Maria Enrica Pasini

Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant

The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.

Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa.

Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.

Glycosidases are present on the surface of Drosophila melanogaster spermatozoa

We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, β‐N‐acetylglucosaminidase (GlcNAc′ase) and α‐mannosidase (Man′ase), have been evidenced. They have an optimal pH of 6–6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of α‐mannose and β‐N‐acetylglucosamine residues present on the egg shell at the site of sperm entry. Mol. Reprod. Dev. 48:276–281, 1997.

Hypocretin (Orexin) Regulates Glutamate Input to Fast-Spiking Interneurons in Layer V of the Fr2 Region of the Murine Prefrontal Cortex

We studied the effect of hypocretin 1 (orexin A) in the frontal area 2 (Fr2) of the murine neocortex, implicated in the motivation-dependent goal-directed tasks. In layer V, hypocretin stimulated the spontaneous excitatory postsynaptic currents (EPSCs) on fast-spiking (FS) interneurons. The effect was accompanied by increased frequency of miniature EPSCs, indicating that hypocretin can target the glutamatergic terminals. Moreover, hypocretin stimulated the spontaneous inhibitory postsynaptic currents (IPSCs) on pyramidal neurons, with no effect on miniature IPSCs. This action was prevented by blocking 1) the ionotropic glutamatergic receptors; 2) the hypocretin receptor type 1 (HCRTR-1), with SB-334867. Finally, hypocretin increased the firing frequency in FS cells, and the effect was blocked when the ionotropic glutamate transmission was inhibited. Immunolocalization confirmed that HCRTR-1 is highly expressed in Fr2, particularly in layer V–VI. Conspicuous labeling was observed in pyramidal neuron somata and in VGLUT1+ glutamatergic terminals, but not in VGLUT2+ fibers (mainly thalamocortical afferents). The expression of HCRTR-1 in GABAergic structures was scarce. We conclude that 1) hypocretin regulates glutamate release in Fr2; 2) the effect presents a presynaptic component; 3) the peptide control of FS cells is indirect, and probably mediated by the regulation of glutamatergic input onto these cells.

Interspecific analysis of the glycosidases of the sperm plasma membrane in Drosophila

We have studied the presence of four sperm glycosidases, α‐mannosidase, α‐l‐ fucosidase and two β‐hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha‐mannosidase and β‐hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha‐l‐fucosidase is restricted to the species of the genus Drosophila. Alpha‐mannosidase and β‐hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross‐species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta‐hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of β‐hexosaminases and α‐l‐fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface. Mol. Reprod. Dev. 76: 85–100, 2009.

Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse

We studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 µM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 µM dihydro‐β‐erythroidine (which blocks α4‐containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7‐containing receptors). Excitatory postsynaptic currents s were also stimulated by 5‐iodo‐3‐[2(S)‐azetidinylmethoxy]pyridine, selective for β2‐containing receptors, in a dihydro‐β‐erythroidine ‐sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4β2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels. Synapse 00:000–000, 2013.

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37 °C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL(+)/rpsL31 Escherichia coli strain in which the dominant rpsL(+) allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA-TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.

Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa.

Fruit flies in the family Tephritidae are rated among the worlds most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a β-N-acetylhexosaminidase, an α-mannosidase and an α-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for β-N-acetylhexosaminidase, 310 kDa for α-mannosidase and 140 kDa for α-l-fucosidase. SDS-PAGE showed that β-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, α-mannosidase consists of six subunits with different molecular weights and α-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding β-N-acetylhexosaminidase and α-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition.

Expression study of an α-l-fucosidase gene in the Drosophilidae family

The plasma membrane of Drosophila (Sophophora) melanogaster spermatozoa contains an alpha-l-fucosidase that might be involved in fertilization by interacting with alpha-l-fucose residues on the micropyle of the eggshell. D. (S.) melanogaster has a single gene called CG6128 or Fuca encoding for a putative alpha-l-fucosidase. Two transcripts have been annotated, RA of 3514 bp, and RB of 1673 bp. While both transcripts encode an alpha-l-fucosidase, RA contains an upstream open reading frame, translated into a polypeptide containing a predicted BTB/POZ domain. We demonstrate that Fuca is expressed in male and female germ lines. RT-PCR analysis indicated a broader tissue expression. Homologous genes are expressed in the same tissues in several drosophilid flies belonging to the genera Drosophila and Scaptodrosophila. However, the long transcript is restricted to species belonging to the subgenus Sophophora. The presence of two transcripts in species of the subgenus Sophophora and only one in species belonging to the subgenus Drosophila might be related to the phylogenetic relationships of these subgenera. Immunofluorescence demonstrated that the gene product, localized to the sperm plasma membrane, is absent from Scaptodrosophila lebanonensis spermatozoa. These findings support the hypothesis that the enzyme is involved in the molecular events of primary gamete interactions that are conserved among drosophilids belonging to Drosophila genus.

Identification and expression profiling of Ceratitis capitata genes coding for β-hexosaminidases.

The goal of this study was to identify the genes coding for β-N-acetylhexosaminidases in the Mediterranean fruit fly (medfly) Ceratitis capitata, one of the most destructive agricultural pests, belonging to the Tephritidae family, order Diptera. Two dimeric β-N-acetylhexosaminidases, HEXA and HEXB, have been recently identified on Drosophila sperm. These enzymes are involved in egg binding through interactions with complementary carbohydrates on the surface of the egg shell. Three genes, Hexosaminidase 1 (Hexo1), Hexosaminidase 2 (Hexo2) and fused lobes (fdl), encode for HEXA and HEXB subunits. The availability of C. capitata EST libraries derived from embryos and adult heads allowed us to identify three sequences homologous to the D. melanogaster Hexo1, Hexo2 and fdl genes. Here, we report the expression profile analysis of CcHexo1, CcHexo2 and Ccfdld in several tissues, organs and stages. Ccfdl expression was highest in heads of both sexes and in whole adult females. In the testis and ovary the three genes showed distinct spatial and temporal expression patterns. All the mRNAs were detectable in early stages of spermatogenesis; CcHexo2 and Ccfdl were also expressed in early elongating spermatid cysts. All three genes are expressed in the ovarian nurse cells. CcHexo1 and Ccfdl are stage specific, since they have been observed in stages 12 and 13 during oocyte growth, when programmed cell death occurs in nurse cells. The expression pattern of the three genes in medfly gonads suggests that, as their Drosophila counterparts, they may encode for proteins involved in gametogenesis and fertilization.