RESULTS
十 一 十 一 十 一 十
Time after dilution （min）
Fig． 5．． The ratio of motile spermatozoa of the Nile tilapia 1， 2， 4 and 8 min after dilution with the DB with （＋） or without （一） 1／100 Volume of the ovarian
fluid． Dark shaded column， spermatozoa di ssolved in Ca2＋一free ASP gQntaining O．5mM EGTA was suspended in the DB containing 1 mhil EGTA．
Light shaded column， spermatozoa dissolved in Ca2＋・一free ASP was suspended in the DB． Open column， spermatozoa dissolved in ASP was suspended in the DB containing O．2mM Ca2＋． N．D．， motile spermatozoa was not detected． Asterisks represent significant difference from the value without
the ovarian fluid （p〈 O．Ol）．
31
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r：M一一一一一111一！一・一一一一一P一一 一 一一一rxl OO xl OOO xl OOOO
Ovarian fEuid dilution
Buffer
Fig． 6 Concentration−dependency of the prolongation effect of the ovarian fluid on the sperm motility of the Nile tilapia． Control， the DB containing O．2mM Ca2＋
without the ovarian fluid．
32
灘．灘灘鰯．欝欝・： 一躍灘翻
り ゴ．・
P灘
1懇
．＿・・灘 要
．置ｾ・獅ワ
Effect of the ovaian fluid concentrations on sperm motility was shown in figure 6． Sperm suspension in ASP was dissolved in the DB containing O．2mM CaC12 with various concentrations of the ovarian fluid， and motile spermatozoa were measured 4 min after dilution． The prolongation of sperm motility by the ovarian fluid was in a concentration−dependent manner， and the ovarian fluid kept the elongation effect until about 500×
dilution．
2） Partial characterization of the substance in the ovarian fluid
which affects sperm motility
The collected fractions of the ovarian fluid by the gel filtration were each examined for its activity to prolongation of sperm motility． As can be seen in figure 7， the elongation activity was recognized mainly in fraction No． 40 to 44． ln considering the eluant fractions of the standard materials， the molecular weight of the sperm motility prolongation substance （SMPS）
showed the activity estimated to be approximately 2000． Also in fraction No． 20 and 60， although a low level， but the activity was recognized．
The ovarian fluid was incubated in boiling water for 5 min． After the incubation， whether the ovarian fluid maintained the above observed
activity was analyzed． The result was shown graphically in figure． 8． The ovarian fluid maintained the activity which elongated sperm motility even
after the boiling for 5 min．
3） Effect of the TAT−10 on sperm motility prolongation
The TAT−10 and TAT−21 as control antibody treated sperm suspensions and non−treated sperm suspension were dissolved in the pooled fractions
（No．40−44） of the ovarian fluid by the gel filtration containing 1％ BSA， or the DB containing O．2mM Ca2＋． The TAT−10 at the concentrations O．5pg to 50yg significantly inhibited prolongation effect of sperm motility by the
3f
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Aprotinine
6500
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pe ptide 1400
Tryptophan
240
o
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10 20 30 40 50 60 70
Fraction number
Fig． 7 Sperm motility prolongation activity in each fraction of the ovarian fluid of the Nile tilapia separated by the gel filtration with Sephadex G−25
（1．5×16cm） equilibrated with the DB containing O．2mM Ca2＋． Bars show that the positions of these materials are eluted． Numbers beneath the names of the materials represent the molecular weight of the materials．
34
1て一
．鰻
蜩蜩蛯P灘 1灘騨1灘麗麗醗醗懸盤1
撚論
螺彗
ににコ．灘
・灘欝灘
．菱．i
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20
to
o Buffer OF heat＋
F．ig．一8 Heat stability of the sperin motility prolongation activity of the ovarian fluid ．of the． Nile tilapia． Sperm solutions were suspended in the diluent containing O．2mM Ca2＋， the diluent containing O．2niM Ca2＋ with 1／100 volume of the ovarian fluid （OF） and the diluent containing O．2mM Ca2＋ with
1／100 volume of the ovarian fluid incubated at 1000C for 5 min （OF heat＋）．
Motile spermatozoa was estimated 4 min after the dilution．
35
．＝ ・・1vr
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灘・ 蛛E @・灘・、
自繋『蛹怐E−灘．
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・灘懸灘羅
SMPS， although the TAT−21 did not inhibited the effect of the SMPS．
There were no visible effect of both the TAT−10 and the TAT−21 treatment
on p ercent motility （Fig． 9）．
4） Tissue specificity of the TAT−10 antigen
The localization of TAT−10 recognized antigen in testis， olfactory organ，
brain and ovary of the Nile tilapia were examined． As shown in figure 10，
the antigen was also existed as single polypeptide of the same molecular weight 27kDa．
5） lmmunohistochemical localizations of the TAT−10 antigen
Immunohistochemical localization of the antigen on the olfactorysystem were shown in Fig． 11． The nucleoplasm of olfactory neurons were strongly stained by TAT−10， and the cytoplasm faced to free surface of olfactory neurons （Fig．11A） and olfactory nerve bundles were also stained by TAT−10． On the sagital section of the brain， fibres of olfactory bulb were especially strongly stained by TAT−10 （Fig． 11B）． In the ovary， TAT−10 antigen first emerged in the cytoplasm of oogonia． The cytoplasm of
perinucleolus stage oocyte had especially strong immunoreactivity to TAT−
10 （Fig． 12A）． ln the late perinucleolus stage oocyte， the nucleoplasm was also stained weakly by TAT−10， and only nucleoplasm was stained by
TAT−10 in the early yolk vesicle stage oocyte （Fig． 12B）． No
immunoreactivity to TAT−10 was observed in vitellogenic oocyte （Fig． 12C）．
Immunoelectron microscopic study on testis showed that especially the cytoplasm of spermatid at initial and middle phase of chromatin
condensation was strongly stained in patches， and also the TAT−10
antigen localized on the plasma membrane of these spermatids （Fig． 13）．
3．6
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