TEM, FISH and molecular studies in infertile men with pericentric inversion of chromosome 9.

Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants of human karyotype. A number of investigators have suggested that chromosomal anomalies can contribute to human infertility causing spermatogenetic derangement. The present study was aimed at verifying the influence of chromosome 9 inversion on human spermatogenesis. Semen samples of 18 male carriers of chromosome 9 inversion, analysed by light microscopy, revealed that five patients were azoospermic. PCR analysis demonstrated that two of them also had Y microdeletions. The other 13 showed generally normal sperm concentrations and reduced motility. The morphological characteristics of sperm were studied by TEM and the data were elaborated by a mathematical formula. Sperm pathologies resulted more frequently in the studied group compared to controls, particularly apoptosis. Partial sequences of the A-kinase anchoring protein (Akap) 4 and 3 genes were performed in all patients, as a previous study by our group highlighted Dysplasia of Fibrous Sheath (DFS) defect in two men with inv 9 investigations. The possible effect of chromosome 9 inversion on meiotic chromosome segregation was investigated by FISH, which showed an increased incidence of diploidy. We hypothesized that this inversion could have variable effects on spermatogenesis, from azoospermia to severely altered sperm morphology, motility and meiotic segregation.

Ultrastructural sperm study in infertile males with microdeletions of Y chromosome.

A retrospective study to detect specific Y chromosome microdeletions and to evaluate sperm ultrastructural characteristics in infertile men was set up. We selected 219 infertile men referred to Regional Referral Center for Male Infertility, Siena, Italy for semen analysis from January 1999 to April 2004. Family history, lymphocyte karyotype determination, Y microdeletion screening, physical examination, hormonal assays, semen analysis were carried out. Sperm concentration and progressive motility, ultrastructural analysis of sperm organelles, PCR amplification of sequence tagged sites for Y microdeletion screening were performed. Different Y-chromosome deletions were found, mainly in the AZFb and AZFc regions. Severe alterations of sperm ultrastructure, affecting whole sperm population, were detected in carriers of Y-deletions. Our data confirms the highest frequency of Y deletions in azoospermic patients. In all other patients with Y microdeletions, sperm ultrastructural defects affected the whole sperm population and were mainly related to apoptosis or immaturity.

Gene deletions in an infertile man with sperm fibrous sheath dysplasia.

BACKGROUND: Asthenozoospermia may sometimes be related to genetic structural defects of the sperm tail detectable by transmission electron microscopy. Dysplasia of the fibrous sheath (DFS) is a genetic sperm defect, characterized by dysplastic development of the axonemal and periaxonemal cytoskeleton. We report the case of an infertile man with normal sperm count and total sperm immotility in which dysplasia of the fibrous sheath, Akap3, Akap4 gene deletions, meiotic segregation of chromosomes 18, X and Y and Y microdeletions were investigated. METHODS: A 32-year-old man with a 3-year history of primary infertility presented at our Regional Referral Center for Male Infertility. Family medical history, lymphocyte karyotype, PCR analysis, physical examination, hormone assays and semen analysis were performed. RESULTS: Ultrastructural sperm evaluation showed dysplasia of the fibrous sheath. Immunostaining of AKAP4 protein was negative in sperm tails. PCR analysis revealed intragenic deletions of the Akap3 and Akap4 genes. Fluorescence in situ hybridization on sperm showed a high frequency of XY disomy. CONCLUSION: In this infertile patient, our results suggest a possible relationship between dysplasia of the fibrous sheath, partial deletions in the Akap3 and Akap4 genes and absence of AKAP4 protein in the fibrous sheath. These findings, however, were not detected in another four patients with dysplasia of the fibrous sheath. Our results require future confirmatory molecular analyses.

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 4. The bovine fertilization (Notulae seminologicae 10).

In this paper we apply a modification of the formula of Baccetti et al. (1995) in the evaluation of submicroscopical characteristics of bull spermatozoa used in assisted reproduction. In the present experiment sperm quality is proposed as a useful parameter in predicting the success of fertilization. Our results demonstrate that the percentage of spermatozoa devoid of submicroscopic defects, according to the particular Bayesan formula proposed by us, is clearly correlated with the result of artificial insemination. In fact, the parameter concerning sperm quality obtained in variously successful donors shows a large correlation with fertility power. The synthetic parameters observed are therefore a good tool in the prediction of sperm power in artificial fertilization. The evaluation is mainly concerned with the quality of the acrosomal characters, the status of the chromatin, the shape of mitochondria, the position of the postacrosomal sheath, the perinuclear space and the axonemal pattern. All these characters are expressed with different means in ejaculates. All these data confirm that submicroscopic-mathematical evaluation offers a convincing and reliable diagnosis based upon sperm structure and functions such as acrosomal reaction and cell motility. It has been also demonstrated that sperm quality is a major factor in the success of artificial insemination and it is clearly revealed in the integrity of most of sperm organelles.

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte.

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.

Studies on varicocele. II. The inhibin secretion.

In this paper a previous interpretation given by the authors concerning one of the ways varicocele can affect fertility is confirmed. Moreover, it is definitely demonstrated that the high temperature stimulates inhibin secretion (and probably the testosterone-estradiol conversion) in the Sertoli cells, while the somatomedin secretion in vitro seems to be unaffected. It means that the action of the temperature on the germinal cells seems to be mediated by the pathway: inhibin (plus estradiol)-->pituitary-->FSH. Inhibin in the Golgi complex of Sertoli and germinal cells has been detected by electron microscopical immunocytochemical techniques.

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells.

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

Effect of cytidine analogs on cell growth and differentiation on a human neuroblastoma line.

The cytidine analog 5-AZA-2'-deoxycytidine (5-AZA-CdR) has been demonstrated to induce cellular differentiation; on the other hand, induction of differentiation has been suggested as a possible form of therapy for leukemic cells. We have evaluated the possibility that the neuroblastoma malignant tumor growth could be controlled by treatment that promotes the differentiation of immature tumor cells. We have previously reported on differentiation of murine neuroblastoma cells (41A3) treated with 5-AZA-CdR. In this paper, we describe the effect of 5-AZA-CdR on human neuroblastoma cell line CHP-100. The drug-treated cells show some degree of differentiation, demonstrated by morphological and biochemical markers. A significant DNA hypomethylation and partial inhibition of DNA synthesis and cell proliferation is also observed. This effect is more stable than that caused by another cytidine analog, Cytosine-beta-D-Arabinofuranoside (ARA-C).

5-Aza-2'-deoxycytidine as inducer of differentiation and growth inhibition in mouse neuroblastoma cells.

We studied the effect of 5-aza-2'-deoxycytidine (5-AZA-CdR) on the differentiation of murine 41A3 neuroblastoma cells. Neuroblastoma cells treated with 0.1-1.0 microM 5-AZA-CdR underwent differentiation; markers of neuronal functions, such as acetylcholinesterase activity and growth of nerve fibers, were expressed at a higher level in the drug-treated cells than in the controls. This increased expression was accompanied by significant hypomethylation of newly synthesized DNA. A secondary event seemed to be a partial inhibition of DNA synthesis, cell proliferation and colony-forming activity. These effects were more pronounced than those caused by the related cytidine analog, 1-beta-D-arabinosil-cytosine (ARA-C). The results obtained suggest that 5-AZA-CdR may be an effective agent for the growth control of human neuroblastoma cells.

Interneurons versus efferent neurons: heterogeneity in their neurite outgrowth response to glia from several brain regions.

We have studied in vitro the morphology of two populations of dopaminergic neurons from mouse embryos: the periglomerular interneurons from the olfactory bulb (DOBI) and the efferent neurons from the substantia nigra (DENN). The intrinsic potential of both neuronal types has been studied by comparing process outgrowth in a predominantly neuronal environment or in a glial environment that is endogenous or from other brain regions. Both populations exhibit in vitro different characteristics that reflect their phenotype in situ. In addition they greatly differ in their response to glial signals. DOBI maintain a constant stellate morphology with short processes under all culture conditions tested, whereas DENN exhibit a great plasticity and in particular respond to olfactory bulb glia with a striking increase in neurite length. The olfactory bulb glia differs from other brain region glia in two aspects: (a) in addition to type I astrocytes, common to all the glial monolayers that we have studied, it contains a population of fusiform astrocytes (GFAP+) that might represent the superficial glia (Raisman, 1985); and (b) both astrocytes and fusiform cells produce large amounts of laminin that is secreted in a thick extracellular matrix. DENN outgrowth on olfactory bulb glia, however, is not blocked by antilaminin antibodies that block outgrowth on a laminin substrate. Our results demonstrate that two neuronal populations sharing the same neurotransmitter present intrinsic differences in the control of cell shape. The fact that glia harvested from different brain regions supports varying extent of DENN neurite outgrowth suggests a heterogeneity of environmental signals throughout the developing brain.

Positive control of target cerebellar cells on norepinephrine uptake in embryonic brainstem cultures in serum-free medium.

Brainstem cells from 15-day-old mouse embryos (E 15) grown for 8-10 days in dissociated primary cell culture in serum-free medium show high affinity uptake for [3H]norepinephrine (NE) that is specifically inhibited by desmethylimipramine (DMI), and also show high affinity binding for [3H]DMI. Brainstem cell uptake capacity for NE is increased at least 2-fold when cocultured with target cerebellar or striatal cells of the same embryonic age. The stimulation exerted by the cerebellum appears to be developmentally regulated since more mature cerebella (from 16-17-day-old embryos) exerted a greater stimulation than younger structures (from 14-15-day-old embryos). This stimulatory effect is also correlated with the number of available target sites since increasing the amount of cerebellar cells result in an increased stimulation of uptake capacity. The high affinity binding for [3H]DMI is also enhanced in coculture. The number of noradrenergic neurons, detected by autoradiography, remains unchanged in coculture indicating that added cerebellar cells did not take up NE, suggesting that the number of uptake sites per noradrenergic neuron is increased in the presence of target cells. These results indicate that interactions between afferent and target neurons, which normally take place during in vivo development, also occur in vitro and may result in a modification of neurotransmitter uptake in the afferent neuron.

Cell surface modifications in neuronal maturation.

Changes in carbohydrate composition of the cell surface related to neuronal maturation have been studied on neuroblastoma and embryonic dorsal root ganglia (DRG) cultures by using fluorescein conjugated lectins. In neuroblastoma cells, it has been found that the surface of the fibers differs from that of the cell body as shown by concanavalin A (Con A) and WGA binding. In primary cultures of embryonic DRG, lectin binding has also shown that the neuron surface undergoes changes during maturation. In fact, lectin binding which is absent at early stages (5--6 day old embryos) becomes first detectable at the 7th day and then increases progressively. At day 7, the Con A binding pattern resembles that observed in neuroblastoma cells. The possibility of correlating these surface changes with cell adhesive properties and cell differentiation is discussed.