Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.

[ICSI treatment in severe asthenozoospermia]. / Asthénozoospermie sévère à vitalité normale et ICSI.

In the management of asthenozoospermia, the spermogram-spermocytogram plays an important role during diagnosis. It is of major importance to distinguish between necrozoospermia and sperm vitality. An ultrastructural study of spermatozoa is processed in the case of primary infertility without female implication, severe, unexplained and irreversible asthenozoospermia, sperm vitality at least 50 % and normal concentration of spermatozoa. Ultrastructural flagellar abnormalities are numerous and involve most spermatozoa. ICSI provides a suitable solution for patients with sperm flagellar defects to conceive children with their own gametes but the rate of ICSI success may be influenced by the type of flagellar abnormality. Some fertilization and birth rate failures which are related to some flagellar abnormalities might occur.

Bacterial flora of the low male genital tract in patients consulting for infertility.

The physiological aerobic bacterial flora of the low male genital tract was determined. This prospective study was performed on 600 semen specimens collected from 543 asymptomatic males consulting for infertility. Semen cultures were sterile in 28.8%, with a polymicrobial flora and/or absence or low titres of Ureaplasma urealyticum in 49.3%, and with one or two aerobic and facultative bacteria > or =1 x 10(3) CFU ml(-1) and/or U. urealyticum with titres > or =10(4) CCU ml(-1) (colour changing units) in 21.8%. In standard aerobic cultures, Gardnerella vaginalis was the most commonly isolated species (26.1%), followed by coagulase-negative staphylococci (15.7%) and Streptococcus anginosus (14.2%). Ureaplasma urealyticum was absent in 84.5% of semen samples, but when recovered, high (> or =10(4) CCU ml(-1)) and low titres (< or =10(3) CCU ml(-1)) were counted in 7.2% and 8.3% respectively. Of 48 patients, the follow-up of semen cultures showed marked variations in time. This study shows that (i) there was no relationship between the bacterial flora and the leucocytospermia; (ii) low titres of U. urealyticum in semen were not associated with a disturbance of the ecosystem; (iii) the critical threshold for U. urealyticum should be raised to > or =10(4) CFU ml(-1) and (iv) a positive semen culture should be repeated before any treatment.

Dystrophic neuropeptidergic neurites in senile plaques of Alzheimer's disease precede formation of paired helical filaments.

The relationship between peptidergic neurites and paired helical filaments (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SP) was studied using combined fluorescence and bright field optics. Cryostat sections of AD hippocampi were first stained by thioflavine-S and immunolabeled with antisera raised against different neuropeptides: somatostatin 28(1-12) (som 28(1-12)), somatostatin 14 (som 14), neuropeptide Y (NPY), cholecystokinin (CCK) and substance P (sP). Secondly, using the elution-restaining procedure, sections were immunolabeled with anti-tau/PHF. In immature SP, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained less PHF than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SP without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHF. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore this phenomenon seems to precede any formation of PHF in SP.

Dystrophic peptidergic neurites in senile plaques of Alzheimer's disease hippocampus precede formation of paired helical filaments.

The relationship between peptidergic dystrophic neurites and paired helical filament (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SPs) was studied using combined fluorescence and bright-field optics. Cryostat sections of AD hippocampi were first stained with thioflavine-S and immunolabelled with antisera raised against different neuropeptides: somatostatin-28(1-12), somatostatin-14, neuropeptide Y, cholecystokinin (CCK) and substance P. Secondly, using the elution-restaining procedure, sections were immunolabelled with anti-tau/PHF. In immature SPs, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained fewer PHFs than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SPs without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHFs. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore, this phenomenon seems to precede any formation of PHFs in SPs.

Cortical angiopathy in Alzheimer's disease: the formation of dystrophic perivascular neurites is related to the exudation of amyloid fibrils from the pathological vessels.

We studied the organization of dystrophic neurites around pathological vessels in Alzheimer cortex. Two techniques were used simultaneously on serial sections: thioflavine staining of amyloid substance and immunohistochemistry with immune sera against Paired Helical Filaments (anti-PHF) and native Tau proteins (anti-Tau). We observed different distributions of dystrophic neurites (immunolabelled with anti-PHF or anti-Tau) around thioflavine-stained angiopathic arterioles. The wall of the vessels with large diameter (greater than 100 microns) presented a congophilic angiopathy without neuropil reaction. In vessels with lesser diameter (less than 100 microns), dystrophic neurites constituted a discontinuous sleeve around vessels, always in close contact with amyloid substance outside the wall (dysphoric angiopathy). We observed structures similar to senile plaques around capillaries (diameter: 10-15 microns). The sleeve of dystrophic neurites with aggregated Tau proteins were always observed in the close vicinity of the amyloid substance which exuded from the pathological blood vessels. Thus, the exudation of these amyloid fibrils seems to induce the formation of dystrophic neurites (neuritic reaction).

Observation of morphological relationships between angiopathic blood vessels and degenerative neurites in Alzheimer's disease.

Two main techniques are used to stain the three types of brain lesions characteristic of Alzheimer's disease: Neurofibrillary tangles (NFT), senile plaques (SP) and congophilic angiopathy. Thioflavine-S is an histochemical marker of the amyloid substance located essentially in the central core of senile plaques and in the walls of the pathological blood vessels. Specific antibodies against Paired Helical Filaments (PHF), the ultrastructural elements of NFT, reveal neuron cell bodies with NFT and numerous dystrophic neurites, mostly around neuritic plaques. Using simultaneous histochemical and immunohistochemical labellings on the same tissue sections of Alzheimer cortex (association cortex and hippocampus), the different lesions were stained with great sensitivity and specificity. Moreover, an unusual morphological relationship between two types of lesions was detected in two Alzheimer brains with prominent congophilic angiopathy: we observed a well marked concentration of dystrophic neurites, immunolabelled with anti-PHF, around blood vessels with Thioflavine-S stained amyloid angiopathy. These lesions were distributed like a sleeve around 1/10 of dyshoric or congophilic blood vessels. The significance of such lesions is unknown but they probably represent a step of the pathogenesis of Alzheimer brain lesions and may explain the general mechanism of lesion formation in Alzheimer's disease.

In vitro and in vivo incorporation of 63Ni[II] into lung and liver subcellular fractions of Balb/C mice.

The binding of nickel to proteins in lung and in liver was investigated by analysis of 63Ni[II] incorporation into the nuclear, mitochondrial, microsomal and soluble fractions. Three different procedures were performed: (i) in vitro incorporation, (ii) a time course study of in vivo incorporation where the animals were sacrificed after 30 min, 1, 2 and 4 h after a single i.p. injection of 63NiCl2, (iii) in vivo incorporation after 7 successive i.p. injections of 63NiCl2 every 24 h and the animals were sacrificed 24 h after the last injection. In all cellular fractions (except the nuclear fractions) we could observe several nickel-binding proteins regardless of the type of incorporation performed. Most of these proteins were revealed after in vitro as well as in vivo incorporation, some of them, however, were labelled only after in vitro incorporation, others only after in vivo incorporation. Some proteins can only be revealed after successive injections. In addition, the 63Ni-labelled proteins are not all the same at the beginning of the incorporation (30 min) as after longer periods (1 and 2 h). The lung fractions (especially the mitochondrial fraction) were always more highly labelled than the liver fractions. These biochemical investigations not only confirm that the lung is a target organ for nickel-retention, but also demonstrate that Ni is preferentially bound to its mitochondrial and microsomal fractions. It is shown here that several cellular proteins are implicated in the transport and the metabolism of nickel in the cell.

63Ni[II]-incorporation into lung and liver cytosol of Balb/C mouse. An in vitro and in vivo study.

The kinetic studies of 63Ni[II]-incorporation in whole tissue show that after 6 days lung has the highest affinity to nickel of all studied organs. The same observation was made when 63Ni[II]-incorporation was carried out by 7 daily injections. In both experiments, kidneys take the second place in the relative distribution of nickel. For the studies of Ni-binding proteins in lung and liver cytosols, three different types of 63Ni[II]-incorporation were performed: (i) in vitro incorporation, (ii) kinetic study of in vivo incorporation where the animals were sacrificed after 30 min, 1, 2 and 4 h after a single i.p. injection of 63NiCl2, (iii) in vitro incorporation by 7 daily i.p. injections of 63NiCl2 with sacrifice of the animals 24 h after the last injection. Several Ni-binding proteins could be observed without regard to the type of incorporation performed. In liver cytosol most of these proteins can be revealed in vitro as well as in vivo. The in vivo labelled proteins are not the same at the beginning of the incorporation (30 min) and after an elapsed period (1 to 2 h). In lung cytosol in vitro and in vivo incorporation gave different results: although an intense labelling occurs after in vitro incubation, only few proteins are labelled after in vivo incorporation, two of which are only revealed after continuous exposure to 63Ni[II]. Most of the labelled proteins of lung and liver cytosols can be recovered after different fractionation experiments. These investigations confirm that nickel is preferentially bound to lung and demonstrate that nickel-incorporation is different in lung and in liver cytosols. It is shown here that several cellular proteins are implicated in the nickel-transport. The evolution of this phenomenon suggests the existence of a nickel-metabolism in the cell.

Structures of fifteen oligosaccharides isolated from new-born meconium.

New born meconium contains at least a hundred oligosaccharides. In this study the isolation and characterization of the major constituents is described. The structure elucidation of 15 neutral and acidic oligosaccharides was carried out by methylation analysis, mass spectrometry and 360-MHz 1H-NMR spectroscopy. The results show that the oligosaccharides accumulating in human meconium are probably products of the catabolism of the O- and N-linked carbohydrate chains of glycoproteins. It is proposed that endo-N-acetyl-alpha-D-galactosaminidase, endo-beta-D-galactosidase and endo-N-acetyl-beta-D-glucosaminidase are involved in the production of these compounds.

Structure of seven oligosaccharides excreted in the urine of a patient with Sandhoff's disease (GM2 gangliosidosis-variant O).

The urine of a patient with Sandhoff's disease (GM2 gangliosidosis-variant O) contains 10--12 N-acetylglucosamine-rich oligosaccharides in high amounts. The structures of seven of these have been determined: beta-GlcNAc(1--2)-alpha-Man-(1--3)-beta-man-(1--4)-GlcNAc; beta-GlcNAc-(1--4)-alpha-Man-(1--3)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--6)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--4)-alpha-Man-(1--6)-beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--3)-[beta-GlcNAc-(1--2)-alpha-Man-(1--6)]beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man-(1--3)[beta-GlcNAc-(1--2)-alpha-Man-(1--6)][beta-GlcNAc-(1--4)]beta-Man-(1--4)-GlcNAc; beta-GlcNAc-(1--2)-alpha-Man(1)-(1--3)[beta-GlcNAc-(1--2)-alpha-Man(2)-(1--6)]beta-Man-(1--4)-GlcNAc, with additional beta-GlcNAc, with additional beta-GlcNAc-(1--4) on mannose (1) or (2). An unusual oligosaccharide, with a tri-branched beta-mannose, has been characterized as the major component excreted in urine.