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ABSTRACT: Kisspeptins, acting via GPR54, are new players in the control of reproductive axis. They have the ability to communicate with GnRH neurons sending environmental, metabolic and gonadal signals, with the induction of GnRH and LH secretion as final effect. At present, the physiological significance of kisspeptin signalling in the gonad is poorly investigated. We cloned GPR54 receptor from the anuran amphibian, Rana esculenta, testis and investigated its expression in several tissues (brain, spinal cord, ovary, muscle and kidney). In particular, the expression analysis was carried out in pituitary and testis during the annual sexual cycle. Pituitary and testicular GPR54 mRNA increased at the end of the winter stasis (February) and reached high levels during the breeding season (April). The analysis of GPR54 expression in testis was reinforced by in situ hybridization that revealed GPR54 presence in the interstitial compartment and in proliferating germ cells. Testicular GPR54 expression in February and in June was indicated to be estradiol dependent. Furthermore, in February, kisspeptin-10 (Kp-10) induced the testicular expression of both GPR54 and estrogen receptor alpha (ERalpha) in a dose dependent manner. Conversely, in March, Kp-10 had a biphasic effect upon the expression of ERalpha, being inhibitory at short (1 h) and stimulatory at longer (4 h) incubation time. In conclusion, our results demonstrate that frog testis expresses GPR54 in estradiol dependent manner and that Kp-10 modulates the testicular expression of ERalpha; thus kisspeptin/GPR54 system might be locally involved in the regulation of estrogen dependent testicular functions such as germ cell proliferation and steroidogenesis.
Biology of Reproduction 01/2013; · 4.01 Impact Factor
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ABSTRACT: 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.
AJP Endocrinology and Metabolism 06/2012; 303(4):E475-87. · 4.75 Impact Factor
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ABSTRACT: Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.
Molecular and Cellular Endocrinology 01/2012; 355(1):1-14. · 4.19 Impact Factor
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ABSTRACT: 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.
General and Comparative Endocrinology 07/2011; 173(3):389-95. · 3.27 Impact Factor
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ABSTRACT: Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates.
General and Comparative Endocrinology 03/2011; 171(1):17-27. · 3.27 Impact Factor
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Teresa Chioccarelli,
Giovanna Cacciola,
Lucia Altucci,
Sheena E M Lewis,
Luke Simon,
Giulia Ricci,
Catherine Ledent, Rosaria Meccariello,
Silvia Fasano,
Riccardo Pierantoni,
Gilda Cobellis
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ABSTRACT: Marijuana smokers and animals treated with Δ9-tetrahydrocannabinol, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Because the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of wild-type, Cnr1+/-, and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, whereas histone displacement was disrupted to a lesser extent. Furthermore, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA packaging during spermiogenesis and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.
Endocrinology 10/2010; 151(10):5017-29. · 4.46 Impact Factor
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Gilda Cobellis,
Giulia Ricci,
Giovanna Cacciola,
Pierangelo Orlando,
Stefania Petrosino,
Maria Grazia Cascio,
Tiziana Bisogno,
Luciano De Petrocellis,
Teresa Chioccarelli,
Lucia Altucci,
Silvia Fasano, Rosaria Meccariello,
Riccardo Pierantoni,
Catherine Ledent,
Vincenzo Di Marzo
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ABSTRACT: During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile ("start-up"). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the 2-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.
Biology of Reproduction 10/2009; 82(2):451-8. · 4.01 Impact Factor
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ABSTRACT: The effects of cannabinoids on human health have been known since the antiquities when the extract of the plant Cannabis sativa was used because of its psychoactivity. The scientific story of the cannabinoids started in the 1960s with the isolation and characterization of the active component of the plant. After the synthesis of cannabinoid analogues, the analysis of structure-effect relationships was implemented, and this had a similar effect to a positive "Pandora's box" opening. To date, numerous roles have been ascribed to the "endocannabinoid system." Here we describe its involvement in the control of male reproduction, taking into consideration possible evolutionary speculations. Indeed, the endocannabinoid system is a very ancient signaling system, being clearly present from the divergence of the protostomian/deuterostomian.
Annals of the New York Academy of Sciences 05/2009; 1163:112-24. · 3.15 Impact Factor
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ABSTRACT: 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.
Annals of the New York Academy of Sciences 05/2009; 1163:279-91. · 3.15 Impact Factor
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ABSTRACT: The importance of the endocannabinoid system (ECBS) and its involvement in several physiological processes is still increasing. Since the isolation of the main active compound of Cannabis sativa, Delta(9)-THC, several lines of research have evidenced the basic roles of this signaling system mainly considering its high conservation during evolution. In this chapter the attention is focussed on the involvement of the ECBS in the control of male reproductive aspects at both central and local levels which are both considered from a comparative point of view.
Vitamins & Hormones 02/2009; 81:367-87. · 2.19 Impact Factor
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Stefania Crispi,
Raffaele A Calogero,
Mario Santini,
Pasquale Mellone,
Bruno Vincenzi,
Gennaro Citro,
Giovanni Vicidomini,
Silvia Fasano, Rosaria Meccariello,
Gilda Cobellis,
Simona Menegozzo,
Riccardo Pierantoni,
Francesco Facciolo,
Alfonso Baldi,
Massimo Menegozzo
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ABSTRACT: The goal of our study was to molecularly dissect mesothelioma tumour pathways by mean of microarray technologies in order to identify new tumour biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets.
We performed Affymetrix HGU133A plus 2.0 microarray analysis, containing probes for about 39,000 human transcripts, comparing 9 human pleural mesotheliomas with 4 normal pleural specimens. Stringent statistical feature selection detected a set of differentially expressed genes that have been further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes, never associated before to mesotheliom that could be involved in tumour progression. Notable is the identification of MMP-14, a member of matrix metalloproteinase family. In a cohort of 70 mesothelioma patients, we found by a multivariate Cox regression analysis, that the only parameter influencing overall survival was expression of MMP14. The calculated relative risk of death in MM patients with low MMP14 expression was significantly lower than patients with high MMp14 expression (P = 0.002).
Based on the results provided, this molecule could be viewed as a new and effective therapeutic target to test for the cure of mesothelioma.
PLoS ONE 01/2009; 4(9):e7016. · 4.09 Impact Factor
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ABSTRACT: Endocannabinoids are lipidic modulators able to bind cannabinoid receptors (CNRs). Two types of CNRs have been cloned, CNR1 (central) and CNR2 (peripheral). The objectives of the present study were to investigate the expression pattern of CNR1 in the rat testis during prepubertal development and to define the CNR1 spatiotemporal pattern. From 31 to 60 days of age, CNR1 was immunolocalized in round elongating spermatids and spermatozoa, suggesting an important role for this receptor in spermatogenesis. From 14 to 60 days of age, adult Leydig cells (ALCs) at different developmental stages were positive for CNR1. In particular, CNR1 expression in differentiating ALCs was negatively correlated to cell division. Bromodeoxyuridine uptake experiments on serial sections showed that immature Leydig cells in mitosis were negative for CNR1; in contrast, immature nonmitotic Leydig cells were positive for CNR1. A further observation of few ALCs in CNR1KO mice validates the role of CNR1 during proliferative activity involved in ALC differentiation. In addition, starting from 41 days of age, a faint CNR1 signal was also observed in Sertoli cells. Taken together, these results demonstrate the first clear evidence (to our knowledge) of CNR1 in mammalian germinal epithelium, ALCs, and Sertoli cells and indicate that differentiation of ALCs may depend on the endocannabinoid system.
Biology of Reproduction 08/2008; 79(4):758-65. · 4.01 Impact Factor
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ABSTRACT: 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.
Endocrinology 06/2008; 149(5):2149-58. · 4.46 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Endocrinology 05/2008; 286(1-2 Suppl 1):S46-51. · 4.19 Impact Factor
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ABSTRACT: Prevailing studies emphasize on endocannabinoid activity in brain. However, sporadic evidences hint that endocannabinoid system controls male reproduction ranging from invertebrates to vertebrates. Although N-arachidonoylethanolamine is described in rat testis, its activity is still poorly known. Type-1 cannabinoid receptor and fatty acid amide hydrolase are particularly expressed in elongating spermatids and spermatozoa suggesting that endocannabinoids affect spermiogenesis and sperm physiology. Aim of this paper is to provide an analysis of the information available in vertebrates on male germ cell progression and sperm maturation mediated by the endocannabinoid system.
Molecular and Cellular Endocrinology 05/2008; 286(1-2 Suppl 1):S24-30. · 4.19 Impact Factor
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ABSTRACT: Endogenous and environmental estrogens have been proved to affect male reproduction in vertebrates. Both positive and negative effects in the regulation of the reproductive tract have been described. Since it is well known that amphibians represent a useful model to study several aspects concerning reproductive activity, we have taken advantage of the frog, Rana esculenta, to study the involvement of estrogens in sperm release. We show here that pituitary hormones increased the number of peritubular myoid cells (PMCs) expressing Fra-1 and induced testicular morphological changes related to sperm release. The estrogen antagonist ICI182-780 counteracted the hypophysis driven effects. In vivo and in vitro experiments demonstrated that 17beta-Estradiol acted directly on the testis to switch-on Fra-1 in PMCs. Furthermore, impairment of estrogen activity significantly reduced sperm release mainly affecting the detachment of spermatozoa from Sertoli cells (spermiation). Therefore, estrogens can be considered a new entry in the list of substances involved in spermiation.
General and Comparative Endocrinology 03/2008; 155(3):838-46. · 3.27 Impact Factor
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ABSTRACT: The endocannabinoid system is a conserved system involved in the modulation of several physiologic processes, from the activity of the central nervous system to reproduction. Type 1 cannabinoid receptor (CNR1) cDNA was cloned from the brain and testis of the anuran amphibian, the frog Rana esculenta. Nucleotide identity ranging from 62.6% to 81.9% is observed among vertebrates. The reading frame encoded a protein of 462 amino acids (FCNR1) with all the properties of a membrane G-coupled receptor. Alignments of FCNR1 with those of other vertebrates revealed amino acid identity ranging from 61.9% to 88.1%; critical domains for CNR1 functionality were conserved in the frog. As nucleotide differences of cnr1 cDNA were observed in brain and testis, the genomic sequence of the cnr1 gene was also determined in the same tissue preparations. Nucleotide changes in codons 5, 30, 70, 186, 252 and 408 were observed when cDNA and genomic DNA were compared; the nucleotide differences did not affect the predicted amino acid sequences, except for changes in codons 70 and 408. Interestingly, the predicted RNA folding was strongly affected by different nucleotide sequences. Comparison of cnr1 mRNA sequences available in GenBank with the corresponding genomic sequences revealed that also in human, rat, zebrafish and pufferfish, nucleotide changes between mRNA and genomic sequences occurred. Furthermore, amino acid sequences deduced from both mRNA and the genome were compared among vertebrates, and also in pufferfish the nucleotide changes corresponded to modifications in the amino acid sequence. The present results indicate for the first time that changes in nucleotides may occur in cnr1 mRNA maturation and that this phenomenon might not be restricted to the frog.
FEBS Journal 07/2007; 274(11):2909-20. · 3.79 Impact Factor
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ABSTRACT: 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.
Biology of Reproduction 08/2006; 75(1):82-9. · 4.01 Impact Factor
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ABSTRACT: Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.
Molecular Reproduction and Development 06/2006; 73(5):551-8. · 2.53 Impact Factor
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ABSTRACT: 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.
Biology of Reproduction 06/2005; 72(5):1101-8. · 4.01 Impact Factor
