Yolk synthesis in the marine shrimp, Penaeus vannamei

Camp. Biochem. Physiol. Vol. 103A,No. 4, pp. 71I-714, 1992

0300-9629/92$5.00+ 0.00 0 1992Pergamon Press Ltd

Printed in Great Britain

YOLK SYNTHESIS

IN THE MARINE

PENAEUS

SHRIMP,

VANNAMEI

L. SCOTTQUACKENBWSH Department of Biological Sciences, Florida International University, Miami, FL 33199, U.S.A. (Tel. 305 348-3101) (Received 7 April 1992; accepted 15 May 1992)

Abstract-l. In uifro yolk synthesis was measured in fragments of the ovary of developing shrimp, Penueus vannamei. 2. Progesterone and estradiol stimulated yolk synthesis in vitro, while ecdysterone, testosterone and estrogen had no effect. 3. A peptide factor from the eyestalks of crayfish stimulated yolk synthesis in vitro. A peptide factor from shrimp eyestalks inhibited yolk synthesis in vitro.

INTRODUCHON Current methods in penaeid shrimp culture use eyestalk ablation (removal) to induce yolk production in

captive female shrimp (Chamberlain et al., 1985; Primavera, 1985). This operation removes the major neurohormone center in the animal, and in particular removes the natural source of gonad inhibiting hormone (GIH) (Fingerman, 1987; Quackenbush, 1986). The eyestalk ablated shrimp will respond to this operation with a rapid and unstoppable gonadal development. Recent basic research has focussed on the process of vitellogenesis (egg yolk protein production) in crustaceans (Quackenbush, 1989a,b; Rankin et al., 1989; Tom et al., 1987a,b; Yano and Chinzei, 1987). GIH regulates vitellogenesis through an inhibitory process (Charniaux-Cotton, 1985; Meusy et al., 1987; Quackenbush, 1989). Gonadal stimulation via the neuroendocrine system has been postulated and tested in crabs and crayfish (Eastman-Reks and Fingerman, 1984; Kulkarni et al., 1991). However, there is no demonstration of a neuroendocrine factor which can stimulate vitellogenesis in the ovary of shrimp. Steroid hormone-stimulation of yolk synthesis was demonstrated for some species (Fyhn et al., 1977; Fingerman, 1987; Quackenbush, 1986). The present study investigates the effects of exogenous steroid hormones on the in vitro yolk synthesis of shrimp ovarian fragments. The primary objective of this study was to determine if stimulation of yolk synthesis could be induced using steroid hormones. Based on a survey of the literature of crustacean endocrinology, candidate hormones were selected (Quackenbush, 1986, 1989; Fingerman, 1987). Additionally, peptide hormones from crayfish eyestalks and shrimp neural tissue were also assayed for their effects on in vitro yolk synthesis. MATERIALS AND METHODS Animals

Adult female shrimp, Penaeus vannumei (3WSg) were purchased from GMSB Inc., a shrimp hatchery located in

Summerland Key, Florida. The shrimp were maintained in 550gal seawater aquaria (36%; 25°C; light dark cycle of 14:lO) and fed a mixture of frozen squid and dry pellets (Rangen, Inc., Buhl, Idaho). Shrimp were unilaterally eyestalk ablated using scissors and the resultant wound cauterized. One week after eyestalk ablation, ovarian development was staged using a 4-point scale (Liao and Chen, 1983) and the shrimp with beginning yolk deposition (stage 2) were used for the in vitro assay of yolk protein synthesis. In vitro assay Ovaries used in the assay of hormone effects on in vitro protein synthesis were always obtained from eyestalk ablated shrimp. In order to maintain consistent assays for all the different hormones tested, ovaries that had egg diameters of 0.16 f 0.05 mm were used. This size of egg is about one half of the size of a fully mature egg. Ovaries with either larger or smaller eggs were rejected and not used in the assay system. Ovaries were removed from shrimp via dissection under chilled saline (Cooke et al., 1977). Fragments of the ovaries (2-3 mm in size) were then placed into sterile culture media (Media-199, Sigma Chemical Co., St Louis, MO) in sterile culture dishes. Sugars, salts and antibiotics were added to the incubation media for the crustacean tissues (Quackenbush, 1989a,b). Radioactive leucine ([‘4C]leucine: ICN Radiochemicals, Irvine, CA) was then added to the media containing tissue fragments in a dose that would allow for incorporation of leucine into newly synthesized proteins (0.05 pCij5 nmol/lO ~1) (Quackenbush, 1989a). Finally, the hormones to be assayed were added to the incubation media in either olive oil vehicle (for steroid hormones) or saline vehicle (for peptide hormones) at the required dose. Tissues were incubated for 4 hr at room temperature while being gently agitated (4&70 rpm). After, incubation, tissue fragments were removed from the culture dishes and rinsed with cold saline, then homogenized in phosphate buffer. Three volumes of ice cold saturated ammonium sulphate was added to the homogenates to precipitate all the proteins. The mixture was centrifuged at lO,OOOg for 1Omin. The resultant pellet of protein was resolubilized in cold phosphate buffer and then aliquoted into two fractions. The first fraction was used to measure total protein (Bio-Rad Protein Reagent Assay) and total leucine incorporation (scintillation spectrophotometry). The second fraction was used to measure incorporation into yolk proteins. This assay used an immunoprecipitation procedure to separate the yolk proteins from all the other proteins present in the homogenate of the ovarian tissues. An 711

712

L. SCOT-~QUACKENBUSH

to the purified egg yolk protein from the shrimp, Penaeus vannomei was made in rabbits. A lOy1 sample of this antisera was added to each fraction of ovarian homoantibody

genate. After immunopellet

an 18 hr incubation at 4”C, the resulting contained only yolk proteins, and it was

analysed for incorporation of radioactive leucine, and the amount of total protein in the pellet. Using these methods the effects of hormone on the genera1 protein synthesis (fraction one) and specific yolk protein synthesis (fraction two) are examined. The results from these determinations were analysed by comparing the incorporation of radioactive leucine into tissues incubated with a dose of hormone versus tissues incubated with a control of saline or olive oil and cholesterol. Thus, stimulation and inhibition of general protein synthesis and yolk protein synthesis was measured at the same time on the same tissues. The results are reported as a percentage of the control, values greater than 120% indicated significant stimulation, while values less than 80% indicated significant inhibition. Again, either stimulation or inhibition was measured at the same time on the same tissues. Statistical tests were performed on the data to determine the significance of the differences between the doses and controls (I -test for percentages; Sokal and Rohlf, 1981). Hormones

The steroid hormones: progesterone (4-pregnene-3,20dione), testosterone (17rY-hydroxy-4-androsten-3-one 17acetate), estradiol(3,17-/?-dihydroxyl-3,5(10)-estratriene-17acetate), estrogen (3-hydroxy-1,2,5(10)-estratriene-17-one3-acetate) and ecdysterone (28,3j7,14a,20r!7,22,35-hexahydroxy-7-cholestene-6-one) were purchased from Sigma and stored according to packaged directions. Gonad inhibiting hormone (GIH) was isolated from the eyestalks of shrimp as described in Quackenbush (1989a). Fractions were tested as described above. Extracts of brain, thoracic ganglion were dissected from fresh shrimp and prepared as the eyestalk extracts described above. In collaboration with Dr Albert0 Huberman, Instituto National de la Nutrition, Univ. de Mexico several peptides purified from the eyestalks of the crayfish, Procambarus bouueri, were tested. The peptides were isolated using protocols already

described (Huherman et al., 1989;Aguilar et al.,

in press). We tested five different peptides (labelled HI-HS) for their activities in the in vitro assay. RESULTS Steroids

Three steroids: testosterone, estrogen and ecdysterone were found to have no effect on general protein synthesis nor on yolk protein synthesis. When tested at several doses (10-‘-10-9 M) the responses of the tissue fragments were not significantly different

0 ; 0

Dose (M) 10-3 10-d 10-5 10-e IO-’ lo-* 10-g Vehicle control N=

% of control value (crude protein/yolk protein) Testosterone Estrogen Ecdysterone 97/100 87/93 101/96 lO2/88 95194 95197 102/97 100/100 9/9

87189 S9/88 86192 93196 97196 102/101 103/93 96/87 93198 96194 102/100 101/98 101/102 loo/94 loO/lOO 100/100 919 919 % Control is calculated by: Test value d.p.m. (mg) divided by control value d.p.m. (mg) multiplied by 100. Values 120% or
200

I

Estradlol Crude A Pellet

0

-8

-9

-7

Dose

-6

: Log

-5

-4

-3

Molar

Fig. 1. The effect of exogenous estradiol on the incorporation of labelled leucine into isolated fragments of the ovary from Penaeus vannamei. Open triangles are the values (mean + SD) for crude or total protein. Qpen circles are the values for the immunopellet which contains only vitellogenin. The dose of steroid hormone is indicated as a final volume for the incubation media.

from controls treated with cholesterol in olive oil (Table 1). Estradiol had little effect on the general protein synthesis of ovary tissue in vitro when tested at doses from lo-’ to 1O-9 M. However, this steroid did stimulate yolk protein synthesis in vitro at the high dose range from 10e3 to 10e5 M (t-test, P < 0.05). The lower doses of estradiol affected neither yolk or general protein synthesis (Fig. 1). Progesterone stimulated both general protein synthesis and yolk protein synthesis at doses higher than 10m6M (Fig. 2). At lower concentrations of this steroid hormone the ovarian fragments responded no differently from olive oil controls given cholesterol. Progesterone produced the greatest increase in protein synthesis of all the steroid hormones tested. The effective in oitro dose of progesterone suggests that it might be used to regulate yolk synthesis in intact animals. In summary, progesterone was the most potent stimulator of general and yolk protein synthesis in these in vitro assays. Estradiol only affected the yolk protein synthesis, and that was only at relatively high doses. Ecdysone, testosterone and estrogen had little effect on the general and yolk protein production in uitro. It should also be noted that even high doses of

250

Table 1. Effect of steroid hormones on protein synthesis in the isolated ovarian fragments of the shrimp, Penaeus uannamei

250

L

0 =.

200 t

Progesterone Crude

I -9

A

I

I

I

-8

-7

-6

Dose:

Log

I -5

I -4

I 3

Molar

Fig. 2. The effect of exogenous progesterone on the incorporation of labelled leucine into isolated fragments of the ovary from Penaeus vannumei. Symbols as in Fig. 1.

Yolk in shrimp Table 2. Effects of shrimp tissue extracts on protein synthesis isolated ovarian fragments of the shrimp, Penaeus vnnnomei %

Dose (M) 10-3 10-4 10-S 10-h N=

of control Eyestalk GIH

in

(crude protein/yolk protein) Thoracic Brain extract extract

97130 96145 87160 loo/98 919

97196 9s/99 96197 95198 919

87196 SE/89 96192 93197 919

Dose is in eyestalk, brain or thoracic equivalents. One eyestalk has one eyestalk equivalent dose of hormone or extract.

steroid hormone were not inhibitory, that is the high doses of hormone did not block either protein synthesis or yolk synthesis in vitro. Peptides

Yolk synthesis was inhibited by the eyestalk peptide extract containing GIH as already demonstrated. As before, the GIH only affected the yolk protein synthesis, with little effect on general protein synthesis compared to controls. Both the extracts of brain tissue and thoracic nerve tissue had no significant effects on the general or protein synthesis in vitro (Table 2). It seems that only the eyestalk contained any hormones which inhibited the yolk synthesis. Also, neither brain nor thoracic nerve extracts had any stimulatory effect on the assays. Peptide Hl from Huberman was thought to be a native GIH, and it produced a significant inhibition of yolk synthesis, as well as inhibiting general protein synthesis. Peptide H2 was thought to be a peptide that inhibited the molt cycle called MIH, but in this assay system it was found to stimulate yolk protein synthesis dramatically (Table 3). Peptide H2 did not, however, effect the general protein synthesis. The remaining fractions peptides H3, H4 and H5 had no biological activity in the assays (Table 3). In summary, the shrimp eyestalk contains a peptide hormone that is specific for the inhibition of yolk synthesis in vitro. Additionally, a peptide from crayfish eyestalk (H2) can stimulate by as much as 300% the yolk synthesis in vitro. Extracts of brain and thoracic ganglia have neither stimulatory nor inhibitory effects in these assays. DISCUSSION

The success of a shrimp hatchery is determined by the consistent output of viable shrimp larvae. This in turn depends on the reliable induction of maturation of shrimp in captivity. In the hatcheries that produce Peruzeus uannamei larvae, eyestalk ablation of female broodstock to induce reproduction is the only reliable method or technique available to meet these demands. However, it has been suggested that eyestalk ablation causes too much disruption of the shrimp’s endocrine system, because the larval viability from ablated female shrimp decreases with subsequent spawning. Clearly, new methods must be explored and tested to regulate captive shrimp reproducton in order for hatcheries to produce dependable larvae in commercial quantities. Most animals regulate their reproductive processes with both steroid and peptide hormones. In shrimp,

713

there has been no clear demonstration of which of a list of perhaps 10 steroid hormones actually regulate ovarian maturation (Fingennan, 1987; Quackenbush, 1986, 1989). The present study found that in vitro assays measured significant stimulation of both general protein synthesis and specific yolk protein synthesis when the steroid hormone progesterone was present. This response was specific to progesterone, and steroid hormones with a similar chemical structure like ecdysone, estrogen and testosterone had no effect on in vitro protein synthesis. This suggests but does not yet prove that progesterone may have a natural biological role in shrimp ovarian maturation. The steroid hormone estradiol was found to stimulate only yolk protein synthesis, with no significant effect on the general protein synthesis. This suggests that this compound had a specific action on the ovarian tissue, that of yolk protein stimulation. This is in contrast to progesterone which stimulated both processes. Both estradiol and progesterone stimulated yolk synthesis about equally, producing a 50% increase in incorporation in 4 hr of incubation. Since this assay is an artificial in uitro assay, it is possible that both hormones may be involved in the natural regulation of ovarian maturation in shrimp. It should also be noted that this assay was for a relatively brief incubation of 4 hr, longer durations of incubation may prove to produce increased response in the tissues. Future work should test these two compounds, estradiol and progesterone, in assays of reproductive induction in intact shrimp. The eyestalk neuroendocrine system of crustaceans has long been known to produce a peptide hormone that inhibits gonadal development in both males and females. It has been speculated that the neuroendocrine system may also produce an antagonist of GIH that would be a natural stimulator of gonadal development, the putative Gonad Stimulating Hormone or GSH. The presence of GSH in any neuroendocrine tissue of crustaceans has never been demonstrated. Using the peptide isolated from crayfish we now have proven the existence of a peptide hormone stored in the eyestalk which has a direct stimulatory action on the ovary in vitro. This hormone was specific to the stimulation of yolk synthesis, it produced a 300% increase in incorporation into yolk proteins in vitro. The GSH had little effect on general protein synthesis, like the GIH already isolated from the eyestalks of penaeid shrimp (Quackenbush, 1989a,b). The new GSH must be sequenced and synthesized before it can be tested in whole intact female shrimp. Table 3. Effects of pcptides isolated from the eyestalks of the crayfish, Prorambarus bouueri on the protein synthesis of isolated fragments of the ovary of the marine shrimp, Penaeus oannamei Dose of peptide

I

10-j lo-’ 10-S 10-e N =

30142 41162 86172 88189 12/12

% of Control (crude protein/yolk protein) 2 3 4

1071370 861303 961280

I IO/l00

96197 98/99 lOOi 86189

97194 96195 87189 93195

5 94196 96198 u/s9 92194

all cases

Doses are in sinus gland equivalents, where one sinus gland from one eyestalk would have one equivalent. % Control was calculated as described above. Values 120% or ~80% are statistically significant at P c 0.05.

L. SCOTTQu

714

This peptide hormone, GSH may play a key regulatory role in the development of competent eggs and viable larvae in penaeid shrimp. It might be possible in the future to add back the GSH lost after eyestalk ablation, and thereby restore some endocrine control of shrimp reproduction after eyestalk ablation. Additionally, if the hormone was available, it might be used to stimulate reproduction without eyestalk ablation. In summary, this project was designed to evaluate the potential of both steroid and peptide hormones as regulators and stimulators of yolk protein synthesis in commercially important penaeid shrimp. Two well known steroid hormones, estradiol and progesterone can stimulate yolk protein production in vitro. A new peptide hormone from crayfish eyestalks also stimulates yolk protein production in oitro. Future work will determine if these hormones can be made to effect yolk protein production in intact whole shrimp in a hatchery situation. Acknowledgements-l thank Dr Albert0 Huberman, Instituto National de la Nutrition for providing peptide samples. This work was supported by the U.S. Dept of Commerce, NOAA, Florida-Sea Grant NA89AA-D-SGO53: RLRA 16 and RLRA 12 L.S.O. and bv the state of Florida Department of Agriculture, AMDkP grant 89200008 to L.S.Q. I would also thank Chris Collier, Donna Palchick and April Runnel for their help.

REFERENCES Aguilar M. B., Quackenbush L. S., Hunt D. T., Shabanowitz J. and Huberman A. (1992) Identification, purification and initial characterization of the vitellogenesis inhibiting hormone from the mexican crayfish, Procambarus bouvieri. Comp. R~ochem. Physioi. 1023, 491-498.

Chamberlain G. W., Haby M. G. and Miget R. J. (1985) Texas Shr~p Farming ~anauI, pp. I-lS8. Texas Agricultural Extension Service, Corpus Christi, Texas. Charniaux-Cotton H. (1985) Vitellogenesis and its control in malacostracan crustacea. Am. 2001. 25, 197-206. Cooke 1. M., Haylett B. and Weatherby T. (1977) Electrically elicited nemosecretory and electrical responses of the isolated crab sinus gland in normal and reduced calcium salines. J. exp. Biol. 70, 125-149. Eastman-Reks S. and Fingerman M. (1984) Effects of neuroendocrine tissue and cyclic AMP on ovarian growth in uivo and in virro in the fiddler crab, Uca pugilaror. Comp. Biochem. Physiol. 79A,6799684.

Fingerman M. (1987) Endocrine mechanisms in crustacea. .I. Crust. Biol. 7, l-24. Fyhn U. E. H., Fyhn H. J. and Costlow J. D. (1977) Cirripede vitellogenesis, effect of ecdysterone in vitro. Gen. camp. Endocr. 32, 266-277.

Liao C. I. and Chen Y. P. (1983) Maturation and spawning of penaeid prawns. In Handbook of Maricultnre: Crustacean Aquaculture (Edited by McVey J, P.), pp. 155-160. CRC Press, Boca Raton, Florida. Huberman A., Hernandez-Arana A., Augilar M. 8. and Rojo-Dominguez A. (1989) Secondary structure of a crustacean neuropeptide family by means of CD. Peptides 10, lll3~I115.

Kulkarni G. K., Glade L. and Fingerman M. (1991) Oogenesis and the effect of neuroendocrine tissue on in vitro synthesis of protein by ovary of the red swamp crayfish, Procambarus clarkii (Girard). J. Crust. Biol. 11, 5133522. Meusy J. J., Martin G., Soyez D., VanDeijnen J. E. and Gallo J. M. (1987) Immunochemical and immunocytochemical studies of the crustacean vitellogenesis inhibiting hormone (VIH). Gen. camp. Endocr. 67, 333-341. Primavera J. H. {198S) A review of the maturation and reproduction in closed thelycum penaeids. Proc. First Int. Conf Culture of Penaeid Prawn, Iloilo City, Philippines. Quackenbush L. S. (1986) Crustacean endocrinoiogy: a review. Can. J. Fish. Aqua. Sci. 43, 2271-2282. Quackenbush L. S. (1989a) Vitellogenesis in the shrimp, Penaeus vannamei. In vitro studies of the isolated hepatopancreas and ovary. Comp. Biochem. Physiol. 948, 253-261. Quackenbush L. S. (1989b) Yolk protein production in the marine shrimp, Penaeus vannamei. J. Crust. Biol. 9, 509-S 16. Rankin S. M., Bradfield J. Y. and Keeley L. L. (1989) Ovarian protein synthesis in the South American white shrimp, Penaeus vunnamei, during the reproductive cycle. Int. J. Reprod. Dev. IS, 27733. Sokal R. and Rohlf F. F. (1981) Biometry. Freeman Press, San Francisco. Tom M., Menachem G. and Ovadia M. (l987a) Purification and partial characterization ofvitellin from the ovaries of Parapenaeus longirostris. Comp. Biochem. Physiol. 87B, 17723. Tom M., Menachem G. and Ovadia M. (1987b) Localization of the vitellin and its possible precursors in various organs of Parapenaeus longirostris. Int. J. Invert. Reprod. Deu. 12, 1-12.

Yano I. and Chinzei Y. (1987) Ovary is the site of vitellogenin synthesis in Kuruma prawn, Penaeus japonicus. Comp. Biochem. Physiol. 86B, 2133218.