Normal live birth after vitrified/warmed oocytes intracytoplasmic sperm injection with immotile spermatozoa in a patient with Kartagener's syndrome.

The present article is a report on two cases of male Kartagener's syndrome enrolled in intraconjugal IVF programme due to akinetospermia. Viable spermatozoa were selected using a hypo-osmotic swelling test (HOST) and pentoxifylline activation and subsequently microinjected into vitrified/warmed oocytes. The treatment enabled one of these two couples to achieve a pregnancy and to give birth to a healthy baby girl.

Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus.

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.

The N-acetylglucosamine-specific receptor of the thyroid: purification, further characterization, and expression patterns on normal and pathological glands.

The N-acetylglucosamine receptor of the thyroid has been putatively described as both a prohormonal receptor that could play a role in the intrafollicular retention of immature thyroglobulin and a vectorial conveyor of these immature molecules to the iodination site. To further characterize this receptor, we have developed a purification procedure yielding nanomolar amounts of N-acetylglucosamine receptor. This thyroid lectin appeared to have an isoelectric point near 5.2 and to be composed of 51-kilodalton monomers with no Asn-linked glycoconjugates. Recognition of the receptor by antipeptide antibodies (Ab/ROV1) raised against a preselected sequence of cation-dependent lectins indicated immunological kinship with the Gal/GalNAc-specific hepatic lectin. Affinity-purified Ab/ROV1 and polyclonal antibodies against the purified receptor (TGRD-Ab) were used to study the location and expression pattern of the receptor on animal and human thyroid tissue. On porcine slices, positive labeling was observed in various intracellular vesicular compartments with both antibodies and was particularly intense in the apical membrane and subapical compartments. The same pattern was observed in normal human thyroid. In contrast, the receptor 1) could not be found on epithelial cells from thyroid papillary carcinoma; 2) was abundant, but concentrated in the subnuclear region of the thyrocytes in adenomatous goiter; and 3) was almost exclusively located at the basolateral membrane in follicular carcinoma as well as in thyrocytes from glands treated with antithyroid drug before surgery. These observations indicate that expression of the N-acetylglucosamine receptor is characteristic of the fully differentiated phenotype, and its potential function as a thyroglobulin conveyor back to the lumen would be either impaired or abolished in some disease processes.

Development of cholinergic properties in nerve cell cultures in the presence of brain extract.

Cells dissociated from cerebral hemispheres of 6-day-old chick embryos were cultured either in standard nutrient medium or in the presence of a brain extract from 8-day-old chick embryo. Morphological observations showed the development of bipolar and multipolar neurons in both culture conditions and acetylcholinesterase activity was found in all neuronal cells. Brain extract stimulated the morphological maturation of neurons, expressed by the formation of fiber bundles, fine structural maturation and development of synapses rich in clear vesicles. Furthermore, acetylcholinesterase and choline acetyltransferase activities were higher in the cultures treated with brain extract. In these cultures, the values of choline acetyltransferase activity reached a peak at 10 days and then decreased. These observations are discussed with particular reference to proliferation, maturation and degeneration of cholinergic neurons.

Expression of Ia antigens on freshly dissociated mouse thyroid follicles demonstrated by immunofluorescence.

Iak antigens were detected by indirect immunofluorescence on CBA mouse thyroid follicles. Isolated thyroid follicles, free of lymphocyte contamination, were obtained by collagenase treatment and mechanical disruption; they were then cytocentrifuged on glass slides. This material was incubated with polyclonal or monoclonal anti Iak antibodies followed by FITC-conjugated F(ab')2. Subsequent microscopic observations of these open thyroid follicles revealed that Iak antigens were spontaneously expressed. Labelling was distributed preferentially on the thyrocyte cell surface and, to a lesser extent, on intracellular organelles. Conversely, nuclei were never stained. In some cases, labelling was mottled. Ia antigens may have been previously unobserved because, beside autoimmune disorders they are present in such small amounts that detection is very difficult. The fact that Ia expression is independent of morphological signs of spontaneous autoimmune thyroiditis (particularly, lymphocyte infiltration of follicles), raises the problem of correlation between the autoimmune disorders and Ia expression.

Effects of chick brain extract on the proliferation of chick neuroblasts cultured in media supplemented with low and high serum concentrations.

The proliferative activity of chick neuroblasts cultured in a medium containing a low (5%) or a high (20%) concentration of fetal calf serum (FCS) was analyzed and the influence of a chick brain extract was investigated. Morphological observations and tritiated thymidine incorporation measurements have shown that neuroblasts from 6 day-old chick embryo cerebral hemispheres proliferate more actively in the medium with 5% FCS compared to the medium with 20% FCS. The medium containing 5% FCS favoured the maintenance of neuronal cells in a neuroblast stage as shown by electron microscopy. The stimulatory effect of brain extract on the proliferation of neuroblasts is stronger in the low serum culture condition. These findings indicate that a low serum-containing medium is an adequate condition to study neuronal proliferation and effects of growth factors on these cells.

The catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope-mediated membrane fusion after CD4 cell binding.

Protein disulfide isomerase (PDI) is a multifunctional protein with thiol-disulfide redox-isomerase activities. It catalyzes thiol-disulfide interchange reactions on the cell surface that may cause structural modifications of exofacial proteins. PDI inhibitors alter human immunodeficiency virus (HIV) spread, and it has been suggested that PDI may be necessary to trigger HIV entry. This study examined this hypothesis by using cell-to-cell fusion assays, in which the HIV envelope (Env) expressed on the cell surface interacts with CD4(+) lymphocytes. PDI is clustered at the lymphocyte surface in the vicinity of CD4-enriched regions, but both antigens essentially do not colocalize. Anti-PDI antibodies and 2 inhibitors of its catalytic function altered Env-mediated membrane fusion at a post-CD4 cell binding step. The fact that the PDI catalytic activity present on lymphocytes is required for fusion supports the hypothesis that catalysts assist post-CD4 cell binding conformational changes within Env.

Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.

Developmental changes in cultured neurones from chick embryo cerebral hemispheres. An ultrastructural and neurochemical study.

The acquisition of ultrastructural and neurochemical properties was studied during the development of neurones from 8-day-old chick embryo cerebral hemispheres, cultivated on polylysine-coated surfaces. The first contacts suggestive of synaptic profiles were observed after 3 days of culture. From the 6th day, junctions with significant thickening of the postsynaptic element and many presynaptic vesicles could be seen. Synaptic endings contained predominantly dense-cored vesicles. These cultured neurones acquired dopaminergic properties during their development: (1) 95% of the cells exhibited glyoxylic acid-induced fluorescence; (2) tyrosine hydroxylase and dopa decarboxylase activities were present and increased with time in culture, but dopamine-beta-hydroxylase activity was undetectable. Choline acetyltransferase activity remained at a very low level at all stages of culture. It is suggested that when cultured in the total absence of glial cells, the neurones of embryonic chick cerebral hemispheres give no evidence of cholinergic mechanisms, but display a number of dopaminergic characteristics.

Preparation and partial characterization of polyclonal antibodies raised against a preselected sequence of calcium dependent lectins.

The synthetic-peptide strategy was used to generate antibodies raised against calcium-dependent lectins of vertebrates. We demonstrate that a synthetic peptide predicted from the amino acid sequence of the carbohydrate recognition domain can induce blocking antibodies which would react with, or in close vicinity of, the binding site of the parent molecule. As the preselected sequence was chosen in a consensus sequence region, we also present preliminary investigations of the use of specific antisera as a common biological probe against calcium dependent lectins. The availability of monospecific polyclonal sera opens new possibilities in biochemical and structural studies as well as immunodection of calcium dependent lectins.

Identification of the membrane receptor binding domain of thyroglobulin. Insights into quality control of thyroglobulin biosynthesis.

The last stages of thyroglobulin maturation occur in the thyroid follicular lumen and include thyroid hormone formation and glycan completion. In this compartment, newly secreted thyroglobulins interact with a thyrocyte membrane receptor that prevents their premature lysosomal transfer and degradation. Both GlcNAc moieties and thyroglobulin peptide determinants are involved in receptor interaction. Here we used monoclonal antibodies (mAbs) directed against human thyroglobulin either to inhibit (mAb78) or to enhance (mAb240) the thyroglobulin binding and to identify the region of the thyroglobulin involved in the receptor recognition. Peptides containing the mAb epitopes were obtained by immunoscreening cyanogen bromide-derived native human thyroglobulin peptides and a cDNA thyroglobulin expression library. Three peptides, localized in the thyroglobulin N-terminal domain, were obtained. Peptides N1 (Ala1148-Gln1295) and N2 (Ser789-Met1008) were recognized by mAb240 and mAb78, respectively. None of them bound the receptor. The third peptide, N3 (Ser789-Met1172), (i) overlapped all or part of the N1 and N2 peptide sequences and was recognized by both mAbs, (ii) carried two complex glycans at Asn797 and Asn928, of which a subset presented accessible GlcNAc residues, and (iii) inhibited the thyroglobulin binding to FRTL5 cell membrane preparations. The N3 peptide includes tyrosine residues that have been reported to be involved in hormone formation. These results suggest that structural modifications closely associated with hormone formation within this domain act as sensors for the receptor interaction and thus for the intrafollicular retention or lysosomal homing of the prohormone.

Protein-disulfide isomerase (PDI) in FRTL5 cells. pH-dependent thyroglobulin/PDI interactions determine a novel PDI function in the post-endoplasmic reticulum of thyrocytes.

Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser(789)-Met(1172) TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an "escort" for immature TG in acidic post-endoplasmic reticulum compartments is discussed.