Vincenza Ciaramella

The contribution of lower vertebrate animal models in human reproduction research.

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.

Kisspeptin Receptor, GPR54, as a Candidate for the Regulation of Testicular Activity in the Frog, Rana esculenta

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 signaling 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 on 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 an estradiol-dependent manner and that Kp-10 modulates the testicular expression of ERalpha; thus, the kisspeptin/GPR54 system might be locally involved in the regulation of estrogen-dependent testicular functions such as germ cell proliferation and steroidogenesis.

Intra-Testicular Signals Regulate Germ Cell Progression and Production of Qualitatively Mature Spermatozoa in Vertebrates

Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic–pituitary control, being regulated by the secretion of pituitary gonadotropins, follicle stimulating hormone, and luteinizing hormone, in response to stimulation exerted by gonadotropin releasing hormone from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intra-gonadal signals sustains the production of high quality mature spermatozoa. In this review, we focus on the recent advances in the area of the intra-gonadal signals supporting sperm development.

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.

Anandamide modulates the expression of GnRH-II and GnRHRs in frog, Rana esculenta, diencephalon.

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.

Cannabinoids and Reproduction: A Lasting and Intriguing History

Starting from an historical overview of lasting Cannabis use over the centuries, we will focus on a description of the cannabinergic system, with a comprehensive analysis of chemical and pharmacological properties of endogenous and synthetic cannabimimetic analogues. The metabolic pathways and the signal transduction mechanisms, activated by cannabinoid receptors stimulation, will also be discussed. In particular, we will point out the action of cannabinoids and endocannabinoids on the different neuronal networks involved in reproductive axis, and locally, on male and female reproductive tracts, by emphasizing the pivotal role played by this system in the control of fertility.

Kisspeptin drives germ cell progression in the anuran amphibian Pelophylax esculentus: A study carried out in ex vivo testes

Kisspeptin, via Gpr54 receptor, regulates puberty onset in most vertebrates. Thus, the direct involvement of kisspeptin activity in testis physiology was investigated in the anuran amphibian, Pelophylax esculentus. In this vertebrate gpr54 mRNA has been localized in both interstitial compartment and spermatogonia (SPG), whereas SPG proliferation requires the cooperation between estradiol and testicular Gonadotropin releasing hormone (Gnrh). In the pre-reproductive period, dose response curve to assess the effects of Kisspeptin-10 (Kp-10) was carried out in vitro (dose range: 10(-9)-10(-6)M; incubation times: 1 and 4h); proliferative activity and germ cell progression were evaluated by expression analysis of proliferating cell nuclear antigen (pcna), estrogen receptor beta (erβ), Gnrh system (gnrh1, gnrh2, gnrhr1, r2, r3) and by the count of empty, mitotic and meiotic tubules. All selected markers were up regulated at 4h Kp-10 incubation. Histological analysis also proved the increase of mitotic activity and the progression of spermatogenesis. Besides Kp-10 modulation of testicular Gnrh system, in vitro treatment with 17β-estradiol (10(-6)M) ± the antagonist ICI182-780 (10(-5)M) revealed gnrh2 and gnrhr3 estrogen dependent expression. In the reproductive period, testes were incubated for 1 and 4h with Kp-10 (10(-7)M) or Kp-10 (10(-7)M)+kisspeptin antagonist [Kp-234 (10(-6)M)]. Results obtained in the pre-reproductive period were confirmed and Kp-234 completely counteracted Kp-10 effects. In conclusion, Kp-10 modulated the expression of pcna, erβ, gnrhs and gnrhrs, inducing the progression of the spermatogenesis.

Molecular Chaperones, Cochaperones, and Ubiquitination/Deubiquitination System: Involvement in the Production of High Quality Spermatozoa

Spermatogenesis is a complex process in which mitosis, meiosis, and cell differentiation events coexist. The need to guarantee the production of qualitatively functional spermatozoa has evolved into several control systems that check spermatogenesis progression/sperm maturation and tag aberrant gametes for degradation. In this review, we will focus on the importance of the evolutionarily conserved molecular pathways involving molecular chaperones belonging to the superfamily of heat shock proteins (HSPs), their cochaperones, and ubiquitination/deubiquitination system all over the spermatogenetic process. In this respect, we will discuss the conserved role played by the DNAJ protein Msj-1 (mouse sperm cell-specific DNAJ first homologue) and the deubiquitinating enzyme Ubpy (ubiquitin-specific processing protease-y) during the spermiogenesis in both mammals and nonmammalian vertebrates.

Kisspeptins, Estrogens and Male Fertility

BACKGROUND The control of male fertility requires accurate endocrine, paracrine and autocrine communications along the hypothalamus-pituitary-gonad (HPG) axis. In this respect, the possible interplay between upcoming/classical modulators of reproductive functions deserves attention in that may be a successful tool for the future exploitation of new potential therapeutic targets in the treatment of fertility disorders. METHODS In this review we will discuss upcoming data concerning the role of kisspeptins, the products of the Kiss1 gene, and estrogens - classically considered as female hormones - as well as their possible interplay in testis. RESULTS Kisspeptins, via the activation of kisspeptin receptor Gpr54 represent the main gatekeeper of the hypothalamic Gonadotropin Releasing Hormone (GnRH) centrally modulating the onset and maintaining reproductive functions. As a consequence, the loss of kisspeptin signalling causes hypogonadotrophic hypogonadism in humans and animal models. In spite of the well recognized functions at hypothalamic levels, recent data strongly support direct production and activity of kisspeptin in testis and its involvement in the control of Leydig cells, germ cells progression and sperm functions. Similarly, estrogens exhibit high impact on proliferative/apoptotic/differentiative events in testis, thus resulting as local key modulators for the production - but also for the release, transport and maturation - of high quality spermatozoa. CONCLUSION This review summarizes the upcoming data from experimental models and humans concerning the testicular activity of kisspeptins and estrogens to preserve male fertility. Mutual enhancement of kisspeptin and estradiol signalling for the progression of spermatogenesis has also been discussed.

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.