Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats.

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.

Effects of differential pulse frequencies of chicken gonadotrophin-releasing hormone-I (cGnRH-I) on laying hen gonadotrope responses in vitro.

The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at -70° C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells' features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.

Ultrastructural study of spermatogenesis in Eisenia foetida (Annelida, Oligochaeta)

La espermatogénesis y ultraestructura del espermatozoide siguen un patrón común en los oligoquetos terrestres, sin embargo, muchos de sus aspectos son distintivos de cada especie y tienen importancia para los estudios filogenéticos y taxonómicos. Con el objetivo de profundizar sobre aspectos de la espermatogénesis y, en especial, sobre la ultraestructura del espermatozoide maduro de Eisenia foetida, se estudiaron sus células germinales mediante técnicas convencionales de microscopía electrónica. La espermatogénesis ocurre en las vesículas seminales y sigue un patrón comparable con el de otras especies de oligoquetos. Los detalles ultraestructurales del espermatozoide revelaron que es una célula filiforme, con el acrosoma situado en posición anterior, seguido por el núcleo, pieza intermedia y cola, la que posee el flagelo con la configuración microtubular típica de 9+2. Podemos concluir que el espermatozoide de Eisenia foetida es del tipo apomórfico,ya que presenta varias características consideradas evolucionadas, como son: acrosoma muy largo, vesícula acrosómica primaria contenida dentro del tubo acrosómico, varilla axial muy extendida y terminada en un capítulo desarrollado y el número de mitocondrias de la pieza intermedia igual a seis.

Spermatogenesis and the spermatozoon ultrastructure follow a common pattern in terrestrial oligochaetes. However, many of their characteristics are distinctive of each species and they are of great importance for phylogenetic and taxonomic studies. Germinal cells were studied through conventional electronic microscope techniques in order to go deep on spermatogenesis, especially on the mature spermatozoon ultrastructure of Eisenia foetida. Spermatogenesis occurs in seminal vesicles and follows a pattern comparable to that of some other oligachaetes species. Spermatozoon ultrastructural details revealed that it is a filiform cell, with the acrosome placed in anterior position, followed by the nucleus, the midpiece and the tail which flagellum has a 9+2 typical microtubular configuration. We may conclude that Eisenia foetida spermatozoon is of a apomorphic type since it presents several evolved characteristics such as: a very large acrosome; the primary acrosomic vesicle is held within the acrosomal tube, the axial rod is very elongated and ended in a developed capitulum and a number of mitochondria of the midpiece equal to six.

Relative Concentrations of Placental Lactogen II and PRL-Like Protein-A in Stressed Rats Placenta / Concentraciones del Lactógeno Placentario II y la Proteína A Ligada a Prolactina en Placentas de Ratas Estresadas

The chronic stress induces functional adaptations in the hypothalamo-pituitary- adrenocortical (HPA) and in the sympathetic-medullary-adrenal axis (SAM). Both axis are considered vital regulators of the homeostasis in vertebrates (Seyle, 1936; Ostrandrer et al, 2006. On the other hand, the placenta provides highly specialized functions during gestation that are critical for the normal development of the embryo/fetus (Soares et al., 1991). We hypothesized that the chronic immobilization (IMO) stress in pregnancy rats produces alterations in prolactin concentrations in placental tissue and also changes in the response of SAM axis. Chronic stress by IMO was applied on days 12, 17 and 21 of pregnancy rats. Relative concentrations and localization of placental lactogen-II (PL-II) and the PRL- like protein A (PLP-A) in chorioalantoic placenta were estimated by Immunoblotting and Immunocytochemical analysis. The levels of catecholamines metabolite, acid 3-metoxi 4-hidroximandélico (VMA), were analyzed in stressed rats urines on 6,12,17,21 days of pregnancy, by HPLC, in order to determine the response of SAM axis. During the days of the pregnancy studied, chronic stress did not induce any changes neither in the localization nor in placental concentrations of PL-II and PLP-A. The VMA values in stressed mothers urines increased on the day 6 respecting the control ones at the same time of pregnancy. VMA values in stressed rats at 21 days of pregnancy are smaller than the respective controls. We conclude that the chronic stressed mothers activated the SAM axis at the beginning of pregnancy and then they diminished the metabolites catecholamines that were interpreted as a stress adaptation coincident with normal concentrations of both placentary prolactines at this stage of the pregnancy.

El estrés crónico induce adaptaciones funcionales en los ejes hipotálamo-pituitario-adrenal (UPA) y en el simpático médulo adrenal (SAM). Ambos ejes son considerados reguladores vitales de la homeostasis en los vertebrados (Seyle, 1936; Ostrandrereí al., 2006). Por otro lado, el desarrollo y crecimiento fetal de los mamíferos dependen en gran medida del buen funcionamiento de la placenta (Soares, 1991). Nosotros hipotetizamos que el estrés crónico por inmovilización (IMO) aplicado a las ratas gestantes produce alteraciones en las concentraciones de las prolactinas en el tejido placentario y cambios en la respuesta del eje SAM. Se le aplicó estrés crónico por IMO a las hembras en los días 12, 17 y 21 de la preñez y se analizó por inmunocitoquímica e inmunoblotting la localización y concentraciones del lactógeno placentario dos (PL-II) y la proteína A ligada a la prolactina (PLP-A) en la placenta. Se analizaron por HPLC, en las orinas de ratas preñadas (6,12,17,21 días), los niveles del metabolito de las catecolaminas, (ácido 3-metoxi 4-hidroximandélico) (VMA), a fin de determinar la respuesta del eje SAM al tratamiento. El estrés crónico no indujo cambios tanto en la localización como en las concentraciones de PL-II y PLP-A en las placentas en los días de la preñez estudiados. Los valores de VMA en las orinas de las madres estresadas se incrementaron en el día 6 con respecto al control del mismo tiempo de preñez. Mientras que a los 21 días los valores de VMA de las ratas estresadas son menores que los controles respectivos. Concluimos que en las madres estresadas crónicamente, no se alteraron las concentraciones de ambas prolactinas placentarias. En cambio se activó el eje SAM al comienzo de la preñez ante el primer estímulo estresante y luego una reducción de la respuesta del eje ante el estrés crónico, a medida que avanza la preñez.

Chronic stress effects on the apoptotic index of the adrenal cortex of pregnant rats

Chronic stress by immobilization during gestation can alter several mechanisms that maintain homeostasis in adrenal gland. The aim of this work was to quantify the apoptotic index of adrenal cortex during mid-pregnancy and to prove cytological characteristics by electron microscopy. The apoptotic index did not present significant differences between the adrenal cortex areas of control and experimental rats in any of the three ages studied. The day of gestation influenced significantly on the apoptotic index in both groups. This index increased as gestation progressed. It may be concluded that chronic stress by immobilization might induce the increase of apoptotic index in adrenal cortex as gestation progresses which might be related variations of plasmatic corticosterone and prolactin, and to the decrease of specific growth factors. On the other hand, it might be concluded that each zone of adrenal cortex behaves independently in regards to apoptosis and cellular proliferation via paracrine and/or autocrine regulatory mechanisms without being affected by other zones.

Chronic stress effects on the apoptotic index of the adrenal cortex of pregnant rats

Chronic stress by immobilization during gestation can alter several mechanisms that maintain homeostasis in adrenal gland. The aim of this work was to quantify the apoptotic index of adrenal cortex during mid-pregnancy and to prove cytological characteristics by electron microscopy. The apoptotic index did not present significant differences between the adrenal cortex areas of control and experimental rats in any of the three ages studied. The day of gestation influenced significantly on the apoptotic index in both groups. This index increased as gestation progressed. It may be concluded that chronic stress by immobilization might induce the increase of apoptotic index in adrenal cortex as gestation progresses which might be related variations of plasmatic corticosterone and prolactin, and to the decrease of specific growth factors. On the other hand, it might be concluded that each zone of adrenal cortex behaves independently in regards to apoptosis and cellular proliferation via paracrine and/or autocrine regulatory mechanisms without being affected by other zones.(AU)

Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, Xenarthra).

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were-stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes.

[Effects of parathion on acetylcholinesterase activity in rat kidney]. / Efectos del paration sobre la actividad de acetilcolinesterasa en riñón de rata.

In this work the effects of parathion (a competitive acetylcholinesterase inhibitory agent) on the enzyme activity was studied by cytochemical methods in the kidney of rats; being the doses used not inhibitory of red blood cells acetylcholinesterase (subtoxic dose). Two groups of rats were used: control (CR) and parathion-treated (TR) groups, both submitted to a water deprivation period of 24 h. for induction of primary thirst. The treated-rats group received parathion i.p. at doses of 600 micrograms/100g body weight. For demonstration of acetylcholinesterase activity, renal tissue incubation was performed by the Karnovsky and Roots method with the Tsuji Larabi variation. The results of the five assays of incubation enable us to conclude that there exists a noticeable activity of acetylcholinesterase in the renal cortex of control rats, which is blocked by subtoxic doses of organophosphorus pesticide. We suggest that the natriuretic effect of parathion can be explained by this mechanism.