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Gonadotropin-releasing hormone (either GnRH1 or GnRH2) exerts a local activity in vertebrate testis, including human testis. Relationships between endocannabinoid (eCB) and GnRH systems in gonads have never been elucidated in any species so far. To reveal a cross-talk between eCBs and GnRH at testicular level, we characterized the expression of GnRH (GnRH1 and GnRH2) as well as GnRH receptor (GnRH-R1, -R2, and -R3) mRNA in the testis of the anuran amphibian Rana esculenta during the annual sexual cycle; furthermore, the corresponding transcripts were localized inside the testis by in situ hybridization. The possible endogenous production of the eCB, anandamide (AEA), was investigated in testis by analyzing the expression of its biosynthetic enzyme, Nape-pld. Incubations of testis pieces with AEA were carried out in the postreproductive period (June) and in February, when a new spermatogenetic wave takes place. In June, AEA treatment significantly decreased GnRH1 and GnRH-R2 mRNA, stimulated the transcription of GnRH2 and GnRH-R1, and did not affect GnRH-R3 expression. In February, AEA treatment upregulated GnRH2 and GnRH-R3 mRNA, downregulated GnRH-R2, and did not affect GnRH1 and GnRH-R1 expression. These effects were mediated by type 1 cannabinoid receptor (CB1) since they were fully counteracted by SR141716A (Rimonabant), a selective CB1 antagonist. In conclusion, eCB system modulates GnRH activity in frog testis during the annual sexual cycle in a stage-dependent fashion.

In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs. Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1). In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Since the end of the 1970s, studies have shown that, besides the endocrine route, a chemical mediator may also act through autocrine and/or paracrine mechanisms. This has opened new frontiers for research as a result of a redefinition of what endocrinology represents. Apart from androgens within the male gonad, testicular gonadotropin-releasing hormone, estrogens, molecular chaperones, proto-oncogenes, and, very recently, the endocannabinoid system have been shown to play important roles. Their activities to regulate spermatogenesis, including spermiogenesis and sperm maturation, will be discussed from the comparative viewpoint to describe adaptive phenomena and to speculate on evolution.

The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.

Endocannabinoids, via cannabinoid receptors (CB1 and CB2), affect reproductive functions at both local and central level. Due to the high complexity of the endocannabinoid system, to the widespread distribution outside the nervous system and to the high degree of evolutionary conservation, a deep CB1 molecular characterization among species may be useful to elucidate the activity of endocannabinoids at multiple levels. In this review we report CB1 characterization in non-mammalian animal models and, in particular, in the anuran amphibian, the frog, Rana esculenta; we also describe its expression during the annual sexual cycle. Moreover, since reproductive functions are under control of gonadotropin releasing hormone (GnRH), cb1 mRNA and protein expression profile in the forebrain has been compared to those of GnRH-I, the mammalian form primarily involved in gonadotropin release.

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c-jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.

This review emphasizes the comparative approach for developing insight into knowledge related to cellular communications occurring in the hypothalamus-pituitary-gonadal axis. Indeed, research on adaptive phenomena leads to evolutionary tracks. Thus, going through recent results, we suggest that pheromonal communication precedes local communication which, in turn, precedes communication via the blood stream. Furthermore, the use of different routes of communication by a certain mediator leads to a conceptual change related to what hormones are. Nevertheless, endocrine communication should leave out of consideration the source (glandular or not) of mediator. Finally, we point out that the use of lower vertebrate animal models is fundamental to understanding general physiological mechanisms. In fact, different anatomical organization permits access to tissues not readily approachable in mammals.

Gonadotrophin-releasing hormone (GnRH) immunoreactivity is detectable in the brain, ovary, and testis of the newt, Triturus carnifex, collected during February (reproductive phase), May, and July (nonreproductive phase). In the brain of May animals, chicken GnRH-II positive cell bodies are located within the terminal nerve, the anterior preoptic area, and the preoptic nucleus, which appears to be devoid of immunoreactive mammalian GnRH cell bodies. During February and July, both chicken GnRH-II and mammalian GnRH are detected only within the terminal nerve and anterior preoptic area. Generally, in the reproductive as well as the nonreproductive periods, chicken GnRH-II fibers are widely distributed in the brain; however, the distribution of fibers of both molecular forms suggests that they exert hypophysiotropic activity. High-pressure liquid chromatography (HPLC) coupled with radioimmunoassay indicates the presence of an early-eluting GnRH peak in brains and gonads but not in plasma. Using chicken GnRH-II antiserum, immunoreactivity is observed in spermatocytes, spermatozoa, and the external theca layer. Seasonal changes of the GnRH-like material are observed in both sexes, and its high concentration detectable during February is in good correlation with the timing of reproduction.

In the amphibian brain four molecular forms of GnRH have been identified so far: mammalian GnRH (m- and hydroxyproline9m-), chicken II GnRH (cII), and a salmon (s) GnRH-like peptide. In Rana esculenta, cII- and s-GnRH-like molecules have been partially characterized in the brain extracts using HPLC combined with radioimmunoassay. Moreover, since cII-GnRH-like material has been detected in Rana esculenta testis, the present study describes the localization of the above peptides in the brain and testis of the frog. Immunoreactive cII-GnRH and m-GnRH neurons and fibers were identified in the anterior preoptic area (APOA) and in the median septal area (MSA). A population of cells located on the dorsal side of the caudal preoptic region was also stained. Immunopositive fibers were seen to overlap the median eminence before ending within the pars nervosa. Moreover, densely packed fibers made close contact with the vascular complex in the median eminence. Conversely, immunoreactive s-GnRH-like material was absent in APOA and MSA, but weakly scattered elements were detected by the anti-s-GnRH serum in the dorsal side of the caudal preoptic region. Using m-GnRH antiserum, a strong immunopositivity was observed in the median eminence but not within the pars nervosa, indicating that, besides cII-GnRH and s-GnRH-like material, also m-GnRH-like material is present in Rana esculenta brain. In the testis, cells of the interstitial and germinal compartment were detected by anti-cII-GnRH during different periods of the annual cycle. In particular, in October and February interstitial tissue was intensely stained, coinciding with periods of increased androgen production and the onset of the new spermatogenic wave, respectively.

In the testis of the frog, Rana esculenta, mitotic activity of primary spermatogonia is regulated by gonadotropins and synergistically by testosterone. In addition GnRH-like material directly stimulates gonadal activity. Intact animals were treated with a GnRH agonist (GnRHa, buserelin, Hoechst) and/or a GnRH antagonist giving injections intraperitoneally on alternate days for 15 days. Moreover, testes were treated in vitro for 24 hr with GnRHa. 3H-thymidine and colchicine were used to assess the labelling and the mitotic index (LI and MI) of primary spermatogonia. Both LI and MI were increased by the treatment with GnRHa but the rate of cells measured by LI was significantly higher than that of cells measured by MI. Therefore, our results confirm the role of GnRH-like material as local regulator of the testicular activity in vertebrates and show its involvement in promoting the G1-S transition of spermatogonial cell cycle in the frog, Rana esculenta.

In the frog, Rana esculenta, two molecular forms of GnRH, coeluting with chicken (c) GnRH-II and salmon (s) GnRH, have been detected using HPLC and radioimmunoassay. Mammalian (m) GnRH seems to be also present. In amphibians the role of cGnRH-II seems to be primarily the involvement in the regulation of neuroendocrine processes and, while the mGnRH has been postulated to act as a neurotransmitter and/or neuromodulator, the activity of sGnRH-like material has not been investigated. Therefore, we have treated the frogs with single or multiple injections of cGnRH-II or sGnRH (6 micrograms) or both peptides (6 micrograms of each) to detect differences in the response measured as testicular or plasma androgen (testosterone plus 5 alpha-dihydrotestosterone) concentration during the annual reproductive cycle. The basal profile of testicular and plasma androgen shows that the spring peak disappeared in control animals given multiple injections and kept in short-term captivity. We show in the treatment with cGnRH-II and/or sGnRH that the effects of the peptides depend on the season, the experimental design, and the tissue in which androgen levels were measured. In particular, both peptides strongly stimulate androgen production during the autumn-winter period, the time of the greater response to the GnRHs when basal levels of steroids are highest.

Two molecular forms of GnRH (chicken GnRH II and a second variant) are present in the brains of species from all the major vertebrate groups. Their differential distribution in the brain and temporal expression during development suggests that have different functional roles. We investigated the nature of GnRH molecular forms in the brain, plasma, testis, and ovary of adult and juvenile lungfish (Protopterus annectens), using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. In the brain of adult and juvenile lungfish, two peptides with identical chromatographic and immunologic properties to mammalian GnRH and chicken GnRH II were detected. Chicken GnRH II predominated in both the adult and juvenile brain, and the percentage of chicken GnRH II relative to mammalian GnRH was greater in the juvenile brain. In the plasma, only mammalian GnRH was present. Immunoreactive GnRH was not detected in the testis and ovary. Chicken GnRH II and mammalian GnRH were found in the cells of the preoptic nucleus and in the ganglion of the nervus terminalis. Fibers were seen in the ventral hypothalamus, and chicken GnRH II immunoreactivity was detected within the neural lobe of the pituitary. The finding of chicken GnRH II in a sarcopterygian fish adds further support to our hypothesis that this ubiquitous structural variant is highly conserved and likely to have an important functional role. Mammalian GnRH, previously described in several early-evolved actinopterygian fish, also has a fairly widespread distribution and early evolutionary origin. The immunocytochemical distribution of mammalian GnRH and chicken GnRH II fibers in the lungfish brain suggests that both forms are hypophysiotropic. In addition, the presence of mammalian GnRH in the plasma of the lungfish suggests that this molecular form of GnRH has a hypophysiotropic function reaching target organs (pituitary and gonads) via the general circulation.

The distribution of different molecular forms of gonadotropin-releasing hormone (GnRH) in the brain and serum of the spotted dogfish, Scyliorhinus canicula, was investigated by an indirect immunofluorescence method, using antisera against salmon (s-), chicken-II (cII-) and mammalian (m-) GnRHs, and by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with radioimmunoassays. Five GnRH molecular forms were demonstrated on the basis of the retention time in the RP-HPLC system. The characteristics of four of these GnRH peptides are consistent with those of m-, cII-, dogfish (df-), and sGnRH. The fifth form appears to be novel. Immunoreactive sGnRH structures were confined to the diencephalon; whereas cIIGnRH and mGnRH were found in the telencephalon and diencephalon. cIIGnRH- and dfGnRH-like molecules were detected in the serum. Moreover, a specific, low-affinity GnRH binding protein (GnRH-BP) was found in the serum of the spotted dogfish. The binding of [125I]sGnRHA to the serum GnRH-BP was dependent on incubation time, equilibrium being reached within 1 hr at 4 degrees; binding was rapid and completely reversible. Scatchard analysis yielded a linear plot with a Kd of 7.9 x 10(-7) M. The presence of a GnRH-BP in spotted dogfish serum suggests a probable action of GnRH via the general circulation.

GnRH-like material has been identified using HPLC followed by RIA in the ovary of Rana esculenta. During the reproductive cycle three immunoreactive GnRH peaks were eluted. One of them coeluted with s-GnRH, the other two forms between GnRH and cII-GnRH. During the recovery phase s-GnRH immunoreactivity disappears. By immunocytochemistry, cII-GnRH immunostaining was localized to granulosa cells while s-GnRH was present in the perinuclear zone of the oocytes.

ChickenII-(cII-) and salmon (s-) GnRH levels have been measured in the male frog Rana esculenta during the annual cycle. The presence of pituitary binding activity for both peptides and plasma androgen levels has been investigated in order to give insight into the significance of the dual control exerted by the GnRH forms present in the R. esculenta brain. ChickenII- and s-GnRHs showed high values during the spring-summer period. Conversely, while cII-GnRH peaked in February, s-GnRH declined slowly from February until May. Plasma androgen levels increased as the peptides decreased during the autumn-winter period. Still high androgen levels (but significantly lower as compared with winter concentrations) were found during spring. Using iodinated cII-GnRH, GnRH binding sites were detected in pituitary preparations when the corresponding peptide concentration decreased in the brain. On the contrary, no binding sites were found using labeled s-GnRH. Our results indicate that cII-GnRH has a hypophysiotropic activity, while the role of s-GnRH needs to be further investigated.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.

The effects of vertebrate gonadotropin-releasing hormone (GnRH) variants on plasma and testicular androgen level in intact and hypophysectomized (PDX) male frogs, Rana esculenta, have been investigated. In intact animals, mammalian (m)-GnRH, m-GnRH analog (buserelin), salmon (s)-GnRH, chicken (c) I-GnRH, cII-GnRH, D-Arg6-cII-GnRH (cII-GnRHA), and lamprey (l)-GnRH (1.5 micrograms and 6 micrograms, total dose given on alternate days for 5 days) were able to enhance androgen production showing that specificity of pituitary responsiveness to GnRH variants appears to be low. Chicken II-GnRH was more effective than s-GnRH in eliciting testicular and circulatory androgen level increase. Moreover, in animals treated with 6 micrograms of cII-GnRH and s-GnRH in combination, androgens decreased as compared with animal treated with cII-GnRH only, suggesting that GnRH receptors bind preferentially the s-GnRH form. In PDX animals, buserelin (1.5 and 6 micrograms), cII-GnRH, and its analog (6 micrograms) were able to increase plasma androgen levels whereas testis androgen concentrations were increased by cII-GnRH (1.5 and 6 micrograms), D-Arg6-cII-GnRHA, and buserelin (6 micrograms). Since androgen production in PDX animals is influenced especially by peptides sharing cII-GnRH structure, it is suggested that a testicular cII-GnRH-like material play a role as local modulator of the gonadal activity in Rana esculenta.

The possible physiological role of a putative testicular gonadotropin-releasing hormone (GnRH)-like material was studied in the frog, Rana esculenta. We have investigated (a) changes of mitotic index (MI) of primary spermatogonia (SPG) in GnRH agonist (GnRH-Ag)-treated testes in vitro; (b) changes of androgen concentrations in testes of intact frogs treated with a GnRH antagonist (GnRH-Ant); (c) variations of mitotic index of primary SPG in intact GnRH-Ant-injected animals and in testes incubated with GnRH-Ant; (d) changes of MI in hypophysectomized (PDX) animals treated with hypophysis (PD) homogenate, and hCG alone or in combination with GnRH-Ant; and (e) changes of androgen concentrations in plasma PDX frogs treated with hCG and hCG plus GnRH-Ant. Our results indicate that while GnRH-Ag induced accumulation of primary SPG mitosis, GnRH-Ant inhibited androgen production and natural occurring mitosis accumulation. Therefore, GnRH-Ant may counteract an endogenous peptide working into the testis.