GENERAL AND COMPARATIVE ENDOCRINOLOGY 59, 308-3 15 (1985)

Pituitary Responsiveness to Superfused GnRH in Two Species of Ranid Frogs

DAVID A. PORTER AND PAULLICHT

Department of Zoology, University qf‘Calijornia, Berkeley, California, 94720

Accepted November 20, 1984

Dynamics of pituitary responsiveness to gonadotropin-releasing hormone (GnRH) as mea- sured by LH and FSH secretion rates were examined for two species of ranid frogs in an in vitro superfusion system. The influence of sex was studied with juvenile bullfrogs, Rana catesbeiana, and responsiveness to long-term continuous superfusion with GnRH was ex- amined in R. catesbeiana and R. pipiens. Female juvenile bullfrogs showed virtually no response to doses of GnRH up to 1000 &ml, whereas males responded to doses of GnRH as low as 10 &ml, and very large increments in LH and FSH were observed with 100 and 1000 rig/ml GnRH (responsiveness in the males was dependent on body size). Adult R.

pipiens were more sensitive than bullfrogs to GnRH; 0.2 @ml GnRH was an effective dose. These data indicate that, relative to mammals, sensitivity to GnRH in frogs is higher than suggested by previous in vivo studies. As in mammals, “self-priming” in response to GnRH was evident; but unlike mammals, the “self-priming” occurred only after 7-12 hr of con- tinuous superfusion with GnRH, and the R. pipiens glands were more resistant to desen- sitization during 48 hr of GnRH treatment. This study confirms that sex- and age-dependent differences in responsiveness to GnRH as well as “self-priming” found in previous in vivo studies are evidently dependent on properties of the anterior pituitary per se. o 1985 Academic

Press, Inc.

Numerous aspects of the physiology of responsiveness to gonadotropin-releasing hormone (GnRH) in the amphibian have been characterized by in viva studies in the bullfrog, Rana catesbeiana. As in mam- mals, frogs infused with GnRH exhibited “self-priming” in terms of plasma levels of LH and FSH, but frogs were distinctive in retaining responsiveness to GnRH during long-term infusions or when treated with long-acting GnRH agonist (McCreery and Licht, 1983a, b). In vivo studies in the frog also demonstrated sexual and ontogenetic differences; males were more responsive than females to GnRH, and juveniles were far less sensitive to GnRH than adults (McCreery and Licht, 1984). Steroid effects on pituitary responsiveness were evident, but unlike mammals, it appears that the re- sponsiveness to GnRH is augmented by the androgen, Sa-dihydrotestosterone (DHT) rather than estrogens (McCreery and Licht, 1984).

Unfortunately, such in vivo studies do not allow accurate determination of what aspects of GnRH responsiveness are re- lated to direct effects at the level of the an- terior pituitary. Since plasma clearance rates for LH and FSH differ greatly be- tween gonadectomized and intact male R. catesbeiana (McCreery and Licht, 1983c), it is not clear whether dissimilar values of plasma LH and FSH necessarily imply dif- fering gonadotropin secretion rates. Also, in vivo approaches cannot discern if ob- served differences are at the level of the pituitary or due to differing steroidal milieu; and other factors peripheral to the pituitary and the involvement of endogenous hypo- thalamic factors are difficult to control.

To help resolve these problems, an in vitro approach was utilized in this study to examine directly the characteristics of pi- tuitary responsiveness to GnRH in the frogs R. pipiens and R. catesbeiana. Atten- tion was focused on sexual differences in

0016~6480/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

308

GnRH SUPERFUSION IN FROGS 309

pituitary responsiveness in young R. cates- beiana and studies in adult R. pipiens and R. catesbeiana focused on long-term con- tinuous superfusion with GnRH to study possible “self-priming” and “desensitiza- tion.” The overall objective was to deter- mine if previous in vivo results pertaining to sexual differences, self-priming and de- sensitization, and GnRH responsiveness could be demonstrated directly at the level of the pituitary.

MATERIALS AND METHODS

General Methods Animals and hormones. R. catesbeiana were ob-

tained from California and adult R. pipiens from Wis- consin from commercial suppliers. They were kept in running tap water at 22” and 12L: 12D, and fed crickets and small mice; body weights were maintained or in- creased. The GnRH used here was the same synthetic mammalian GnRH as used in previous in vivo studies with Rana in this laboratory.

In vitro superfusion system. Dulbecco’s modified Eagle’s medium (DME) diluted 2 to 1 with deionized water, containing 10 mM Hepes was used for all su- perfusions. Frogs were decapitated for removal of pi- tuitaries. Pituitaries (intact or fragments) were imme- diately transferred into siliconized columns made from disposable l-ml plastic syringes that had been trimmed to a total volume of 300 pl. A polyethylene mesh was fitted into the bottom of each column. The outflow of a peristaltic pump was connected to the bottom of the columns via a 20-gauge hypodermic needle. The sy- ringe plunger, through which another 20-gauge needle had been inserted, was placed in the column to give an effective column volume of 100 pl. The column superfusate was then directed toward fraction collec- tors. During superfusions, the columns were placed in water baths regulated at 25”, and medium to be super- fused was held in a refrigerated water bath at 4-5”. The medium warmed to within 0.5” of the column water bath temperature by the time it reached the column, even at flow rates far in excess of those used in the superfusions. Flow rates for the superfusions ranged between 50 and 75 pl/min.

Rate of responsiveness to the onset of GnRH. To determine the rate of responsiveness by pituitaries to GnRH, it was necessary to ascertain the clearance characteristics of the superfusion system. A 6-min bolus of trypan blue dye dissolved in distilled water was introduced at the same flow rate as used in the experiments. The bolus was followed by a rinse with distilled water, and clearance was measured by deter- mining O.D. at 500 nm.

Assessment of pituitary responsiveness. Gonado- tropin (LH, FSH) content of superfusates were deter- mined with double antibody RIAs homologous for R. catesbeiana gonadotropins (Daniels et nl., 1977) which have been validated for R. pipiens (Farmer et al., 1977). Potencies of LH and/or FSH were determined through the use of a logit-log computer program.

Histological procedure. To assess histological con- dition of tissues, samples superfused for periods of 8- 48 hr were fixed in Kamovsky’s fluid, postfixed in osmium tetroxide, dehydrated in propylene oxide, and embedded in Spurr’s plastic. Sections were cut at 1 km, and stained with hematoxylin and eosin.

Dose-response in adult male R. pipiens. Anterior pituitaries from adult males (26.7 + 1.32 g body wt) were bisected longitudinally, and each half was placed in a separate superfusion column. Following a 3-hr wash with DME alone, one hemipituitary was then treated with three different doses of GnRH in as- cending order, the other half gland with the same three doses in descending order. The mode of delivery was a 20-min treatment with GnRH followed by a lOO-min wash of DME alone before the next dose of GnRH was given.

Continuous superfusion of anterior pituitaries with GnRH. Half or whole anterior pituitaries of R. pipiens were placed in individual columns, and superfused ini- tially for 3 hr with DME alone. GnRH superfusion was then begun with 20 or 100 &ml. The length of super- fusion varied, and at times GnRH treatment was in- terrupted by periods of superfusion with DME alone. Limited tests with one subadult and three adult R. catesbeiana followed a similar protocol except that glands were cut into more pieces and doses of lOO- 200 &ml of GnRH were employed. In some experi- ments, hemipituitaries were superfused with DME alone to serve as additional controls.

Dose-response experiments of juvenile R. cates- beiana. Whole glands from subadult bullfrogs weighing < 100 g were placed individually in columns, with four glands superfused per experiment. Based on preceding results for R. pipiens, five doses of GnRH were chosen and delivered in ascending order. Each dose of GnRH was delivered for 6 mitt, followed by 54 min of DME superfusion (i.e., one dose for 6 min/hr).

RESULTS

Histological preparations studied under the light microscope indicated only small areas of centrally located necrotic tissue after over 24 hr of superfusion. The area of necrotic tissue gradually increased beyond 24 hr, so some decline in output of LH and FSH may be attributable to cell death.

310 PORTER AND LICHT

Hemipituitaries superfused only with DME showed no significant changes in go- nadotropin secretion following the first 3 hr of super-fusion. Turning off and restarting DME superfusion (in a manner resembling the procedure whereby doses of GnRH were changed during super-fusion) did not alter the basal secretion. Moreover, when- ever GnRH super-fusion was stopped and DME started, gonadotropin secretion in- variably declined. Therefore, to allow for treating hemipituitaries from the same gland in different manners, each pituitary served as its own control in the following tests.

Dose-Response in Adult Male R. pipiens

The mean output of LH from hemipitui- taries of male R. pipiens (N = 6) increased progressively for the three doses between 0.2 and 20.0 rig/ml GnRH (Fig. 1). How- ever, the magnitude of the response to each dose depended on the order in which it was administered: the response to 0.2 and 20.0 &ml tended to be greater when the dose was administered as the last (i.e., doses given in ascending order) rather than the first (descending order) in the sequence. For example, at the higher dose, LH levels in the first case were nearly twice those seen in the second (P = 0.053). Thus, the responses to the three doses cannot be treated independently of each other.

Continuous Superfusion of Anterior Pituitaries with GnRH

The output of LH with prolonged contin- uous GnRH treatment is shown in Figures 2 and 3 for male R. pipiens. Basal output varied between 40 and 209 rig/ml (variation was due in part to differences in flow rates and sizes of animals). With both doses of GnRH, rates of gonadotropin secretion in- creased within 10 min after initiation of GnRH superfusion, but LH output then gradually increased to peak values between 8 and 14 hr after the start of superfusion.

FIG. 1. Increment in LH output above baseline levels @g/ml) in response to three different doses of GnRH by R. p&ens hemipituitaries. For 0. I and 20.0 &ml GnRH, the first bar of the set of three indicates LH output when the particular dose is given first, the shaded bar indicates responsiveness when the dose is given last. The cross-hatched bar represents the mean of the frst and last bars. Vertical lines represent SEM.

With 20 rig/ml GnRH, initial output was maintained for several hours but the ratio of mean LH output (&ml) for hours lo- 12 of GnRH superfusion to mean output during the first 3 hr ranged from 1.3 to 1.8 (Fig. 2); similar temporal patterns were ob- served for FSH. In eight other experiments with this dose (data not shown), glands were shown to retain responsiveness to GnRH wet1 in excess of 24 hr with final levels of stimulation approximately equal to those obtained in the first few hours of

HRS OF GnRH SUPERFUSlOW

FIG. 2. LH (ngiml) output for whole anterior pitui- taries from five male R. pipiens continuously super- fused with 20 rig/ml GnRH for 17-20 hr.

GnRH SUPERFUSION IN FROGS 311

0 h 1 b b SUPER&ON

‘I2 i4 ‘15 HRS OF

FIG. 3. LH (t&ml) output for three male R. pipiens hemipituitaries continuously superfused with 100 ngl ml GnRH for 13 hr and then again for 1 hr following a 2-hr “rinse” with medium alone. Periods of GnRH treatment are indicated by horizontal bars above ab- scissa.

GnRH superfusion. Results were similar with 100 rig/ml GnRH, except for a more distinct depression in output after the first hour, and initial levels were not reached again until after 10 hr (Fig. 3).

When long-term GnRH superfusion was discontinued or interrupted by DME alone, LH and FSH output dropped rapidly. After the lower doses of GnRH, output quickly returned to baseline levels (Fig. 4), but this decline was retarded after high doses of GnRH (Fig. 3). In all cases, when the glands were challenged again with GnRH, the response was typically comparable to

HOURS OF SUPERFUSl on

FIG. 4. Representative levels of LH and FSH (ng/ ml) during long-term superfusion of one R. pipiens pi- tuitary with 20.0 rig/ml GnRH. Periods of GnRH treat- ment (22, 3, and 0.5 hr in duration) are indicated by the bold lines directly above the abscissa.

that observed at the start of the experiment and output rates often exceeded even the previous peak response (Fig. 3).

Pituitaries from three R. catesbeiana (two adult females and one juvenile male) super-fused for 14- 17 hr with 100 or 200 ng/ ml GnRH showed a temporal response pat- tern similar to that observed with the higher dose of GnRH in R. pipiens. Since the ab- solute levels of secretion were widely dis- parate (e.g., basal levels ranged between ~1 and 370 &ml) values were normalized on the basis of maximal secretion rates ob- served throughout the superfusion (Fig. 5). If anything, the decline and secondary peak in output, again after ca. 7- 12 hr, was more apparent in the bullfrogs than in R. pipiens. The mean LH output (&ml) for the last 3 hr of GnRH superfusion ranged from 1.2 to 8 times that measured during the first 3 hr.

Dose-Response of Juvenile R. catesbeiana

Mean rates of LH and FSH secretion (corrected for body weight to reduce inter- animal variation) for glands from six males and six females in response to increasing doses of GnRH are shown in Fig. 6. Males are clearly more responsive to GnRH than are females, and this divergence in respon- siveness between the sexes increases as

0 b i b b ’ i ’ 18 Ts HRS OF GnRH ;~PER;lJSIzi

FIG. 5. LH secretion by three R. cat~sbeiana an- terior pituitaries in response to continuous superfusion with 200 ngiml GnRH expressed as percentage of the maximum observed level of LH (&ml) for two adult females (0, +) and a juvenile male (A).

312 PORTER AND LICH’I

FSH

FIG. 6. Mean (six male, six female R. catesbeiana) values of peak LH and FSH output rate (&ml/kg) in response to five different doses of GnRH administered in ascending order; each dose consisted of a 6-min pulse followed by a .54-min “rinse” with medium alone. Males are indicated by the shaded bars; vertical lines represent SEM.

body weight increases (Fig. 7). The only cases in which the response of male R. ca- tesbeiuna to 1000 rig/ml GnRH approached the low values of females was with the two smallest (19.6 and a 21.4 g) animals (Fig. 7). This correlation of body weight with go- nadotropin output, although not statisti- cally significant (Y = 0.66, P = 0.156 for males, r = 0.02 for females) led to consid- erable variation among males.

Rate of Responsiveness to the Onset of GnRH The bullfrog glands responded more rap-

(1.01 a m

0 A 20 28

rJd Wfl d’(p) 2 60

FIG. 7. Peak rate of output of LH (ng/min/g) for six male (0) and six female (A) R. catesbeiana corrected for body weight in response to superfusion with 1000 rig/ml GnRH.

YI WUTES

FIG. 8. LH (ngiml) released from a typical male R. caresbeinna anterior pituitary in response to three dif- ferent doses of GnRH compared to the clearance pro- file of a 6-min bolus of trypan blue. The horizontal line below the x-axis represents the period of trypan blue delivery. 0 = 1000, A = 100, and 0 = 10 &ml GnRH. Line without symbols represents clearance pattern of trypan blue.

idly to the 1000 rig/ml than to 100 rig/ml dose (Fig. 8). The test of physical charac- teristics (turnover rate) of the system with trypan blue showed there was a 2-min lag time in the system from when a bolus enters the base of the super-fusion column to when it begins to appear at the fraction collector. Therefore, the rapid response phase to 1000 rig/ml GnRH began 4 min after the peak concentration of GnRH was expected to have been reached in the superfusion column.

DISCUSSION

The superfusion system described has proven effective for characterizing the dy- namic responses of frog anterior pituitaries to secretagogues, and thus offers the op- portunity to study the actions of GnRH at the level of the pituitary without the poten- tially confounding extrapituitary effects en- countered in vivo. The prolonged gonado- tropin output and histological condition in- dicate that frog tissues remain viable for at least 30-48 hr. We recognize that the use of pituitary fragments as opposed to dis- persed cells may have certain limitations in terms of sample repeatability and diffu- sional limitations, but has the advantages

GnRH SUPERFUSION IN FROGS 313

of maintaining attachment of cells to acel- lular substrates which facilitates retention of differentiated characteristics (Gospodar- wicz et al., 1978), preserving cell-cell con- tact to maintain normal function (Fletcher et al., 1976), and eliminating the prolonged preincubation periods often required after cell dispersion that may result in the al- tering of initial cellular conditions. Also, the use of tissue fragments facilitates the study of individual glands which is requisite for elucidating the effects of physiological state because the variable and unknown history of feral animals hinders control of physiological condition.

Sensitivity and Rate of Responsiveness of the Frog Glands to GnRH These results confirm that some of the

differences noted between mammals and frogs in vivo are indeed properties of the tissues while others may be an “artifact” of in vivo conditions (i.e., due to extrapi- tuitary factors). Previous in vivo studies suggested that the frog gland was less sen- sitive to GnRH than the mammalian gland (McCreery and Licht, 1983a), but the present results indicate only small differ- ences among these species. The doses em- ployed in this study are in the same range as those used in superfusions with rats in which 10 &ml produced submaximal re- sponsiveness by hemipituitaries (e.g., Waring and Turgeon, 1980). Thus, although juvenile R. catesbeiana may be somewhat less sensitive to GnRH than are rats, sen- sitivity of R. pipiens is comparable to rats, and certainly the disparity between anurans and mammals is less than was suggested by the infusion studies. This discrepancy be- tween in vivo and in vitro studies might be related to a higher clearance rate of GnRH in frogs than in mammals. It is not yet clear whether the greater sensitivity of R. pipiens compared to R. catesbeiana represents a fundamental interspecific difference or is related to age effects.

The time lag between onset of GnRH su- perfusion and appearance of elevated LH in the superfusate which exceeds that ac- counted for by the physical parameters of the superfusion system alone (Fig. 8), sug- gests a delay of ca. 4 min in the response of the glands to 1000 rig/ml GnRH. In rats, elevated LH levels are also seen within minutes (Evans et al., 1984; Smith and Vale, 1981; Turgeon and Waring, 1981; Waring and Turgeon, 1980). Since in mam- malian studies both the flow rates and the temperature (37”) used were higher than in the frog superfusions, the rate of onset of the initial response of the frog pituitaries does not appear to be appreciably slower than in mammalian pituitaries. The lower temperatures may affect the rate at which peak output of gonadotropin occurs. We cannot say with certainty whether the greater lag time in responsiveness to 1000 vs 100 rig/ml GnRH in R. catesbeiana rep- resents a dose-response effect or diffu- sional limitations.

Sex and Age Effects

Pituitaries from juvenile (20-60 g) R. ca- tesbeiana exhibit sexual differences in re- sponsiveness to in vitro superfusion with GnRH comparable to those seen in vivo (Figs. 6, 7). Furthermore, the smaller males were much less responsive than were larger animals, as in vivo (McCreery and Licht, 1984). These results establish that both sex and ontogenic related differences in respon- siveness to GnRH are due to substantial differences at the level of the anterior pi- tuitary.

Priming and Desensitization to GnRH Results for both acute and chronic re-

sponses of pituitaries to GnRH in vitro in- dicate that the tissues of both ranids were altered by previous exposure to GnRH. As observed in vivo, FSH and LH secretion rates typically paralleled one another, al- though the two hormones were not always stimulated to the same extent. In all 17

314 PORTER AND LICHT

adult R. pipiens tested, LH levels and re- sponses were higher than FSH as was ob- served with bullfrogs in vivo (McCreery et al., 1982; McCreery and Licht, 1983a, b, 1984); however, the FSH response in ju- venile bullfrogs in vitro was higher than ex- pected (cf. Fig. 6). Pituitary fragments treated with three different doses could not be treated as independent samples because responses were augmented by previous ex- posure to GnRH (cf. Fig. 1). A feature of the response to chronic exposures to GnRH in both species was gradual increase in LH and FSH secretion until ca. lo- 14 hr of su- perfusion (Figs. 2-5). Finally, responses to GnRH following a brief period of “rest” after long-term superfusion were markedly enhanced compared to previous levels of output (cf. Figs. 4, 5). Such results suggest GnRH “self-priming” of the frog pituitary tissues.

Self-priming by GnRH is well known for mammals (e.g., rats: Aiyer and Fink, 1974; Evans et al., 1984; Fink et al., 1976; Tur- geon and Waring, 1981; Waring and Tur- geon, 1980), and was previously suggested by in vivo studies with R. catesbeiana (McCreery and Licht, 1983a, b, 1984). The pattern and, in particular, the timing of the self-priming effects in vitro are similar to those observed with continuous in vivo in- fusion in bullfrogs (McCreery and Licht, 1983a, b). Thus, data from the present su- perfusions confirm that previous in vivo re- sults were indicative of direct effects of GnRH at the level of the anterior pituitary in the frog.

The ability of the bullfrog to show a pro- longed response to continuous GnRH in- fusion or injection of long-acting GnRH ag- onists distinguished it from mammals which typically show rapid desensitization to such GnRH treatments (McCreery et al., 1982; McCreery and Licht, 1983a). There is evi- dence for desensitization in the frog in vitro, but it is not like that seen in mam- mals. We believe the declining levels of LH and FSH observed after ca. lo-14 hr of

continuous GnRH superfusion (Fig. 4) are indicative of meaningful attenuation in re- sponsiveness to GnRH rather than deteri- oration of tissue. First, when GnRH super- fusion was interrupted and then restarted after these long periods, gonadotropin output returned to levels equivalent to those observed before the interruption; and second, the histological appearance of tis- sues after up to 48 hr of superfusion was generally indicative of viable cells. How- ever, gonadotropin output of frog glands never declined to pre-GnRH treatment values as in mammals. Marked desensiti- zation characterized by a decline of gonad- otropin output to “baseline” values has been observed in the rat after 12 hr of GnRH super-fusion (Smith and Vale, 1981), after 4-6 hr in rams (Bremner et al., 1976), and after 16 hr in the ewe (Chakraborty et al., 1974). In one R. pipiens pituitary LH and FSH output was still highly elevated (four- and eightfold, respectively) after 47 hr continuous GnRH superfusion (data not shown); this level of stimulation was greater than the initial response to GnRH. Thus, in general, the pituitary tissues of both ranids show greater resistance to de- sensitization than any of the mammalian species studied.

ACKNOWLEDGMENTS We thank Raymond Pang, Ken Finn, and Jeff Liu

for technical assistance. GnRH was generously pro- vided by Dr. J. Rivier of the Peptide Biology Labo- ratory, The Salk Institute for Biological Studies, La Jolla, Calif. This work was supported by NSF Grant PCM 840675 1.

REFERENCES Aiyer, M. S., and Fink, G. (1974). The role of sex

steroid hormones in modulating the responsive- ness of the anterior pituitary gland to luteinizing hormone releasing factor in the female rat. J. En- docrinol. 62, 553-572.

Bremner, W. .I., Findlay, .I. K., Cumming, 1. A., Hudson, B., and de Kretser, D. M. (1976). Pitu- itary-testicular response in rams to prolonged in- fusion of luteinizing hormone-releasing hormone (LHRH). Rio/. Reprod. 15, 141- 146.

GnRH SUPERFUSION IN FROGS 315

Chakraborty, P. K., Adams, T. E., Tamovsky, G. K., and Reeves, J. J. (1974). Serum and pituitary LH concentrations in ewes infused with LHRH/FSH- RH. J. Anim. Sci. 39, 1150-I 157.

Daniels, E. L., Licht, P., Farmer, S. W., and Papkoff, H. (1977). Immunochemical studies on the pitu- itary gonadotropins (FSH and LH) from the bull- frog, Rana catesbeiana. Gen. Camp. Endocrinol. 32, 146-157.

Evans, W. S., Uskavitch, D. R., Kaiser, D. L., Hell- mann, P., Borges, J. L. C., and Thomer, M. 0. (1984). The self-priming effect of gonadotropin- releasing hormone on luteinizing hormone re- lease: Observations using rat anterior pituitary fragments and dispersed cells continuously peri- fused in parallel. Endocrinology 114, 861-867.

Farmer, S. W., Licht, P., Papkoff, H., and Daniels, E. (1977). Purification of gonadotropins in the leopard frog (Rana pipiens). Gen. Comp. Endo- crinol. 32, 158-162.

Fink, G., Chiappa, S. A., and Aiyer, M. S. (1976). Priming effect of luteinizing hormone releasing factor elicited by preoptic stimulation and by in- travenous infusion and multiple injections of the synthetic decapeptide. J. Endocrinol. 69, 359- 372.

Fletcher, W. H., Anderson, N. C., Jr., and Everett, J. W. (1976). Extracellular communication in the rat anterior pituitary gland. An in vivo and in vitro study. .I. Cell Biol. 67, 469-476.

Gospodarwicz, Ci., Vlodavsky, I., Greenburg, G., and Johnson, L. K. (1978). Cellular shape is deter- mined by the extracellular matrix and is respon- sible for the control of cellular growth and func- tion. In G. Sato and R. Ross, eds., “Hormones and Cell Culture” pp. 561-592. Cold Spring Harbor Laboratory, Cold Spring Harbor.

McCreery, B. R., Licht, P., Barnes, R., Rivier, J., and Vale, W. (1982). Actions of agonistic and antago- nistic analogs of gonadotropin releasing hormone (Gn-RH) in the bullfrog, Rana catesbeiana. Gen. Camp. Endocrinol. 46, 51 l-520.

McCreery, B. R., and Licht, P. (1983a). Pituitary and gonadal responses to continuous infusion of go-

nadotropin releasing hormone in the male bull- frog, Rana catesbeiana. Biol. Reprod. 29, 129- 136.

McCreery, B. R., and Licht, I? (1983b). Induced ovu- lation and changes in pituitary responsiveness to continuous infusion of gonadotropin-releasing hormone (GnRH) during the ovarian cycle in the bullfrog, Rana catesbeiana. Biol. Reprod. 29, 863-871.

McCreery, B. R., and Licht, P. (1983~). Effects of go- nadectomy on polymorphism in stored and cir- culating gonadotropins in the bullfrog, R. cates- beiana. I. Clearance profiles. Biol. Reprod. 29, 637-645.

McCreery, B. R., and Licht, P. (1984). The role of an- drogen in the development of sexual differences in pituitary responsiveness to gonadotropin re- leasing hormone (GnRH) agonist in the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 54, 350-359.

Nansel, D. D., Aiyer, M. S., Meinzer, W. H., II, and Bogdanove, E. M. (1979). Rapid direct effects of castration and androgen treatment on luteinizing hormone-releasing hormone-induced luteinizing hormone release in the phenobarbitol-treated male rat: Examination of the role direct and in- direct androgen feedback mechanisms might play in the physiological control of luteinizing hormone release. Endocrinology 104, 524-531.

Smith, M. A., and Vale, W. W. (1981). Desensitization to gonadotropin-releasing hormone observed in superfused pituitary cells on cytodex beads. En- docrinology 108, 752-759.

Turgeon, J. L., and Waring, D. W. (1981). Acute pro- gesterone and 17B-estradiol modulation of lutein- izing hormone secretion by pituitaries of cycling rats superfused in vitro. Endocrinology 108, 413- 419.

Waring, D. W., and Turgeon, J. L. (1980). Luteinizing hormone releasing hormone-induced luteinizing secretion in vitro: Cyclic changes in responsive- ness and self-priming. Endocrinology 106, 1430- 1436.