Annls Soc. r. zool. Belg. — T. 113 (1983) — suppl. 1 — pp. 327-334 — Bruxelles 1983
(Manuscript received on 02.03.1983)
V IT E L L O G E N IN S
IN
GASTEROSTEUS AGULEATUS
by
F. OL,LE V IE R and M. COVENS
Zoological Institute,
Naamsestraat 59, B - 3000 Louvain (Belgium)
SU M M A RY
Oestradiol treatment of sexually immature three-spined sticklebacks induced four
specific lipoproteins in the plasma. This vitellogenin induction was dose dependent. An
antibody prepared against the most concentrated vitellogenic protein reacted positively
with two proteins from ovarian homogenates. Immunocytochemical treatment of ovarian
sections with this antibody revealed the presence of immunopositively staining granules.
This positive reaction was limited to the oestradiol treated fishes and appeared only
in oocytes from stage 5 on. These findings are discussed.
IN T R O D U C T IO N
I n teleosts o o c y t e g ro w th goes th ro u g h fo u r p e r io d s : p r im a ry g ro w th , v ite llo genesis b y e n d og en ou s y o lk syn th esis, v itellog en esis b y u p ta k e o f e x o g e n o u s y o lk
p recu rsors an d m a tu ra tio n ( R e i n b o t h , 19 72 ; C l e m e n s , 19 74 ; V a n B o h e m e n , 1981).
G o n a d o tr o p in is a m a jo r c o n tr o l fa c to r in all stages e x c e p t in p r im a ry g ro w th
( B a l i n s k y , 1975; K h o o , 1979). P e r io d ic a c id S c h iff p o s itiv e y o lk vesicles p r o d u c e d b y
th e fo llicle s th em selv es, ap p ea r d u rin g th e first g o n a d o tr o p in d e p e n d e n t p e rio d ,
co rre sp o n d in g t o stage 4 a c c o rd in g t o T r o m p - B l o m (1959). T h ese v esicles g iv e rise
t o c o rtica l a lv e o li ( W a l l a c e a n d S e l m a n , 19 81 ; V a n B o h e m e n , 1981).
O estrog en s are essential fo r th e in d u c tio n o f sy n th esis in th e liv e r o f v ite llo g e ­
nins. T h ese p r o te in s are fem a le sp ecific lip o p h o s p h o g ly c o p ro te in s . T h e y are secreted
in to th e b lo o d , tr a n sp o rte d t o th e o v a r ie s an d th ere se le ctiv e ly ta k en u p b y th e
follicles d u rin g th e th ir d g ro w th ph ase o r th e ph ase o f tru e v itellog en esis as referred
t o b y W a l l a c e a n d S e l m a n (1981) o r stage 5 as r e fe rre d t o b y T r o m p - B l o m (1959).
D u rin g m a tu ra tio n th e o o c y t e fu rth e r in creases in size d u e to h y d ra ta tio n
(stage 6 in T r o m p - B l o m , 1 9 5 9 ).
W e p rep a red an a n tib o d y ag ain st p u rified stick le b a ck v ite llo g e n in a n d u sed
im m u n o c y to ch e m ica l m e th o d s to estab lish th e site a n d m o m e n t o f v ite llo g e n in
u p ta k e in th e o o c y t e s o f u n tre a te d a n d oe stra d io l-tre a tm e n t anim als.
M ETHODS
Animals
F u lly -p la te d th ree-sp in ed stick leb a ck s, Gasterosteus aculeatus fo r m a trachurus,
w ere k e p t in th e la b o r a to r y in d ech lo rin a te d ru n n in g ta p w a ter a t 11 ± 1° C u n d er
n orm a l d a y lig h t c o n d itio n s. T h e e x p erim en ts w ere p e r fo rm e d fr o m O cto b e r to
F eb ru a ry . S ex u a lly im m a tu re 3<S a n d $9 stick leb a ck s w ere in je c te d 6 tim es, o n ce
e v e r y tw o d a y s . T h ree ex p e rim e n ta l g rou p s w ere tr e a te d w ith 6.6 n g, 66 n g o r
660 n g oestra d io l-3 -b e n zo a te p er g b o d y w eig h t resp ectiv ely . O estrad iol w as d issolv ed
in p ea n u t o il a n d th e co n tro ls r e ce iv e d o n ly p ea n u t oil. B lo o d o b ta in e d b y ta il
sev era n ce w as c o lle c te d in h ep arin ised m icr o h e m a to crit tu bes an d cen trifu g ed
im m ed ia tely .
Electrophoresis
P la sm a p rotein s w ere sep a ra ted e le c tr o p h o re tica lly in p o ly a c r y la m id e (P A A )
g ra d ien t gels (5-15 % ) o r in agarose gels (1 % C orn in g u n iversal electrop h oresis
film ). P ro te in s w ere stain ed w ith C oom assie brillian t blu e R 250 (0.01 % S e r v a )
a n d lip id s w ith S u d a n b la c k B (1 % M e r c k ) .
Immunology
A fte r P A A g ra d ien t gel e lectrop h oresis an d stain in g w ith C oom assie b rillia n t
blu e fo r 15 m in , th e cen tra l p a rt o f th e m ost c o n ce n tra te d v ite llo g e n in b a n d w a s
c o lle c te d an d sto re d a t — 20 ° C. P r io r to in je c tio n , th e v itellog en in co n ta in in g gel
p ieces w ere h om og en ised in R in g e r a n d th en m ix e d w ith an eq u a l a m o u n t o f F reu n d
co m p le te a d ju v a n s in an u ltra son e a p p aratu s (M S E , son ip rep 150).
A r a b b it w as in je c te d 5 tim es o v e r a 4 m o n th p e r io d : a first in trad erm a l
in je c tio n w as fo llo w e d b y 4 su b cu ta n ou s b o o s te r in jection s.
A ft e r c o lle c tin g b lo o d th e seru m fr a c tio n w as tre a te d w ith a m m on iu m su lp h a te
in o r d e r t o p u r ify th e l g G fr a c tio n . T h e presen ce o f a n tib o d ie s w as v erified b y
im m u n o d iffu s io n in agarose. T issu es w ere e m b e d d e d in pa ra p la st an d 5 urn section s
w ere prep a red . T h e im m u n o c y to ch e m ica l p e r o x id a s e -a n ti-p e ro x id a s e te ch n iq u e
(P A P ) as d e s cr ib e d b y V a n d e s a n d e (1978) w as used. A ran ge o f a n tib o d y d ilu tio n s
1/100 to 1/3 000 w as used.
RESU LTS
Electrophoresis
U p o n tre a tm e n t o f sex u a lly im m a tu re stick leb a ck s w ith d oses > 6.6 n g
o e stra d io l-3 -b e n zo a te , fo u r lip o p ro te in s, w h ich are a b sen t in im m a tu re an d u n trea ted
c?cJ a n d
a p p ea red in th e p lasm a. T h e m o s t in te n siv e ly stain ed lip o p ro te in b a n d is
ca lled V g 1 a n d th e m olecu la r w eig h t is 640,000 ^ 20 ,000 D a lto n (P l. I : 1). T h e
P LA TE I
1 A . PAAG (5-15 % ) stained with Sudan black B. a : plasma from an oestradiol-3benzoate treated stickleback, b : plasma from a control animal; 1 = vitellogenin 1,
2 = vitellogenin 2, 3 = vitellogenin 3, 4 = vitellogenin 4.
1 B. PAAG (5-15 % ) stained with Coomassie brilliant blue, a : plasma from an oestradiol3-benzoate treated stickleback, b : plasma from a control animal; 1 = vitellogenin 1,
2 = vitellogenin 2 ; - - - - = region where Vg 3 and Vg 4 are situated.
2. P A A G (5-15 % ) stained with Coomassie brilliant blue. Induction of vitellogenesis in
non-vitellogenic ÇÇ by injections of oestradiol-3-benzoate (6 injections). A : control
animal, B : 6.6 ng oestradiol per g body weight, C : 66 ng oestradiol per g body weight,
D : 660 ng oestradiol per g body weight.
B
D
I” *
■Vg1
H
*
©
th r e e o th e r p ro te in s (V g 2, 3 a n d 4) w ere m u ch less co n ce n tra te d . T w o o f th em
(V g 3 a n d 4) a p p a r e n tly co n ta in e d m u ch lip id ; th e y c o u ld be sta in ed ea sily w ith
S u da n b la c k B (PI. I : 1A ), a lth ou g h th e y c o u ld n o t ea sily be sta in ed w ith C oom assie
b rillia n t b lu e (PI. I : I B ). V g 2 lip o p ro te in , h ow ev er, w as m ore r e a d ily d e te cte d
w ith p r o te in stain th a n w ith lip id stain (PI. I : 1).
T h e m in im a l d ose re q u ire d t o in d u ce v ite llo g e n in sy n th esis in th e th ree-sp in ed
stic k le b a ck a m ou n ts t o a b o u t 6.6 n g o e stra d io l p er g b o d y w eigh t. T h e 66 n g d ose a l­
w a y s g a v e v e r y clear results. T h e respon se in creased fu rth e r a t th e 660 n g d ose
(P l. I : 2). W h e n h om og en a tes o f v ite llo g e n ic ov a rie s w ere electrop h oresed in agarose,
tw o p r o te in b a n d s a p p ea red . T h e m o s t c o n ce n tra te d on e (L p ) sh ow ed n ea rly the
sa m e m o b ility as V g 1, th e o th e r o n e (P h ) m o v e d slig h tly tow a rd s th e ca th o d e
(P l. I I : 3b an d c).
I n P A A g ra d ien t gel th ese tw o p ro te in s fr o m o v a ria n h om og en a tes o f v ite llo g e n ic
o v a r ie s m ig ra ted m o r e to w a rd s th e a n od e th an V g 1 (P l. I I : 4). O n ly th e m ost
c o n c e n tra te d b a n d c o u ld be stain ed w ith S u da n b la ck B.
Imm unology
Im m u n o e le ctro p h o re s is in agarose gels o f pla sm a p rotein s o f o e stra d iol treated
<?c? a n d $$ in c o m b in a tio n w ith a n ti-V g 1 -A b rev e a le d th e presen ce o f a pla sm a
p r o te in w h ich re a c te d w ith th e an tiseru m (P l. I I : 3a). N o p recip itin arcs w ere
fo u n d in th e pla sm a o f u n trea ted
an d
Im m u n o e le ctro p h o re s is in ag arose w ith h om og en a tes o f ov a ries fro m v itellog en ic
s tic k le b a ck s a gain st a n ti-V g 1 -A b sh ow ed tw o p r e cip itin arcs : on e fo r b o th m a jo r
p rotein s (P l. I I : 3d).
Im m u n o c h e m ica l tre a tm e n t o f o v a r ia n section s with an tiseru m d ilu tio n s o f
1 /1 200 to 1 /2 000 rev ea led th a t im m u n o p o s itiv e sta in in g gra n u les w ere presen t in
th e « p erip h era l » C ytoplasm a (P l. I I , 5), a t least in stage 5 o o c y t e s fr o m v ite llo ­
g en ic
I n ov a ries o f n o n -o e s tr a d io l tre a te d
n o p o s itiv e re a c tio n w as fo u n d
(P l. I l l : 7). In ov a rie s w ith stage 4 follicles n u m erou s n e g a tiv e ly stain in g v a cu o le s
w ere presen t. V ite llo g e n in clea rly a ccu m u la te d a rou n d th e o o c y t e s b u t d id n o t
e n te r (P l. I I : 6 ).
P LA TE II
3. 1 % agarose gel stained with Coomassie brilliant blue, a : precipitin arc from vitello­
genic plasma with anti-Vg 1-Ab, b : separation of vitellogenic plasma, Vg = vitello­
genin 1, c : separation of ovarian homogenate, Lp = lipovitellin, Ph = phosvitin,
d : precipitin arcs from the yolk proteins with anti-Vg 1-Ab.
4. P A AG (5-15 % ) stained with Coomassie brilliant blue, a : plasma from an oestradiol-3-benzoate treated stickleback, b : ovarian homogenate from an oestradiol-3benzoate treated stickleback; 1 = vitellogenin 1, Lp = lipovitellin, Ph = phosvitin.
5. PAP-treated ovarian section (5 fim) from an oestradiol-3-benzoate treated animal.
5 = stage 5 oocyte, G = positive staining granules in the peripheral ooplasma.
6. PAP-t.reated ovarian section (5 (j.m) from an oestradiol-3-benzoate treated animal,
detail : stage 4 oocyte with negatively staining vacuoles. O = oolemma, T = thecacell
layer stains positive.
Ph
Lp
+
a
b
c
d
®
©
; ]‘Z, ,
•
'
'
\
©
P LA TE III
7. PAP-treated ovarian section (5 [im) from a control animal. 1 = stage 1 oocyte,
2 = stage 2 oocyte, 3 = stage 3 oocyte, 4 = stage 4 oocyte.
D IS C U S S IO N
T h e v ite llo g e n in in d u cin g c a p a c ity o f o e stra d io l in th e th reesp in ed stick le b a ck
is clea rly d ose d ep e n d en t. T h is has also b e e n sh ow n in r a in b o w tr o u t Sahno gairdneri
( V a n B o h e m e n , 1 9 8 1).
S ep a ra tion in P A A g ra d ien t gel is ba sed o n charge a n d M W ( M a u e r , 1971 ; S a r ­
g e n t a n d G e o r g e , 19 75 ; T a n a k a , 1981) w h ile sep a ra tion in agarose is p rim a rily
ba sed o n ch a rge o f th e p rotein . T h e re fo re th e d ifferen t m ig ra tion p osition s in agarose
a n d P A A in d ic a te th a t th e y o lk p rotein s fr o m th e ov a ries — o f v ite llo g e n ic
—
are d ifferen t fr o m V g 1. O n th e o th e r h a n d th e tw o p re cip itin arcs cau sed b y th e
re a c tio n o f o v a r ia n h o m o g e n a te w ith a n ti-V g 1 -A b in d ic a te th a t th e y o lk p rotein s
are im m u n o lo g ic a lly id e n tica l t o V g 1 in th e pla sm a ( B a r r e t t , 1978). T h ese results
su p p o rt th e gen eral a ssu m p tion th a t V g 1 is tran sferred fr o m th e pla sm a in to th e
o o c y t e s a n d tra n sfo rm e d in to th e y o lk p rotein s, lip o v ite llin a n d p h o sv itin : it is a
p recu rsor to th e y o lk p ro te in s ( W a l l a c e a n d S e l m a n , 19 79 ; C l e m e n s , 19 74 ;
D e V l a m i n g et al., 1980).
O n ly th e m o s t c o n c e n tra te d b a n d c a n b e sta in ed w ith S u da n b la ck B an d
th erefore it con ta in s lip o v ite llin . T h e less c o n c e n tra te d b a n d is p r o b a b ly p h o sv itin .
T h e im m u n o lo g ic a l r e a ctio n o f a n ti-V g 1 -A b w ith v ite llo g e n ic pla sm a p rotein s
sh ow s th a t th e an tiseru m w as in d e e d d ir e c te d a gain st an oe stra d io l in d u ce d pla sm a
p r o te in (V g 1), as w e e x p e cte d .
T h e o o c y t e s in th e stick leb a ck s in crease fr o m 0 .1 2 -0 .1 4 m m in stage 1 t o
0 .3 5 -0 .5 6 m m in stage 4 ( T r o m p - B l o m , 1959). I n th ese stages th e y d o n o t con ta in
p rotein s w h ich rea ct w ith th e a n ti-V g 1 -A b . W e m a y th e r e fo re co n c lu d e th a t th e
g ro w th d u rin g th ese stages is n o t du e t o a ccu m u la tio n o f su bsta n ces th a t are im m u n o lo g ica lly rela ted to V g 1 o r t o th e y o lk protein s.
T h e p o s itiv e ly stain in g gra n u les in stage 5 are p e rip h e ra lly situ a ted in th e
o o c y t e s a n d th is is in a g reem en t w ith a u to r a d io g r a p h ic resu lts o b ta in e d in ov a rie s
o f th e zebrafish ( K o e e s m e i e b , 1966).
O u r results lea d to th e c o n c lu sio n th a t stick leb a ck s, in w h ich v itellog en esis is
in d u ce d b y oe stra d io l, h a v e th e c a p a c ity to tra n sfer v ite llo g e n in fr o m th e pla sm a
in to th e o o c y t e s , a t least d u rin g stage 5. W e ca n n o t e x clu d e th a t th e in je c te d o e s tra ­
d io l also stim u la tes th e u p ta k e eith er d ir e c tly o r in d ir e ctly . I t is h o w e v e r g en era lly
a c c e p te d th a t teleost p itu ita r y g o n a d o tr o p in s en h a ce th e tra n sfer o f v ite llo g e n in
fr o m th e b lo o d in to th e v ite llo g e n ic o o c y t e s ( W a h l i et al., 1981), a p p a r e n tly b y
stim u la tin g e x te n siv e m ic r o p in o c y to t ic a c t iv it y a t th e o o c y t e su rface ( W a l l a c e
a n d S e l m a n , 1981, 1982).
S in ce th e o o c y t e s in stage 4 sh ow an a ccu m u la tio n o f v ite llo g e n in ou tsid e th e
o o c y t e , it seem s as i f in th ese fo llicle s th e u p ta k e m ech a n ism is n o t y e t a c tiv a te d .
T h ese results con firm a se le ctiv e a c tiv a tio n o f o o c y te s .
C O N C L U S IO N
T h e m in im al d ose t o in d u ce v ite llo g e n in sy n th esis in m a le as w ell as in fem a le
th ree-sp in ed stick le b a ck s a m ou n ts t o 6.6 n g oe stra d io l-3 -b e n z o a te p er g b o d y w eigh t.
A t least on e o f th e o e stra d io l in d u ce d lip o p ro te in s (V g 1) in th e pla sm a o f th e
th re e-sp in ed stick le b a ck is im m u n o lo g ic a lly rela ted t o th e y o lk p rotein s in th e
o o c y te s . T h is lip o p ro te in is o n ly ta k en u p b y th e o o c y t e s fr o m stage 5 on . Y o u n g e r
o o c y t e s d o n o t possess th e c a p a c ity o f ta k in g u p v itellog en in s, n eith er d o th e y
co n ta in im m u n o lo g ic a lly rela ted protein s.
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