Transcriptional activity and splicing factors are preserved during physiological apoptosis.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Immunohistochemical and ultrastructural study of the lamellae of oocytes in atretic follicles in relation to different processes of cell death.

Atresia is the process through which non-selectable oocytes are eliminated; it involves apoptosis and/or autophagy. This study used immunohistochemical and ultrastructural techniques to characterize the lamellae present in the cytoplasm of oocytes in follicles in the process of atresia in prepubertal and adult Wistar rats. The results indicate that the lamellae are positive to tubulin and myosin immunodetection under light and electron microscopy. Labeling is greater with anti-tubulin and lesser with anti-myosin. Our observations indicate that lamellae are present in oocytes at the initial antral stage in prepubertal rats; that is, from day 14 post-birth to adult age. We were able to determine that the increase in altered lamellae principally occurs in the apoptotic cells rather than in the autophagic cells.

Analysis of different cell death processes of prepubertal rat oocytes in vitro.

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

Chromatin structure contribution to the synaptonemal complex formation.

Meiosis is a key cellular and molecular process for sexual reproduction contributing to the genetic variability of organisms. This process takes place after DNA replication and consists in a double cellular division, giving rise to four haploid daughter cells or gametes. Meiotic recombination between homologous chromosomes, in the meiotic prophase I, is mediated by a tripartite structure named Synaptonemal Complex (SC). The SC is a peptidic scaffold in which the chromatin of homologous chromosomes is organized during the pachytene stage, holding chromosomes together until the meiotic recombination and genetic exchange have taken place. The role of chromatin structure in formation of the SC and the meiotic recombination at meiotic prophase I remain largely unknown. In this review we address the epigenome contribution to the SC formation at meiotic prophase I, with particular attention on the chromatin structure modifications occurring during the sub-stages of meiotic prophase I.

Combined apoptosis and autophagy, the process that eliminates the oocytes of atretic follicles in immature rats.

We studied the alterations of dying oocytes in 1-28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.

Beta-lactam activity against penicillin-resistant Streptococcus pneumoniae strains exhibiting higher amoxicillin versus penicillin minimum inhibitory concentration values: an in vitro pharmacodynamic simulation.

BACKGROUND: Activity of simulated serum concentrations after oral therapy with 400 mg cefditoren pivoxil b.i.d., 500 mg cefuroxime axetil b.i.d. and 875/125 mg amoxicillin/clavulanic acid b.i.d. and t.i.d. regimens was explored over 24 h against Streptococcus pneumoniae. METHODS: Computerized pharmacodynamic simulations were performed against strains with penicillin/amoxicillin/cefuroxime/cefditoren minimum inhibitory concentrations (MICs, microg/ml) and serotypes: strain 1 (0.25/0.12/1/0.12; serotype 6A), strain 2 (2/4/ 2/0.25; serotype 6B), strain 3 (4/16/4/0.5; serotype 14), and strain 4 (4/16/8/1; serotype 14). RESULTS: Bactericidal activity (> or =3 log(10) reduction) at 12 and 24 h was obtained against all strains with cefditoren, against strains 1 and 2 with cefuroxime and amoxicillin/clavulanic acid t.i.d., but only against strain 1 with amoxicillin/clavulanic acid b.i.d.. Bactericidal activity at 24 h was related to T > MIC of >30% dosing interval, 1.7-2.0 log(10) reductions with T > MIC of 20-30%, and <1 log(10) reduction or regrowth with T > MIC of 0%. CONCLUSIONS: It is difficult to achieve pharmacodynamic coverage and bactericidal activity by physiological concentrations of oral beta-lactams against penicillin-resistant pneumococcal strains exhibiting higher amoxicillin versus penicillin MICs. Cefditoren may offer alternatives.

influence of TEM-1 beta-lactamase on the pharmacodynamic activity of simulated total versus free-drug serum concentrations of cefditoren (400 milligrams) versus amoxicillin-clavulanic acid (2,000/125 milligrams) against Haemophilus influenzae strains exhibiting an N526K mutation in the ftsI gene.

The aim of this study was to explore bactericidal activity of total and free serum simulated concentrations after the oral administration of cefditoren (400 mg, twice daily [bid]) versus the oral administration of amoxicillin-clavulanic acid extended release formulation (2,000/125 mg bid) against Haemophilus influenzae. A computerized pharmacodynamic simulation was performed, and colony counts and beta-lactamase activity were determined over 48 h. Three strains were used: ampicillin-susceptible, beta-lactamase-negative ampicillin-resistant (BLNAR) (also resistant to amoxicillin-clavulanic acid) and beta-lactamase-positive amoxicillin-clavulanic acid-resistant (BLPACR) strains, with cefditoren MICs of < or =0.12 microg/ml and amoxicillin-clavulanic acid MICs of 2, 8, and 8 microg/ml, respectively. Against the ampicillin-susceptible and BLNAR strains, bactericidal activity (> or =3 log(10) reduction) was obtained from 6 h on with either total and free cefditoren or amoxicillin-clavulanic acid. Against the BLPACR strain, free cefditoren showed bactericidal activity from 8 h on. In amoxicillin-clavulanic acid simulations the increase in colony counts from 4 h on occurred in parallel with the increase in beta-lactamase activity for the BLPACR strain. Since both BLNAR and BLPACR strains exhibited the same MIC, this was due to the significantly lower (P < or = 0.012) amoxicillin concentrations from 4 h on in simulations with beta-lactamase positive versus negative strains, thus decreasing the time above MIC (T>MIC). From a pharmacodynamic point of view, the theoretical amoxicillin T>MIC against strains with elevated ampicillin/amoxicillin-clavulanic acid MICs should be considered with caution since the presence of beta-lactamase inactivates the antibiotic, thus rendering inaccurate theoretical calculations. The experimental bactericidal activity of cefditoren is maintained over the dosing interval regardless of the presence of a mutation in the ftsI gene or beta-lactamase production.

Pharmacodynamics of simulated total versus free-drug serum concentrations of a low versus a high protein bound third-generation oral cephalosporin (Cefpodoxime versus cefditoren) against Streptococcus pneumoniae.

Pharmacodynamic parameters and bactericidal activity against Streptococcus pneumoniae were investigated by simulating total and free serum concentrations of cefpodoxime versus cefditoren. Total drug T>MIC against the penicillin-intermediate (PISP) and resistant (PRSP) strains were 70.6% and 42.9% for cefpodoxime, and 89.6% and 62.5% for cefditoren, respectively. Comparing activity of free versus total cefpodoxime, there were reductions of 8.5% and 19.1% in T>MIC, related to bactericidal activity reductions from approximately 4.5 to 3 log(10), and from 3 to 2.5 log(10 )against PISP and PRSP, respectively, at 10-12h. For cefditoren, reductions of 45.4% and 100% in T>MIC, were related to bactericidal activity reductions from approximately 5.5 to 2-2.5 log(10 )and from approximately 2.5 to 1.5 log(10 )against PISP and PRSP, respectively, at 10-12h. Higher differences in activity were found against the less resistant strains when comparing total versus free-drug profile.

Effects of human albumin and serum on the in vitro bactericidal activity of cefditoren against penicillin-resistant Streptococcus pneumoniae.

OBJECTIVES: Attempts to interpret antibiotic pharmacodynamics using reported protein binding may underestimate true activity. To elucidate this issue we examined bacterial killing kinetics at cefditoren concentrations equal to C(max) in the presence of 90% human serum or albumin at physiological concentrations. METHODS: Killing curves (final inocula of approximately 10(7) cfu/mL, cefditoren concentration of 4.2 mg/L) were performed against Streptococcus pneumoniae strains exhibiting cefditoren MICs (mg/L) of 0.12 (strain 1), 0.25 (strain 2) and 0.5 (strain 3) in different media: (i) C(max)-MH, Mueller-Hinton broth plus 5% lysed horse blood (MH), (ii) C(max)-HS, MH broth with a final human serum concentration of 90%; and (iii) C(max)-HAlb, MH broth with 4 g/dL human albumin. Killing curves were also performed with a final cefditoren concentration of 0.5 mg/L (similar to free-drug C(max) considering 88% protein binding) in MH broth (12% C(max)). RESULTS: No significant differences were found between the different media or concentrations with strain 1 (log(10) reductions >or=4 at 24 h). Against strains 2 and 3, we observed significantly higher initial inocula decreases at 24 h for C(max)-HS as compared with C(max)-HAlb or 12% C(max). Bactericidal activity (>or=3 log(10) reduction) was obtained at 24 h against the three strains only with C(max)-HS and C(max)-MH. CONCLUSIONS: The presence of physiological concentrations of human albumin, but not 90% human serum, limited bactericidal activity as did the use of concentrations similar to free-drug C(max), suggesting that extrapolation of active drug from total drug by using the protein binding rate is not an accurate method to study antibacterial activity.

Are beta-lactam breakpoints adequate to define non-susceptibility for all Haemophilus influenzae resistance phenotypes from a pharmacodynamic point of view?

OBJECTIVES: To investigate the bactericidal activity, against Haemophilus influenzae strains exhibiting different resistance phenotypes, of simulated serum concentrations obtained in humans after administration of 400 mg of cefditoren twice daily, 500 mg of cefuroxime twice daily, 875/125 mg of co-amoxiclav twice daily or 875/125 mg of co-amoxiclav three times daily. METHODS: An in vitro computerized pharmacodynamic simulation was carried out and colony counts determined over 24 h. Four H. influenzae strains were used, one ampicillin-susceptible strain (Strain 1) and three ampicillin-resistant strains following CLSI and BSAC breakpoints: one beta-lactamase-positive strain with an MIC of co-amoxiclav of 0.5 mg/L (Strain 2), one beta-lactamase-negative ampicillin-resistant strain (BLNAR; ampicillin MIC = 16 mg/L) (Strain 3) and one beta-lactamase-positive strain with an MIC of co-amoxiclav of 4 mg/L (Strain 4). All strains were susceptible to cefuroxime and co-amoxiclav according to current CLSI breakpoints, but Strains 3 and 4 were resistant according to BSAC breakpoints. All strains exhibited cefditoren MIC or= 6 log(10) reduced with all antibiotics tested at 12 and 24 h. Against Strains 3 and 4, log(10) reductions at 12 and 24 h were significantly higher for cefditoren versus cefuroxime (P < 0.01) (although both exhibited bactericidal activity, i.e. >or= 3 log(10) reduction) and versus the two co-amoxiclav regimens (P < 0.001) (that exhibited negligible initial inocula reductions). CONCLUSIONS: Cefditoren exhibited the highest bactericidal activity maintained over time against ampicillin-resistant H. influenzae, regardless of beta-lactamase production and/or BLNAR phenotype. From the pharmacodynamic perspective, BSAC breakpoints seem more adequate to define or detect BLNAR strains.

Levofloxacin vs. azithromycin pharmacodynamic activity against S. pneumoniae and H. influenzae with decreased susceptibility to amoxicillin/clavulanic acid.

Resistant clones/phenotypes are putting into question the activity of commonly used beta-lactams, thus prompting the need for alternative options. A 500 mg levofloxacin vs. azithromycin once daily pharmacodynamic simulation was performed against 10(8) cfu/ml of four Streptococcus pneumoniae strains (exhibiting higher amoxicillin than penicillin MIC) and four Haemophilus influenzae strains: beta-lactamase producing, BLNAR (beta-lactamase-negative ampicillin-resistant) and BLPACR (beta-lactamase-positive amoxicillin/clavulanate-resistant). High levofloxacin AUC/MIC values for H. influenzae, and values of 50-100 for S. pneumoniae produced a >5 log(10) reduction at 24h for all strains. Azithromycin AUC/MIC values of approximately 10 were needed to obtain a 2-3 log(10) reduction of S. pneumoniae initial inocula, but lower AUC/MIC values (of approximately 6) obtained > or =3 log(10) reduction against all strains of H. influenzae. While in vitro simulated serum concentrations of levofloxacin were bactericidal at the end of the dosing interval against all S. pneumoniae strains and azithromycin against the susceptible ones, both antimicrobials achieved this endpoint against the BLNAR and BLPACR strains.

Influencia del desplazamiento de la unión a proteínas por ibuprofeno en la actividad de una cefalosporina de tercera generación frente a Streptococcus pneumoniae / Influence of the displacement of protein binding by ibuprofen in the activity of a third-generation cephalosporin against Streptococcus pneumoniae

El significado clínico de la unión de los antibacterianos a las proteínas plasmáticas es un fenómeno aún no bien definido. El objetivo de esteestudio fue valorar, mediante curvas de muerte bacteriana, el efecto en la actividad bactericida in vitro del cefditoren, a concentracionespróximas a la Cmax, de una administración de 400 mg, frente a tres cepas de Streptococcus pneumoniae (CMI de cefditoren de 0,12, 0,25y 0,5 mg/l) en combinación con albúmina humana (4 g/dl) e ibuprofeno, a la concentración máxima detectada después de la administraciónde 400 mg (32,3 mg/l) y de 10 veces la Cmax (323 mg/l). El cefditoren fue rápidamente bactericida (reducción de 3 log10 UFC/ml del inóculoinicial) frente a todas las cepas empleadas a concentraciones de 4,2 mg/l, en medios de cultivo convencionales. En presencia de albúminahumana este efecto sólo se mantuvo sobre la cepa más sensible (CMI = 0,12 mg/l), detectando recrecimientos más o menos acusadoscon valores de CMI superiores. La presencia de ibuprofeno (32,3 mg/l) retrasó ligeramente la aparición de recrecimientos, mientras queel aumento de la concentración a 10 veces la Cmax de ibuprofeno recuperó la actividad bactericida en todas las cepas. En conclusión, la actividadde un antibacteriano de elevada unión a las proteínas plasmáticas no debería relacionarse exclusivamente con la fracción libre teóricaextrapolada desde las concentraciones plasmáticas. El papel de los antagonistas de la unión a las proteínas merece ser analizado dado sufrecuente uso en procesos respiratorios, particularmente asociados con la administración de cefalosporinas

The clinical significance of protein binding remains to be fully elucidated. The aim of this study was to evaluate the effect in the in vitrobactericidal activity of cefditoren through killing curves at Cmax concentrations against three Streptococcus pneumoniae strains (cefditorenMICs of 0.12, 0.25 and 0.5 mg/l) with or without human albumin (4 g/dl) and ibuprofen at Cmax concentrations (32.3 mg/l) and 10times the Cmax (323 mg/l). Cefditoren was rapidly bactericidal (3 log10 CFU/ml reduction) against the three strains at 4.2 mg/l concentrationin Mueller-Hinton broth plus 5% lysed horse blood. In presence of human albumin, this effect was maintained against the mostsusceptible strain (MIC = 0.12 mg/l). Regrowths were observed with higher MIC values. The presence of ibuprofen (32.3 mg/l) slightly delayedregrowth while the increase of ibuprofen concentration up to 10 x Cmax recovered the bactericidal activity against all strains. Theactivity of an antimicrobial with high protein binding should not be linked exclusively with the theoretical unbound fraction extrapolatedfrom the plasma concentration. The role of protein binding antagonists merits analysis due to their frequent use associated with cephalosporinsin respiratory tract infections

Integration of morphological and cytophysiological studies on estrogen receptor.

Ultrastructural and immunocytochemical studies of an intra-nuclear particle, the perichromatin granule (PCG), demonstrated the presence of processed mRNA in this structure. Ovariectomy caused an increase in the number of PCGs in uterine cells and administration of estradiol drastically reduced the nuclear pool of PCGs in 15 min. In vitro studies demonstrated that this depletion was accompanied by an increase of the export of previously synthesized RNA. Similar quantitative changes of the abundance of PCG and of the rate of the export of RNA were found in ventral prostate after orchiectomy and testosterone restitution, as well as in the target cells of FSH, LH, TSH, and ACTH. These results taken together led us to conclude that PCGs constitute an intra-nuclear compartment of a few processed mRNA in equilibrium with transcription and export. This mRNA is rapidly transferred to the cytoplasm by specific hormone signals.

[Influence of the displacement of protein binding by ibuprofen in the activity of a third-generation cephalosporin against Streptococcus pneumoniae]. / Influencia del desplazamiento de la unión a proteínas por ibuprofeno en la actividad de una cefalosporina de tercera generación frente a Streptococcus pneumoniae.

The clinical significance of protein binding remains to be fully elucidated. The aim of this study was to evaluate the effect in the in vitro bactericidal activity of cefditoren through killing curves at Cmax concentrations against three Streptococcus pneumoniae strains (cefditoren MICs of 0.12, 0.25 and 0.5 mg/l) with or without human albumin (4 g/dl) and ibuprofen at Cmax concentrations (32.3 mg/l) and 10 times the Cmax (323 mg/l). Cefditoren was rapidly bactericidal (3 log(10) CFU/ml reduction) against the three strains at 4.2 mg/l concentration in Mueller-Hinton broth plus 5% lysed horse blood. In presence of human albumin, this effect was maintained against the most susceptible strain (MIC = 0.12 mg/l). Regrowths were observed with higher MIC values. The presence of ibuprofen (32.3 mg/l) slightly delayed regrowth while the increase of ibuprofen concentration up to 10 x Cmax recovered the bactericidal activity against all strains. The activity of an antimicrobial with high protein binding should not be linked exclusively with the theoretical unbound fraction extrapolated from the plasma concentration. The role of protein binding antagonists merits analysis due to their frequent use associated with cephalosporins in respiratory tract infections.

Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats.

The process of cell death of oocytes was studied in atretic ovarian follicles of rats aged from 1 to 28 days using light and electron microscope and cytochemical methods. These methods were TUNEL procedure for DNA breaks, active caspase-3 and lysosome-associated membrane protein 1 (LAMP-1) immunolocalizations. The structural features of the process of oocyte death are mainly characterized by the presence of abundant clear vacuoles and autophagosomes, as well as by the absence of large clumps of compact chromatin associated to the nuclear envelope and apoptotic bodies. These features are common to oocytes in all types of follicles studied. Cytochemical features consisting in positive reactions to TUNEL method, active caspase-3 and LAMP-1 immunolocalizations, are common to the cell death of oocytes in all types of follicles. Particular features of the process of cell death of oocytes are found in different types of follicles. Two morphological patterns of cell death occur in pre-follicular oocytes of the new born and in primordial follicles in 1 to 5 days old rats. One is distinguished by clear nucleoli and moderate compaction of chromatin in clumps frequently resembling meiotic bivalents. The second pattern is characterized by nucleolar condensation and by the absence of compact chromatin. The process of cell death of oocytes in antral follicles is characterized by ribonucleoprotein ribbon-like cytoplasmic structures, pseudo-segmentation, and loss of contact with granulosa cells.

Niveles de hemoglobina en varones fumadores / Haemoglobin levels in smoking men

Debido a la importancia de la hemoglobina como indicador nutricional, así como su papel en la oxigenación de los tejidos, fue realizado este estudio con el objetivo de observar la existencia o no de alteraciones en los niveles de hemoglobina de individuos fumadores donantes de sangre, de tal manera que dichas alteraciones causadas por el cigarrillo sean consideradas, al evaluar el estado de salud de un individuo fumador, así como en el diagnóstico de las patologías asociadas. Fueron seleccionados 121 varones, aparentemente sanos, fumadores y no fumadores, entre 20 y 60 años de edad, donantes de cinco bancos de sangre de Asunción, Paraguay. Los niveles promedios de hemoglobina en fumadores fueron 150 ± 8 g/L y en los no fumadores 148 ± 9 g/L, no siendo la diferencia significativa (p>0.05); sin embargo dentro del grupo de fumadores, la diferencia fue significativamente mayor en aquellos que fumaban de 11 a 20 cigarrillos /día (152 ± 9 g/L) que en aquellos que fumaban de 1 a 10 cigarrillos /día (148 ± 7 g/L) (p<0.05). Por lo tanto, el nivel medio de hemoglobina se incrementa con el número de cigarrillos consumidos por día. El coeficiente de correlación hallado fue de 0,38 con un nivel de confianza del 95%. Debemos seguir investigando, en nuestra población, la influencia del cigarrillo sobre los parámetros hematológicos, y el efecto que producen dichas alteraciones en el estado de salud de los fumadores.

Due to the important role haemoglobin plays as nutritional marker and in tissue oxygenation, this study was carried out to determine if there are alterations in haemoglobin levels of smoking blood donors. In this way, these alterations caused by cigarettes should be considered when the health status of smoking men is evaluated, as well as in the diagnosis of associated pathologies. One hundred and twenty one male individuals were selected for this study. They were apparently healthy, smoking and non­smoking men, between 20 and 60 years old, blood donors from five blood banks in Asunción, Paraguay. The mean haemoglobin level in smokers was 150 ± 8 g/L and in non­smokers 148 ± 9 g/L. The difference was not significant (p>0.05) but the difference was statistically greater in individuals smoking 11 to 20 cigarettes /day (152 ± 9 g/L) than in those who smoked 1 to 10 cigarettes /day (148 ± 7 g/L) (p<0.05). Thus, the mean value of haemoglobin increases with the number of cigarettes smoked per day. The correlation coefficient was 0.38 with a confidence interval of 95%. Further studies about the influence of cigarettes, the haematological parameters and the effect produced by alterations on the health status of smokers are necessary in this population.

Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol.

The localization and abundance of the estrogen receptor activation factor (E-RAF) and a small nuclear ribonucleoprotein (snRNP) complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II), have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing) and with clusters of interchromatin granules (storage of splicing-related molecules). The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

Fine structural cytochemical analysis of homologous chromosome recognition, alignment, and pairing in Guinea pig spermatogonia and spermatocytes.

The nuclei of guinea pig spermatogonia and spermatocytes were studied by means of quantitative autoradiography and electron microscopic methods such as high-resolution cytochemistry, immunocytochemistry, and in situ hybridization. Our observations reveal, in the nucleus of spermatogonia type B, small lampbrush structures of extended chromatin not found in nonmeiotic cells. During meiotic interphase, pairs of parallel lampbrush structures become associated by numerous filaments. The formation of the synaptonemal complex is simultaneous with the extension of chromosomal axes in a continuous leptotene-zygotene stage. Some chromosomes do not recognize their homologs before the onset of the leptotene-zygotene stage and undergo classical leptotene and zygotene stages. The immunocytochemical localization of Dmc1 and Rad51 supports the idea that these proteins are not involved in homology search and final pairing. Immunolocalization of DNA, RNA polymerase II, heterogeneous nuclear ribonucleoproteins, small nuclear ribonucleoproteins, and the trimethyl-guanosin cap of small nuclear RNAs suggests that the chromatin of lampbrush structures transcribe hnRNA and that splicing is scarce. The results of quantitative autoradiography after [3H]uridine labeling show an intense transcription accompanied by a very slow export of RNA. In situ hybridization demonstrates the presence of RNA in the regions of homology recognition and pairing. These results lead us to propose that the RNA synthesized in the lampbrush structures is involved in the process of homology searching and recognition.

Ultrastructural and immunocytochemical analysis of the XY body in rat and Guinea pig.

The formation of the XY body involves the compaction of the extended chromatin to form a mesh of fibrogranular structures. During this process the ribonucleoprotein particles (RNP), which were associated with the chromatin filaments progressively disappear. High resolution immunolocalization indicates that the mature XY body does not contain RNA polymerase II, hnRNPs, or snURNPs. Occasionally chromatin fibrils extend outside of the XY body. These fibrils are frequently associated with nascent RNP fibrils and granules indicating that not all the DNA of the sex chromosomes is transcriptionally inactive. However, transcription is located outside the sex body. The recombination protein Dmc1 is present in nodules associated with the unpaired chromosomal axes of the sex chromosomes located in the XY body. Cytochemical staining methods and in situ hybridization at electron microscopic level show that RNA is present in the unpaired chromosomal axes suggesting that the presence of RNA in the chromosomal axes and in forming synaptonemal complexes is related with the process of final pairing. The sex body and the nucleoli associated with it do not interweave and do not exchange RNA or DNA-containing filaments. These observations indicate that the spatial relation between these structures is just a close proximity, which is, however, very frequent.

Cytochemical study of the distribution of RNA and DNA in the synaptonemal complex of guinea-pig and rat spermatocytes.

The distribution of DNA and RNA in the synaptonemal complex and related structures, was studied using high resolution cytochemical methods and in situ hybridization, in guinea pig and rat testis. Serial sectioning demonstrates that frequently the formation of the synaptonemal complex (SC) occurs without a previous development of isolated chromosomal axes. The lateral elements of the forming SC are in continuity with pairs of DNA-containing thin filaments. These chromatin filaments fold in numerous short loops just before incorporating to the lateral elements. Some of these loops are included in the ribbon-like structure of the lateral elements of the mature SC. We propose that these short loops contain the DNA attachment sequences associated with the proteins of the LE. During the formation of the SC one of the two chromatin filaments incorporates at the central surface of the forming lateral element (LE) and the other is located at the external side of the LE. This unexpected distribution does not correspond to the pair of thick filaments previously discerned in structure of the LE. The presence of RNA associated with the DNA-containing thin filaments, as well as with the axial chromatin elements of the forming SC, may be related with the transcription occurring during meiotic prophase, specially during zygotene stage. We propose that RNA is involved in a still uncharacterized process essential for pairing.