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Effects of cycloheximide on in vitro testosterone secretion from Rana catesbeiana ovaries
0300-9629/86$3.00 + 0.00 Pergamon Journals Ltd
Camp. Blochem. Physiol. Vol. 84A, No. 3, pp. 401403, 1986 Printed in Great Britain
EFFECTS OF CYCLOHEXIMIDE ON IN VITRO TESTOSTERONE SECRETION FROM RANA CATESBEIANA OVARIES GUYLAINE
M.
HUBBARD*
and
PAUL
LICHT
Department of Zoology and Group in Endocrinology, University of California, Berkeley,CA 94720, USA
(Received 11 October 1985) Abstract-l. A 1 hr exposure to 20 pg/ml of the protein synthesis inhibitor, cycloheximide (CHX), essentially abolished secretion of testosterone (T) by bullfrog ovarian fragments during simultaneous
administration of homologous pituitary extract and CHX. 2. Removal of CHX from the medium after 4 hr of treatment reversed the inhibition of T secretion, allowing it to attain control levels. 3. Pre-exposure of ovarian fragments to CHX was not required to obtain an inhibition of T secretion. 4. These data supported the hypothesis that protein synthesis is required for acute and chronic gonadotropic stimulation of steroidogenesis by the bullfrog ovary.
INTRODUCTION
Information on the cellular processes that link gonadotropin stimulation to the activation of steroidogenic enzymes and steroidogenesis is still limited. When rat adrenal glands were treated with cycloheximide (CHX), an inhibitor of protein synthesis, stimulation of corticosterone by adrenocorticotropic hormone was inhibited (Pearce et al., 1981). Protein synthesis is also necessary for follicular testosterone (T) production in rabbits (Losier and YoungLai, 1981), and a role for protein synthesis has been implicated in steroid production by rat Graafian follicles (Tsafriri et al., 1973), rat Leydig cells (Cooke et al., 1975) and hamster follicle cells (Greenwald and Limback, 1984). Among non-mammalian species, the dependence of steroidogenesis on protein synthesis has been shown only for chicken granulosa cells (Zakar and Hertelendy, 1980) and the salmon ovary (Nagahama et al., 1985). Thus, the generality of this phenomenon cannot be evaluated at this time. Accordingly, the present study seeks to shed light on whether protein synthesis is a general requirement for steroid secretion by the tetrapod ovary by examining its role in testosterone secretion by the bullfrog ovary. MATERIALS
AND METHODS
Female Rma catesbeiuna were purchased from a commercial supplier (Central Valley Biologicals). Frogs were maintained at 24°C in running tap water, fed mice and used within a 2 month period. Ovarian fragments (containing 20-30 large oocytes) were obtained by laparotomy under anesthesia, and placed into columns for superfusion as previously described (Hubbard and Licht, 1985). Ovaries used ranged from late stage III Gust prior to the separation of hemispheres) to stage V (preovulatory) (Penkala, 1978).
The general protocol involved superfusion of ovarian fragments with media (diluted Dulbecco’s MEM) or media containing 20 pg/ml cycloheximide (Sigma) for I hr, before addition of homologous pituitary extract for an additional 9 hr. The pituitary extract was prepared from a mixture of adult male and female whole pituitary glands, which were placed in amphibian physiological -saline consisting of 82.5 mM NaCI. 2.5mM KCl. l.OmM CaCl,. l.OmM MgCl,, 1.0 mM Na,HPO,, 5.0 AM HEPES, 3.8 GM NaOH at pH 7.4 (O-R2 solution: Wallace ef al., 1973). The glands were alternately homogenized at high speed (Ultra-Turrax, Janke and Kunkel) and centrifuged several times. A working solution of pituitary extract was made by diluting the stock solution in media to contain approximately 400 ng/ml equivalent immunoreactive luteinizing hormone (LH), as determined by homologous RIA (Daniels et al., 1977). The dose of CHX was chosen based on the functional dose used for inhibition of protein synthesis in the pituitary of a closely related species (Porter, 1985). To determine whether CHX effects were due to general, non-specific toxicity, recovery from CHX treatment was tested by its removal after 3 hr of concurrent administration with pituitary extract. Control tissues were superfused for 4 hr with medium alone prior to initiation of superfusion with pituitary extract. Protocols are summarized in Table 1. Ovarian fragments were boiled upon completion of superfusion to facilitate counting of the oocytes. Testosterone (T) is the major ovarian steroid secreted in the bullfrog; the amount secreted is more than an order of magnitude greater than that of estradiol (Licht et al., 1983). Fractions were analyzed for T content by RIA (Muller, 1977). Rates of T secretion (pg/oocyte/lO min fraction) in each experiment (in Figs 1 and 2) were normalized to a percentage of the maximal secretion rate observed in control tissues (Group 1, Table 1) because ovaries varied considerably in the absolute amount of T produced, due to inter-animal variation. Data were analyzed by the MANOVA test for repeated measures, ANOVA and Duncan’s multiple range test. When P > 0.05, differences were considered to be insignificant. RESULTS
An illustration of the marked stimulation of T secretion by pituitary extract alone (Group 1, Table 1) in control tissues is shown in Fig. 1. In contrast,
*Present address: Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA 92093, USA. 401
GUYLAINE
402
M. HUBBARDand PAUL LIGHT
Table I. General protocol for cycloheximide experiments Treatment group
1 2 3 4 5 A
-.---..I o-l A-DME CHX CHX A-DME A-DME
Time (hr) I4
.~_&IO
N
PE CHX + PE CHX+PE A-DME CHX+PE
PE CHX+PE PE PE -
IO 10 5 5 2
DME = amphibian (diluted) Dulbecco’s MEM; PE = homologous pituitary extract; CHX = Cycloheximide (20 &ml).
simultaneous administration of pituitary extract and CHX after a 1 hr exposure to 20 pg/ml CHX (Group 2, Table I) prevented T secretion over a 9 hr period (Fig. 1). Cycloheximide treatment alone resulted in non-detectable amounts of T as did superfusion with medium alone (data not shown). The effects of CHX on ovarian steroidogenesis (T secretion) were reversible (Group 3, Table 1) (Fig. 2). The initial response to pituitary extract after CHX was removed from the medium was delayed by approximately 45 min relative to that in the control tissue, but peak levels of T secretion 3-4 hr after the beginning of exposure to gonadotropin were not significantly different (P > 0.05) from that seen in controls (Group 4, Table 1). Several experiments were run to determine if preexposure to CHX was necessary to block T secretion (Group 5, Table 1). The results in Fig. 3 indicate that the effect of CHX is rapid, since there was no detectable T secretion when it was given concomitantly with pituitary extract. DISCUSSION
The results presented here indicate that there is a 100
801
g
ca
BO-
so-
d
=
IO-
;I (0
30-
O-
b
4
’
b
Tk
b
b
‘I
lb
b
IHRI
Fig. 2. Recovery of ovarian T secretion from cycloheximide treatment (0 = Group 3, Table 1) compared to an initial exposure to pituitary extract (A = Group 4, Table 1). The arrow indicates the time when cycloheximide was removed (0) and pituitary extract was introduced (A). Data show means and SEM from five experiments. Statistically significant (P < 0.05) differences between means are indicated by *. Non-detectable levels of T secretion are represented by a stimulation of 9% or less. (See Fig. 1 for definition of response units.)
requirement for protein synthesis for steroid secretion in the bullfrog ovary. There is no detectable T secretion from ovarian fragments exposed to CHX prior to, or in conjunction with, exposure to homologous pituitary extract. This finding suggests that gonadotropins stimulate at least one rapidly metabolized protein that is necessary for steroid synthesis. Ovarian fragments exposed to CHX for a total of 4 ht exhibit normal responsiveness (T secretion) to pituitary extract upon removal of CHX, indicating that its effects are not due to general toxicity to the tissue. The fact that there is no indication of T secretion when ovarian fragments are not exposed to CHX prior to concurrent administration of CHX and pituitary extract (Fig. 3) implies that the time course
BOIOBOW403020-
Fig. 1, Effects of cycloheximide (20 pg/ml) on gonadotropin stimulation of T secretion by R. cutesbeiuna ovarian fragments (A = Group 2, Table 1) and stimulation of T secretion by pituitary extract alone (0 = Group 1, Table 1). Ovarian fragments were pretreated for 1 hr with cycloheximide (A) before superfusion with a combination of cycloheximide and pituitary extract, indicated by the arrow. Controls (0) received similar stimulation without cycloheximide. The data are a composite of 10 experiments. Non-detectable levels of T are equivalent to 9% stimulation on the graph. Values for each experiment were normalized to a percentage of the maximal secretion rate of control tissues.
/
* 0 0.0
.
.
_
_
_
_
.
t 0:s
1:o
I.3
r:o
2:s
3:o
3:5
4'0
TIME IHRI 3. Effects of cycloheximide superfused concurrently with pituitary extract following pre-exposure only to media (A = Group 5, Table 1). Data for controls to the same dose of pituitary extract without cycloheximide are indicated by (0). Tissues were superfused continuously with hormone starting at 1 hr (see arrow). Data represent the means from two experiments. Non-detectable T levels are those < 3 pgfoocyte.
CHX
and testosterone
of its action is equal to or less than the time it takes for gonadotropin to exert an effect on T secretion. This also indicates that the protein(s) involved may not be stored within the cells and must be synthesized each time the ovary is stimulated to produce steroids. The existence of a putative regulatory protein with a short half-life has been suggested by other investigators of this phenomenon (rat follicles, Lieberman et al., 1975; rat testis, Cooke et al., 1975; chicken granulosa cells, Zakar and Hertelendy, 1980). It is not clear whether the required protein(s) are necessary for gonadotropic stimulation of CAMP or one of the steroidogenic enzymes, such as 3B-HSD, which has been suggested by Nagahama et al. (1985) for the salmon ovary. Another hypothesis suggests that gonadotropin stimulates the phosphorylation of a pre-existing protein and CHX blocks synthesis of this protein (Janszen et al., 1977; Losier and YoungLai, 1981). In summary, this study has shown that gonadotropic stimulation of steroidogenesis by the bullfrog ovary is sensitive to a protein synthesis inhibitor, cycloheximide, thereby suggesting a protein-synthetic requirement for acute steroid secretion in this species. The precise localization and role of the protein(s) involved remains to be elucidated. Acknowledgements-The authors would like to thank Dr Howard A. Bern and Dr Charles S. Nicoll for reviewing the manuscript during its preparation. In addition, we appreciate the assistance with assays by Mr Raymond Pang. This work was supported by NSF grant PCM 840651. REFERENCES Cooke B. A., Janszen F. H. A., Clotscher W. F. and Van Der Molen H. J. (1975) Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations. Biochem. J. 150, 413418. Daniels E., Licht P., Farmer S. W. and Papkoff H. (1977) Immunochemical studies on the pituitary gonadotropins (FSH and LH) from the bullfrog, Rana cafesbeiana. Gen. camp. Endocr. 32, 146157. Greenwald G. S. and Limback D. (1984) EtTects of treatment with cycloheximide at proestrus on subsequent in vitro follicular steroidogenesis in the hamster. Biol. Reprod. 30, 110551116. Hubbard G. M. and Licht P. (1985) In vitro study of the direct ovarian effects of gonadotropin releasing hormone (GnRH) in the frogs, Runa pipiens and Rana catesbeiuna. Gen. camp. Endocr. 60, 154161.
secretion
by frog ovaries
403
Janszen F. H. A., Cooke B. A. and Van Der Molen H. J. (1977) Specific protein synthesis in isolated rat testis Leydig cells: Influence of luteinizing hormone and cycloheximide. Biochem. J. 162, 341-346. Licht P., McCreery B. R., Barnes R. and Pang R. (1983) Seasonal and stress related changes in plasma gonadotropins, sex steroids and corticosterone in the bullfrog, Rana catesbeiana. Gen. camp. Endocr. 50, 124145. Lieberman M. F., Barnea A., Bauminger S., Tsafriri A., Collins W. P. and Lindner H. R. (1975) LH effect on the pattern of steroidogenesis in cultured Graafian follicles of the rat: Dependence on macromolecular synthesis. Endocrinology 96, 153331542. Losier A. J. and YoungLai E. V. (1981) Role of protein synthesis in rabbit follicular testosterone production. J. Steroid Biochem. 14, 285-293. Muller C. H. (1977) Plasma 5x-dihydrotestosterone and testosterone in the bullfrog, Rana catesbeiana: Stimulation by bullfrog LH. Gen. camp. Endocr. 33, 122-132. Nagahama Y., Young G. and Adachi S. (1985) Effect of actinomycin D and cycloheximide on gonadotropininduced 17a,20p-dihydroxy-4-pregnen-3-one production by intact follicles and granulosa cells of the amago salmon, Oncorhynchus rhodurus. Dec. Growth Dir. 27, 213-221. Pearce R. B., Cronshaw J. and Holmes W. N. (1981) Changes in corticotropic responsiveness and mitochondrial ultrastructure of adrenocortical cells from the inner zone of the duck (Anas piutyrhynchos) adrenal gland: The effects of cycloheximide, puromycin and chloramphenicol. Cell Tiss. Res. 221, 45-57. Penkala J. E. (1978) Some environmental and endocrine factors regulating ovarian cycling in Runa catesbeiana. M.S. Thesis, Louisiana State University, Baton Rouge. Porter D. A. (1985) The effects of cycloheximide on in vitro response of Rana pipiens pituitaries to continuously superfused gonadotropin-releasing hormone. Eioi. Reprod. 33, 393400. Porter D. A. and Licht P. (1985) Pituitary responsiveness to superfused GnRH in two species of ranid frogs. Gen. camp. Endocr. 59, 308-315. Tsafriri A., Lieberman M. E., Barnea A., Bauminger S. and Lindner H. R. (1973) Induction by luteinizing hormone of ovum maturation and steroidogenesis in isolated Graafian follicles of the rat: Role of RNA and protein synthesis. Endocrinology 93, 137881386. Wallace R. A., Jared D. W., Dumont J. N. and Sega M. W. (1973) Protein incorporation by isolated amphibian oocytes III. Optimum incubation conditions. J. exp. Zool. 184, 321-334. Zakar T. and Hertelendy F. (1980) Steroidogenesis in avian granulosa cells: Early and late kinetics of oLH- and dibutyryl cyclic AMP-promoted progesterone production. Biol. Reprod. 23, 974980.
Camp. Blochem. Physiol. Vol. 84A, No. 3, pp. 401403, 1986 Printed in Great Britain
EFFECTS OF CYCLOHEXIMIDE ON IN VITRO TESTOSTERONE SECRETION FROM RANA CATESBEIANA OVARIES GUYLAINE
M.
HUBBARD*
and
PAUL
LICHT
Department of Zoology and Group in Endocrinology, University of California, Berkeley,CA 94720, USA
(Received 11 October 1985) Abstract-l. A 1 hr exposure to 20 pg/ml of the protein synthesis inhibitor, cycloheximide (CHX), essentially abolished secretion of testosterone (T) by bullfrog ovarian fragments during simultaneous
administration of homologous pituitary extract and CHX. 2. Removal of CHX from the medium after 4 hr of treatment reversed the inhibition of T secretion, allowing it to attain control levels. 3. Pre-exposure of ovarian fragments to CHX was not required to obtain an inhibition of T secretion. 4. These data supported the hypothesis that protein synthesis is required for acute and chronic gonadotropic stimulation of steroidogenesis by the bullfrog ovary.
INTRODUCTION
Information on the cellular processes that link gonadotropin stimulation to the activation of steroidogenic enzymes and steroidogenesis is still limited. When rat adrenal glands were treated with cycloheximide (CHX), an inhibitor of protein synthesis, stimulation of corticosterone by adrenocorticotropic hormone was inhibited (Pearce et al., 1981). Protein synthesis is also necessary for follicular testosterone (T) production in rabbits (Losier and YoungLai, 1981), and a role for protein synthesis has been implicated in steroid production by rat Graafian follicles (Tsafriri et al., 1973), rat Leydig cells (Cooke et al., 1975) and hamster follicle cells (Greenwald and Limback, 1984). Among non-mammalian species, the dependence of steroidogenesis on protein synthesis has been shown only for chicken granulosa cells (Zakar and Hertelendy, 1980) and the salmon ovary (Nagahama et al., 1985). Thus, the generality of this phenomenon cannot be evaluated at this time. Accordingly, the present study seeks to shed light on whether protein synthesis is a general requirement for steroid secretion by the tetrapod ovary by examining its role in testosterone secretion by the bullfrog ovary. MATERIALS
AND METHODS
Female Rma catesbeiuna were purchased from a commercial supplier (Central Valley Biologicals). Frogs were maintained at 24°C in running tap water, fed mice and used within a 2 month period. Ovarian fragments (containing 20-30 large oocytes) were obtained by laparotomy under anesthesia, and placed into columns for superfusion as previously described (Hubbard and Licht, 1985). Ovaries used ranged from late stage III Gust prior to the separation of hemispheres) to stage V (preovulatory) (Penkala, 1978).
The general protocol involved superfusion of ovarian fragments with media (diluted Dulbecco’s MEM) or media containing 20 pg/ml cycloheximide (Sigma) for I hr, before addition of homologous pituitary extract for an additional 9 hr. The pituitary extract was prepared from a mixture of adult male and female whole pituitary glands, which were placed in amphibian physiological -saline consisting of 82.5 mM NaCI. 2.5mM KCl. l.OmM CaCl,. l.OmM MgCl,, 1.0 mM Na,HPO,, 5.0 AM HEPES, 3.8 GM NaOH at pH 7.4 (O-R2 solution: Wallace ef al., 1973). The glands were alternately homogenized at high speed (Ultra-Turrax, Janke and Kunkel) and centrifuged several times. A working solution of pituitary extract was made by diluting the stock solution in media to contain approximately 400 ng/ml equivalent immunoreactive luteinizing hormone (LH), as determined by homologous RIA (Daniels et al., 1977). The dose of CHX was chosen based on the functional dose used for inhibition of protein synthesis in the pituitary of a closely related species (Porter, 1985). To determine whether CHX effects were due to general, non-specific toxicity, recovery from CHX treatment was tested by its removal after 3 hr of concurrent administration with pituitary extract. Control tissues were superfused for 4 hr with medium alone prior to initiation of superfusion with pituitary extract. Protocols are summarized in Table 1. Ovarian fragments were boiled upon completion of superfusion to facilitate counting of the oocytes. Testosterone (T) is the major ovarian steroid secreted in the bullfrog; the amount secreted is more than an order of magnitude greater than that of estradiol (Licht et al., 1983). Fractions were analyzed for T content by RIA (Muller, 1977). Rates of T secretion (pg/oocyte/lO min fraction) in each experiment (in Figs 1 and 2) were normalized to a percentage of the maximal secretion rate observed in control tissues (Group 1, Table 1) because ovaries varied considerably in the absolute amount of T produced, due to inter-animal variation. Data were analyzed by the MANOVA test for repeated measures, ANOVA and Duncan’s multiple range test. When P > 0.05, differences were considered to be insignificant. RESULTS
An illustration of the marked stimulation of T secretion by pituitary extract alone (Group 1, Table 1) in control tissues is shown in Fig. 1. In contrast,
*Present address: Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA 92093, USA. 401
GUYLAINE
402
M. HUBBARDand PAUL LIGHT
Table I. General protocol for cycloheximide experiments Treatment group
1 2 3 4 5 A
-.---..I o-l A-DME CHX CHX A-DME A-DME
Time (hr) I4
.~_&IO
N
PE CHX + PE CHX+PE A-DME CHX+PE
PE CHX+PE PE PE -
IO 10 5 5 2
DME = amphibian (diluted) Dulbecco’s MEM; PE = homologous pituitary extract; CHX = Cycloheximide (20 &ml).
simultaneous administration of pituitary extract and CHX after a 1 hr exposure to 20 pg/ml CHX (Group 2, Table I) prevented T secretion over a 9 hr period (Fig. 1). Cycloheximide treatment alone resulted in non-detectable amounts of T as did superfusion with medium alone (data not shown). The effects of CHX on ovarian steroidogenesis (T secretion) were reversible (Group 3, Table 1) (Fig. 2). The initial response to pituitary extract after CHX was removed from the medium was delayed by approximately 45 min relative to that in the control tissue, but peak levels of T secretion 3-4 hr after the beginning of exposure to gonadotropin were not significantly different (P > 0.05) from that seen in controls (Group 4, Table 1). Several experiments were run to determine if preexposure to CHX was necessary to block T secretion (Group 5, Table 1). The results in Fig. 3 indicate that the effect of CHX is rapid, since there was no detectable T secretion when it was given concomitantly with pituitary extract. DISCUSSION
The results presented here indicate that there is a 100
801
g
ca
BO-
so-
d
=
IO-
;I (0
30-
O-
b
4
’
b
Tk
b
b
‘I
lb
b
IHRI
Fig. 2. Recovery of ovarian T secretion from cycloheximide treatment (0 = Group 3, Table 1) compared to an initial exposure to pituitary extract (A = Group 4, Table 1). The arrow indicates the time when cycloheximide was removed (0) and pituitary extract was introduced (A). Data show means and SEM from five experiments. Statistically significant (P < 0.05) differences between means are indicated by *. Non-detectable levels of T secretion are represented by a stimulation of 9% or less. (See Fig. 1 for definition of response units.)
requirement for protein synthesis for steroid secretion in the bullfrog ovary. There is no detectable T secretion from ovarian fragments exposed to CHX prior to, or in conjunction with, exposure to homologous pituitary extract. This finding suggests that gonadotropins stimulate at least one rapidly metabolized protein that is necessary for steroid synthesis. Ovarian fragments exposed to CHX for a total of 4 ht exhibit normal responsiveness (T secretion) to pituitary extract upon removal of CHX, indicating that its effects are not due to general toxicity to the tissue. The fact that there is no indication of T secretion when ovarian fragments are not exposed to CHX prior to concurrent administration of CHX and pituitary extract (Fig. 3) implies that the time course
BOIOBOW403020-
Fig. 1, Effects of cycloheximide (20 pg/ml) on gonadotropin stimulation of T secretion by R. cutesbeiuna ovarian fragments (A = Group 2, Table 1) and stimulation of T secretion by pituitary extract alone (0 = Group 1, Table 1). Ovarian fragments were pretreated for 1 hr with cycloheximide (A) before superfusion with a combination of cycloheximide and pituitary extract, indicated by the arrow. Controls (0) received similar stimulation without cycloheximide. The data are a composite of 10 experiments. Non-detectable levels of T are equivalent to 9% stimulation on the graph. Values for each experiment were normalized to a percentage of the maximal secretion rate of control tissues.
/
* 0 0.0
.
.
_
_
_
_
.
t 0:s
1:o
I.3
r:o
2:s
3:o
3:5
4'0
TIME IHRI 3. Effects of cycloheximide superfused concurrently with pituitary extract following pre-exposure only to media (A = Group 5, Table 1). Data for controls to the same dose of pituitary extract without cycloheximide are indicated by (0). Tissues were superfused continuously with hormone starting at 1 hr (see arrow). Data represent the means from two experiments. Non-detectable T levels are those < 3 pgfoocyte.
CHX
and testosterone
of its action is equal to or less than the time it takes for gonadotropin to exert an effect on T secretion. This also indicates that the protein(s) involved may not be stored within the cells and must be synthesized each time the ovary is stimulated to produce steroids. The existence of a putative regulatory protein with a short half-life has been suggested by other investigators of this phenomenon (rat follicles, Lieberman et al., 1975; rat testis, Cooke et al., 1975; chicken granulosa cells, Zakar and Hertelendy, 1980). It is not clear whether the required protein(s) are necessary for gonadotropic stimulation of CAMP or one of the steroidogenic enzymes, such as 3B-HSD, which has been suggested by Nagahama et al. (1985) for the salmon ovary. Another hypothesis suggests that gonadotropin stimulates the phosphorylation of a pre-existing protein and CHX blocks synthesis of this protein (Janszen et al., 1977; Losier and YoungLai, 1981). In summary, this study has shown that gonadotropic stimulation of steroidogenesis by the bullfrog ovary is sensitive to a protein synthesis inhibitor, cycloheximide, thereby suggesting a protein-synthetic requirement for acute steroid secretion in this species. The precise localization and role of the protein(s) involved remains to be elucidated. Acknowledgements-The authors would like to thank Dr Howard A. Bern and Dr Charles S. Nicoll for reviewing the manuscript during its preparation. In addition, we appreciate the assistance with assays by Mr Raymond Pang. This work was supported by NSF grant PCM 840651. REFERENCES Cooke B. A., Janszen F. H. A., Clotscher W. F. and Van Der Molen H. J. (1975) Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations. Biochem. J. 150, 413418. Daniels E., Licht P., Farmer S. W. and Papkoff H. (1977) Immunochemical studies on the pituitary gonadotropins (FSH and LH) from the bullfrog, Rana cafesbeiana. Gen. camp. Endocr. 32, 146157. Greenwald G. S. and Limback D. (1984) EtTects of treatment with cycloheximide at proestrus on subsequent in vitro follicular steroidogenesis in the hamster. Biol. Reprod. 30, 110551116. Hubbard G. M. and Licht P. (1985) In vitro study of the direct ovarian effects of gonadotropin releasing hormone (GnRH) in the frogs, Runa pipiens and Rana catesbeiuna. Gen. camp. Endocr. 60, 154161.
secretion
by frog ovaries
403
Janszen F. H. A., Cooke B. A. and Van Der Molen H. J. (1977) Specific protein synthesis in isolated rat testis Leydig cells: Influence of luteinizing hormone and cycloheximide. Biochem. J. 162, 341-346. Licht P., McCreery B. R., Barnes R. and Pang R. (1983) Seasonal and stress related changes in plasma gonadotropins, sex steroids and corticosterone in the bullfrog, Rana catesbeiana. Gen. camp. Endocr. 50, 124145. Lieberman M. F., Barnea A., Bauminger S., Tsafriri A., Collins W. P. and Lindner H. R. (1975) LH effect on the pattern of steroidogenesis in cultured Graafian follicles of the rat: Dependence on macromolecular synthesis. Endocrinology 96, 153331542. Losier A. J. and YoungLai E. V. (1981) Role of protein synthesis in rabbit follicular testosterone production. J. Steroid Biochem. 14, 285-293. Muller C. H. (1977) Plasma 5x-dihydrotestosterone and testosterone in the bullfrog, Rana catesbeiana: Stimulation by bullfrog LH. Gen. camp. Endocr. 33, 122-132. Nagahama Y., Young G. and Adachi S. (1985) Effect of actinomycin D and cycloheximide on gonadotropininduced 17a,20p-dihydroxy-4-pregnen-3-one production by intact follicles and granulosa cells of the amago salmon, Oncorhynchus rhodurus. Dec. Growth Dir. 27, 213-221. Pearce R. B., Cronshaw J. and Holmes W. N. (1981) Changes in corticotropic responsiveness and mitochondrial ultrastructure of adrenocortical cells from the inner zone of the duck (Anas piutyrhynchos) adrenal gland: The effects of cycloheximide, puromycin and chloramphenicol. Cell Tiss. Res. 221, 45-57. Penkala J. E. (1978) Some environmental and endocrine factors regulating ovarian cycling in Runa catesbeiana. M.S. Thesis, Louisiana State University, Baton Rouge. Porter D. A. (1985) The effects of cycloheximide on in vitro response of Rana pipiens pituitaries to continuously superfused gonadotropin-releasing hormone. Eioi. Reprod. 33, 393400. Porter D. A. and Licht P. (1985) Pituitary responsiveness to superfused GnRH in two species of ranid frogs. Gen. camp. Endocr. 59, 308-315. Tsafriri A., Lieberman M. E., Barnea A., Bauminger S. and Lindner H. R. (1973) Induction by luteinizing hormone of ovum maturation and steroidogenesis in isolated Graafian follicles of the rat: Role of RNA and protein synthesis. Endocrinology 93, 137881386. Wallace R. A., Jared D. W., Dumont J. N. and Sega M. W. (1973) Protein incorporation by isolated amphibian oocytes III. Optimum incubation conditions. J. exp. Zool. 184, 321-334. Zakar T. and Hertelendy F. (1980) Steroidogenesis in avian granulosa cells: Early and late kinetics of oLH- and dibutyryl cyclic AMP-promoted progesterone production. Biol. Reprod. 23, 974980.