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Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants
Comparative Biochemistry and Physiology Part A 136 (2003) 693–700
Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants Pei-San Tsaia,*, Suzanne M. Moenterb, Jason M. Cavolinac a
Department of Integrative Physiology, Campus Box 354, University of Colorado, 114 Clare Small, Boulder, CO 80309-0354, USA b Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA c Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269-4156, USA Received 13 June 2003; received in revised form 24 July 2003; accepted 25 July 2003
Abstract Gonadotropin-releasing hormone (GnRH) is critical for the initiation and maintenance of reproduction in vertebrates. Information regarding GnRH release is abundant in mammals, but absent in poikilothermic tetrapods. In this study, we established a novel GnRH enzyme immunoassay (EIA) to measure GnRH release over time from hypothalamic explants isolated from mature field-caught and commercially-acquired male bullfrogs, Rana catesbeiana. Hypothalamic explants from rats were used as a positive control to test the sensitivity and accuracy of our EIA and to ensure our in vitro system could detect GnRH pulses. Prominent GnRH pulses were present in the majority (9y10) of rat hypothalamic explants, but absent in all (17y17) of the commercial bullfrogs and the majority (5y8) of field-caught bullfrogs. In three cases where GnRH pulses were observed in field-caught bullfrogs, there was only one pulse during the 2-h incubation period; high-frequency pulses similar to those observed in rats were not observed. Veratridine, which opens voltagegated sodium channels, stimulated GnRH release in all explants cultured in the presence of Ca2q , demonstrating explant viability. The levels of both spontaneous and veratridine-induced GnRH release were significantly higher in field-caught than commercial bullfrogs. This study demonstrated, for the first time, the temporal pattern of GnRH release in a poikilothermic tetrapod. Further, our results suggest the levels and patterns of GnRH output in bullfrogs are subject to the dynamic regulation by physiological and environmental cues. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Bullfrog; Rat; Gonadotropin-releasing hormone release; Pulsatility; Enzyme immunoassay; Hypothalamic explants; Veratridine
1. Introduction Successful reproduction in vertebrates depends upon the action of a decapeptide, gonadotropinreleasing hormone (GnRH). In tetrapods, GnRH released from axon terminals in the median emi*Corresponding author. Tel.: q1-303-735-1877; fax: q1303-492-4009. E-mail address: [email protected] (P.-S. Tsai).
nence stimulates the secretion of gonadotropins from the pituitary. Gonadotropins then act as primary regulators of gonadal functions. As an upstream neuroendocrine regulator of reproductive function, GnRH neurons are under the influence of diverse factors and respond to changing physiological and environmental conditions. The proper level and pattern of GnRH secretion ensure the proper progression of reproduction.
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00221-6
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The pattern and regulation of GnRH secretion have been extensively studied in a number of vertebrates, most notably in mammals. However, there are significant gaps in our knowledge regarding the secretion of GnRH in poikilothermic tetrapods. The majority of studies on GnRH in amphibians and reptiles focused on the characterization of GnRH actions and identification of molecular forms of GnRH, with little emphasis on the release of GnRH from the neuronal terminals. Studies that examined changes in the amphibian or reptilian GnRH system in relation to the reproductive status often measured GnRH contents in brain regions (Tsai and Licht, 1993a; Li and Lin, 2000) or the cytology of GnRH neurons (Iela et al., 1994). These measurements, although providing some insights into release, often lead to ambiguous interpretations. For instance, a reduction in GnRH content could be interpreted as enhanced release because rapid release depletes GnRH stores. Conversely, it could be interpreted as attenuated release because the pool of GnRH available for release is smaller. To our knowledge, the secretion of GnRH in a poikilothermic tetrapod has never been directly measured. In this study, we established a novel enzyme immunoassay (EIA) to measure the in vitro GnRH release from hypothalamic explants isolated from sexually mature male bullfrogs, Rana catesbeiana. This EIA employs an O-biotinylated native GnRH tracer that can be easily generated in the laboratory within hours. Further, the EIA is easy to perform and has the sensitivity that rivals, even exceeds, the conventional GnRH radioimmunoassay (RIA). We utilized this EIA to quantify the in vitro secretion of GnRH in sexually mature bullfrogs freshly caught from the field during the breeding season, and for comparison, those from a commercial supplier during the non-breeding season. In addition, we also measured GnRH release from rat hypothalamic explants to ensure our EIA possesses the sensitivity, precision and consistency for measuring GnRH release over time in a species with well-documented GnRH release patterns (Bourguignon and Franchimont, 1984; Bourguignon et al., 1989). Our results demonstrate the establishment of a highly sensitive and novel EIA, and for the first time, the direct measurement of GnRH release over time in a poikilothermic vertebrate.
2. Methods 2.1. Animals All experimental procedures complied with animal protocols approved by the Institutional Animal Care and Use Committee at the University of Connecticut and University of Colorado. 2.1.1. Frogs Mature male bullfrogs were obtained from two sources. Commercially-available frogs were purchased during the non-breeding season (March– May) from Connecticut Valley Biological (Southampton, MA). These frogs were kept under 12L:12D photoperiod at the University of Connecticut Frog Facility, and fed newborn rats every week. All commercial animals were euthanized within 2 weeks of arrival. Field-caught bullfrogs were collected in June and July of 1999 from the upper Bolton Lake in Vernon, CT, and quickly transported to the University of Connecticut in holding sacks. Field-caught frogs were euthanized within 3 h of capture for hypothalamic incubation. All field-caught frogs were chorusing at the time of capture, indicating sexual maturity and reproductive readiness. Male frogs from both sources had an average gonadal somatic index (GSI; milligram gonad weightygram body weight) of 1.65 and had well-developed thumb pads. The observation that GSI did not differ between breeding and non-breeding bullfrogs was consistent with a previous observation of Licht et al. (1983), when they reported little monthly variations in testis weight of wild bullfrogs throughout the year. When testicular histology of representative animals were examined, all stages of germ cells were found in testes of both groups, as reported previously (Licht et al., 1983), although breeding frogs had more cysts containing primary spermatocytes in the process of meiotic division (data not shown). 2.1.2. Rats Two- to 6-month-old mature male Wistar rats were obtained from the University of Connecticut breeding colony. They were kept under 12L:12D photoperiod and fed water and rat chow ad libitum. 2.2. In vitro incubation of hypothalamic explants 2.2.1. Incubation media Both Ca2q-free and Ca2q-supplemented incubation media were prepared from the same base
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medium, a Ca2q-free Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Grand Island, NY). For culturing rat explants, DMEM was supplemented with 10 mM HEPES, 0.01% BSA, 1 mM pyruvic acid, and 4 mM L-glutamine. DMEM was diluted with an additional 50% water and given the same supplements for the culture of frog explants. When required, both mammalian and frog media were supplemented with 1.85 mM CaCl2. All media were sterilized by filtration through 0.22 mm filters and the pH adjusted to 7.4. All media supplements were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2.2. Creation of explants Hypothalamic explants from frogs and rats were obtained immediately after decapitation of animals. Frog hypothalamic explants were created by four cuts: a coronal cut 2-mm rostral to the optic chiasm, a coronal cut on the caudal border of the optic tectum, and two sagittal cuts along the lateral margins of the median eminence. Rat hypothalamic explants were created by five cuts: a coronal cut 2-mm rostral to the optic chiasm, a coronal cut on the rostral border of the mammillary bodies, two sagittal cuts along the lateral sulci and a transverse cut 2-mm dorsal to the median eminence. GnRH immunocytochemistry confirmed both rat and bullfrog explants contain abundant GnRH neuronal perikarya and terminals (data not shown). 2.2.3. Hypothalamic incubation All explants were pre-incubated for 30 min at 37 8C (for rats) and 28 8C (for bullfrogs) to establish baseline GnRH secretion. Following preincubation, hypothalamic incubations were carried out for an additional 125 min, during which incubation medium was collected every 5 min. Hypothalamic explants were incubated in 24-well plates or borosilicate glass tubes in their appropriate media. Rat explants were incubated in 500 ml Ca2q-supplemented mammalian culture medium pregassed with 95% O2:5% CO2. Some rat explants were incubated in Ca2q-free medium containing 5 mM ethylene glycol-bis-(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid (EGTA), a calcium chelator, to remove residual Ca2q from the medium. Frog explants were incubated in 350 ml Ca2q-supplemented frog culture medium pregassed with 95% O2:5% CO2. All explants were given a challenge of 20 mM veratridine (an agent that opens voltage-gated Naq channel) after the
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125-min incubation period for 10–20 min to test tissue viability. All samples were snap frozen on dry ice and stored at y70 8C until GnRH measurement by EIA. 2.3. EIA 2.3.1. Generation and purification of O-biotinylated GnRH tracer The procedure for O-biotinylating GnRH was adopted from Miller et al. (1992). Briefly, a 10:1 molar ratio of sulfosuccinimidyl-6-(biotinamido) hexanoate (biotinylation reagent, Vector Laboratories, Burlingame, CA) and mammalian GnRH (mGnRH, Peninsula Laboratories, Belmont, CA) were dissolved in 50 mM sodium bicarbonate. The solution was vortexed and left at room temperature for 17 min with occasional mixing. The reaction was stopped by the addition of trifluoroacetic acid (TFA, 0.1% final concentration), filtered, and injected through a 200 ml injection loop into a Waters Symmetry C18 high-performance liquid chromatography (HPLC) column (3.9=150 mm, particle size 5 mm). The solvent gradient was delivered by Waters 515 HPLC pumps and the UV absorbance monitored by a Waters 2487 dual wavelength detector. The mobile phase was 0.05% TFA in acetonitrile with a flow rate of 1 mlymin. The separation program consisted of an initial isocratic elution of 15% acetonitrile for 10 min, followed by a linear gradient to 40% acetonitrile in the next 24 min. Using this program, three biotinylated forms of GnRH (serine-biotinylated, tyrosine-biotinylated, and serine, tyrosine-biotinylated) were isolated. Further analysis revealed that all three forms of biotinylated GnRH were functional as tracers; however, serine-biotinylated GnRH resulted in the best assay sensitivity (data not shown). Thus, serine-biotinylated GnRH was used as the tracer for all subsequent EIAs. 2.3.2. Incubation and color development Ninety-six well Immulon 4 HB plates (Dynex, Chantilly, VA) were pre-coated with 100 ml of 5 mgyml donkey anti-rabbit antibody (Jackson Immuno Research, West Grove, PA) in 0.1 M sodium carbonate (pH 9.6) for 8 h at room temperature. The wells were washed 6 times with EIA buffer (0.01 M phosphate buffer, 0.15 M NaCl, 0.1% Tween 20) and coated with 100 ml of the anti-GnRH antiserum (R1245, generously provided by Dr Terry Nett, Colorado State University,
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1:200 000) overnight at 4 8C. This antiserum detects specifically the mammalian form of GnRH (mGnRH), which is the native hypothalamicpreoptic form in R. catesbeiana (Wang et al., 2001). Following well washing, samples and standards (100 ml) were added to the wells and the incubation allowed to proceed for an additional 24 h at 4 8C. After 24 h, 50 ml of HPLC-purified Obiotinylated native GnRH (1:40 000) was added to the wells and allowed to incubate for an hour at room temperature. The wells were washed extensively, and 100 ml of avidin D-horseradish peroxidase (1:5000, Vector Laboratories) was added and allowed to incubate at room temperature for 1 h. The wells were washed extensively and 100 ml of 3,39,5,59-tetramethylbenzidine (Vector Laboratories) was added and the incubation allowed to proceed for 30–60 min. The color reaction was stopped by the addition of 100 ml of 1 N H2SO4. The plates were read at 450 nm using an ELISA plate reader. Standard curves and sample doses were calculated using the four-logistics parameter curve fit. 2.4. Analysis GnRH pulses were identified by the computer algorithm Cluster analysis developed by Velduis and Johnson (1986). A constant coefficient of variation was set for each experiment. Other parameters included a 1=1 cluster configuration and a t-statistic value of 3.76 for the upstroke and 1 for the down stroke to achieve a 1% false positive rate. For the comparison between two groups, the Mann–Whitney U test was used. Differences between groups were considered significant when P-0.05. 3. Results Our initial validation of the GnRH EIA revealed the assay was very specific to mGnRH, the native form of GnRH present in the bullfrog hypothalamic-preoptic area (Wang et al., 2001). The crossreactivities of this EIA to chicken-I (cGnRH-I), chicken-II (cGnRH-II), lamprey I (lGnRH-I), salmon (sGnRH) and tunicate I (tI-GnRH) forms of GnRH were 0.32, 0.1, 0.17, 0.16 and less than 0.01%, respectively. In addition, the EIA matched the sensitivity of even the most sensitive GnRH RIAs, with detectability in the range of 0.1 pgy well. ED80, ED50 and ED20 of the EIA were 0.22
Fig. 1. A representative EIA standard curve using the serinebiotinylated GnRH as the tracer. Percent ByB0s% (boundytotal bound). For the standard, mGnRH was used.
pgywell, 1.62 pgywell and 9.06 pgywell, respectively. Intra- and inter-assay coefficients of variation were 13.82"9.9% and 7.4%"3.0%, respectively. A representative EIA standard curve is presented (Fig. 1). To verify the effectiveness of the EIA in detecting GnRH release over time in our in vitro incubation system, hypothalamic explants from reproductively mature male rats were used as positive controls. Ninety percent (9y10) of rat hypothalamic explants studied, including ones cultured in Ca2q-free medium, released GnRH in discrete pulses that were detectable by Cluster analysis (Fig. 2). In the presence of Ca2q (Fig. 2a,b), the level of GnRH secreted by the explants was approximately 6–10 times higher than the level secreted in the absence of Ca2q (Fig. 2c,d). The mean interpulse interval was 29.2"12.6 min, and the average duration and amplitude of pulses were 16.75"2.7 min and 32.2"9.3 pgyml, respectively. Explants were challenged with veratridine at the end of each experiment to test tissue viability. All explants cultured in the presence of Ca2q responded to veratridine (Fig. 2a, b, insets). In the absence of Ca2q, however, the veratridine response was abolished or significantly reduced (Ps0.04, ns9; Fig. 2c, d, insets). Overall, incubation in Ca2q-free medium decreased pulse amplitude but had no significant effect on either the pulse frequency or duration.
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(data not shown). All bullfrogs, commercial and field-caught, responded robustly to a veratridine challenge at the end of the experiment. The mean spontaneous GnRH secretion and GnRH secreted during the first 5 min of veratridine challenge were significantly higher in the field-caught bullfrogs than in commercially-purchased bullfrogs (Fig. 4a, b). 4. Discussion
Fig. 2. The temporal pattern of GnRH secretion from four representative male rat hypothalamic explants incubated in the presence (a, b) and absence (c, d) of Ca2q. *Significant peaks defined by cluster analysis. Insets: responsiveness of explants to a 20 mM veratridine challenge. Black horizontal bars in insets represent the duration of veratridine challenge. Note that multiple significant peaks were detected in rat explants.
Measurements of GnRH released from bullfrog hypothalamic explants (Fig. 3) revealed that the spontaneous GnRH secretion in the field-caught bullfrog was approximately 6–10 times lower than rats. GnRH secretion was detectable in all (8y8) field-caught bullfrogs, but pulses were detected in only three of these frogs (Fig. 3a, b, c). In each case, only a single significant pulse was detected over the 2-h incubation period, a pattern that differs markedly from the pattern of pulses observed in rats (Fig. 2). In explants from commercially-purchased bullfrogs (ns17), spontaneous GnRH release was not consistently detectable with our EIA, and none of the explants exhibited GnRH pulsatility detectable by Cluster analysis
We have established a sensitive GnRH EIA with the precision and accuracy needed to measure GnRH release from vertebrate hypothalamic explants. For the first time, we investigated the levels and temporal pattern of GnRH release in a reproductively mature poikilothermic tetrapod, the bullfrog, R. catesbeiana. Overall, GnRH release in field-caught breeding bullfrogs differs from rats in two respects. First, bullfrog GnRH release was 6– 10 times lower than rats. Second, the prominent GnRH pulses observed in rats were rarely seen in bullfrogs. These results suggest the levels and patterns of GnRH release required for the proper stimulation of pituitary gonadotropin secretion may vary substantially from species to species. To our knowledge, our GnRH EIA was the first to employ an O-biotinylated GnRH as a homologous tracer. Currently, the most widely used GnRH EIAs employ an N-biotinylated D-Lys6-GnRH as the tracer (Li et al., 1994; Maurer and Wray, 1999). The D-Lys6-GnRH analogue was chosen as the substrate for biotinylation because the native GnRH, with its blocked amino terminal, could not be N-biotinylated. D-Lys6-GnRH was one of the few GnRH analogues containing a free amine group available for N-biotinylation. However, the use of this heterologous GnRH tracer results in a considerably less sensitive assay (Maurer and Wray, 1999). An alternative to N-biotinylation was reported by Miller et al. (1992), in which successful O-biotinylation of native GnRH occurred on either serine or tyrosine residues. This tracer yielded a highly specific EIA with a sensitivity that rivals the most sensitive of GnRH RIAs. The ease of O-biotinylating and purifying the biotinylated products, as well as the sensitivity and specificity of this tracer in our EIA make it an attractive alternative to the use of enzyme-coupled tracer, biotinylated GnRH analogues and radioactive assays.
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Fig. 3. The temporal pattern of GnRH release from the hypothalamic explants of three representative male field-caught breeding bullfrogs (a, b and c). *Significant peaks defined by cluster analysis. Insets: responsiveness of explants to a 20 mM veratridine challenge. Black horizontal bars in insets represent the duration of veratridine challenge. A single GnRH pulse was detected by cluster analysis in each of the three explants.
The characteristics of pulsatile GnRH release from rat explants in our study are similar to those reported previously for adult male rats, with GnRH interpulse intervals averaging approximately 30 min (Bourguignon et al., 1989; Rasmussen, 1993; Purnelle et al., 1997). In sharp contrast with rats, GnRH pulses were not detected using the same analysis criteria in explants from non-breeding commercial frogs and in all but three explants from breeding field-caught frogs, which each exhibited a single pulse. The most-likely explanation is that GnRH pulse frequency in bullfrogs is so low that the 2-h sampling window used was insufficient to observe multiple pulses. In this regard, such low pulse frequencies have been reported in mammals. Caraty and Locatelli (1988) reported a frequency of one GnRH pulse every 2– 4 h in intact adult male rams, and even lower frequencies have been observed in luteal phase ewes and ewes during the non-breeding season (Moenter et al., 1991; Barrell et al., 1992). Of further interest, GnRH pulses were observed in three out of eight (37.5%) explants taken from field-caught breeding bullfrogs and none (0y17) from commercial non-breeding bullfrogs, suggesting the presence of discrete GnRH pulses might be a phenomenon uniquely associated with reproductive activation. Unfortunately, we had to limit our incubation period to 2 h because the failure of bullfrog explants to respond consistently to a veratridine challenge after 3 h in culture indicated viability was beginning to decline at this time (data not shown). The low number of bullfrogs
exhibiting GnRH pulses and the presence of only a single pulse during the incubation precludes general conclusions on the pulsatile characteristics of GnRH release in these animals. Nevertheless, these results raise the tantalizing possibility that bullfrog GnRH release during the reproductive season, like mammals, might be episodic. It was well established that pituitaries of mammals, birds, some fishes and reptiles became rapidly desensitized to continuous exposure to GnRH, resulting in decreased gonadotropin secretion (Belechetz et al., 1978; Nett et al., 1981; Smith and Vale, 1981; Licht and Porter, 1985; King et al., 1986; Habibi, 1991; Tsai and Licht, 1993b). To maintain elevated gonadotropin output in these systems, GnRH must be applied in a pulsatile fashion. The pulsatile release of GnRH into hypothalamic–pituitary portal circulation has been extensively documented in mammals (cf. Carmel et al., 1976; Clarke and Cummins, 1982). In marked contrast to mammals, the episodic mode of GnRH stimulation is not required for the maintenance of gonadotropin secretion in ranid frogs. In the bullfrog, plasma gonadotropin levels remained elevated for at least 4 days in response to constant GnRH infusion (McCreery and Licht, 1983). Most unexpectedly, continuous GnRH stimulation was much more effective than pulsatile GnRH administration in stimulating in vitro gonadotropin secretion from bullfrog pituitaries (Porter and Licht, 1986). Such unusual preference of bullfrog pituitaries for continuous GnRH stimulation suggests the secretory dynamics of GnRH in
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Fig. 4. The mean GnRH secretiony5 min sampling period during a 2-h incubation (a), and GnRH secreted during the first 5 min of the veratridine challenge (b) in commercial (ns17) and field-caught (ns8) bullfrogs. Each bar represents the mean"S.E.M. *P-0.05.
bullfrogs may differ considerably from mammals. This notion was partially supported by our current observation that bullfrogs lack robust, mammalianlike GnRH pulses. In addition, our results suggest the bullfrog pituitary may not have the same dependency on pulsatile GnRH stimulation as mammals. Mean levels of spontaneous GnRH release are significantly higher in field-caught breeding bullfrogs than in commercial non-breeding bullfrogs. Field-caught bullfrogs and commercial bullfrogs differ in two respects: reproductive status and the duration in captivity. Field-caught bullfrogs were obtained at the peak of the breeding season, which spanned only approximately 2 weeks between the months of June and July. All behavioral signs in
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field-caught bullfrogs point to the maximal activation of the reproductive axis, including the establishment of individual territories, active mating calls and amplexus with females. In contrast, commercial bullfrogs did not call or amplex with females in the laboratory. Thus, the higher levels of GnRH release in field-caught bullfrogs most likely reflect the reproductive readiness of these animals. In support of this notion, field-caught bullfrogs in the breeding condition also had a significantly more robust response to the veratridine challenge than did non-breeding commercial bullfrogs, perhaps suggesting greater amounts of GnRH in readily releasable pools in the breeding frog. Unfortunately, we cannot rule out the possible suppression of GnRH release by captive stress in commercial bullfrogs. Bullfrogs are extremely sensitive to stress induced by capture and handling (Licht et al., 1983). Levels of gonadal steroids and gonadotropins in bullfrogs declined within hours of capture (Licht et al., 1983), and became undetectable in most bullfrogs kept in captivity after 16 h. Although the captive history of these commercial frogs was not known, it is possible that they had been in the holding facilities for weeks, even months, before shipment to our laboratory. This long-term captive stress could shut down the GnRH system, leading to minimal hormone release. In support of this, both corticotropinreleasing hormone (CRH; Petraglia et al., 1987) and corticosterone (Moore and Zoeller, 1985) have been shown to potently suppress the GnRH system in vertebrates. In sum, we have established a specific and highly sensitive EIA to measure GnRH release from rat and bullfrog hypothalamic explants over time. Our study is the first to report the temporal pattern of GnRH release in a poikilothermic tetrapod. We showed that the levels and patterns of GnRH release in bullfrogs contrasted sharply with rats. The differences in GnRH secretion between these two species may reflect different functional requirements for the maximal activation of pituitary gonadotropin secretion. Further, the differences in GnRH secretory dynamics between field-caught and commercial bullfrogs are indicative of the ability of physiological and environmental cues to potently alter the output of this neurohormone.
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Acknowledgments We thank Leif Saul, Lihong Zhang and Hector Postigo for the field assistance in bullfrog collection. This work was supported by NSF grants IBN9707446 and IBN-9733237 to P.-S.T. and NIH HD 34860 to S.M.M. References Barrell, G.K., Moenter, S.M., Caraty, A., Karsch, F.J., 1992. Seasonal changes of gonadotropin-releasing hormone secretion in the ewe. Biol. Reprod. 46, 1130–1135. Belechetz, P., Plant, T.M., Nakai, Y., Keogh, E.J., Knobil, E., 1978. Hypophysial response to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202, 631–633. Bourguignon, J.P., Franchimont, P., 1984. Puberty-related increase in episodic LHRH release from rat hypothalamus in vitro. Endocrinology 114, 1941–1943. Bourguignon, J.P., Gerard, A., Mathieu, J., Simons, J., Franchimont, P., 1989. Pulsatile release of gonadotropin-releasing hormone from hypothalamic explants is restrained by blockade of N-methyl-D,L-Aspartate receptors. Endocrinology 125, 1090–1096. Caraty, A., Locatelli, A., 1988. Effect of time after castration on secretion of LHRH and LH in the ram. J. Reprod. Fertil. 82, 263–269. Carmel, P.W., Araki, S., Ferin, M., 1976. Pituitary stalk portal blood collection in rhesus monkeys: evidence for pulsatile release of gonadotropin-releasing hormone (GnRH). Endocrinology 99, 243–248. Clarke, I.J., Cummins, J.T., 1982. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111, 1737–1739. Habibi, H.R., 1991. Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in goldfish pituitary: effects of native GnRH peptides and synthetic GnRH antagonist. Biol. Reprod. 44, 275–283. Iela, L., D’Aniello, B., di Meglio, M., Rastogi, R.K., 1994. Influence of gonadectomy and steroid hormone replacement therapy on the gonadotropin-releasing hormone neuronalsystem in the anterior preoptic area of the frog (Rana esculenta) brain. Gen. Comp. Endocrinol. 95, 422–431. King, J.A., Davidson, J.S., Millar, R.P., 1986. Desensitization to gonadotropin-releasing hormone in perifused chicken anterior pituitary cells. Endocrinology 119, 1510–1518. Li, Q., Paciotti, G.F., Tamarkin, L., Ottinger, M.A., 1994. LHRH-I release from quail hypothalamic slices measure by specific EIA. Gen. Comp. Endocrinol. 95, 13–24. Li, Y., Lin, H., 2000. Differences in mGnRH and cGnRH-II contents in pituitaries and discrete brain areas of Rana rugulosa W. according to age and stage of maturity. Comp. Biochem. Physiol. C 125, 179–188. Licht, P., McCreery, B.R., Barnes, R., Pang, R., 1983. Seasonal and stress related changes in plasma gonadotropins, sex
steroids and corticosterone in the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 50, 124–145. Licht, P., Porter, D.A., 1985. LH secretion in response to gonadotropin-releasing hormone by superfused pituitaries from two species of turtles. Gen. Comp. Endocrinol. 59, 442–448. Maurer, J., Wray, S., 1999. Luteinizing hormone-releasing hormone quantified in tissues and slice explant cultures of postnatal rat hypothalami. Endocrinology 140, 791–799. McCreery, B.R., Licht, P., 1983. Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin-releasing hormone during the ovarian cycle in the bullfrog, Rana catesbeiana. Biol. Reprod. 29, 863–871. Miller, B.T., Collins, T.J., Nagle, G.T., Kurosky, A., 1992. The occurrence of O-acylation during biotinylation of gonadotropin-releasing hormone and analogs. J. Biol. Chem. 267, 5060–5069. Moenter, S.M., Caraty, A., Locatelli, A., Karsch, F.J., 1991. Pattern of gonadotropin-releasing hormone (GnRH) secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge. Endocrinology 129, 1175–1182. Moore, F.L., Zoeller, R.T., 1985. Stress-induced inhibition of reproduction: evidence of suppressed secretion of LH-RH in an amphibian. Gen. Comp. Endocrinol. 60, 252–258. Nett, T.M., Crowder, M.E., Moss, G.E., Duello, T.M., 1981. GnRH-receptor interaction. V. Down regulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous hormone. Biol. Reprod. 24, 1145–1155. Petraglia, F., Sutton, S., Vale, W., Plotsky, P., 1987. Corticotropin-releasing factor decreases plasma luteinizing hormone levels in female rats by inhibiting gonadotropin-releasing hormone release into hypophysial-portal circulation. Endocrinology 120, 1083–1088. Porter, D.A., Licht, P., 1986. Effects of temperature and mode of delivery on responses to gonadotropin-releasing hormone by superfused frog pituitaries. Gen. Comp. Endocrinol. 63, 236–244. Purnelle, G., Gerard, A., Czajkowski, V., Bourguignon, J.P., 1997. Pulsatile secretion of gonadotropin-releasing hormone by rat hypothalamic explants of GnRH neurons without cell bodies. Neuroendocrinology 66, 305–312. Rasmussen, D., 1993. Episodic gonadotropin-releasing hormone release from the rat isolated median eminence in vitro. Neuroendocrinology 58, 511–518. Smith, M.A., Vale, W.W., 1981. Desensitization to gonadotropin-releasing hormone observed in superfused pituitary cells on cytodex beads. Endocrinology 108, 752–759. Tsai, P.-S., Licht, P., 1993a. Differential distribution of chickenI and chicken-II GnRH in the turtle brain. Peptides 14, 221–226. Tsai, P.-S., Licht, P., 1993b. GnRH-induced desensitization of in vitro luteinizing hormone secretion in the turtle, Trachemys scripta. Gen. Comp. Endocrinol. 89, 238–247. Velduis, J.D., Johnson, M.L., 1986. Cluster analysis; a simple, versatile and robust algorithm for endocrine pulse detection. Am. J. Physiol. 250, E486–E493. Wang, L., Yoo, M.S., Kang, H.M., Im, W.B., Choi, H.S., Bogerd, J., et al., 2001. Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana). J. Exp. Zool. 289, 190–201.
Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants Pei-San Tsaia,*, Suzanne M. Moenterb, Jason M. Cavolinac a
Department of Integrative Physiology, Campus Box 354, University of Colorado, 114 Clare Small, Boulder, CO 80309-0354, USA b Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA c Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269-4156, USA Received 13 June 2003; received in revised form 24 July 2003; accepted 25 July 2003
Abstract Gonadotropin-releasing hormone (GnRH) is critical for the initiation and maintenance of reproduction in vertebrates. Information regarding GnRH release is abundant in mammals, but absent in poikilothermic tetrapods. In this study, we established a novel GnRH enzyme immunoassay (EIA) to measure GnRH release over time from hypothalamic explants isolated from mature field-caught and commercially-acquired male bullfrogs, Rana catesbeiana. Hypothalamic explants from rats were used as a positive control to test the sensitivity and accuracy of our EIA and to ensure our in vitro system could detect GnRH pulses. Prominent GnRH pulses were present in the majority (9y10) of rat hypothalamic explants, but absent in all (17y17) of the commercial bullfrogs and the majority (5y8) of field-caught bullfrogs. In three cases where GnRH pulses were observed in field-caught bullfrogs, there was only one pulse during the 2-h incubation period; high-frequency pulses similar to those observed in rats were not observed. Veratridine, which opens voltagegated sodium channels, stimulated GnRH release in all explants cultured in the presence of Ca2q , demonstrating explant viability. The levels of both spontaneous and veratridine-induced GnRH release were significantly higher in field-caught than commercial bullfrogs. This study demonstrated, for the first time, the temporal pattern of GnRH release in a poikilothermic tetrapod. Further, our results suggest the levels and patterns of GnRH output in bullfrogs are subject to the dynamic regulation by physiological and environmental cues. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Bullfrog; Rat; Gonadotropin-releasing hormone release; Pulsatility; Enzyme immunoassay; Hypothalamic explants; Veratridine
1. Introduction Successful reproduction in vertebrates depends upon the action of a decapeptide, gonadotropinreleasing hormone (GnRH). In tetrapods, GnRH released from axon terminals in the median emi*Corresponding author. Tel.: q1-303-735-1877; fax: q1303-492-4009. E-mail address: [email protected] (P.-S. Tsai).
nence stimulates the secretion of gonadotropins from the pituitary. Gonadotropins then act as primary regulators of gonadal functions. As an upstream neuroendocrine regulator of reproductive function, GnRH neurons are under the influence of diverse factors and respond to changing physiological and environmental conditions. The proper level and pattern of GnRH secretion ensure the proper progression of reproduction.
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00221-6
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The pattern and regulation of GnRH secretion have been extensively studied in a number of vertebrates, most notably in mammals. However, there are significant gaps in our knowledge regarding the secretion of GnRH in poikilothermic tetrapods. The majority of studies on GnRH in amphibians and reptiles focused on the characterization of GnRH actions and identification of molecular forms of GnRH, with little emphasis on the release of GnRH from the neuronal terminals. Studies that examined changes in the amphibian or reptilian GnRH system in relation to the reproductive status often measured GnRH contents in brain regions (Tsai and Licht, 1993a; Li and Lin, 2000) or the cytology of GnRH neurons (Iela et al., 1994). These measurements, although providing some insights into release, often lead to ambiguous interpretations. For instance, a reduction in GnRH content could be interpreted as enhanced release because rapid release depletes GnRH stores. Conversely, it could be interpreted as attenuated release because the pool of GnRH available for release is smaller. To our knowledge, the secretion of GnRH in a poikilothermic tetrapod has never been directly measured. In this study, we established a novel enzyme immunoassay (EIA) to measure the in vitro GnRH release from hypothalamic explants isolated from sexually mature male bullfrogs, Rana catesbeiana. This EIA employs an O-biotinylated native GnRH tracer that can be easily generated in the laboratory within hours. Further, the EIA is easy to perform and has the sensitivity that rivals, even exceeds, the conventional GnRH radioimmunoassay (RIA). We utilized this EIA to quantify the in vitro secretion of GnRH in sexually mature bullfrogs freshly caught from the field during the breeding season, and for comparison, those from a commercial supplier during the non-breeding season. In addition, we also measured GnRH release from rat hypothalamic explants to ensure our EIA possesses the sensitivity, precision and consistency for measuring GnRH release over time in a species with well-documented GnRH release patterns (Bourguignon and Franchimont, 1984; Bourguignon et al., 1989). Our results demonstrate the establishment of a highly sensitive and novel EIA, and for the first time, the direct measurement of GnRH release over time in a poikilothermic vertebrate.
2. Methods 2.1. Animals All experimental procedures complied with animal protocols approved by the Institutional Animal Care and Use Committee at the University of Connecticut and University of Colorado. 2.1.1. Frogs Mature male bullfrogs were obtained from two sources. Commercially-available frogs were purchased during the non-breeding season (March– May) from Connecticut Valley Biological (Southampton, MA). These frogs were kept under 12L:12D photoperiod at the University of Connecticut Frog Facility, and fed newborn rats every week. All commercial animals were euthanized within 2 weeks of arrival. Field-caught bullfrogs were collected in June and July of 1999 from the upper Bolton Lake in Vernon, CT, and quickly transported to the University of Connecticut in holding sacks. Field-caught frogs were euthanized within 3 h of capture for hypothalamic incubation. All field-caught frogs were chorusing at the time of capture, indicating sexual maturity and reproductive readiness. Male frogs from both sources had an average gonadal somatic index (GSI; milligram gonad weightygram body weight) of 1.65 and had well-developed thumb pads. The observation that GSI did not differ between breeding and non-breeding bullfrogs was consistent with a previous observation of Licht et al. (1983), when they reported little monthly variations in testis weight of wild bullfrogs throughout the year. When testicular histology of representative animals were examined, all stages of germ cells were found in testes of both groups, as reported previously (Licht et al., 1983), although breeding frogs had more cysts containing primary spermatocytes in the process of meiotic division (data not shown). 2.1.2. Rats Two- to 6-month-old mature male Wistar rats were obtained from the University of Connecticut breeding colony. They were kept under 12L:12D photoperiod and fed water and rat chow ad libitum. 2.2. In vitro incubation of hypothalamic explants 2.2.1. Incubation media Both Ca2q-free and Ca2q-supplemented incubation media were prepared from the same base
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medium, a Ca2q-free Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Grand Island, NY). For culturing rat explants, DMEM was supplemented with 10 mM HEPES, 0.01% BSA, 1 mM pyruvic acid, and 4 mM L-glutamine. DMEM was diluted with an additional 50% water and given the same supplements for the culture of frog explants. When required, both mammalian and frog media were supplemented with 1.85 mM CaCl2. All media were sterilized by filtration through 0.22 mm filters and the pH adjusted to 7.4. All media supplements were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2.2. Creation of explants Hypothalamic explants from frogs and rats were obtained immediately after decapitation of animals. Frog hypothalamic explants were created by four cuts: a coronal cut 2-mm rostral to the optic chiasm, a coronal cut on the caudal border of the optic tectum, and two sagittal cuts along the lateral margins of the median eminence. Rat hypothalamic explants were created by five cuts: a coronal cut 2-mm rostral to the optic chiasm, a coronal cut on the rostral border of the mammillary bodies, two sagittal cuts along the lateral sulci and a transverse cut 2-mm dorsal to the median eminence. GnRH immunocytochemistry confirmed both rat and bullfrog explants contain abundant GnRH neuronal perikarya and terminals (data not shown). 2.2.3. Hypothalamic incubation All explants were pre-incubated for 30 min at 37 8C (for rats) and 28 8C (for bullfrogs) to establish baseline GnRH secretion. Following preincubation, hypothalamic incubations were carried out for an additional 125 min, during which incubation medium was collected every 5 min. Hypothalamic explants were incubated in 24-well plates or borosilicate glass tubes in their appropriate media. Rat explants were incubated in 500 ml Ca2q-supplemented mammalian culture medium pregassed with 95% O2:5% CO2. Some rat explants were incubated in Ca2q-free medium containing 5 mM ethylene glycol-bis-(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid (EGTA), a calcium chelator, to remove residual Ca2q from the medium. Frog explants were incubated in 350 ml Ca2q-supplemented frog culture medium pregassed with 95% O2:5% CO2. All explants were given a challenge of 20 mM veratridine (an agent that opens voltage-gated Naq channel) after the
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125-min incubation period for 10–20 min to test tissue viability. All samples were snap frozen on dry ice and stored at y70 8C until GnRH measurement by EIA. 2.3. EIA 2.3.1. Generation and purification of O-biotinylated GnRH tracer The procedure for O-biotinylating GnRH was adopted from Miller et al. (1992). Briefly, a 10:1 molar ratio of sulfosuccinimidyl-6-(biotinamido) hexanoate (biotinylation reagent, Vector Laboratories, Burlingame, CA) and mammalian GnRH (mGnRH, Peninsula Laboratories, Belmont, CA) were dissolved in 50 mM sodium bicarbonate. The solution was vortexed and left at room temperature for 17 min with occasional mixing. The reaction was stopped by the addition of trifluoroacetic acid (TFA, 0.1% final concentration), filtered, and injected through a 200 ml injection loop into a Waters Symmetry C18 high-performance liquid chromatography (HPLC) column (3.9=150 mm, particle size 5 mm). The solvent gradient was delivered by Waters 515 HPLC pumps and the UV absorbance monitored by a Waters 2487 dual wavelength detector. The mobile phase was 0.05% TFA in acetonitrile with a flow rate of 1 mlymin. The separation program consisted of an initial isocratic elution of 15% acetonitrile for 10 min, followed by a linear gradient to 40% acetonitrile in the next 24 min. Using this program, three biotinylated forms of GnRH (serine-biotinylated, tyrosine-biotinylated, and serine, tyrosine-biotinylated) were isolated. Further analysis revealed that all three forms of biotinylated GnRH were functional as tracers; however, serine-biotinylated GnRH resulted in the best assay sensitivity (data not shown). Thus, serine-biotinylated GnRH was used as the tracer for all subsequent EIAs. 2.3.2. Incubation and color development Ninety-six well Immulon 4 HB plates (Dynex, Chantilly, VA) were pre-coated with 100 ml of 5 mgyml donkey anti-rabbit antibody (Jackson Immuno Research, West Grove, PA) in 0.1 M sodium carbonate (pH 9.6) for 8 h at room temperature. The wells were washed 6 times with EIA buffer (0.01 M phosphate buffer, 0.15 M NaCl, 0.1% Tween 20) and coated with 100 ml of the anti-GnRH antiserum (R1245, generously provided by Dr Terry Nett, Colorado State University,
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1:200 000) overnight at 4 8C. This antiserum detects specifically the mammalian form of GnRH (mGnRH), which is the native hypothalamicpreoptic form in R. catesbeiana (Wang et al., 2001). Following well washing, samples and standards (100 ml) were added to the wells and the incubation allowed to proceed for an additional 24 h at 4 8C. After 24 h, 50 ml of HPLC-purified Obiotinylated native GnRH (1:40 000) was added to the wells and allowed to incubate for an hour at room temperature. The wells were washed extensively, and 100 ml of avidin D-horseradish peroxidase (1:5000, Vector Laboratories) was added and allowed to incubate at room temperature for 1 h. The wells were washed extensively and 100 ml of 3,39,5,59-tetramethylbenzidine (Vector Laboratories) was added and the incubation allowed to proceed for 30–60 min. The color reaction was stopped by the addition of 100 ml of 1 N H2SO4. The plates were read at 450 nm using an ELISA plate reader. Standard curves and sample doses were calculated using the four-logistics parameter curve fit. 2.4. Analysis GnRH pulses were identified by the computer algorithm Cluster analysis developed by Velduis and Johnson (1986). A constant coefficient of variation was set for each experiment. Other parameters included a 1=1 cluster configuration and a t-statistic value of 3.76 for the upstroke and 1 for the down stroke to achieve a 1% false positive rate. For the comparison between two groups, the Mann–Whitney U test was used. Differences between groups were considered significant when P-0.05. 3. Results Our initial validation of the GnRH EIA revealed the assay was very specific to mGnRH, the native form of GnRH present in the bullfrog hypothalamic-preoptic area (Wang et al., 2001). The crossreactivities of this EIA to chicken-I (cGnRH-I), chicken-II (cGnRH-II), lamprey I (lGnRH-I), salmon (sGnRH) and tunicate I (tI-GnRH) forms of GnRH were 0.32, 0.1, 0.17, 0.16 and less than 0.01%, respectively. In addition, the EIA matched the sensitivity of even the most sensitive GnRH RIAs, with detectability in the range of 0.1 pgy well. ED80, ED50 and ED20 of the EIA were 0.22
Fig. 1. A representative EIA standard curve using the serinebiotinylated GnRH as the tracer. Percent ByB0s% (boundytotal bound). For the standard, mGnRH was used.
pgywell, 1.62 pgywell and 9.06 pgywell, respectively. Intra- and inter-assay coefficients of variation were 13.82"9.9% and 7.4%"3.0%, respectively. A representative EIA standard curve is presented (Fig. 1). To verify the effectiveness of the EIA in detecting GnRH release over time in our in vitro incubation system, hypothalamic explants from reproductively mature male rats were used as positive controls. Ninety percent (9y10) of rat hypothalamic explants studied, including ones cultured in Ca2q-free medium, released GnRH in discrete pulses that were detectable by Cluster analysis (Fig. 2). In the presence of Ca2q (Fig. 2a,b), the level of GnRH secreted by the explants was approximately 6–10 times higher than the level secreted in the absence of Ca2q (Fig. 2c,d). The mean interpulse interval was 29.2"12.6 min, and the average duration and amplitude of pulses were 16.75"2.7 min and 32.2"9.3 pgyml, respectively. Explants were challenged with veratridine at the end of each experiment to test tissue viability. All explants cultured in the presence of Ca2q responded to veratridine (Fig. 2a, b, insets). In the absence of Ca2q, however, the veratridine response was abolished or significantly reduced (Ps0.04, ns9; Fig. 2c, d, insets). Overall, incubation in Ca2q-free medium decreased pulse amplitude but had no significant effect on either the pulse frequency or duration.
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(data not shown). All bullfrogs, commercial and field-caught, responded robustly to a veratridine challenge at the end of the experiment. The mean spontaneous GnRH secretion and GnRH secreted during the first 5 min of veratridine challenge were significantly higher in the field-caught bullfrogs than in commercially-purchased bullfrogs (Fig. 4a, b). 4. Discussion
Fig. 2. The temporal pattern of GnRH secretion from four representative male rat hypothalamic explants incubated in the presence (a, b) and absence (c, d) of Ca2q. *Significant peaks defined by cluster analysis. Insets: responsiveness of explants to a 20 mM veratridine challenge. Black horizontal bars in insets represent the duration of veratridine challenge. Note that multiple significant peaks were detected in rat explants.
Measurements of GnRH released from bullfrog hypothalamic explants (Fig. 3) revealed that the spontaneous GnRH secretion in the field-caught bullfrog was approximately 6–10 times lower than rats. GnRH secretion was detectable in all (8y8) field-caught bullfrogs, but pulses were detected in only three of these frogs (Fig. 3a, b, c). In each case, only a single significant pulse was detected over the 2-h incubation period, a pattern that differs markedly from the pattern of pulses observed in rats (Fig. 2). In explants from commercially-purchased bullfrogs (ns17), spontaneous GnRH release was not consistently detectable with our EIA, and none of the explants exhibited GnRH pulsatility detectable by Cluster analysis
We have established a sensitive GnRH EIA with the precision and accuracy needed to measure GnRH release from vertebrate hypothalamic explants. For the first time, we investigated the levels and temporal pattern of GnRH release in a reproductively mature poikilothermic tetrapod, the bullfrog, R. catesbeiana. Overall, GnRH release in field-caught breeding bullfrogs differs from rats in two respects. First, bullfrog GnRH release was 6– 10 times lower than rats. Second, the prominent GnRH pulses observed in rats were rarely seen in bullfrogs. These results suggest the levels and patterns of GnRH release required for the proper stimulation of pituitary gonadotropin secretion may vary substantially from species to species. To our knowledge, our GnRH EIA was the first to employ an O-biotinylated GnRH as a homologous tracer. Currently, the most widely used GnRH EIAs employ an N-biotinylated D-Lys6-GnRH as the tracer (Li et al., 1994; Maurer and Wray, 1999). The D-Lys6-GnRH analogue was chosen as the substrate for biotinylation because the native GnRH, with its blocked amino terminal, could not be N-biotinylated. D-Lys6-GnRH was one of the few GnRH analogues containing a free amine group available for N-biotinylation. However, the use of this heterologous GnRH tracer results in a considerably less sensitive assay (Maurer and Wray, 1999). An alternative to N-biotinylation was reported by Miller et al. (1992), in which successful O-biotinylation of native GnRH occurred on either serine or tyrosine residues. This tracer yielded a highly specific EIA with a sensitivity that rivals the most sensitive of GnRH RIAs. The ease of O-biotinylating and purifying the biotinylated products, as well as the sensitivity and specificity of this tracer in our EIA make it an attractive alternative to the use of enzyme-coupled tracer, biotinylated GnRH analogues and radioactive assays.
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Fig. 3. The temporal pattern of GnRH release from the hypothalamic explants of three representative male field-caught breeding bullfrogs (a, b and c). *Significant peaks defined by cluster analysis. Insets: responsiveness of explants to a 20 mM veratridine challenge. Black horizontal bars in insets represent the duration of veratridine challenge. A single GnRH pulse was detected by cluster analysis in each of the three explants.
The characteristics of pulsatile GnRH release from rat explants in our study are similar to those reported previously for adult male rats, with GnRH interpulse intervals averaging approximately 30 min (Bourguignon et al., 1989; Rasmussen, 1993; Purnelle et al., 1997). In sharp contrast with rats, GnRH pulses were not detected using the same analysis criteria in explants from non-breeding commercial frogs and in all but three explants from breeding field-caught frogs, which each exhibited a single pulse. The most-likely explanation is that GnRH pulse frequency in bullfrogs is so low that the 2-h sampling window used was insufficient to observe multiple pulses. In this regard, such low pulse frequencies have been reported in mammals. Caraty and Locatelli (1988) reported a frequency of one GnRH pulse every 2– 4 h in intact adult male rams, and even lower frequencies have been observed in luteal phase ewes and ewes during the non-breeding season (Moenter et al., 1991; Barrell et al., 1992). Of further interest, GnRH pulses were observed in three out of eight (37.5%) explants taken from field-caught breeding bullfrogs and none (0y17) from commercial non-breeding bullfrogs, suggesting the presence of discrete GnRH pulses might be a phenomenon uniquely associated with reproductive activation. Unfortunately, we had to limit our incubation period to 2 h because the failure of bullfrog explants to respond consistently to a veratridine challenge after 3 h in culture indicated viability was beginning to decline at this time (data not shown). The low number of bullfrogs
exhibiting GnRH pulses and the presence of only a single pulse during the incubation precludes general conclusions on the pulsatile characteristics of GnRH release in these animals. Nevertheless, these results raise the tantalizing possibility that bullfrog GnRH release during the reproductive season, like mammals, might be episodic. It was well established that pituitaries of mammals, birds, some fishes and reptiles became rapidly desensitized to continuous exposure to GnRH, resulting in decreased gonadotropin secretion (Belechetz et al., 1978; Nett et al., 1981; Smith and Vale, 1981; Licht and Porter, 1985; King et al., 1986; Habibi, 1991; Tsai and Licht, 1993b). To maintain elevated gonadotropin output in these systems, GnRH must be applied in a pulsatile fashion. The pulsatile release of GnRH into hypothalamic–pituitary portal circulation has been extensively documented in mammals (cf. Carmel et al., 1976; Clarke and Cummins, 1982). In marked contrast to mammals, the episodic mode of GnRH stimulation is not required for the maintenance of gonadotropin secretion in ranid frogs. In the bullfrog, plasma gonadotropin levels remained elevated for at least 4 days in response to constant GnRH infusion (McCreery and Licht, 1983). Most unexpectedly, continuous GnRH stimulation was much more effective than pulsatile GnRH administration in stimulating in vitro gonadotropin secretion from bullfrog pituitaries (Porter and Licht, 1986). Such unusual preference of bullfrog pituitaries for continuous GnRH stimulation suggests the secretory dynamics of GnRH in
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Fig. 4. The mean GnRH secretiony5 min sampling period during a 2-h incubation (a), and GnRH secreted during the first 5 min of the veratridine challenge (b) in commercial (ns17) and field-caught (ns8) bullfrogs. Each bar represents the mean"S.E.M. *P-0.05.
bullfrogs may differ considerably from mammals. This notion was partially supported by our current observation that bullfrogs lack robust, mammalianlike GnRH pulses. In addition, our results suggest the bullfrog pituitary may not have the same dependency on pulsatile GnRH stimulation as mammals. Mean levels of spontaneous GnRH release are significantly higher in field-caught breeding bullfrogs than in commercial non-breeding bullfrogs. Field-caught bullfrogs and commercial bullfrogs differ in two respects: reproductive status and the duration in captivity. Field-caught bullfrogs were obtained at the peak of the breeding season, which spanned only approximately 2 weeks between the months of June and July. All behavioral signs in
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field-caught bullfrogs point to the maximal activation of the reproductive axis, including the establishment of individual territories, active mating calls and amplexus with females. In contrast, commercial bullfrogs did not call or amplex with females in the laboratory. Thus, the higher levels of GnRH release in field-caught bullfrogs most likely reflect the reproductive readiness of these animals. In support of this notion, field-caught bullfrogs in the breeding condition also had a significantly more robust response to the veratridine challenge than did non-breeding commercial bullfrogs, perhaps suggesting greater amounts of GnRH in readily releasable pools in the breeding frog. Unfortunately, we cannot rule out the possible suppression of GnRH release by captive stress in commercial bullfrogs. Bullfrogs are extremely sensitive to stress induced by capture and handling (Licht et al., 1983). Levels of gonadal steroids and gonadotropins in bullfrogs declined within hours of capture (Licht et al., 1983), and became undetectable in most bullfrogs kept in captivity after 16 h. Although the captive history of these commercial frogs was not known, it is possible that they had been in the holding facilities for weeks, even months, before shipment to our laboratory. This long-term captive stress could shut down the GnRH system, leading to minimal hormone release. In support of this, both corticotropinreleasing hormone (CRH; Petraglia et al., 1987) and corticosterone (Moore and Zoeller, 1985) have been shown to potently suppress the GnRH system in vertebrates. In sum, we have established a specific and highly sensitive EIA to measure GnRH release from rat and bullfrog hypothalamic explants over time. Our study is the first to report the temporal pattern of GnRH release in a poikilothermic tetrapod. We showed that the levels and patterns of GnRH release in bullfrogs contrasted sharply with rats. The differences in GnRH secretion between these two species may reflect different functional requirements for the maximal activation of pituitary gonadotropin secretion. Further, the differences in GnRH secretory dynamics between field-caught and commercial bullfrogs are indicative of the ability of physiological and environmental cues to potently alter the output of this neurohormone.
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