Christophe Arnoult

Role of TLR9 in Anti-Nucleosome and Anti-DNA Antibody Production in lpr Mutation-Induced Murine Lupus

Systemic lupus erythematosus is characterized by the production of autoantibodies directed against nuclear Ags, including nucleosome and DNA. TLR9 is thought to play a role in the production of these autoantibodies through the capacity of nuclear immunogenic particles to interact both with BCR and TLR9. To determine the role of TLR9 in SLE, C57BL/6-lpr/lpr-TLR9−/− and TLR9+/+ mice were analyzed. The abrogation of TLR9 totally impaired the production of anti-nucleosome Abs, whereas no difference was observed in the frequency of anti-dsDNA autoantibodies whose titer was strikingly higher in TLR9−/− mice. In addition a higher rate of mesangial proliferation was observed in the kidney of TLR9-deficient animals. These results indicate that in C57BL/6-lpr/lpr mice, TLR9 is absolutely required for the anti-nucleosome Ab response but not for anti-dsDNA Ab production which is involved in mesangial proliferation.

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca2+ from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP3) receptors (InsP3Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP3R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion–induced calcium entry, whereas its NH2 terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation

A T‐type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the eggs extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole‐cell patch–clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage‐dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage‐dependent facilitation of the current. These data are consistent with the presence of a two‐state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.

Critical Role of TLR2 and TLR4 in Autoantibody Production and Glomerulonephritis in lpr Mutation-Induced Mouse Lupus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathogenic autoantibodies directed against nuclear Ags and immune complex deposits in damaged organs. Environmental factors have been thought to play a role in the onset of the disease. The recognition of these factors is mediated by TLRs, in particular TLR2 and TLR4 which bind pathogen-associated molecular patterns of Gram+ and Gram− bacteria, respectively. We attempted to determine the role of these TLRs in SLE by creating TLR2- or TLR4-deficient C57BL/6lpr/lpr mice. These mice developed a less severe disease and fewer immunological alterations. Indeed, in C57BL/6lpr/lpr-TLR2 or -TLR4-deficient mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and antinuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 and TLR4 in the production of Abs directed against only certain categories of SLE-related autoantigens. Analysis of B cell phenotype showed a significant reduction of marginal zone B cells, particularly in C57BL/6lpr/lpr-TLR4-deficient mice, suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease.

Skeletal muscle ATP-sensitive K+ channels recorded from sarcolemmal blebs of split fibers: ATP inhibition is reduced by magnesium and ADP.

SummaryA new, nonenzymatically treated preparation of amphibian sarcolemmal blebs has been used to study the regulation of skeletal muscle ATP-sensitive K+ [K(ATP)] channels.When a frog skeletal muscle fiber is split in half in a Ca2+-free relaxing solution, large hemispherical membrane blebs appear spontaneously within minutes without need for Ca2+-induced contraction or enzymatic treatment. These blebs readily formed gigaseals with patch pipettes, and excised inside-out patches were found to contain a variety of K+ channels. Most prominent were K(ATP) channels similar to those found in the surface membrane of other muscle and nonmuscle cells. These channels were highly selective for K+, had a conductance of ≈ 53 pS in 140mmK+, and were blocked by internal ATP. The presence of these channels in most patches implies that split-fiber blebs are made up, at least in large part, of sarcolemmal membrane.In this preparation, K(ATP) channels could be rapidly and reversibly blocked by glibenclamide (0.1–10 μm) in a dose-dependent manner. These channels were sensitive to ATP in the micromolar range in the absence of Mg. This sensitivity was noticeably reduced in the presence of millimolar Mg, most likely because of the ability of Mg2+ ions to bind ATP. Our data therefore suggest that free ATP is a much more potent inhibitor of these channels than MgATP. Channel sensitivity to ATP was significantly reduced by ADP in a manner consistent with a competition between ADP, a weak inhibitor, and ATP, a strong inhibitor, for the same inhibitory binding sites.These observations suggest that the mechanisms of nucleotide regulation of skeletal muscle and pancreatic K(ATP) channels are more analogous than previously thought.

Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.

Mutations in DNAH1, which Encodes an Inner Arm Heavy Chain Dynein, Lead to Male Infertility from Multiple Morphological Abnormalities of the Sperm Flagella

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Teratozoospermia: spotlight on the main genetic actors in the human

BACKGROUND Male infertility affects >20 million men worldwide and represents a major health concern. Although multifactorial, male infertility has a strong genetic basis which has so far not been extensively studied. Recent studies of consanguineous families and of small cohorts of phenotypically homogeneous patients have however allowed the identification of a number of autosomal recessive causes of teratozoospermia. Homozygous mutations of aurora kinase C (AURKC) were first described to be responsible for most cases of macrozoospermia. Other genes defects have later been identified in spermatogenesis associated 16 (SPATA16) and dpy-19-like 2 (DPY19L2) in patients with globozoospermia and more recently in dynein, axonemal, heavy chain 1 (DNAH1) in a heterogeneous group of patients presenting with flagellar abnormalities previously described as dysplasia of the fibrous sheath or short/stump tail syndromes, which we propose to call multiple morphological abnormalities of the flagella (MMAF). METHODS A comprehensive review of the scientific literature available in PubMed/Medline was conducted for studies on human genetics, experimental models and physiopathology related to teratozoospermia in particular globozoospermia, large headed spermatozoa and flagellar abnormalities. The search included all articles with an English abstract available online before September 2014. RESULTS Molecular studies of numerous unrelated patients with globozoospermia and large-headed spermatozoa confirmed that mutations in DPY19L2 and AURKC are mainly responsible for their respective pathological phenotype. In globozoospermia, the deletion of the totality of the DPY19L2 gene represents ∼ 81% of the pathological alleles but point mutations affecting the protein function have also been described. In macrozoospermia only two recurrent mutations were identified in AURKC, accounting for almost all the pathological alleles, raising the possibility of a putative positive selection of heterozygous individuals. The recent identification of DNAH1 mutations in a proportion of patients with MMAF is promising but emphasizes that this phenotype is genetically heterogeneous. Moreover, the identification of mutations in a dynein strengthens the emerging point of view that MMAF may be a phenotypic variation of the classical forms of primary ciliary dyskinesia. Based on data from human and animal models, the MMAF phenotype seems to be favored by defects directly or indirectly affecting the central pair of axonemal microtubules of the sperm flagella. CONCLUSIONS The studies described here provide valuable information regarding the genetic and molecular defects causing infertility, to improve our understanding of the physiopathology of teratozoospermia while giving a detailed characterization of specific features of spermatogenesis. Furthermore, these findings have a significant influence on the diagnostic strategy for teratozoospermic patients allowing the clinician to provide the patient with informed genetic counseling, to adopt the best course of treatment and to develop personalized medicine directly targeting the defective gene products.

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

Biophysical and pharmacological characterization of spermatogenic T-type calcium current in mice lacking the CaV3.1 (α1G) calcium channel: CaV3.2 (α1H) is the main functional calcium channel in wild-type spermatogenic cells

Mammalian acrosome reaction (AR) requires successive activation of three different types of calcium channels (T‐type channels, Inositol‐3‐phosphate (InsP3) receptors, and TRPC2 channels). All the calcium signaling is under the control of the activation of the first‐one, a T‐type calcium channel. The molecular characterization of the T‐type calcium channel is still a matter of debate, previous reports showing the presence of transcripts for CaV3.1 and CaV3.2 subunits. Using mice deficient for CaV3.1 subunit, we show that the T‐type current density in spermatogenic cells is not reduced in deficient mice versus control mice. We characterized the biophysical and pharmacological properties of T‐type current in spermatogenic cells from CaV3.1 deficient mice. Biophysical and pharmacological properties of spermatogenic T‐type current from wild‐type and CaV3.1 deficient mice demonstrate that CaV3.3 does not contribute to T‐type current. Moreover, nickel and amiloride inhibit T‐type currents in deficient and wild‐type mice with similar potencies. These results demonstrate that T‐type currents in spermatogenic cells is due to CaV3.2 subunit and that CaV3.1 contributes to a very negligible extent to the T‐type currents. Thus, the deficient CaV3.1 mouse model allows the characterization of native CaV3.2 currents in spermatogenic cells. Spermatogenic CaV3.2 currents present specific feature in comparison to the cloned CaV3.2 current so far. More particularly, the time‐dependence of recovery from short‐term inactivation of native spermatogenic CaV3.2 is close to 100 millisecond, a value expected for CaV3.1 current.