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Biology of Reproduction 60, 1217-1223 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

In Vitro Nitric Oxide Effects on Basal and Gonadotropin-Releasing Hormone-Induced Gonadotropin Secretion by Pituitary Gland of Male Crested Newt (Triturus carnifex) during the Annual Reproductive Cycle1

Anna Gobbetti2,a, and Massimo Zerania

a Department of Molecular, Cellular, and Animal Biology, University of Camerino, 62032 Camerino, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to test the possible nitric oxide (NO) involvement in pituitary gonadotropin secretion in the male crested newt, Triturus carnifex. Pituitaries were incubated in vitro with medium alone, GnRH, NO donor (NOd, sodium nitroprusside), NO synthase inhibitor (NOSi, N{omega}-nitro-L-arginine methyl ester), cGMP analogue (cGMPa, 8-bromo-cGMP), soluble guanylate cyclase inhibitor (sGCi, cystamine), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi during the annual reproductive cycle: pre-reproduction, reproduction (noncourtship and courtship), and the refractory, recovery, and estivation periods. To determine pituitary gonadotropin secretion indirectly, newt testes were superfused in vitro with preincubated pituitaries, and androgen release was determined. NO synthase (NOS) activity and cGMP levels were assessed in the preincubated pituitaries. Medium alone- and GnRH-preincubated pituitary increased androgen secretion during pre-reproduction, noncourtship, courtship, and recovery; the GnRH-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOd and cGMPa increased androgens in all reproductive phases considered except courtship; the NOd- and cGMP-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOS activity was highest during courtship and lowest during the refractory and estivation periods. GnRH increased NOS activity during pre-reproduction, noncourtship, and recovery. Cyclic GMP levels were highest during courtship and lowest during the refractory period and estivation. GnRH increased cGMP levels during pre-reproduction, noncourtship, and recovery, while NOd did so during all reproductive phases considered. These results suggest that basal and GnRH-induced gonadotropin secretion are up-regulated by NO in the pituitary gland of the male Triturus carnifex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The involvement of hypothalamic GnRH in the neurohormonal control of reproduction, through the stimulation of pituitary gonadotropin secretion, is ubiquitous in vertebrates [1]. In the amphibian brain, several forms of GnRH that are structurally related to mammalian, chicken II, and salmon GnRH have been identified [2]. As in other vertebrates, the main role of these GnRH forms in amphibians is modulation of pituitary gonadotropin secretion. In fact, these neuropeptides induce gonadotropin release that in turn determines gonadal steroidogenesis both in vivo and in vitro [3].

At the pituitary level, GnRH interacts with specific G protein-coupled receptors of the gonadotroph surface, triggering the activation of several intracellular pathways. The GnRH-receptor interaction mainly activates phospholipase C with the ensuing hydrolysis of membrane polyphosphoinositides, which are converted into diacylglycerol and inositol phosphates (InPs). These are postreceptor transducing messengers that are responsible for the activation of protein kinase C and the mobilization of intracellular Ca2+, respectively [4–8]. Other studies suggest that the GnRH action is mediated by cGMP and Ca2+, and that the site at which Ca2+ is required is at a postreceptor locus before cGMP formation [9, 10].

Nitric oxide (NO) is a diffusive free radical involved, as an intra- and/or intercellular messenger, in a wide range of physiological events [11]. NO synthase (NOS) is the enzyme responsible for production of NO from L-arginine [12]. Up to date, three forms of NOS have been described: type I (neuronal) and type III (endothelial) are constitutive (Ca2+-calmodulin-dependent), while type II is inducible (Ca2+-independent) [13]. Various evidence suggests that NO develops its intracellular messenger role mainly by activating soluble guanylate cyclase and the subsequent cGMP synthesis [14]. Among the various physiological effects of NO, it has been suggested that NO modulates endocrine activity in mammals [15]. The presence of NOS has been identified in hypothalamic areas [16–20] and pituitary lobes [21], in particular in gonadotrophs [22], suggesting that NO mainly acts as a neuroendocrine modulator of the reproductive processes through regulation of the releasing factors, such as GnRH, and that of gonadotropins.

As regards amphibians, little is known about involvement of NO in reproductive processes. In the urodele crested newt, Triturus carnifex, NO regulates male courtship; NOS activity, measured in the newt male brain, significantly increases during the courtship phases [23]. In the anuran water frog, Rana esculenta, NADPH-diaphorase activity is widely distributed in the hypothalamo-pituitary system [24], and NO is involved in regulation of postreproduction through stimulation of prostaglandin E2-9-ketoreductase activity in GnRH-dependent prostaglandin F2{alpha} synthesis by the frog interrenal gland [25]. More recently, we found that NO mediates basal and GnRH-induced gonadotropin in vitro secretion by the pituitary of the female Rana esculenta [26].

To verify the possible conservative role of NO over all amphibians, we have studied another amphibian species, the urodele crested newt, Triturus carnifex. The population of this species studied has a discontinuous cycle similar to those described in other newts living in temperate zones [27, 28]. The animals breed during cold months (January to March, reproduction); in the spring, breeding is interrupted (postreproduction); in the summer, the newts disappear underground (estivation); at the beginning of the autumn, they return to the pond; recrudescence of the sexual organs occurs until December (pre-reproduction). The male is characterized by a postnuptial gametogenesis; new germ cells are found from April to October; during autumn, sperm accumulate in testicular ampullae where they are held until the reproductive period. Secondary sexual characteristics (crest height, abdominal gland weight) develop through autumn and appear to have completed development in January. The breeding period is characterized by high plasma androgen levels, and the postreproductive period (refractory) by the highest estradiol plasma values [28–30]. In the context of the "associated/dissociated" model of reproductive cycles proposed by Crews [31], the reproductive cycle of this newt is difficult to locate, because gametogenesis is "dissociated," while hormone levels are "associated."

In this work we studied the in vitro effects of NO on basal and GnRH-induced pituitary gonadotropin secretion in the male crested newt, Triturus carnifex, during the annual reproductive cycle. To this end, because specific gonadotropin antisera are not available for this species, we indirectly monitored pituitary secretion through testicular androgen release induced by gonadotropins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

In November (pre-reproduction), February (reproduction), April (refractory period), June (recovery period), and August (estivation), Triturus carnifex males (45 animals for each month) were captured in the field and decapitated. In February, newts were divided into two groups: courtship and noncourtship males. Brains and testes were rapidly removed and placed in cold Dulbecco's Modified Eagle's medium (DMEM; Sigma Chemical Co., St. Louis, MO) containing 10 mM Hepes, 0.1 mg/ml penicillin, and 0.2 mg streptomycin; they were then transferred to the laboratory on ice. The pituitaries were removed under the dissection microscope and placed in DMEM.

In Vitro Superfusion

The superfusion system previously described by Zerani et al. [32] was followed. A bottle containing medium was connected to the inflow of a peristaltic pump; the pump outflow was connected to the cap of a column (column A) (glass barrel, 5 mm diameter x 4 cm length, with a porous polyethylene disk at the bottom; Bio-Rad, Hercules, CA). Column A was then directed toward a fraction collector; in some experimental groups, another column (column B) was connected between the pump and column A, and thus column B was superfused before column A. The effective volume of the columns was 1 ml. The flow rate for the superfusion was 100 µl/min, and fractions were collected at 10-min intervals; at this rate, a bolus of material passed completely through the first column in about 4 min and through the second in about 6 min. The bottle with medium was placed in a water bath at 19°C, and the columns were kept at room temperature (about 19°C). The collected medium fractions were frozen and kept at -20°C until assay. One testis, randomly chosen, was transferred into column A. One pituitary was placed into column B. Before superfusion, pituitaries, randomly chosen, were preincubated in vitro with 1) DMEM alone; 2) DMEM plus GnRH (50 nM, Sigma); 3) DMEM plus NO donor (NOd; sodium nitroprusside, 100 nM, Sigma); 4) DMEM plus NO synthase inhibitor (NOSi; N{omega}-nitro-L-arginine methyl ester, 50 nM; Sigma); 5) DMEM plus cGMP analogue (cGMPa, 8-bromo-cGMP, 75 nM; Calbiochem-Novabiochem Corp., San Diego, CA); 6) DMEM plus soluble guanylate cyclase inhibitor (sGCi; cystamine, 50 nM, Sigma); 7) DMEM plus GnRH (50 nM) plus NOSi (50 nM); 8) DMEM plus GnRH (50 nM) plus sGCi (50 nM); 9) DMEM plus NOd (100 nM) plus sGCi (50 nM). Multiwell tissue culture plates (Becton Dickinson Co., Clifton, NJ) were used; the final volume of each well was 1 ml. Culture plates were incubated at room temperature (about 19°C). The pituitaries of each well were removed after 30 min of incubation and immediately transferred into column B for later superfusion. In all experimental groups, column B, containing the pituitary, was connected 30 min (arrows in the figures) after the beginning of testis superfusion with medium alone. The superfusions were stopped after 90 min. An experimental group with testis only was processed in order to determine the basal secretion of androgens. There were five replications for all the experimental groups. The whole superfusion experiment was repeated for all reproductive phases considered. Preliminary evidence led to our choice of the superfusion conditions and the minimum effective doses of the substances used in the present study (doses tested: 1, 25, 50, 75, 100, 250, and 500 nM; data not shown).

NOS Activity Determination

NOS activity was determined in the preincubated pituitaries by monitoring the conversion of [3H]L-arginine into [3H]L-citrulline, with a modified method previously described [33–35], and the data were expressed as fmol/mg protein. The pituitaries were homogenized in 1 ml of cold fresh homogenating buffer (50 mM Tris, 1 mM EDTA, and 1 mM EGTA, pH 7.4) and centrifuged at 20 000 x g for 60 min at 4°C. Supernatant (25 µl) and incubation buffer (100 µl; 1.5 mM NADPH, 1 mM CaCl2) containing 100 000 dpm [2,3-3H]L-arginine (specific activity, 30–40 Ci/mmol; Sigma) were added to the incubation tube. After 30-min incubation at room temperature (about 19°C), the enzymatic reaction was stopped by addition of 2 ml of blocking buffer (20 mM Hepes, 2 mM EDTA, pH 5.5). The mixture was applied to a preequilibrated column (20 mM sodium acetate, 2 mM EDTA, 0.2 mM EGTA, pH 5.5; 1-cm diameter) containing 1 ml of Dowex AG50W-X8 (Sigma), and the material was eluted with 2 ml of water. [3H]L-Citrulline was quantified in a liquid scintillation system (LS 1801; Beckman Instr., Fullerton, CA). Additional determinations were performed in the presence of excess of NOSi to verify the specificity of the assay for production of [3H]L-citrulline by NOS (data not shown). Protein concentration was determined by a commercial assay kit (Bio-Rad).

Cyclic GMP Determination

The concentrations of cGMP in the preincubated pituitaries were measured using an ACE enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the data were expressed as fmol/mg protein. The cross-reactivities > 0.01% were 100% acetylated cGMP, 9% cGMP, 0.05% acetylated cAMP.

Androgen Determination

Androgen contents of the superfusion medium samples were determined according to an RIA method previously described [29]. The sensitivity was 13.5 pg (intraassay variability: 9%; interassay variability: 17%). The parallelism among the standard curve in buffer, a standard curve in superfusion medium (then extracted), and a serial dilution of a single superfusion medium sample (extracted) was constant. Testosterone antiserum was provided by Dr. G.F. Bolelli (CNR-Institute of Normal and Pathologic Cytomorphology, University of Bologna, Italy) and Dr. F. Franceschetti (CNR-Physiopathology of Reproduction Service, University of Bologna, Italy). Testosterone was not separated from 5{alpha}-dihydrotestosterone; therefore, since the antiserum used is not specific, the data are expressed as androgens. The tritiated hormone was purchased from Amersham Int. (Buckinghamshire, UK) and the nonradioactive hormone from Sigma.

Statistical Analysis

All data were submitted to ANOVA and Duncan's multiple-range test; the superfusion data were examined by Kruskal-Wallis test also [36, 37].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Androgens

Basal androgen secretion from testis superfused without pituitary was constant between the start and finish of superfusion in all reproductive phases considered (data not shown).

Medium alone-preincubated pituitary increased (p < 0.01) androgen secretion within 10 min of addition during pre-reproduction, noncourtship, courtship, and recovery; the increase was higher during courtship (p < 0.01) than during all other phases (Figs. 1–4). GnRH-preincubated pituitary increased (p < 0.01) androgen secretion within 10 min after addition during pre-reproduction, noncourtship, courtship, and recovery; the increase induced by GnRH-preincubated pituitary was higher (p < 0.01) than that induced by medium alone-preincubated pituitary during pre-reproduction, noncourtship, and recovery (Figs. 1–4). NOd- and cGMPa-preincubated pituitary increased (p < 0.01) androgen secretion within 10 min of addition in all reproductive phases considered; the increase induced by NOd- and cGMPa-preincubated pituitary was higher (p < 0.01) than that induced by medium alone-preincubated pituitary during pre-reproduction, noncourtship, and recovery (Figs. 1–6). Pituitary preincubated with NOSi, sGCi, GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi did not change the basal secretion of androgens at any of the reproductive phases considered (Figs. 1–6).



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  		FIG. 1. Androgen secretion of testis in vitro superfused with pituitary in the male crested newt, Triturus carnifex, during pre-reproduction. Arrows indicate when the pituitary, preincubated in substances shown, was added. Each point is the mean of 5 values and is expressed as a percentage of basal (mean ± SEM = 111 ± 10 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion; {dagger}p < 0.01 vs. medium alone.



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  		FIG. 2. Androgen secretion of testis in vitro superfused with pituitary in the male crested newt, Triturus carnifex, during reproduction (noncourtship). Arrows indicate when the pituitary, preincubated in substances as shown, was added. Each point is the mean of 5 values and is expressed as a percentage of the basal secretion (mean ± SEM = 236 ± 27 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion; {dagger}p < 0.01 vs. medium alone.



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  		FIG. 3. Androgen secretion of testis in vitro superfused with pituitary in the male crested newt, Triturus carnifex, during reproduction (courtship). Arrows indicate when the pituitary, preincubated in substances as shown, was added. Each point is the mean of 5 values and is expressed as a percentage of the basal secretion (mean ± SEM = 194 ± 24 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion.



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  		FIG. 4. Androgen secretion of testis in vitro superfused with pituitary in the male crested newt, Triturus carnifex, during recovery. Arrows indicate when the pituitary, incubated in substances as shown, was added. Each point is the mean of 5 values and is expressed as a percentage of the basal secretion (mean ± SEM = 124 ± 14 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion; {dagger}p < 0.01 vs. medium alone.



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  		FIG. 5. Androgen secretion of testis in vitro superfused with pituitary in the male crested newt, Triturus carnifex, during the refractory period. Arrows indicate when the pituitary, incubated in substances as shown, was added. Each point is the mean of 5 values and is expressed as a percentage of the basal secretion (mean ± SEM = 35 ± 4 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion; {dagger}p < 0.01 vs. medium alone.



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  		FIG. 6. Androgen secretion of testis in vitro superfused with pituitary in male crested newt, Triturus carnifex, during estivation. The white arrows indicate when the pituitary, preincubated in substances as shown, was added. Each point is the mean of 5 values, and is expressed as a percentage of the basal secretion (mean ± SEM = 29 ± 5 pg/mg protein per 10 min). *p < 0.01 vs. 10, 20, and 30 min of superfusion; {dagger}p < 0.01 vs. medium alone.

NOS Activity

Basal NOS activity was highest (p < 0.01) during courtship and lowest (p < 0.01) during the refractory period and estivation in relation to that during pre-reproduction, noncourtship, and recovery (Table 1). GnRH and GnRH plus sGCi increased (p < 0.01) NOS activity during pre-reproduction, noncourtship, and recovery. NOSi and GnRH plus NOSi decreased (p < 0.01) NOS activity at all reproductive phases considered (Table 1). NOd, cGMPa, sGCi, and NOd plus sGCi did not change basal NOS activity at any of the phases considered (Table 1).


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  		TABLE 1. In vitro effects of GnRH, NOd, NOSi, cGMPa, and sGCi on NOS activity by pituitaries of male crested newt, Triturus carnifex, during the annual reproductive cycle.

Cyclic GMP

Basal cGMP levels were highest (p < 0.01) during courtship and lowest (p < 0.01) during the refractory period and estivation in relation to those during pre-reproduction, noncourtship, and recovery (Table 2). GnRH increased (p < 0.01) cGMP levels during pre-reproduction, noncourtship, and recovery (Table 2). NOd increased (p < 0.01) cGMP levels at all reproductive phases considered; NOd-increased levels were higher (p < 0.01) than GnRH-increased levels (Table 2). In all other experimental groups, cGMP levels decreased (p < 0.01) at all reproductive phases considered; sGCi-, GnRH plus sGCi-, and NOd plus sGCi-decreased levels were lower (p < 0.01) than NOSi- and GnRH plus NOSi-decreased levels (Table 2).


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  		TABLE 2. In vitro effects of GnRH, NOd, NOSi, and sGCi on cGMP contents of pituitaries of male crested newt, Triturus carnifex, during the annual reproductive cycle.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several studies have shown opposite effects of NO on pituitary gonadotropin secretion in mammals: some authors have suggested that basal LH secretion is not affected by NO [38, 39], and others that GnRH-induced LH secretion is inhibited by this free radical [22, 40]. In contrast, Chiodera et al. [41] found that GnRH-induced secretion of both LH and FSH is decreased by a NOS inhibitor; Yamada et al. [42] found that a potent long-lasting GnRH antagonist completely blocked the castration-induced increase of pituitary NOS; and Shi et al. [43] found that NO may attenuate the gonadotropin response to castration as a local balancing mediator. More recently, Garrel et al. [44] reported that there is an up-regulation of neuronal NOS by GnRH in gonadotrophs of rat pituitary; in fact, GnRH stimulates gene expression of NOS, resulting in an increased protein level and enzymatic activity.

The present study shows, first, that the gonadotropin secretion of male Triturus carnifex pituitary follows the reproductive annual cycle; in fact, the highest gonadotropin effects were found during the reproductive and recovery periods. These data mirror those of gonadotropin plasma levels found in a sympatric population of Rana esculenta [45]. In addition, our data show that the reproductive period gonadotropin secretion was increased by courtship, in agreement with what was reported for female Rana esculenta, whereas amplexus increases gonadotropin secretion during the reproductive period [26].

As previously found for another amphibian species [3], also in male Triturus carnifex, GnRH stimulated the pituitary to release gonadotropin secretion. In addition, the present data indicate that NO and cGMP mediate the gonadotropin secretion induced by GnRH, since the addition of inhibitors of NOS and sGC counteracted the effects of GnRH and since both NOS activity and cGMP production of the pituitary were stimulated by GnRH.

NOS activity and cGMP production were evoked by GnRH during pre-reproduction, noncourtship, and recovery, but not during courtship, the refractory period, and estivation. During courtship there were no GnRH effects, probably because the highest basal gonadotroph activity characterizes this phase; thus the addition of GnRH may be ineffective in increasing this activity. In fact, the highest activity of NOS and the highest production of cGMP were found in the pituitary during courtship. In agreement with this, the pronounced surges in gonadotropins are evident around the time of courtship in the bullfrog Rana catesbeiana [46] and the time of amplexus in the male toad Bufo japonicus [47], and the highest levels of NOS activity and cGMP production are present during amplexus in the pituitary of female Rana esculenta [26]. The unavailability of GnRH receptors in the gonadotrophs and/or inhibition of the GnRH intracellular mechanism that activates NOS could explain the ineffectiveness of GnRH during the refractory period and estivation. In agreement with this idea, NOd and cGMPa stimulated gonadotropin secretion in all phases considered except courtship; however, as reported above, the highest gonadotroph activity is characteristic of this phase.

Differently from the findings in mammals, but in accordance with what was found in female Rana esculenta [26], the present results showed that basal gonadotropin secretion is mediated by NO in male Triturus carnifex. In fact, the pituitary effects in inducing androgen secretion were increased by NOd during all reproductive phases except courtship, while these effects were decreased by NOSi during pre-reproduction, noncourtship, courtship, and recovery.

In summary, in amphibians, both basal and GnRH-induced pituitary gonadotropin secretion seem to be up-regulated by NO. In particular, a GnRH cellular mechanism of action on the regulation of gonadotropin secretion by the pituitary, previously found in female Rana esculenta [26], may be proposed for male Triturus carnifex as well: the GnRH-receptor interaction activates membrane phospholipase C favoring diacylglycerol and InP3 production and, in turn, protein kinase C activation and intracellular Ca2+ mobilization. One or both of these activate NOS with a consequent increase of NO, which intervenes in the secretion of gonadotropins, through sGC activation.


   ACKNOWLEDGMENTS
 
We are indebted to James Burge of the Camerino University Institute of Linguistics for help with the English.


   FOOTNOTES
 
1 This work was financially supported by MURST. Back

2 Correspondence: A. Gobbetti, Dipartimento di Biologia MCA, University of Camerino, via F. Camerini 1, 62032 Camerino MC, Italy. FAX: 39 737 636216; goze{at}camserv.unicam.it Back

Accepted: December 22, 1998.

Received: August 28, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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