Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells.

BACKGROUND: NRD convertase, also termed Nardilysin, is a Zn(++) metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined. METHODS: To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa. RESULTS: Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme. CONCLUSIONS: The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

OBJECTIFS: La NRD convertase aussi appelée Nardilysine, une Zn++ metalloendopeptidase qui clive spécifiquement dans la région N terminale des résidus arginine et lysine des sites dibasiques, est impliquée dans la transformation/maturation des proprotéines. Le but de cette étude est de localiser cette enzyme durant la spermiogénèse afin de comprendre son rôle au cours de la maturation des spermatides. MÉTHODES: La Nardilysine est révélée par immunohistochimie au niveau ultrastructural chez des souris contrôles fertiles et chez un mutant stérile (ébouriffé : ebo/ebo). Des analyses quantitatives sont effectuées par comptage des grains d'or colloïdal qui permettent de détecter la localisation spécifique de l'enzyme au cours de la croissance des spermatides dans des régions particulières. RÉSULTATS: L'expression de la Nardilysine chez les souris sauvages et stériles ebo/ebo est limitée aux cellules germinales avec une augmentation significative dans les étapes ultimes de la spermiogénèse. L'enzyme est fortement exprimée dans le cytoplasme des spermatides allongées et dans les structures microtubulaires, la manchette et le flagelle. Aucun marquage n'est observé au niveau des organites cellulaires des spermatides. Chez le mutant ebo/ebo, dont le flagelle est anormal, l'enzyme est toujours présente sur les doublets de microtubules du flagelle. La quantification des particules d'or chez la souris sauvage et chez le mutant révèle une association spécifique de l'enzyme avec les microtubules du flagelle. CONCLUSIONS: L'accumulation spécifique de la Nardilysine au niveau de la manchette et du flagelle suggère que cette enzyme pourrait contribuer à l'établissement de ces structures microtubulaires particulières et/ou à leurs propriétés fonctionnelles.

The emerging role of connexin 43 in testis pathogenesis.

Direct intercellular communication is mediated by gap junctions and their constitutive proteins, the connexins, which are organized in a hexameric arrangement forming a channel between adjacent cells. Connexins are essential for cell homeostasis and are also involved in many physiological processes such as cell growth, differentiation and death. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival and apoptosis, in which one connexin isoform, connexin 43, plays an essential role as evidenced by the targeted genetic deletion of Cx43 gene. A controlled balance of germ cell growth is a prerequisite to maintain either normal level of spermatozoa necessary for fertility and/or to limit an uncontrolled and anarchic germ cell proliferation, a major risk for germ cell tumor cell development. In the present review, we highlight the emerging role of connexins in testis pathogenesis, specifically in two intimately interconnected human testicular diseases: azoospermia with impaired spermatogenesis and testicular germ cell tumors, whose incidence increased during the last decades. This review proposes the gap junction protein connexin 43 as a new potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism.

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Chemical connexin impairment in the developing gonad associated with offspring infertility.

A dramatical decline in human male reproductive function has been reported for the past 20 years. Many recent epidemiological, clinical and experimental findings suggest that the reproductive dysfunction could result from prenatal and neonatal chemical compound exposure. Even if numerous studies argue for a relationship between male infertility and environmental and/or occupational exposure, the molecular mechanisms by which these anti-reproductive compounds act are still unclear. Recent findings showed that a family of transmembranous proteins, connexins, regulates numerous physiological functions involved in the development such as cell proliferation, differentiation, migration and apoptosis. In the testis and the ovary, connexins are known to be essential for the establishment and the maintenance of spermatogenesis in males and oogenesis and folliculogenesis in females. Moreover, mutation of connexin genes leads to several developmental human diseases (myelin-related diseases, hearing loss, congenital cataract, skin disorders or more complex syndromes such as the oculodendrodigital dysplasia....) and altered connexin expression, trafficking and degradation are often associated with the tumoral process. We propose, in the present work, to give an overview of connexin expression and intercellular gap junction coupling during development: in preimplantation, implantation and postimplantation embryos. Moreover, we underline the impact of maternal chemical exposure on connexin expression during fetal gonad development and we link this effect to future offspring fertility.

Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects.

Rift Valley fever virus (RVFV) is an emerging, highly pathogenic virus; RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemorrhagic fever in humans, as well as death, abortions, and fetal deformities in animals. RVFV nonstructural NSs protein, a major factor of the virulence, forms filamentous structures in the nuclei of infected cells. In order to further understand RVFV pathology, we investigated, by chromatin immunoprecipitation, immunofluorescence, fluorescence in situ hybridization, and confocal microscopy, the capacity of NSs to interact with the host genome. Our results demonstrate that even though cellular DNA is predominantly excluded from NSs filaments, NSs interacts with some specific DNA regions of the host genome such as clusters of pericentromeric gamma-satellite sequence. Targeting of these sequences by NSs was correlated with the induction of chromosome cohesion and segregation defects in RVFV-infected murine, as well as sheep cells. Using recombinant nonpathogenic virus rZHDeltaNSs210-230, expressing a NSs protein deleted of its region of interaction with cellular factor SAP30, we showed that the NSs-SAP30 interaction was essential for NSs to target pericentromeric sequences, as well as for induction of chromosome segregation defects. The effect of RVFV upon the inheritance of genetic information is discussed with respect to the pathology associated with fetal deformities and abortions, highlighting the main role played by cellular cofactor SAP30 on the establishment of NSs interactions with host DNA sequences and RVFV pathogenesis.

Connexins as precocious markers and molecular targets for chemical and pharmacological agents in carcinogenesis.

Gap junctions, intercellular channels structured by the connexin protein family, have been implicated in the control of cell homeostasis, proliferation, differentiation and death. A loss of the gap junction intercellular communication and/or connexin dysfunction are typical features of cancer per se and have been associated with the effect of many carcinogens. Indeed, many early human neoplasia of various organs and human tumor cell lines exhibit deficient connexin-mediated communication expression mainly related, in a large number of observations, with an aberrant cytoplasmic localization of this membranous protein. Restoration of normal phenotype in transformed cells by restoration of exogenous connexin gave rise to the concept that connexins may act as tumor suppressors. However, the mechanisms by which connexins mediate such a tumor suppressor effect are multiple. They may result from: formation of functional channels; hemichannels or are directly associated with connexin expression. In addition, the literature shows that they may be dependent upon the cell type and the connexin type. In the present review, we analyze all these aspects of connexin/gap junction involvement in the carcinogenesis process, in human cancers and discuss the possibility of using connexins as potential anti-oncogenic targets for cancer chemoprevention and/or chemotherapy.