incubated with PBS containing 1％ BSA （Sigma） to block non−specific protein binding site， and then incubated with the culture supernatant of the TAT−30 hybridoma for 3 hr at room temperature． The sections were further incubated with 10nm−gold conjugated goat anti−mou se lg（］H lgM
（Bio Cell， Cardiff， UK） at a dilution 1： 100 for lhr． The sections were
viewed with a Hitachi H−7000 electron microscope after staining with uranyl acetate．
10） Statistical analysis
In the present study， results were expressed as mean±SE， and differences between groups were compared by paired Student s t 一test．
纏
↓融濃 ﾇ鱒霜
『丁一一岬『■･
⊆﹂⊆O◎oN一6．ωm︽
O．8
O．6
O．4
Oe2
O．O
140kD旧 ←→
@ ●●
X4kD−
U7kD・
S3kD一 ●●
R0kD−
@ Ir軸■＿二司■■一一膨
A B
o 20 40 60 80 100
．．，iiil
120
Fraction number
Fig． 14 Sepharose 6B column chromatography （2．5×80cm） of the TAT−30 positive fraction obtained by the salting out of the seminal plasma of the Nile tilapia． Shaded square shows the SPP （TAT−30 antigen−rich） fraction． lnset：
electrophoretic patterns of the seminal plasma （A） and the SPP fraction （B） on
10％ SDS−PAGE． Arrow shows the TAT−30 antigen． Positions of molecular
weight markers are indicated on the left of the figure．
55
議懸難灘灘灘鐵雛羅灘1鍵翻灘．懸．
に、鑛 ・・i灘．．鑛i灘灘 il ・，、i：＿，、，灘i ．奪
饗辮灘欝騨』，、
． 再・自@ eN
2） lnhibition of sperm motility by the seminal plasma protein The seminal plasma proteins inhibited sperm motility with
concentration−dependent fashion， and the inhibitory effect reached plateau at approximately 100pg／ml （Fig． 15）． The ratio of motile spermatozoa was already significantly suppressed by adding the seminal plasma proteins
O．lpg／ml （p〈 O．05）．
Effects of the antibodies treatments on the inhibition of sperm motility by the seminal plasma proteins were shown in Table 2． ln this
experiment， concentration of the seminal plasma protein was adjusted at 100pg protein／ml． The seminal plasma of the Nile tilapia contained
approximately 400ptg protein／ml by the protein determination using BSA as standard． The treatment of the TAT−30 recovered the ratio of motile spermatozoa concentration−dependent manner in the diluent buffer with the seminal plasma proteins． ln contrary， the treatment of the TAT−21 as control hardly recovered the ratio． The recovery of motility by th e TAT−30 was not observed after 1．5 min after the dilution． ln the diluent buffer without the seminal plasma proteins added the TAT−30 or the TAT−21，
both of the sperm motility were slightly inhibited， and the sperm motility added the TAT−30 in the diluent buffer tend to be lower than that added the TAT−21 in the diluent buffer．
3） Partial characterization of the sperm motility inhibiting factor
（SMIF）
The seminal plasma was centrifuged at 1000g for 10 min to remove insoluble material， and the supernatant was loaded onto the Superose 6 gel filtration． Each eluted fraction was assayed for detection of the TAT−
30 antigen by dot blot analysis in order to determine the molecular weight of the SMIF， comparing with the eluted fractions of standard materials．
56
「ﾔ ・
蛛@鹸灘
．灘鑛．、、灘 懸、・
一 ．，、 欝魯・
灘襲・ 薩． 翻漁 ア 慶 磯∫う き闇闇鑛卍灘磁．，吃
灘．．鑛灘麟懇懇螺i灘懸盤．s，、，，．、 懸盤灘ll灘1撒 騨驚驚
翁︾60No罵⊆ΦΩωΦ50Σ 50 40 30 20 10 o
i
T−0−⊥
白
o Oe1 1 10 100 1000
Seminal plasma protein （lwg／ml）
Fig． 15 Concentration−dependency of the SPP on sperm motility inhibiton of the Nile tilapia
57
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︐97噛一．蔚
叢
灘難
灘﹁・蕪幽肇
甑
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旨﹁オー．
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鑛 難難．
難灘
鱗灘
灘懸鍵
懸難
いO．OVq．︵OO一︶畠畠の十m︻︵︻の﹀ρ．一〇．OVq．︵○〇一︶へ幽の十M田︵︻の＞6．﹄￡づρ一信Oづ一咽℃O一
q刷h℃Oρ唱口dhO．歳山の綴Q邸O脳O︵君＼boユ︶口O帽邸お口OO口OQO一の旨＆O﹄旨Oρd口づ口驚Q刷りのヨロ⑪旨dへ．口哨OりO旨α
d口口の邸﹇α肩口嘱dHのの帆畠畠の ．＜のρq承一bo口璃自欝り信OQ寸．卜国q﹇U閏1の個﹄ト一≧目〇一旨￡づρ一雨の5一刷℃O﹄一．m︻︵︻
◎D．Nn刊eり．eり創
僑O．α判F．Feり
ω①．eり判O．①寸
①．N三寸．⑩α
O①．eり引O．Oeり
Ωeり．寸刊Φ．Φeう
O．F刊①．①r
邸eり．寸引卜．Fめ ︵め．O︶OQり−トくト＋︵OOこ∩＝の＋mO ︵め︶OQつ−↑＜ト＋︵OOr︶氏氏の＋mO
︵Oめ︶Oeり一﹂一＜﹂一十︵OOF︶∩■∩圏の十mO
︵Oの︶F叩↑＜ト＋︵OOr︶氏氏の＋ODO
︵OO︶Oeりート＜﹂一十mO
︵OO︶Fα一﹂一＜ト十mO
︵OOF︶氏氏の＋mO
oDO
︵3︾邸ONO一口⊆﹂﹂①∩鯛ω〇一霜〇一≧一⊆①コ＝ロ
三Φ↑9α価∈の而互σ三∈ΦωΦ￡﹀Ω
﹀彰O∈∈﹂Φαω↑O⊂￡一Ω署55⊂O芒Φ琵8﹄↑台O碧器Φ￡一〇暮Φ↑血．αΦ一Ω醇
58
豫
糊
灘． 聾㌦
・鋸
The SM［F was eluted in the same position as the blue dextran， and was estimated approximately Mr 2，000，000 （Fig． 16A， B）．
The electrophoretic pattern and Western blotting with the TAT−30 and the lectins were shown in figure 17． The seminal plasma contained mainly
120kDa and minor 78kDa， 43kDa， 38kDa and 27kDa polypeptides． The 120kDa protein was only recognized by the TAT−30， however， the minor polyp eptides other than 120kDa were not reacted with the TAT−30． IThe lectin binding characteristics of the seminal plasma showed that LCA recognized mainly the 120kDa polypeptide and 38kDa polypeptides． The 38kDa polypeptide was also stained by the UEA−1 （data not shown）．
These blots by the lectins incubated in the presence of the competing sugar failed to produce bands．
Th e immunoprecipitate was carried out SDS−PAGE in 12．5qo acrylamide with silver staining （Fig．18）． The electric p attern of the
immunoprecipitate was subjected to densitometric analysis． Peak areas of the 120kDa protein， the 27kDa protein and the 18kDa protein were measured and ratio of 1： O．24： O．1 between the peaks were given． Taking the molecular weight of these proteins of the SMIF into account， the molar ratio of these polypeptides was calculated to be 1： 1．1 ： O．7．
4） The TAT−30 antigen localization
Tissue sp ecificity of the TAT−30 antigen was shown in figure 19． The antigen was observed only in testis as a single protein of 120kDa．
Immunohistochemical localization of the antigen on the testicular
tissue were shown in figure 20． The TAT−30 antigen 10calized in the whole cytoplasm of Sertoli cells， epithelial cells of seminal ducts， and
spermatozoa in both seminal lobule lumen and seminal ducts of tilapia testis． All spermatognic cells in cysts were not stained by TAT−30． The
59
戯鰹
tA
磯．
灘
灘i灘
講
Oe2
0 91
⊆﹂⊆OooN一邸．ω口︽
OiO
A）
o 5
Volume
20 25
Φ∈三〇﹀
18 16 14 12 10 8 6
B） BSA （67KD）
Aldolase （1 58KD）
Catalase （232KD）
1 Ferritin（440KD）
Thyroglobulin （669KD）
Blue dextran （2，000KD）
104 105 to6 Molecular weight
107
Fig． 16 A） Superose 6 column chromatography of the seminal plasma of the Nile tilapia． Shaded square shows the TAT−30 antigen−rich fraction． B）
Molecular weight estimation of the SMIF． Closed circles are standards and
open circle is the SMIF．
60
mellwafilkl｝
購鍵慧灘轟轟懸難1鑛騨螺灘灘灘灘蓮麗麗鍵盤灘i譲『
灘鎌︑
140kD
t94kD
67kO
里．ぐ
43kD一
pe
30kO
画一−
2 3
Fig． 17 El ectrophoretic pattern and Western blotting by the TAT−30 and the LCA of the seminal plasma of the Nile tilapia． Lane 1， seminal plasma with Coomassie Brilliant Blue staining； lane 2， TAT−30 staining；
lane 3， LCA staining． Arrow shows the TAT−30 antigen． Positions of molecular weight markers are indicated on the left of the figure．
61
範
・
@灘鰭・
，撒灘鑛黙
席 ，s． m，臣，， ・，i蛤・慈
︒躍菖
顎
τ畿ド︒窩
鱗霧x護 ．，、1・・B・・
O三
．．時1
120kD
1
27kD 18kD
l 1
193，71
77 ，93
e．oo
LllLdi−Qm一一pt一一．L
398 ．oo
．
Fig． 18 Densitmetric analysis of 12．5％ SDS−PAGE of the reduced SMIF of the Nile tilapia． Bars show the positions of the components of the SMIF．
62
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