Indian Journal of Experimental Biology Vol. 37, July 1999, pp. 655-661

Identification of bovine sperm specific polypeptides reactive with antisperm antibodies

o Tripathi, N C Sharma*, S K Singh & Lavleen K Gupta Division of Animal Reproduction, Indian Veterinary Research Institute (IVRI). Izatnagar 243 122. India

Received 15 April 1998: revised 16 March 1999

The present study was taken to characterize molecular weights of sperm specific polypeptides antigenic to rabbits and calf with the aim to assess their immunoreactivity with IgG antihodies in sera from immuno-infertilc Co\\ s.Seropositivity fa r ant isperm IgG antibodies in 75 repeat breeder and 15 pregnant contro l cat1 le was tested by cellular EI.ISA using washed spermatozoa antigen from 4 bulls. Molecular weights of bovine sperm polypeptides antigenic to rabbit and calf wcre determined by 10% SDS-PAGE and Western blotting. Molecular weights of sperm peptides reactive wi th sera from immuno-infCrtile cows were also determined. Seropositivity of antisperm IgG antibodies for bull I. II. III and IV was 23 .6. 14.6. 26.6 and 20%. respectively. /I. total of 16 polypeptides were discernible on gel. Out of these. 7 polypeptides were immunoreactive with sera from hyperimlllunized rabbits as compared to 3 poly-peptides which reacted with sera frolll hyper-imlllunized calf. On ly t\\'o polypeptides were reactive wi th sera from immuno-inferti le cows. Variable number of spenn polypeptides and their imllluno-reactivity have been reported in different species. Antigenicity of ditlerent polypeptides in sperlll needs further investigat ions.

Bovine infertility has been associated with presence of antisperm antibodies which impaire the physiological processess of reproduction . The relati ve importance of anti sperrn immunity as a cause of unexplained infert ility in bov ines is largely unknown I . The high circulatory leve ls of antisperm antibodies have been positively correlated with unexpla ined i'nferti I ity in artificially insem inated infertile cows2 Bovine sperm antigens are capable of eliciting the production of antisperm antibodies. Identification and characterization of these antigens are important for (a) understanding the mechanism in volved in antibody mediated impairment of reproduction ; (b) to develop reliable diagnost ics; and (c) to prevent and treat the cases of unexplained immuno-infertility. Extensive studies have been performed in human sperm specific antigens and associated consequencesJ.4 . Such studies are rather sketchy in farm animals l .

Auto and isoantibodies directed against the surface of spermatozoa may cause infertility through immobi-lization by agglutination or complement fixation or inhibition of cervical mucus penetration4-6 . These antibodies have been conventionally detected by agglutination and prec ipitation techniques which are comparati ve ly less sens iti ve and show considerable degree of cross reactivi ty. The use of ELISA has

Correspontknt mithor

increased the sensitivity and reproducibility of th e diagnosis l .7. Antigens spec iti c to spermatozoa ",ere reported to show genetic polymorphi sm8.9 . Some of them were identica l between the species 10 while in some species, two genetically distinct population of spermatozoa having different antigenic characteristics have been reported II . This polymorphism is rel ated to ge net ic configurati on and may be utilized for prevention of immuno-infertility by changing anti -genically related bull s. Accurate knowledge of these factors may explore the better understand ing of immuno-infertility mechatiism and could great ly aid in developing preventi ve procedures.

Molecular characterization through intervention of electrophoretic separation of sperm polypeptides by SOS-PAGE and detection of their immuno-reactivit\ to hyperimmune sera as well as sera from immuno-infert ile individual may give more deeper in sights. Similar approaches have been made in htlman 11. 1.1 and in buffalo l4 .

In the present study presence of circu l ator~ anti sperm IgG antibod ies as a cause of unexp lained-infertility has been assessed in artific ially insemi-nated repeat breeder cows. Bovine sperm atozoal polypeptides are also characterized by SDS-PACi I: and immunoblotti ng with homologtls/hetero logus hyperi mmune sera and wi th sera frolll immuno-infertil e cows.

656 INDIAN 1. EXP. BIOL., JULY 1999

Materials and Methods Collection of serum samples~erum samples were

collected from cross-bred (Holstein Friesianx Sahiwal) cattle maintained in organised dairy herds under standard conditions of husbandry in Bareilly, U.P., India. Repeat breeder cows (75), 3-8 years old,who failed to become pregnant in 3 or more subsequent inseminations, were examined per rectally and those which appeared normal, were included in this study. Samples were also collected from pregnant (2-4 months) cows (15) and heifers (5) of 18-24 months of age.

Preparation' of spermatozoa antigen-Semen from 4 fertile crossbred (H.F. xSahiwal) bulls was collected by means of artificial vagina. Individual ej aculate containing more than 1 x 109 spennlml and > 70% motility was centrifuged at 50015 for 20 min. at 20C to separate seminal plasma. Spermatozoal pellets were washed four times with phosphate buffer saline (PBS, pH 7.4) by resuspending and centrifuging at 500 g for 20min. Finally prepared spermatozoa were suspended in PBS (containing 1 mM of PMSF and I: 10000 of thiomersal) and concentration was adjusted to 1 x 108 sperm/ml and stored at -80C till further use.

Immunization of rabbits and calf-Female rabbits (4) of Newzealand white strain, 4 months old were immunized by inoculating emulsion containing Freund's complete adjuvants (FCA; Sigma) and I x I 08 spermatozoa. One rabbit was kept as control. Three subsequent boosters were given in same manner at IS days interval with Freund's incomplete adjuvant (FIA).and I x 108 sperm. Blood was collected from all rabbits after one week of last inoculation and separated sera were stored at -80C.

Sperm antisera was also raised in calf against pooled spermatozoa from 4 bulls. The procedure was same as in rabbits except number of spermatozoa which in this case was I x l 09 spermatozoa.

Standardization of cellular ELISA for detection of antisperm antibodies-Different serum dilutions (I :25, I :50, I : 1 00, 1 :200) from known positive (hyper immune calf antibovine sperm sera) and known negative (pooled heifer sera) were analyzed. The best diffferential optical density (0.0.) was obtained at I :50 dilution. To decide seropositivity in test sera, the cut off value of 0.0. was decided as per Cuzppon l5 I.e. posltave if O.O.is>mean 0.0. + 2xSO offertile group.

Detection of antis perm antibodies by cellular ELISA (cELISA)---Detection of IgG class of antibodies was done in serum samples from repeat breeder, pregnant control, heifers, hyperimmunized rabbits and calf as per Lander et all. Washed spermatozoa antigen was diluted in PBS (PH 7.4) to make the dilution of 1 x 106 cells/ml. Hundred III of this was coated in wells of microtitre ELISA plates (Tarson, India). Plates were kept at 20C overnight infront of air blower. Once the wells were completely dried, remaining sites were completely blocked by filling whole wells with 5% skimmed milk in PBS for overnight at 4C.Assay plates were washed thrlice with PBS (PH7.4) containing 0.05% (v/v) Tween-20 (PBS-T) between all incubation steps. After washing with ~BS-T, 100 III test sera (1.50 dilute in PBS-T) in triplicate was added in all wells and then plates were incubated at 37C for 2 hr in humified chamber. Following washing and filling the wells with 100 Il of I: I 0,000 di luted rabbit anti bovine 1 gG-HRPO conjugate ( II OK; Nil, India), plates were again incubated for I hr . After washing 100 Il of substrate solution (0.5 mg orthophenylene diamine/ml of citrate buffer) was added to each well. The enzyme action was stopped by adding 100 III of 5 N H2S04 after 20 min. Absorbance of each well was measured at 450 nm using an anthos Labtec ELISA reader..

Detection of sperm specific polypeptides by SDS-PADE-One dimentional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE) was performed as per Hansen and Bjerrum lO with slight ~odification . Briefly, stacking gels composed of 4% .acrylamide and resolving gels containing 10% acrylamide were run in a vertiCal slab gel unit (Atto, Japan). Spermatozoa protein was obtained by suspending washed spermatozoa in Tris-HCK (6.25 mM) buffer (PH 6.8) containing 2% SOS 10% glycerol, 5%

f3-mercaptoethanol and 0.1 % bromophenol blue and immersing for 5 min in water bath at 95C. Gels were loaded with 100 III well of sperm protein and run for 8 hr at a constant current of 12 rnA. Molecular weight standard at the range of 14.3 to 66 kDa (Sigma) were run in parallel. Gel was stained with coomassie brilliant Blue R-250 (Sigma).

Detection of humoral responsive polypeptides by immunobloting-Humoral responsive polypeptides were detected as per Kanchev et al. 14 with slight modification. Proteins from gel were electro-

TRIPATHI el 01.: IDENTIFICATION OF BOVINE SPERM SPECIFIC POLYPEPTIDES 657

phoretically transfemed to nitrocellulose paper (NCP) by semi-dried system (Atto, Japan) for 90 min at a constant current of 2 mA/cm2. The transfer buffer was used as per instruction manual of the apparatus. Remaining sites in NCP were blocked by placing it in 5% skimmed milk in Tris-NaCI buffer (50 mM tris-HCI, ISO mMNaCI, pH 10.2 and 0.1 % Tween-20) for 2 hr at 37 C. Strips of NCP with transferred spermatozoal proteins were allowed to react with 1:50 dilute hyper immune sera of rabbit/calf or sera of immuno-infertile repeat breeder for 18 hr at 4C followed by three washings for 4min in Tris-NaCI buffer. The strips were then incubated in 1:500 diluted goat antirabbit I gG-HRPO/rabbit antibovine I gG-HRPO conjugate (10K, Nil , India) for 2 hr at 37C. Strips were again washed 4 times and subjected to substrate solution (Di-amino benzidine solution) for 5 min at room temperature. The reaction was terminated by washing the NCP strips with distilled water.

Results Testing of hyperimune sera for antisperm 1 gG

antibodies-The hyperimmune sera raised in rabbits and calf were tested for presence of antisperm I gG antibodies by cellular-ELISA. A titre of I: 1600 was obtained in rabbit sera but it was only I :400 in calf serum.

30

26.67**

Detection of antisperm 1 gG antibodies in test samples-Detection of I gG antisperm antibodies by cELlSA was carried out in repeat breeder catle (75) pregnant control (15) and heifers (5) using sperm antigens from. 4 bulls. The results pertaining to percentage and frequency of seropositivity of antisperm antibodies are given in Figs I and 2.

Detection of !>perm specific polypeptides---Sperm antigens from 4 bulls were subjected to SDS-PAGE to detect various polypeptides. After staining of gel (Fig. 3) 16 polypeptides' were detectable in all 4 bulls . The number of polypeptides were same but comparative difference was there which has been depicted by difference in colour intensity of diferent peptides among bulls.

fmmunoblotting to detect humoral responsiveness of sperm specific antigens-Rabbit anti bovine sperm sera were analysed against spermatozoal proteins by immunoblotting (Fig. 4). Seven spermatozoal poly-peptides were found immuno-reactive with I gG antibodies in rabbit antibovine sperm sera (Table I ) . These reactions were present in immunoblotting by the sera from all rabbits and sperm antigen from all bulls. Serum from control rabbit did not react with any polypeptide. Calf antibovine sperm sera reacted with 3 polypeptides of spermatozoa. These were of 158.5 , 69.2 and 51.3kD (Fig. 5) . Sera from 4 repeat breeder cows which were found seropositive for ant i

25 2533** rl ----------------------------------~ '. Bull I ~ Bull" 0 Bull III lID Bun IV I

VI

~ E c d Q)

> ..... 'iii 10 o

Q.

5

20**

n.33

o 0 0 0 Re peat breeder Pregnant (control) Heifer(control)

Fig. I-Seropositivity for antisperm antibodies in repeat breeder and control cattle[P:*

658 INDIAN J. EXP. SIOL., JULY 1999

sperm antibodies for all 4 bull s, were also subjected for immuno-reactivity. Two peptides of 69.2 and 51.3 kDa molecular weight reacted invariably with these sera (Fig. 6).

Discussion Spermatozoa contains proteins foreign to female

reproductive tract, therefore, there are chances of initiations of immune response against them. The chances are further increased when insemi nat ions are frequently rcpeated in repeat breeder catt le . Variable occurrence of anti-sperm antibodies in repeat breeder have already been reported . The ana lys is of sera from repeat breeders by cELI SA revea led the variable

100

occurrence of antisperm antibodies (14 .67-26 .67%) Wang l6 studied the relationship between sperm antibody reaction of ELISA and infertility in Chinese black and white dairy cows . He found that 34.5% ')f infertile cows had sperm antibodies in blood serum as compared to 6.7% in non-pregnant cows wi th a history of normal fertility . The present findings in repeat breeder group is s lightly lower in ocurrence which may be due to variation in individ Uial susceptibility and number of inseminations rece ived by animal s. However the present findings are in

. h W 16 I agreement Wit ang 111 pregnant contro group except fo r one bull. This may be due to anti genic sim ilari ty between the bull tested and the bull from

100

1_- 8+ 0++ m+++ ~++++ I 90

80

70

60

~ 50 CJ E c CJ 40

\f) 30 o CL

20

'10

80

o 0 o 0 0 0 Repeat Breede r Pregnant ( con trol) He ifer ( co ntr ol)

Fig. 2- Frequen cy of seroposi ti vity fo r ant ispcrm an ti bodies [- = negati ve for all 4 bull s: positi ve for 1(+). 2(++). 3(+++ ) and 4(++++)

bul ls. IP *

TRlPATHI e/ al.: IDENTIFICATION OF BOVINE SPERM SPECIFIC POLYPEPTIDES 659

(kDa) M I

66-.... 45 -

34_7 -./

24 18- 4 ; 14_3

U III IV (kDa)

~27.8 ~25_4 ~22_6 :.---2L4 ~19_0

~ ~~:~ \ ~ 13_2

\~ 11 7

Fig. 3-{X.5)- SOS-PAGE analysis of sperm protein from hull s. [Lane M = known SOS-PAGE marker: Lanes Ito IV = sperm protein from Bull I to IV].

I II III IV (kDa)

/30_5 " / 25_4 t. /21.4

19_0 17_0 14_1 11.7

Fig. 4--(X .5) - Ilumoral responsiveness of sperm specilic polypcptides to rahbi t anti bovinc sperm sera Lane I to I V seropositi vity of polypeptides from hull I to IV I.

which animal has been served earlier or it may be due to presence of cross-reactive antibodies in serum of

. I . 11 7 partlcu ar al11ma . All heifers tested were negative for antisperm

antibodies. These findings are in agreement with findings of Farahani el al. 18 who reported that agglutination re

660 INDIAN J. EXP. BIOL., JULY 1999

B

'f (kDa)

....... 69.2

51.3

Fig. 5----{X. I) Humoral responsiveness of sperm specific polypeptides to calf anti bovine sperm sera. [Lane PB Seroreactive polypeptide of pooled sperm from 4 bull s].

Indentijication of sperm 5pecijic polypeptides by SDS-PAGE-The present finding in cattle regarding similar number of polypeptides in different bulls is in agreement for same in buffalo II. Variation in intensity of bands among spermatozoa from different bulls may be due to differences at the level of expression for different proteins. Variable numbers of polypeptides have been reported in different species.

Detection of humoral responsiveness of po/ypeptides-Reactivity of 7 polypeptides in bovine spermatozoa have been reported by double immuno-diffusion technique with antisera raised in rabbit21. When human sperm specific polypeptides were tested for immunoreactivity with rabbit antihuman sperm sera by SDSPAGE and immunoblotting, 12 poly-peptids were found immunoreactive, whereas, immuno reactivity of on ly 3 human spermatozoal polypeptides could be antigenically correlated to agglutinating activity from immuno-infertile sera 10. Their observations are also supportive of specIes variation regarding antigenicity of human spermatozoa.

Identification and molecular characterization of

Ie. r ., (kDa)

69.2

51.3

Fig. 6-(X .I) - Humoral responsiveness of sperm speci fic polypeptides to sera from immuno-infertil e repeat breeders. [Lane Ie = Seroreactive polypeptides of pooled sperm from 4 bull s].

sperm specific isoantigens in human beings revealed that 2 polypeptides of 44 and 72 kDa reacted significantly more frequently with serum antibodies from immuno infertile female " . Three isoantigens were al so identified and rabbits l2; as compared to 7 antigenic polypeptides of rabbit spermatozoa were found immuno-reactive with rabbit anti mouse sperm sera by SDS-PAGE and immunoblotting2o. These findings are a lso supportive of species variation in antigenicity of rabbit sperm polypeptides.

Immuno-infertility was diagnosed as a cause of unexplained inferti lity in cows but seropositivity was variable for sperm antigen from different bulls .

Antigenicity of sperm polypeptides was also variable in different species and even in between the 2 sexes.

Acknowledgement One of the authors (DT) thanks le AR, New Delhi

for Junior research fellowship .

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TRIPATHIet al. : IDENTIFICATION OF BOVINE SPERM SPECIFIC POLYPEPTIDES 661

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29 (1993)62 .. 15 Czuppon A 8 , J Anal Chem, 317 ( 1994) 728. 16 Wang G L, Anim Breed Abstr, 15 (1990) 5133 Abstr. 17 Tornov A, Veterinanomeditsinki Mamki , 23 ( 1986) 47. 18 Farahani J K, Tompkins W & Wagner W C, Theriogenology.

15 (1985) 605 . J 9 Deo S & Roy D J, Indian Vet J, 48 ( 197 1) 571. 20 Hen J C. & Eddy E M, Bioi Reprod, 22 ( 1980) 1263. 21 Hunter A G & Hafs H D, J Reprod Ferti!, 7 (1964) 357.