Endocannabinoids and endovanilloids: a possible balance in the regulation of the testicular GnRH signalling.

Reproductive functions are regulated both at central (brain) and gonadal levels. In this respect, the endocannabinoid system (eCS) has a very influential role. Interestingly, the characterization of eCS has taken many advantages from the usage of animal models different from mammals. Therefore, this review is oriented to summarize the main pieces of evidence regarding eCS coming from the anuran amphibian Rana esculenta, with particular interest to the morphofunctional relationship between eCS and gonadotropin releasing hormone (GnRH). Furthermore, a novel role for endovanilloids in the regulation of a testicular GnRH system will be also discussed.

Anandamide regulates the expression of GnRH1, GnRH2, and GnRH-Rs in frog testis.

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.

Interplay between the endocannabinoid system and GnRH-I in the forebrain of the anuran amphibian Rana esculenta.

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.

UBPy/MSJ-1 system during male germ cell progression in the frog, Rana esculenta.

mUBPy (mouse ubiquitin specific processing protease) is a de-ubiquitinating enzyme expressed in mouse testis and brain. In testis, it interacts with the DnaJ protein MSJ-1 (mouse sperm cell specific DnaJ first homologue), a molecular chaperone expressed in spermatids and spermatozoa. Since MSJ-1 is conserved among vertebrates, to demonstrate an evolutionarily conserved function of UBPy/MSJ-1 system, we assayed mUBPy presence in the anuran amphibian, the frog, Rana esculenta, during the annual sexual cycle. By Western blot we have detected a specific signal of 126kDa in testis and isolated spermatozoa. During the annual sexual cycle, the signal gradually increases as soon as spermatogenesis resumes after the winter stasis. Using immunocytochemistry, we have localized the protein in spermatids and spermatozoa. In conclusion, UBPy/MSJ-1 system is available in R. esculenta testis suggesting a conserved fundamental function in spermatogenesis and sperm formation.

Endocannabinoid system in frog and rodent testis: type-1 cannabinoid receptor and fatty acid amide hydrolase activity in male germ cells.

N-arachidonoylethanolamide (anandamide [AEA]) is the main endocannabinoid described to date in the testis. It exerts its effects through the activation of G-protein coupled cannabinoid receptors (CNR). However, the activity of AEA in controlling male reproduction is still poorly known. Here we provide direct evidence on the presence of the "endocannabinoid system," constituted by type-1 cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH), in the frog Rana esculenta testis demonstrating its expression in tubular compartment. In fact, during the annual reproductive cycle, both proteins increase in September, when the appearance of spermatids (SPT) occurs. Immunocytochemistry confirms their localization in germ cells and, in particular, in elongated SPT. Signals are still present in spermatozoa (SPZ), as demonstrated by Western blot analysis. Furthermore, the activation of CNR1 reduces sperm motility. Comparative research, carried out using mouse and rat SPZ, definitely indicates that the endocannabinoid system operates in SPZ of phylogenetically distant species. A conserved physiological role of endocannabinoid system in controlling the inhibition of sperm motility is suggested.

Fra-1 activity in the frog, Rana esculenta, testis.

Using an anti-Fos family member antiserum, we previously described, in the testis of Rana esculenta, the presence of a nuclear 43-kDa protein that we hypothesized to be Fra-1. Using an antiserum against Fra-1, we here report on Fra-1 expression, localization, and putative activity in the R. esculenta testis during the annual reproductive cycle. Western blot analysis confirms that the nuclear 43-kDa protein is Fra-1. Immunocytochemistry demonstrates Fra-1 in peritubular myoid cells (PMC), efferent ducts, and blood vessels. We present, for the first time for a vertebrate, experimental evidence that the expression of Fra-1 in PMC is related to its activity during sperm transport from the tubular compartment to the efferent ducts.

Insulin-induced changes in beta-adrenergic response: an experimental study in the isolated rat papillary muscle.

BACKGROUND: The aim was to investigate the ability of insulin to modulate the response to beta-adrenergic action on myocardial contractility, assessed as percentage changes of developed tension, in isolated rat papillary muscle. METHODS: Dose-response curves for isoproterenol, calcium, and forskolin were constructed in an incremental fashion with the presence or absence of insulin at the dose of 50 muU/mL. Dose-response curves for isoproterenol on insulin background were also assessed in the presence and absence of a selective antagonist for beta(2)-adrenoceptor, ICI, at the dose of 5 x 10(-8) mol/L. RESULTS: Insulin did not modify the dose-response curve to calcium (EC(50): 1.4 +/- 0.4 mmol/Lfor insulin, n = 8 v 1.5 +/- 0.3 mmol/L for control, n = 8; P = not significant), whereas it was able to shift to the left the dose-response curve and reduce significantly the EC(50) of isoproterenol (EC(50): 0.2 +/- 0.2 nmol/L for insulin, n = 13 v 1.1 +/- 0.4 nmol/L for control, n = 12; P < .01). ICI shifted to the right dose-response curve of isoproterenol and increased about 10-fold the EC(50) value of isoproterenol, but insulin was still able to shift to the left dose-response curve of isoproterenol and to reduce significantly the EC(50) of isoproterenol also in the presence of ICI (EC(50): 11.0 +/- 1.5 nmol/L for ICI, n = 7 v 1.9 +/- 0.8 nmol/L for ICI + insulin, n = 7; P < .01). Insulin did not modify the dose-response curve to forskolin. CONCLUSIONS: Our results suggest that the insulin-induced modulation of contractility is calcium independent and insulin leads to a supersensitization on the beta(1)-adrenoceptors without effects on beta-adrenoceptor independent adenylate cyclase-related pathway.

Fra1 activity in the frog, Rana esculenta, testis: a new potential role in sperm transport.

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.

Detection of msj-1 gene expression in the frog, Rana esculenta testis, brain, and spinal cord.

MSJ-1 is member of the DnaJ/heat shock protein (Hsp) 40 chaperone protein family. It is present in mouse testis and spinal cord. In particular, MSJ-1 is localized in post-meiotic cells and in motoneurones of the ventral horns. To assess whether the role of this protein is evolutionarily conserved, we have investigated if msj-1 gene is expressed in the frog, Rana esculenta. Using reverse transcription-polymerase chain reaction (RT-PCR), a msj-1-like transcript was detected in testis, brain, and spinal cord. Homology ranging from 42.3 to 46.0% was found as compared with the mammalian counterparts. Muscle did not show any signal. By Western blot analysis, a signal of the predicted size of 30 kDa was evidenced in testis, brain, and spinal cord but not in ovary, heart, liver, kidney, and muscle. MSJ-1 fluctuations in the testis reveal that it appeared in concomitance with post-meiotic events during the annual sexual cycle, as shown in a previous study. The protein is localized in spermatids and is still retained in mature spermatozoa, where it has perinuclear and centriolar localization. MSJ-1 levels did not change in brain and spinal cord. Furthermore, in the brain MSJ-1 was mainly present in diencephalon and mesencephalon, while in spinal cord MSJ-1 was localized into several motoneurones of the cervical and thoracic tract. A putative role in vesicle trafficking is briefly discussed.