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In vitro stimulation of protein synthesis in squid ovary by optic gland extract
GENERAL
AND
COMPARATIVE
ENDOCIUNOLOGY
32,248-251
(1977)
NOTE In Vitro Stimulation of Protein Synthesis Squid Ovary by Optic Gland Extract
in
An in vitro illecebrosus.
bioassay for octopus gonadotropin was modified for use with the squid, Illex An extract of squid optic glands of both sexes stimulated protein synthesis from 3H-leucine by squid ovary.
Wells and Wells (1959) showed that sex- waters from July to November. During this ual maturation in the octopus, Octopus vuf- time, many male squid become sexually gads, is controlled by the endocrine action mature but, even when the squid leave the Newfoundland area, the females have not of the optic glands. All cephalopods examof ined, with the exception of Nautilus, have yet begun the final, rapid development optic glands and in those species investithe ovary and very few eggs are vitellogenic. gated specifically for endocrine function, the optic glands are found to act in a fashion METHODS similar to those of 0. vufgaris (Durchon and Richard, 1967; Wells and Wells, 1972, The optic gland extract was prepared by homogenizing a known number and weight of glands 1975; Rowe, 1976). Wells et al. (1975) have developed an in in 0.025 M Hepes buffer, pH 7.7 [0.075 M NaCl, 0.1 mM dithiothreitol, 0.5 mM disodium EDTA (Idler er vitro bioassay for optic gland gonadotropin al., 1975)] for a final concentration of 2 optic glands/ using 0. vulgaris. Female 0. vulgaris were 100 ~1 buffer. The extract was stored at -70” until caused to mature precociously by cutting used. Optic glands from males and females collected on different dates were treated separately. Each exthe inhibitory nerve supply to the optic glands (Wells and Wells, 1959); the sub- tract used in an assay was prepared from the optic glands of 43-100 different animals of the same sex sequent release of gonadotropin stimulated and size group collected on the same date. vitellogenesis. Isolated groups of eggs and Ovaries for the assays were taken from female I. their follicle cells were incubated in the illecebrosus larger than 240 mm mantle length. The presence of optic gland extract and l*C- ovary of a single female was cut into small pieces and leucine. The uptake of leucine and the in- 20-30 mg portions were weighed and preincubated in medium. The nutrient medium used was as corporation of leucine into protein by the nutrient described in Wells et al. (1975) with Medium 100 refollicle cells were measured; changes in placed by Eagles minimum essential medium which these rates caused by the addition of optic contains less leucine. The number of incubates was 10 treated (OG+) and 10 control (OG-) except in gland extract to the incubation media Assay 2 where n was 6 and Assay 7 where n was 5. served as a biological assay for the optic At the end of the preincubation period, the medium gland gonadotropin. was drawn off and replaced either by fresh nutrient The present investigation was undermedium or by buffered seawater. The 3H-leucine and taken to determine if the optic gland extract aqueous optic gland extract or an equal amount of stimulates protein synthesis in the ovary of incubation medium were added at this time and the incubation period was begun. an oceanic squid, Illex illecebrosus. These incubation was complete a 200 4 ahquot of squid follow a migratory life cycle in the theWhen incubation medium was taken, mixed with 10 ml northwest Atlantic Ocean and, in most Aquasol scintillation fluid and counted with a Packard years, are found in Newfoundland coastal Tri-Carb scintillation spectrometer. The difference 248 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 00166480
NOTE between the initial counts and the ahquots represented uptake of the labeled amino acid from the medium by the tissue. The ovary tissue was placed in test tubes in dry ice, thawed, and homogenized in 0.5 ml distilled water and precipitated overnight in 10% trichloroacetic acid (TCA). The precipitate was washed three times in 5% TCA and the final precipitate was homogenized in 0.5 N saline, suspended in 10 ml Aquasol in scintillation vials and counted. Additional washing in TCA did not significantly change the counts. The total count was divided by the weight of the incubated tissue to give the incorporation of leucine into protein per milligram of ovarian tissue.
249
subpedunculate lobe of the brain, is routinely successful with Octopus (Wells and Wells, 1959, 1972). The animals, however, recover rapidly and completely from anaesthetic and the highly muscular nature of the mantle closes any wounds without the need of stitches. I. illecebrosus appear to be able to recover from light anaesthetic but to date have not survived surgery. The long preincubation time used in the I. illecebrosus assays was to allow for depletion of any endogenous optic gland horRESULTS AND DISCUSSION mone in the ovary. In Assays 6 and 7 (Table 1) the nutrient medium was changed from 4 The bioassay for Octopus gonadotropin (Wells et al., 1975) was carried out at 25”. to 6 times during the preincubation period; Exploratory assays with I. illecebrosus this procedure is known to enhance the rewere performed at 25” according to this sponse of endocrine tissue in vitro, e.g., Idler, 1973). technique (Assays 1-3, Table 1). Protein Incorporation of leucine into protein was synthesis should proceed more rapidly at this high temperature rather than at a more greater when ovary tissue was incubated in physiologically correct temperature. Howbuffered seawater rather than in nutrient ever, no significant differences in the rate of medium (Assay 2, Table 1). Wells et al. protein synthesis were observed between (1975) found that both leucine uptake and control and optic gland treated tissue and it incorporation into protein were lower when was decided to lower the temperature for eggs of 0. vulguris were preincubated in seawater than in nutrient medium (incubaboth the preincubation and incubation periods to lo” in subsequent assays. tion in nutrient medium) but a larger proportion of the radioactive leucine was inWells et al. (1975) used a preincubation period of 3 hr and an incubation period of 3 corporated into protein. In none of the assays with I. illecebrosus was there a sighr in the assays for Octopus gonadotropin. In the work with I. illecebrosus there was nificant increase in uptake of leucine with no significant effect of optic gland extract optic gland extract; however, incorporation on protein synthesis with a similar preincuof 3H-leucine into protein was significantly greater in optic gland treated incubates in bation time and a doubled incubation time (Assay 1, Table 1) and in subsequent assays both buffered seawater (Assay 5, Table 1) longer preincubation and incubation pe- and in nutrient medium (Assay 6, Table 1). Assays were carried out from September riods were used. The longer incubation thus, the ovaries used periods required to demonstrate an effect of to mid-November; optic gland extracts of 1. illecebrosus may were from progressively older animals. Albe, in part, a reflection of the fact that it though female I. illecebrosus are typically throughout the time was not technically feasible to stimulate the sexually immature optic glands of 1. illecebrosus by cutting the spent in Newfoundland waters the ability of inhibitory nerve supply. Stimulation of the the ovary to respond to optic gland extract optic glands in the Octopus resulted in ac- varied. The ovary in which protein synthesis was tively vitellogenic ovaries used in assays by Wells et al. (1975). Surgical manipulation of most significantly stimulated by optic gland the condition of the optic glands, either by extract (Assay 6, Table 1) was from an anisevering the optic nerve or by removing the mal which had been held in the laboratory,
3. 4. 5. 6. 7.
Hr 3 3 3 12 12 12 14 48
T(“)
25” 25” 25b 25* 10* 108 lobe 10”’
25 25 25 25 10 10 10 10
TO
Incubation
D Incubation in medium. * Incubation in buffered seawater. c Medium changed 4-6 times during preincubation. d Optic glands from 61 animals. e--0 Optic glands from 43, 49, 100 animals, respectively.
Ott 3175 Ott lo/76 Ott 31/75 Nov 18/75 Nov 8/76
1. Sept 23175 2. Sept 28175
Assay
Preincubation
6 15 15 15 15 32 24 24
Hr P, 0, 0, 0, 6, d, P, P,
early early early early early early Nov Nov
Septd Septd Septd Septd Sept” Sept’ 7“ 7”
Optic gland source
in Vitro
61 k 3.9 199 ” 31.7 482 k 53.3 356 k 40.2 466 2 45.3 176 -+ 13.6 80 f 3.7 1304 2 131.2
OG+
64 164 401 361 395 118 65 913
” 6.9 AZ 15.0 ” 47.5 k 24.3 k 31.3 f 19.6 k 3.4 -c 325.4
OG-
3H-Leucine incorporation (dpm/mg k SEM)
TABLE 1 THE EFFECT OF OPTIC GLAND EXTRACT ON PROTEIN SYNTHESIS BY THE OVARY OF I. illecebrosus
0.05 0.01 NS
NS NS NS NS NS
P
NOTE
251
without food, for 1 week. Squid held in the lar in female and male cephalopods (Wells laboratory for more than 4 days typically and Wells, 1975). With modifications apshow histological and physical signs of ac- propriate to the specific cephalopod being celerated reproductive development (Rowe investigated, the assay developed by Wells and Mangold, 1975; Rowe, 1976) and the et al. (1975) appears to be applicable to the ovary of this particular squid was likely assay of molluscan “gonadotropin.” more vitellogenic than those used in earlier assays. Similar results were obtained with ACKNOWLEDGMENTS another large, late season female I. illeceWe would like to thank Mr. James Devereaux and brosus kept in the laboratory for 5 days; the Mr. Damien Whitten for assistance in collecting the large standard error in the control incu- squid and the optic glands used in the study. bates, where n = S/group compared to n = 10 in Assays 5 and 6, prevented these reREFERENCES sults from being statistically significant Durchon, M., and Richard, A. (1967). Etude, en cul(Assay 7, Table 1). ture organotypique, du role endocrine de la The stimulation of protein synthesis by glande optique dans la maturation ovarienne chez Sepin oj&+nalis L. (Mollusque: Cephalopode). optic gland extract in Assay 5 can most C.R. Acad. Sci. 264, 1497-1500. likely be attributed to the longer incubation Idler, D. R. (1973). Comments on “Structure and time as compared to Assay 4. However, the Function of the Adrenal Gland of Fishes” by possibility that the female gonad was more David Gordon Butler. Amer. Zool. 13, 881-884. responsive in late October cannot be Idler. D. R., Bazar, L., and Hwang, S. J. (1975). Fish Gonadotropin(s). II. Isolation of gonadotropin(s) excluded. from chum salmon pituitary glands using affinity The optic glands used in the assays were chromatography. Endocrinol. Res. Commun. from squid captured on different dates. Ex2(3), 215-235. tract of optic glands of sexually mature Rowe, V. L. (1976). Reproductive development as a males stimulated leucine incorporation into function of growth and physiology in the squid, lllex illecebrosus Lesueur. PhD thesis, Memorial protein (Assay 5, Table 1) as did extract Univ. of Newfoundland. from the optic glands of large female squid V. L., and Mangold, K. (1975). The effect of collected on November 7, 1975 (Assay 6, Rowe,starvation on sexual maturation in 1lle.u illeceTable 1). brows (Lesueur) (Cephalopoda: Teuthoidea). J. Female I. illecebrusus must be very close Exp. Mar. Biol. Ecol. 17, 157-163. to the final onset of sexual maturation in the Wells, M. J., and Wells, J. (1959). Hormonal control of sexual maturity in Octopus. J. Exp. Biol. 36, late autumn. From histological examination l-33. of the ovaries of 1. illecebrosus at this time Wells, M. J., and Wells, J. (1972). Optic glands and it was seen that oocytes are surrounded the state of the testis in Octopus. Mar. Bebar,. with a thick follicular layer and that the Physiol. 1, 71-83. follicle cells are generally columnar and be- Wells. M. J., and Wells, J. (1975). Optic gland implants and their effects on the gonads of Ocginning secretory activity (Rowe, 1976). topus. .I. Exp. Biol. 62, 579-588. From the results of the assays it is seen Wells, M. J., O’Dor, R. K., and Buckley, S. K. L. that, by early November, the optic glands (1975). An in vitro bioassay for a molluscan goof female I. illecebrosus contain sufficient nadotropin. J. Exp. Biol. 62, 433-446. quantities of “gonadotropin” to stimulate V. LESLIE ROWE vitellogenesis but possibly have not reD. R. IDLER ceived the appropriate releasing stimulus. Marine Sciences Research Laboratory’ The significant increase in leucine incorpoUniversity of Netifoundland ration into protein by ovary tissue in the Memorial St. John’s, Neufoundland. Canada AIC 5S7 presence of extract from male I. ilfeceAccepted.February S. 1977 bvosus optic glands is additional evidence r M.S.R.L. Contribution No. 234. that the optic gland “gonadotropin” is simi-
AND
COMPARATIVE
ENDOCIUNOLOGY
32,248-251
(1977)
NOTE In Vitro Stimulation of Protein Synthesis Squid Ovary by Optic Gland Extract
in
An in vitro illecebrosus.
bioassay for octopus gonadotropin was modified for use with the squid, Illex An extract of squid optic glands of both sexes stimulated protein synthesis from 3H-leucine by squid ovary.
Wells and Wells (1959) showed that sex- waters from July to November. During this ual maturation in the octopus, Octopus vuf- time, many male squid become sexually gads, is controlled by the endocrine action mature but, even when the squid leave the Newfoundland area, the females have not of the optic glands. All cephalopods examof ined, with the exception of Nautilus, have yet begun the final, rapid development optic glands and in those species investithe ovary and very few eggs are vitellogenic. gated specifically for endocrine function, the optic glands are found to act in a fashion METHODS similar to those of 0. vufgaris (Durchon and Richard, 1967; Wells and Wells, 1972, The optic gland extract was prepared by homogenizing a known number and weight of glands 1975; Rowe, 1976). Wells et al. (1975) have developed an in in 0.025 M Hepes buffer, pH 7.7 [0.075 M NaCl, 0.1 mM dithiothreitol, 0.5 mM disodium EDTA (Idler er vitro bioassay for optic gland gonadotropin al., 1975)] for a final concentration of 2 optic glands/ using 0. vulgaris. Female 0. vulgaris were 100 ~1 buffer. The extract was stored at -70” until caused to mature precociously by cutting used. Optic glands from males and females collected on different dates were treated separately. Each exthe inhibitory nerve supply to the optic glands (Wells and Wells, 1959); the sub- tract used in an assay was prepared from the optic glands of 43-100 different animals of the same sex sequent release of gonadotropin stimulated and size group collected on the same date. vitellogenesis. Isolated groups of eggs and Ovaries for the assays were taken from female I. their follicle cells were incubated in the illecebrosus larger than 240 mm mantle length. The presence of optic gland extract and l*C- ovary of a single female was cut into small pieces and leucine. The uptake of leucine and the in- 20-30 mg portions were weighed and preincubated in medium. The nutrient medium used was as corporation of leucine into protein by the nutrient described in Wells et al. (1975) with Medium 100 refollicle cells were measured; changes in placed by Eagles minimum essential medium which these rates caused by the addition of optic contains less leucine. The number of incubates was 10 treated (OG+) and 10 control (OG-) except in gland extract to the incubation media Assay 2 where n was 6 and Assay 7 where n was 5. served as a biological assay for the optic At the end of the preincubation period, the medium gland gonadotropin. was drawn off and replaced either by fresh nutrient The present investigation was undermedium or by buffered seawater. The 3H-leucine and taken to determine if the optic gland extract aqueous optic gland extract or an equal amount of stimulates protein synthesis in the ovary of incubation medium were added at this time and the incubation period was begun. an oceanic squid, Illex illecebrosus. These incubation was complete a 200 4 ahquot of squid follow a migratory life cycle in the theWhen incubation medium was taken, mixed with 10 ml northwest Atlantic Ocean and, in most Aquasol scintillation fluid and counted with a Packard years, are found in Newfoundland coastal Tri-Carb scintillation spectrometer. The difference 248 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 00166480
NOTE between the initial counts and the ahquots represented uptake of the labeled amino acid from the medium by the tissue. The ovary tissue was placed in test tubes in dry ice, thawed, and homogenized in 0.5 ml distilled water and precipitated overnight in 10% trichloroacetic acid (TCA). The precipitate was washed three times in 5% TCA and the final precipitate was homogenized in 0.5 N saline, suspended in 10 ml Aquasol in scintillation vials and counted. Additional washing in TCA did not significantly change the counts. The total count was divided by the weight of the incubated tissue to give the incorporation of leucine into protein per milligram of ovarian tissue.
249
subpedunculate lobe of the brain, is routinely successful with Octopus (Wells and Wells, 1959, 1972). The animals, however, recover rapidly and completely from anaesthetic and the highly muscular nature of the mantle closes any wounds without the need of stitches. I. illecebrosus appear to be able to recover from light anaesthetic but to date have not survived surgery. The long preincubation time used in the I. illecebrosus assays was to allow for depletion of any endogenous optic gland horRESULTS AND DISCUSSION mone in the ovary. In Assays 6 and 7 (Table 1) the nutrient medium was changed from 4 The bioassay for Octopus gonadotropin (Wells et al., 1975) was carried out at 25”. to 6 times during the preincubation period; Exploratory assays with I. illecebrosus this procedure is known to enhance the rewere performed at 25” according to this sponse of endocrine tissue in vitro, e.g., Idler, 1973). technique (Assays 1-3, Table 1). Protein Incorporation of leucine into protein was synthesis should proceed more rapidly at this high temperature rather than at a more greater when ovary tissue was incubated in physiologically correct temperature. Howbuffered seawater rather than in nutrient ever, no significant differences in the rate of medium (Assay 2, Table 1). Wells et al. protein synthesis were observed between (1975) found that both leucine uptake and control and optic gland treated tissue and it incorporation into protein were lower when was decided to lower the temperature for eggs of 0. vulguris were preincubated in seawater than in nutrient medium (incubaboth the preincubation and incubation periods to lo” in subsequent assays. tion in nutrient medium) but a larger proportion of the radioactive leucine was inWells et al. (1975) used a preincubation period of 3 hr and an incubation period of 3 corporated into protein. In none of the assays with I. illecebrosus was there a sighr in the assays for Octopus gonadotropin. In the work with I. illecebrosus there was nificant increase in uptake of leucine with no significant effect of optic gland extract optic gland extract; however, incorporation on protein synthesis with a similar preincuof 3H-leucine into protein was significantly greater in optic gland treated incubates in bation time and a doubled incubation time (Assay 1, Table 1) and in subsequent assays both buffered seawater (Assay 5, Table 1) longer preincubation and incubation pe- and in nutrient medium (Assay 6, Table 1). Assays were carried out from September riods were used. The longer incubation thus, the ovaries used periods required to demonstrate an effect of to mid-November; optic gland extracts of 1. illecebrosus may were from progressively older animals. Albe, in part, a reflection of the fact that it though female I. illecebrosus are typically throughout the time was not technically feasible to stimulate the sexually immature optic glands of 1. illecebrosus by cutting the spent in Newfoundland waters the ability of inhibitory nerve supply. Stimulation of the the ovary to respond to optic gland extract optic glands in the Octopus resulted in ac- varied. The ovary in which protein synthesis was tively vitellogenic ovaries used in assays by Wells et al. (1975). Surgical manipulation of most significantly stimulated by optic gland the condition of the optic glands, either by extract (Assay 6, Table 1) was from an anisevering the optic nerve or by removing the mal which had been held in the laboratory,
3. 4. 5. 6. 7.
Hr 3 3 3 12 12 12 14 48
T(“)
25” 25” 25b 25* 10* 108 lobe 10”’
25 25 25 25 10 10 10 10
TO
Incubation
D Incubation in medium. * Incubation in buffered seawater. c Medium changed 4-6 times during preincubation. d Optic glands from 61 animals. e--0 Optic glands from 43, 49, 100 animals, respectively.
Ott 3175 Ott lo/76 Ott 31/75 Nov 18/75 Nov 8/76
1. Sept 23175 2. Sept 28175
Assay
Preincubation
6 15 15 15 15 32 24 24
Hr P, 0, 0, 0, 6, d, P, P,
early early early early early early Nov Nov
Septd Septd Septd Septd Sept” Sept’ 7“ 7”
Optic gland source
in Vitro
61 k 3.9 199 ” 31.7 482 k 53.3 356 k 40.2 466 2 45.3 176 -+ 13.6 80 f 3.7 1304 2 131.2
OG+
64 164 401 361 395 118 65 913
” 6.9 AZ 15.0 ” 47.5 k 24.3 k 31.3 f 19.6 k 3.4 -c 325.4
OG-
3H-Leucine incorporation (dpm/mg k SEM)
TABLE 1 THE EFFECT OF OPTIC GLAND EXTRACT ON PROTEIN SYNTHESIS BY THE OVARY OF I. illecebrosus
0.05 0.01 NS
NS NS NS NS NS
P
NOTE
251
without food, for 1 week. Squid held in the lar in female and male cephalopods (Wells laboratory for more than 4 days typically and Wells, 1975). With modifications apshow histological and physical signs of ac- propriate to the specific cephalopod being celerated reproductive development (Rowe investigated, the assay developed by Wells and Mangold, 1975; Rowe, 1976) and the et al. (1975) appears to be applicable to the ovary of this particular squid was likely assay of molluscan “gonadotropin.” more vitellogenic than those used in earlier assays. Similar results were obtained with ACKNOWLEDGMENTS another large, late season female I. illeceWe would like to thank Mr. James Devereaux and brosus kept in the laboratory for 5 days; the Mr. Damien Whitten for assistance in collecting the large standard error in the control incu- squid and the optic glands used in the study. bates, where n = S/group compared to n = 10 in Assays 5 and 6, prevented these reREFERENCES sults from being statistically significant Durchon, M., and Richard, A. (1967). Etude, en cul(Assay 7, Table 1). ture organotypique, du role endocrine de la The stimulation of protein synthesis by glande optique dans la maturation ovarienne chez Sepin oj&+nalis L. (Mollusque: Cephalopode). optic gland extract in Assay 5 can most C.R. Acad. Sci. 264, 1497-1500. likely be attributed to the longer incubation Idler, D. R. (1973). Comments on “Structure and time as compared to Assay 4. However, the Function of the Adrenal Gland of Fishes” by possibility that the female gonad was more David Gordon Butler. Amer. Zool. 13, 881-884. responsive in late October cannot be Idler. D. R., Bazar, L., and Hwang, S. J. (1975). Fish Gonadotropin(s). II. Isolation of gonadotropin(s) excluded. from chum salmon pituitary glands using affinity The optic glands used in the assays were chromatography. Endocrinol. Res. Commun. from squid captured on different dates. Ex2(3), 215-235. tract of optic glands of sexually mature Rowe, V. L. (1976). Reproductive development as a males stimulated leucine incorporation into function of growth and physiology in the squid, lllex illecebrosus Lesueur. PhD thesis, Memorial protein (Assay 5, Table 1) as did extract Univ. of Newfoundland. from the optic glands of large female squid V. L., and Mangold, K. (1975). The effect of collected on November 7, 1975 (Assay 6, Rowe,starvation on sexual maturation in 1lle.u illeceTable 1). brows (Lesueur) (Cephalopoda: Teuthoidea). J. Female I. illecebrusus must be very close Exp. Mar. Biol. Ecol. 17, 157-163. to the final onset of sexual maturation in the Wells, M. J., and Wells, J. (1959). Hormonal control of sexual maturity in Octopus. J. Exp. Biol. 36, late autumn. From histological examination l-33. of the ovaries of 1. illecebrosus at this time Wells, M. J., and Wells, J. (1972). Optic glands and it was seen that oocytes are surrounded the state of the testis in Octopus. Mar. Bebar,. with a thick follicular layer and that the Physiol. 1, 71-83. follicle cells are generally columnar and be- Wells. M. J., and Wells, J. (1975). Optic gland implants and their effects on the gonads of Ocginning secretory activity (Rowe, 1976). topus. .I. Exp. Biol. 62, 579-588. From the results of the assays it is seen Wells, M. J., O’Dor, R. K., and Buckley, S. K. L. that, by early November, the optic glands (1975). An in vitro bioassay for a molluscan goof female I. illecebrosus contain sufficient nadotropin. J. Exp. Biol. 62, 433-446. quantities of “gonadotropin” to stimulate V. LESLIE ROWE vitellogenesis but possibly have not reD. R. IDLER ceived the appropriate releasing stimulus. Marine Sciences Research Laboratory’ The significant increase in leucine incorpoUniversity of Netifoundland ration into protein by ovary tissue in the Memorial St. John’s, Neufoundland. Canada AIC 5S7 presence of extract from male I. ilfeceAccepted.February S. 1977 bvosus optic glands is additional evidence r M.S.R.L. Contribution No. 234. that the optic gland “gonadotropin” is simi-