentage of male in fert ility in human is believed to ori ginate from the malfuncti on of the epididymi s~ 5 . Incomplete sperm maturati on w ithi n the ep ididy mi s has been suggested to be the cause of tota l fa ilure of sperm binding to zOlla in unsuccess l"ul human ill I'il ro fe rtili zation cases~6 . Vasectomy also has shown to cause irreversible damage to epididymis and is thought to be one of the causes of infertilit y even after vasovasos tomy"7.4X. Human P34H prote i n wh ieh is in vo l ved in sperm-zona pe llucida interact ion is found to be reduced in certain cases o f idi opathi c in fe rtilit y"9. P2Sb and P2Ib proteins (bull homologues of human P34 H) are associated w ith sub-fertility and their express ion is marh:edly low in subfen ile bul1 5o. It is specu lated th at epididymal antigens play a predominant role in several cases of hu man in krti lit /I . A ll these ev idences point to the important ro le of the epididy mis in bestowing on the spe rm motility and fertili zing abi lity. In view 01" these data it is clear that epididymal proteins have a vital role in maturatioll of spermatozoa. lead ing to success ful reproducti on and, therefore, more attention needs to be focused on identi f ica ti on of epididymal antigens.

Several epididymal protei ns have been ident i fi ed and studi ed for their contribution towards sperm maturation. However. many more still remain to be identified and their functions need to be ascertained. Identi f ica tion of newer epididymal proteins and annonating their functi on will help in understanding the mechanism of sperm maturati on and the sequence of events therein. Thi s will further help in se lecting epididymal targets ror contracepti on w hich will spec i fica ll y alter the ability of the sperm to fertili ze

KHOLE: EPIDIDYMIS AS A TA RG ET FOR CONTRACEPTI ON 767

without any side effects. Hence understanding the epididymi s in more detail s will help in treating certain types of male infertility and also to develop male contracepti ve agents.

Different approaches have been exploited for identification of epididymal proteins such as use of lectin s52

, . ubtracti ve screening of the epididymal cDNA librar/ 3

, use of the ex pressed sequence tag54, . 'is d I I ' . 56 57 N I proteomlcs' an neonata to enzatJon ' eonata

tolerization is a powerful tool for raising monoclonal antibodies to rare or weakly immunogenic anti gens. This approach has been used by several inves ti gators for generating monoc lonal antibodies to rare or less immunogen ic anti gens58

.64 In thi s approach once a

state of tolerance to an anti gen is establ ished , the tolerized animals could be subsequently immunized with a crude preparation of the potential anti gen (i mmunogen). By inducing the immune tolerance to the tolerogen, the immune system will generate an immune response to onl y those epitopes not included in the tolerogen preparati on. This approach increases the probability of obtaining antibodies to functionally significant components that may be weak immunogens.

Identificat ion of ep ididymal proteins by conven­tional immun ization followed by hybridoma techno­logy has not been very successful , probably because the testicular proteins are more immunogeni c than the epididymal proteins. Therefore, the alternate method of, "Neonatal toleri zati on" also called as subtracti ve immunizati on first reported in 199 156 was ex ploited by us with slight modifications. Using thi s method we, were able to generate an immune response to epididymal anti gens in BALBc mi ce57 . In our protocol (Fig la) animals were toleri zed to testi cul ar protein (tolerogen) at birth and were then immunized wi th epididymal sperm protein (immunogen) for raising specific immune response. These studies demonstrated that neonata l tolerizati on with test icular proteins fo llowed by immunization with epididymal sperm proteins enh anced the production of antibodies to epididymal proteins. Serum from these animals locali zed proteins onl y in corpus epithelium and sperm from corpus and cauda ep ididymis but showed no reactivit y with tes ti s (Fig. I b) . Us ing sera from these neonata ll y tolerized and immunized mice, we identified a dominant epididymis specific protein of molecular weight approx imately 27 kDa which was used to raise polyc lonal antibodies in rabbit. The polyclonal ant ibody was very spec ific to epididymis as seen by ELIS A, Western blot and IHe. The antibody iden tifi ed a 27 kDa protein fro m corpus

epididymis as well as sperm from corpus and cauda region . The protein identified was present on the midpiece as seen by indirect immunofluorescence and was androgen regulated and was also developmenta lly regul ated65. These results indicated that thi s protein is secreted mainly in the corpus and acq uired by the spermatozoa during passage through these regions. The presence of the protei n in corpus and cauda may be due to its secretion by the principal cell s of the corpus. The principal ce ll s of the corpus have a well developed endoplasm ic reticulum and golgi apparatus, which indicates th at they are in volved in active protein sy nthesis. There are some reports where principal cells of epididymis have geen shown to incorporate labeled Hmino ac ids and transport radiolabelled molecules through the ce Il 66.li7. Principal ce ll s have been shown to be act ive ly in volved in the physiologica l fu nctions of the epididym is, involv ing endocytosis68 and secretionli9. It is reported that proteins secreted by principal ce ll s may interact with spermatozoa in the lumen of epid idymal duct and enable spermatozoa to develop motilit/ o and fertilit/ I

. It has been shown that corpus/cauda juncti on pl ays a major role in sperm maturat ion72-7.J. Fertilization rate is show n to be higher in spermatozoa from corpus than from caput as seen in in -v it ro fertilization 75.76. Sperm motility and preg nancy rates were also fo und to be significantly improved when the vas was surgicall y joined to the corpus in those pati ents who received specifi c tubule vasoepididymo-

f b · . 77·7~ S' stomy or 0 structJ ve azoospermia . II1ce spermatozoa become motile and fe rtile in the corpus and cauda 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 identifications of sperm antigens (Fig. 2). Monoclonal antibodi es provide a powerful analytical tool, allowing recognition of individual determinants in a co mplex antigenic structure and have becn applied to studi es in reproducti ve biolog/lJ

. Sera from infertile male and femal e or vasectomized 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 epididymal origin and suggested that autoimmune infertility mi ght represent a response to the epididymal rather than tes ti cular sperm. He further suggested that monoclonal anti bodi es ra ised to such unique and immunologica ll y access ible sperm coating an tigens in

768 INDIAN J EXP BIOL. JUL Y 2003

the epididymis rather than in the testi s would seem to preseIll a theoreti ca l solution to male inferti lity.

We generated large number of hybridomas, using Balbc mice which were toleri zed to testicular proteins and immuni zed w ith epididymal sperm prote ins57

.

Majority of clones showed high reactivity w i th epididymal sperm prote in while a small number were fou nd to react with tes ticular sperm proteinsi . Thi s ind icated that neonates were successfull y tol eri zed to testi cular antigen and mounted immune response to epididymal proteins. Immunonuoresccnt loca li za ti on

using polyclonal serum from the T I mouse used for fus ion loca li zed antigens in different reg ions of the sperm such as acrosome. post acrosome, equator, midpiece and tail. Th is indicated that epididymal proteins are located on different reg ions of the sperm and are li kely to play domain speci fic ro les such as sperm-egg interaction , acrosome react ion and motility which are essenti al for fert ili za tion . It was interesting to note th at the pattern of II F locali zati on was seen to be identical in both glu tera ldehyde fixed spermatozoa smcared on glass slide as well as spermatozoa in

Neonatal Tolerization-Immunization Protocol

Mice injected with tolerogen (testicular protein) Day 0

Mice injected with tolerogen (testicular protein) 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 rur lIeonatal to leriza tioll ami illlilluni za ti on

Testis Corpus Cauda

Fig. 1 (b) - llllllluliohi stoc hellli ca l locJ li zat ion of amigens usin g serum from neonat all y toleri zed imilluni zed mouse. Test icular sect ion shows no stai ning. The corpus scction shows localization in the supranuclear region of the epitheliulll and on sperillatozoa. 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: EPIDIDYMIS AS A TARGET FOR CONTRACEPTION 769

Fig. 2 - lrnrnunochemical and funct ional characteri zat ion of antigen using monoclonal alllibod ies. al-;)4 Representati ve pictu rc of immunohistochemica l loca li zation in differclll regions of cpididymis using a monoc lonal antibody. (al ) Proximal caput shows no staining. (a2) Distal caput shows sorne loca li za ti on. (a3) Corpus epi thelium shows strong localizati on. Spermatozoa in thc lumen arc also st ained. (a4) Cauda cpid idymal rcgion shows localizat ion on ci li ary lining and spermatozoa. b J -b3: lmmunolluorcscent locali zat ion of antigcns on spermatozoa using diffcrent monoclonal ant ibodies. (b I ) M ab V2C4E2 stains acrosoma l region. (b2) M ab V3C8, V3F4F4 stain post acrosomal and equatori al region. (b3) M ab V I B8E 10. V3C I 0 stain rnid piece rcgion c l -c2: Agglutinat ion pallern using differclll mAbs. (c I ) Radial pallcrn of agg lutination sccn wi th mAb V2C4E2, V3C8. V3F4F4. (c2) Cornct shape pallcrn of agglutination secn with mAb V I B8E I O. V3C I O.

suspenSion. This observation along with the agglutination pattern indicated that the proteins identified by the monoclonal antibodies are on the surface of sperm. Surface locali zation of sperm antigens is one of the criteria for ideal contraceptive targets.

Earlier studies from our laborator/ 2 have shown that 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 female mi ce, the antibody showed a dose dependent reduction in the antifertility effect and was found to be effective on ly before fertilization . Whereas, in case of male mice, the antibody was able to enter the epididymi s within 24 hI'

and inhibit sperm maturation and sperm fun ction. Fertility was reduced to 100% after about 5 days of antibody administration. Histologically, the effect was partially reversed after a week and completely reversed after two weeks. These studies clearl y indicated that where spermatogenesis is unaffected , the recovery of fertility was faster.

Advantage of using a vaccine based on sperm ant igen is that it can be successfully used both in males and females, as indi viduals who have antisperm antibodies are usuall y infertile but otherwise healthl 3

. Of these ant igens the epid idymal anti gens make better candidates for imIl1unocontraception as they inhibit onl y post testi cular maturation of spermatozoa, mak ing them infertil e without affecting

770 INDIAN J EXP BIOL, JULY 2003

testi cul ar function8~. It is suggested that antigens that do not cause testicular pathology but can access the epididymal lumen and eliminate the spermatozoa or impede their function , with potential reversible antifertility effect would be worth y of consideration for immunocontraception.

Post 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 function. This would be poss ible on ly if we identify target molecules . Once thi s is achieved these molecules could then 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 contributing to some of the studies described in this review.

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