Interphase cytogenetics using fluorescence in situ hybridization: an overview of its application to diffuse and solid tissue

Interphase cytogenetics, utilizing fluorescence in situ hybridization (FISH) techniques, has been successfully applied to diffuse and solid tissue specimens. Most studies have been performed on isolated cells, such as blood or bone marrow cells; a few have been performed on cells from body fluids, such as amniotic fluid, urine, sperm, and sputum. Mechanically or chemically disaggregated cells from solid tissues have also been used as single cell suspensions for FISH. Additionally, intact organized tissue samples represented by touch preparations or thin tissue sections have been used, especially in cancer studies. Advantages and pitfalls of application of FISH methodology to each type of specimen and some significant biological findings achieved are illustrated in this overview.

Genetic variability of Prochilodus lineatus (Characiformes, Prochilodontidae) in the upper Paraná river

The genetic variability of the "curimba", Prochilodus lineatus, from three locations in the Paraná river basin, was investigated by starch gel electrophoresis. A total of 160 specimens were analyzed for 19 enzymes, 12 of which permitted successful interpretation of electrophoretic patterns. Eighteen loci were identified and six of them proved to be polymorphic (EST-1*, EST-2*, IDH-1*, PGM-1*, PGM-2*, LDH-2*). Mean heterozygosity was considered high (13%) by comparison with the literature. A low level of differentiation was found among subpopulations, with mean F ST = 0.018. Values of genetic distance and genetic identity suggest that, at least along this stretch of the river, P. lineatus comprises a single breed with high gene flow. This analysis has important implications for fishery management, aquaculture, and conservation of the stocks

Reproductive isolation between Anastrepha bistrigata and A. striata (Diptera, Tephritidae)

The reproductive isolation between two closely related species, Anastrepha bistrigata and A. striata, was studied in the laboratory. Interespecific copulation attempts were observed, but examination of the spermathecae showed that sperm transference did not occur, even after a prolonged period of contact between the mating pairs. These results indicate prezygotic isolation. The analysis of the hourly distribution of mating activities under laboratory conditions, here described for the first time for A. bistrigata, clearly showed differences for the two species, the activities being concentrated in the afternoon period for A. striata and in the morning for A. bistrigata

Distribution of AMPA-type glutamate receptor subunits in the chick visual system

Several glutamate receptor (GluR) subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex) were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Aversive stimulation during the stress-hyporesponsive period does not affect the number of corticotroph cells in neonatal male rats

Immunohistochemistry was used to evaluate the effects of neonatal handling and aversive stimulation during the first 10 days of life on the number of corticotrophs in the anterior lobe of the pituitary of 11-day-old male Wistar rats. Since adult rats handled during infancy respond with reduced corticosterone secretion in response to stressors and with less behavior inhibition in novel environments, we assumed that neonatal stimulation could affect pituitary morphology during this critical period of cell differentiation. Three groups of animals were studied: intact (no manipulation, N = 5), handled (N = 5) and stimulated (submitted to 3 different aversive stimuli, N = 5). The percentage of ACTH-immunoreactive cells in the anterior lobe of the pituitary (number of ACTH-stained cells divided by total number of cells) was determined by examining three slices per pituitary in which a minimum of 200 cells were counted by two independent researchers. Although animals during the neonatal period are less reactive to stress-like stimulation in terms of ACTH and corticosterone secretion, results showed that the relative number of ACTH-stained cells of neonatal handled (0.25 ± 0.01) and aversive stimulated (0.29 ± 0.03) rats was not significantly different from intact (0.30 ± 0.03) animals. Neonatal stimulation may have a differential effect on the various subpopulations of corticotroph cells in the anterior pituitary

Study of the effects of ß-myrcene on rat fertility and general reproductive performance

ß-Myrcene (MYR) is a monoterpene found in the oils of a variety of aromatic plants including lemongrass, verbena, hop, bay, and others. MYR and essential oils containing this terpenoid compound are used in cosmetics, household products, and as flavoring food additives. This study was undertaken to investigate the effects of MYR on fertility and general reproductive performance in the rat. MYR (0, 100, 300 and 500 mg/kg) in peanut oil was given by gavage to male Wistar rats (15 per dose group) for 91 days prior to mating and during the mating period, as well as to females (45 per dose group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactation up to postnatal day 21. On day 21 of pregnancy one-third of the females of each group were submitted to cesarean section. Resorption, implantation, as well as dead and live fetuses were counted. All fetuses were examined for external malformations, weighed, and cleared and stained with Alizarin Red S for skeleton evaluation. The remaining dams were allowed to give birth to their offspring. The progeny was examined at birth and subsequently up to postnatal day 21. Mortality, weight gain and physical signs of postnatal development were evaluated. Except for an increase in liver and kidney weights, no other sign of toxicity was noted in male and female rats exposed to MYR. MYR did not affect the mating index (proportion of females impregnated by males) or the pregnancy index (ratio of pregnant to sperm-positive females). No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg) did MYR induce an increase in the resorption rate and a higher frequency of fetal skeleton anomalies. No adverse effect of MYR on postnatal weight gain was noted but days of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring. On the basis of the data presented in this paper the no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance can be set at 300 mg of ß-myrcene/kg body weight by the oral route.

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

In situ variation of cervical mucus pH during exposure to atmospheric air

The objective of the present study was to determine if exposure of cervical mucus to air during specular examination could modify mucus pH. Detection of changes is justified because of their possible interference with sperm-mucus interaction, since an acidic pH is unfavorable to sperm penetration and is associated with infertility due to the cervical factor. Twenty women with good quality mucus were evaluated. pH measurements of ecto- and endocervical mucus were made in situ using a glass electrode after 0-, 5- and 10-min exposure to air. There was a progressive alkalinization of mucus pH. Mean values of ectocervical mucus pH were 6.91, 7.16 and 7.27, while mean values of endocervical mucus pH were 7.09, 7.34 and 7.46 at 0, 5 and 10 min, respectively. Significant differences were found between the mean values obtained at 0 and 5 min, and at 0 and 10 min (P<0.05), whereas the differences in mean values at 5 and 10 min were not significant at either site. We conclude that 5 to 10 min of exposure to atmospheric air affects cervical mucus pH in a significant way. Since tests used to evaluate sperm-mucus interaction generally have not considered this possibility, we suggest that they should be performed immediately after mucus collection in order to avoid misinterpretation of the results.

Reproductive ability of pubertal male and female rats

Ten Fisher rats 50 to 55 days of age made up the pubertal group, and ten rats 90 to 95 days of age served as the controls. The testicular and epididymal weights and volumes of the pubertal males were lower than those of the controls (P<0.001). There was also a difference in relative epididymal weight (P<0.001). The sperm of pubertal males was morphologically abnormal in 58.2% of cases, as opposed to only 3.8% in the controls (P<0.001). The mean number of spermatozoa in the control group was 11.9 × 10(6)/ml and their viability was 99.6%, while these values could not be determined for pubertal rats. Serum testosterone was higher in the pubertal animals than in the controls (2.52 ± 1.46 vs 0.92 ± 0.34 nM, P<0.01). The ovaries of control females were heavier than those of pubertal females (P<0.001) but there was no difference in their relative weights. Serum estradiol was similar in both groups (75.5 ± 12.8 vs 81.8 ± 14.7 nM, P>0.05). At the beginning of gestation, the pubertal dams weighed less than the controls (P<0.001) but following uterectomy the body weights were equal. Pubertal dams delivered fewer pups than the controls (8.1 ± 2.5 vs 10.4 ± 1.3, P<0.05). There was no difference in the body weights of their offspring or in the weights of their placentas. The results suggest that, in contrast to their female counterparts, pubertal male rats are not fully mature and have not reached complete reproductive capacity at 50-55 days of age.

A prospective study of hepatitis B virus markers in patients with chronic HBV infection from Brazilian families of Western and Asian origin

The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8%) than in family members of Western origin (36.5%). HBsAg was also more frequent among brothers (79.6 vs 8.5%; P < 0.0001), children (37.9 vs 3.3%; P < 0.0001) and other family members (33.9 vs 16.7%; P < 0.0007) of Asian than Western origin, respectivelly. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7%) and 23/26 (88.5%) of the Asian and Western origin, respectively.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality

It has been reported that mental stress causes abnormality of spermiogram parameters. We investigated the effect of psychological stress on the L-arginine-nitric oxide (NO) pathway. Semen samples were collected from 29 healthy fourth semester medical students just before (stress) and 3 months after (non-stress) the final examinations. Psychological stress was measured by the State Anxiety Inventory questionnaire. After standard semen analysis, arginase activity and NO concentration were measured spectrophotometrically in the seminal plasma. Measurements were made in duplicate. During the stress period, sperm concentration (41.28 ± 3.70 vs 77.62 ± 7.13 x 10(6)/mL), rapid progressive motility of spermatozoa (8.79 ± 1.66 vs 20.86 ± 1.63%) and seminal plasma arginase activity (0.12 ± 0.01 vs 0.22 ± 0.01 U/mL) were significantly lower than in the non-stress situation, whereas seminal plasma NO (17.28 ± 0.56 vs 10.02 ± 0.49 µmol/L) was higher compared to the non-stress period (P < 0.001 for all). During stress there was a negative correlation between NO concentration and sperm concentration, the percentage of rapid progressive motility and arginase activity (r = -0.622, P < 0.01; r = -0.425, P < 0.05 and r = -0.445, P < 0.05, respectively). These results indicate that psychological stress causes an increase of NO level and a decrease of arginase activity in the L-arginine-NO pathway. Furthermore, poor sperm quality may be due to excessive production of NO under psychological stress. In the light of these results, we suggest that the arginine-NO pathway, together with arginase and NO synthase, are involved in semen quality under stress conditions.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.