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London ISSN 1 0 9 6 - 4 9 5 9 V itellogenesis in t h e g ia n t t i g e r p r a w n , P e n a e u s m o n o d o n Fabricius, 1 7 8 9 C h e n , C h e -C h u n ; C h e n , S hiu-N an A n n é e 1 9 9 4 Volum e 1 0 7 B N u m éro 3 P a g e s 4 5 3 - 4 6 0 D o n n é e s a d m in is t r a t iv e s D e m a n d e u r P e rs o n n e à c o n ta c te r S u p p o r t Par voie é le c tro n iq u e ? Email Ariel Fax A d re s s e p o s ta le V laam s I n s t i t u u t v o o r d e Z e e Vismijn Jan H a s p e s la g h ja n h @ v liz .b e 0 5 9 3 4 . 2 1 . 3 0 article Veuillez t r a n s m e t t r e d e m a n i è r e é le c tro n iq u e ja n h @ v liz .b e j a n h @ v liz .b e 0 5 9 34 21 31 V laam s I n s t i t u u t v o o r d e Z e e Vismijn (Jan H a s p e s la g h ) P ak h u izen 4 5 - 5 2 B -8 4 0 0 O o s te n d e L ecteur Cote de r a n g e m e n t Note p o u r le f o u rn is s e u r Etat Im p a la N om bre de fo u r n ls e e u r s a p r è s vous Etat A ttribué le En t r a i t e m e n t le ULg-BST: BST - S c ie n c e s rq :im p a la : 1 9 1 1 7 8 6 0 En t r a i t e m e n t 0 2 / 1 2 / 2 0 0 5 1 2 :0 5 0 2 / 1 2 / 2 0 0 5 1 3 :0 5 A c tio n s (S) A u cu n e action Q R é e x p é d ie r la d e m a n d e á
¡Université d e Liège Bibliothèque g é n é r a l e d e P h ilo so p h ie e t Lettres
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Comp. Biochem. Physiol. Vol. Î07B, N o. 3, pp. 453-460, 1994 © 1994 Elsevier Science Ltd Printed in G reat Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
Vitellogenesis in the giant tiger prawn,
Penaeus
monodon
Fabricius, 1789
Che-Chun Chen* and Shiu-Nan Cheni
D epartm ent o f Aquaculture, N ational Chiayi Institute o f Agriculture, Chiayi, Taiwan 60083, Republic o f China; and tD epartm ent o f Zoology, N ational Taiwan University, Taipei, Taiwan 106, Republic o f China
Vitellogenesis can be induced in the ovaries of the penaeid shrimp, Penaeus monodon, by eyestalk ablation; 80% o f shrimp spawned within 7 days after ablation. Using immunofluorescence, it was observed that vitellin commences to accumulate in the yolk globular stage oocytes. The vitellin of atretic oocytes is reabsorbed and transferred to the newly matured oocytes. Four vitellin-like peptides are synthesized in vitro by the ovaries with mol. wt 220, 168, 130 and 74 kDa, respectively. Amongst them, the 168 and 74 kDa peptides are secreted into the culture medium. The results of immunostaining showed that the anti-Ep antisera is able to react with the ovary extracts of Penaeus japonicus. Each antiserum may react to the compatible peptide of the P. monodon vitellin peptide.
Key words: Penaeus monodon Fabricius; Vitellogenesis.
Comp. Biochem. Physiol. 107B, 453-460, 1994.
Introduction
Vitellin (Vt), a m ajor egg yolk protein, is syn thesized on a large scale in the yolk production tissues which, in vertebrates are the liver and ovary and, in insects, the fat body and ovary. The prim ary translation products are precursor molecules, vitellogenins (Vg), which may be cleaved and modified to yield the m ature yolk proteins in the course o f secretion, transport and deposition in the developing oocyte. In several species of crustaceans, vitellogenin has been isolated and characterized; it has been described as a lipoglycol-carotenopoprotein (Tom et al., 1987; Quinitio et al., 1990; Vazquez- Boucard and Ceccaldi, 1986; Y ano and Chinzei, 1987; R ankin et al., 1989; Browdy et al., 1990). In several species o f decapod crustaceans, the ovary (OV) (Quackenbush, 1989; Y ano and Chinzei, 1987; Browdy et al., 1990; Rankin
et al., 1989), subepidermal adipose tissue (SAT)
(Aiken and Waddy, 1980; Tom et al., 1987) and hepatopancreas (HP) (Vogt et al., 1989; Paulus and Läufer, 1987) have been implicated in
con-Correspondence to : Che-Chun Chen, Rm 203, The Institute of Fishery Biology, N ational Taiwan University, Taipei, Taiwan 106, Republic o f China.
Received 16 June 1993; accepted 30 July ¡993.
tributing to synthesis o f vitellin or its precursor molecule, vitellogenin.
O varian development in crustaceans may be prom oted by eyestalk ablation in several species o f penaeid shrimps. In Penaeus monodon, the isolated vitellin possesses a molecular weight of approximately 540 k D a and is composed of four m ajor subunits o f mol. wt 74, 83, 104 and 168 kD a (Quinitio et al., 1990). Egg extracts are immunologically identical to hemolymph of m aturation stage female shrimp (Chen and Chen, 1993). Previously, we have studied the synthesis o f vitellin in ovarian culture in vitro.
The present study showed that a non-secreted vitellogenin precursor (pre-Vg) is proteolytically cleaved within the ovary into two products with molecular weights o f over 200 kD a and secreted into the culture medium.
Materials and Methods
Animals
Broodstock female P. monodon were ob tained from Tungang, southern Taiwan. Shrimp were m aintained in seawater 2.8% at 28 ± 1°C and fed squid and oysters twice daily. Unilateral eyestalk ablation induces ovarian development. 453
C he-Chun Chen and Shiu-N an Chen
Table 1. The num ber o f nauplius produced from 25 Penaeus monodon females Days after ablation
3 4 5 6 7 8 9 10
Spawning no. 3 5 6 7 1 1 6 3 2 T otal batch no.
(Total N o. 25) 43
Average nauplius no. 55 67 58 60 72 83 56 62 M ean o f nauplius
( X 1000) 6.27/batch The vite using th In vitro Tissue males we L-15 me 7 2 0 + 10 Chen et were lat 200 n Ci/ 1209.3 C medium in an Ej Immunoj. Crude medium A B
Fig. 1. Histologic section o f ovaries o f Penaeus monodon which were taken for in vitro culture (3 days after eyestalk ablation). A, H E stain; B, PAS stain; n, nucleus; nn, nucleoli; fc, follicle cell. Scale: 100 ,um (A),
The vitellogenesis stage was confirmed visually using the criteria described by M otoh (1981). In vitro organ culture
Tissues dissected from early vitellogenic fe males were incubated in 200 ¡x\ (2 x ) Leibovitz’s L-15 medium (Hazleton), with osmolarity of 720 + 10 mmol/kg at 28 + 1°C as described by Chen et al. (1989). New synthetic vitellogenins were labeled in methionine-free medium with 200 /i Ci/ml 35 S-methionine (specific activity 1209.3 Ci/mmol). After incubation, tissue and medium were centrifuged at 15,000 g for 10 min in an Eppendorf microfuge at 4°C.
Immunoprécipitation with anti-Ep -serum
Crude tissue extracts (100 /z 1) or culture medium (100 /il) were added to 50 p.1
anti-Ep-serum and 50 p i protein-A Sepharose CL-6B with incubation at 4°C for 4 hr or overnight. The precipitated pellet, after washing twice with 0.1 M Tris-H C l buffer (p H 7.5), was redissolved in 50^1 SDS sample buffer.
Indirect immunofluorescence microscopy
Paraffin sections were prepared according to standard methods. Tissue sections were incu bated with 200 x anti-Ep-serum for 1 hr at room tem perature, and washed with TBS. FITC-conjugated goat IgG (anti-rabbit-IgG, 1:20 diluted) was applied for 1 hr at room tem perature. The sections were m ounted in 1 M T ris-H C l (p H 8.1) and glycerol (1:9, vol/vol) then observed and photographed with an Olym pus (Model BH2) microscope, with an incident UV attachm ent and fluorescence optics.
456 Che-Chun Chen and Shiu-N an Chen
D
' h S Ï
Fig. 2. Im munofluorescence m icroscopy o f developing ovaries o f P. monodon. A, C, phase contrast; B, D, immunofluorescence. A, B, stage 1; C, D, spent stage. Scale: 50 /tm (A, B); 100 p m (C, D).
Digestion o f Vg with E ndo-H and calf alkaline phosphatase
The culture medium was incubated in 20 mM T ris-H C l buffer plus 20 units of calf alkaline phosphatase (EC 3.1.3.1) (p H 8.0), at 37°C for 2 hr. The reaction was stopped by adding SDS re-educating sample buffered solution and boiling for 3 min. The culture medium was incubated in 20 mM phosphate buffer plus 5 m units endoglycosidase H (EC 3.2.1.96) (p H 5.5) at 25°C for 12 hr. Protease activity was inhibited by 0.5 mM PM SF.
Results
Ovarian development
Eyestalk ablation was used to induce ovarian development; 80% of the eyestalk-ablated ani
mals spawned within 7 days (Table 1). The green color o f the ovaries may be derived from caro- tenoids associated with vitellogenins. Animals in stage I were used. Using hem otoxylin-eosin (HE) stain (Fig. 1A), the ovaries appeared to be in vitellin stage (diameter 40-200 pm ). Using PAS stain (Fig. IB), the vitellinic oocytes (diam eter > 100 p m ) showed an accumulation of large numbers o f glycoproteins.
Immunofluorescence microscopy
The early m aturation oocytes (diameter < 100 /im) neither react to the antiserum nor exhibit fluorescence (Figs 2A,B). The late-stage vitellogenic oocytes had significant fluorescence that was not observed in the early-stage oocyte. The follicular cell around the late-stage oocyte reacted with the anti-Ep antiserum showing strong fluorescence. The reabsorbed oocytes
kD e 220 130 Fig. 3. SDS- F tates from in v (Fig. 2D) ex m aturation Immunoprec Vitellin-lil electrophore tates by anti tissues and SDS-PAGE. extract posse 104, 83 and ively, and o cultured mec lacked the 21 The in vitr polypeptides, respectively. 1 polypeptide v the 95 kD a p Vitellogenii termine carbo Endo-H shov (Fig. 5A). Th( difference wh< may suggest mass glycolati tained little o Black B stair
Vitellogenesis o f P. monodon I, tie green m caro- \n im als in-eosin ed to be . Using s (diam- tion of tiameter um nor te-stage rescence oocyte. : oocyte showing oocytes k D a 2 2 0 -1 3 0
-B
WÊÊÊÊ
k D a 168 104 83 74H
m
Fig. 3. SD S-P A G E (10% acrylamide) o f immunoprecipi- tates from in vitro culture ovary samples. A, ovary extract;
B, culture medium.
(Fig. 2D) exhibited vitellin transport to the new m aturation oocytes.
Immunoprecipitates o f vitellin in ovaries
Vitellin-like proteins o f tissues in vitro were electrophoretically analysed. Im m unoprecipi tates by anti-Vn antiserum and protein from the tissues and medium were tested using SD S-PA G E. Figure 3 shows that the ovary extract possesses five m ajor bands o f 200, 168, 104, 83 and 74 kD a molecular weight, respect ively, and one m inor band o f 130 kD a. The cultured medium revealed similar patterns but lacked the 220 kD a band.
The in vitro culture medium had three new
polypeptides, with mol. wt o f 70, 78 and 95 kD a, respectively. In the proteolytic m ap, the 78 kD a polypeptide was observed as Vp3 (83 kD a), and the 95 kD a peptide as Vp2 (104 kDa) (Fig. 4).
Vitellogenin was treated with Endo-H to de termine carbohydrates. The 220 kD a peptide to E ndo-H showed a reaction o f about 20 kD a (Fig. 5A). The other vitellin protein showed no difference when Endo-H -treated. These results may suggest that the 220 kD a peptide was a mass glycolation, and th at other peptides con tained little or no carbohydrate. In the Sudan Black B stain, the V pl and 220 k D a vitellin
proteins stained positively, as shown in Fig. 5B. After digestion with alkaline phosphatases, Vg revealed no degradation (Fig. 5C).
In vitro protein synthesis
The result obtained from 35S-methionine la beling (Figs 6A,B) showed new synthesis o f ovarian polypeptides in vitro. After 1 h r o f culture, three new synthesized patterns were present in the ovary extract, with mol. wt of 220, 168 and 130 kD a, respectively. Eight hours after culture, the three patterns were m ore intense, and some smaller patterns were present. In the culture medium, 1 hr after culture, two patterns were found with mol. wt 168 and 74 kDa, respectively. There were no other patterns found in culture medium with incubation periods of up to 8 hr. A utoradiography showed that the ovary may synthesize vitellin-like proteins, with mol. wt o f 220, 168, 130 and 74 kD a, plus several smaller polypeptides. In the ovary, there were three patterns of 220, 168 and 130 kDa, respectively. The culture medium contained two secreted polypeptides of 168 and 74 kD a (Fig. 6D), respectively.
Immunoreactions o f P. m onodon vitellin with P.
japonicus vitellin
Results of im munoblotting dem onstrate the cross-reaction o f P. monodon vitellin antiserum with ovarian extracts and hemolymph from m ature female P. japonicus shrimp (Fig. 7). A nti-E pl reacted with one 70 kD a vitellin p ro tein only. The anti-Ep2 reacted with the 80 kD a protein, and also showed weak cross-reaction to the 100 kD a protein and the large vitellin
95 • S
70 •
A
B
Fig. 4. Im m unoblot o f peptide m aps o f in vitro culture m edium after partial proteolysis. A, 10% SD S -P A G E of culture medium; B, partial proteolysis o f the vitellin-like polypeptides. 1, 104 kD a; 2, 95 kD a; 3, 83 kD a; 4, 78 kD a;
458 Che-C hun Chen and Shiu-N an Chen ) 2 z?c
2
3
W H2
3
*220B
C
Fig. 5. Effect o f E ndo-H and calf alkaline phosphatase (CAP) on the P. monodon ovary vitellin subunits. A, Endoglycosidase-H treatm ent: 1, ovary extract; 2, Endo-H digestion, 12 hr. B, Sudan Black B stain: 1, early m aturation stage ovary extract; 2, late m aturation stage ovary extract; 3, egg extract. C, CAP
treatm ent: 1, ovary extract; 2, C A P digestion, 1 hr; 3, C A P digestion, 2 hr.
protein (160 kDa). The anti-Ep3 reacted to the 100 kD a and some larger proteins with mol. wt ranging from 130 to 170 kDa.
Discussion
The eyestalk-ablated broodstock females en tered the vitellogenin stage, which was followed by spawning. The secondary vitellogenin period is less than 4 days; the yolk protein o f the spawning eggs is synthesized in this period. Therefore, the vitellin production tissues were found to be at the peptide translation stage.
Vitellogenesis includes the production of vitellogenin and the accum ulation o f both or ganic and inorganic constituents o f yolk by the oocytes. In the anti-Ep antisera im m unostain
■ ? !!
J88
}£4-(Fig. 2A), vitellin commences to accumulate in the secondary vitellogenesis oocytes (yolk glob ular stage). However, vitellin is not present in the prim ary vitellogenesis oocytes (pre-yolk stage, diam eter < 100 pi). According to Tan- Ferm in and Pudadera (1991), all the m ature oocytes may be spawned out. The atretic oocytes in the secondary vitellogenesis period degenerate and are reabsorbed (H arrison, 1990). In Fig. 2B, the vitellin o f degenerating oocytes is transferred to the newly m atured oocytes. Vitellin appeared to be used no t only for yolk protein form ation, but also for nutritious trans fer in the vitellogenesis stage.
In the immunoprecipitates o f ovary extract and culture, five m ajor vitellin-like peptides occurred. The 220 kD a peptide is only present in c ! 2 3 4 5 6 7 8 I W - » « # - ■ W * “ » » ** — I - » - • -i . . « . . * « » i 2 3 4 5 6 7 8 ,*¡ Mt
B
Fig. 6A and B the ear result c suggest protein newly s in ovai 130 kD culture isolated monodo w t 170 ; similar culture, lymph c 104, 83 the real newly s the ov¡ (Yano ; F ßnits. tain: :a p nmulate in yolk glob- present in (pre-yolk g to Tan- lie m ature he atretic :sis period son, 1990). ng oocytes d oocytes, y for yolk ious trans-iry extract ; peptides present in
1 2 3 4 5 6 7 3
1 2 3 4 5 6 / 8
... ¡0 4 83 m m- - D
í
i
Fig. 6. [35 S]methionine labeling o f in vitro culture ovary proteins. SD S -P A G E fluorography o f imm uno- precipitates for culture ovary samples. A, C, Coomassie Blue R stain; B, D, autoradiography. A, B, ovary extract; C, D , culture medium. The culture times, C, 0 hr; 1, 0.5 hr; 2, 1 hr; 3, 2 hr; 4, 3 hr; 5, 4 hr; 6, 5 hr;
7, 6 hr; 8, 8 hr.
the early secondary vitellogenesis ovary. The result o f Sudan Black B and PAS staining may suggest th at the 220 kD a variant is a glycolipo- protein. In in vitro culture o f the ovary, three newly synthesized vitellin peptides were present in ovary extract with mol. wt 220, 168 and 130 kD a; and two secretory peptides in the culture medium o f mol. wt 168 and 74 kD a. The isolated vitellogenin in hemolymph o f P.
monodon is composed o f two subunits with mol.
wt 170 and 93 kD a (Lee, 1991); th at is, it is very similar to the secretory vitellin found in in vitro
culture. In our present study, P. monodon hem o lymph contained four egg yolk peptides o f 168,
104, 83 and 74 kD a. These may be derived from the reabsorption of vitellin in hemolymph and newly synthesized vitellogenin. In P. japaneus,
the ovary synthesized two vitellin proteins (Yano and Chinzei, 1987). In P. semisulcatus,
vitellin protein can be synthesized by the ovary
in vitro as native vitellin th at consists o f four
subunits (Browdy et al., 1990). Lui and O ’C onnor (1977) concluded that the ovaries o f crab, Pachygrapsus crassipes, are capable o f synthesizing the proteinaceous yolk found in the m ature egg.
O ur results did not show Ep2 and Ep3 to be synthesized in ovaries in vitro. It m ay be th a t the culture time was too short for vitellin to be processed, or th at it cannot be processed in in
vitro culture. From our data, it m ay be con
cluded th at P. monodon ovary synthesizes vitellin in vitro. A 220 kD a pre-vitellin and two interm ediate vitellins (168 and 130 kD a) were found in the ovary, and 168 and 74 kD a vitellins were secretory to culture medium. In vivo, the 168 and 74 kD a peptides m ay constitute vitel logenin and show absorption by oocytes.
I ? 3 A M 1 ? 3 A t 3 A 1 ^ 3 A = — mÊÊÊÊÊÊÊ A . J 9 «Mii M C --- D ■ ■ I ■¡B
Fig. 7. Im m unobiotting showing the cross-reactivity o f antiserum to vitellin o f P. monodon (Pm) with P. japonicus (Pj). A, silver stain; B, C, D , im m unostain. B, anti-Ep2. B, anti-Ep3. C, anti-Ep2. 1, hemolymph
T
460 Che-Chun Chen and Shiu-N an Chen
The vitellin o f P. japonicus has four sub units that are similar to those of P. monodon.
W hen immunostained, they reacted to the anti- Vg antisera of P. monodon. This result may suggest that vitellins in P. monodon and P.
japonicus penaeids are very similar immunolog-
ically and may m ature under the same pro cesses.
References
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