Ultrastructural, immunohistochemical and biochemical analysis of glycosaminoglycans and proteoglycans in the mouse pubic symphysis during pregnancy.

During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.

Involvement of the corticoadrenal hormones in the pheromonal restoration of reproductive activity in aging rats.

The involvement of the adrenal progesterone and corticosterone in the early gonadotropin secretion associated with the pheromonal restoration of ovarian cyclic activity (PRCA) in aging female rats is studied. PRCA is induced by male urinary pheromones and is preceded by an alpha-adrenergic-mediated release of the hypothalamic decapeptide luteinizing hormone-releasing hormone and plasma increases of estradiol, progesterone and the gonadotropins luteinizing hormone and follicle stimulating hormone. Aging reproductive Wistar female rats were used to study the effects of bilateral adrenalectomy and of a subcutaneous injection of the antisteroid RU486 on plasma levels of corticosterone, progesterone and gonadotropins in rats stimulated with nasal spraying of male urine (MU) or saline. The results demonstrate that progesterone and corticosterone released by MU are from adrenal origin, and that these adrenal secretory products are critical for MU-induced increase of gonadotropins. This suggests that olfactory stimulation of ACTH release stimulates adrenal release of progesterone and corticosterone, and both trigger the events that initiate the activation of the hypothalamus-pituitary-ovarian axis that leads to PRCA.

Pinealectomy changes rat ovarian interstitial cell morphology and decreases progesterone receptor expression.

The aim of this study was to evaluate the rat ovarian morphological and function changes after pinealectomy (px). Two months after px, young female Wistar rats were sacrificed and the right ovaries were analysed morphologically and the left ovaries were used for steroid receptor binding experiments. Blood was collected and steroid hormone and melatonin levels were measured using radioimmunoassay kits. Results revealed that in the px group the rat ovaries had an increase in the number of atretic follicles and interstitial cells. These cells showed hyperactivity features on transmission electron microscopy and morphometric analysis (p < 0.05 compared with control and sham groups). Px-group serum showed an increase in estradiol (p < 0.05) and a decrease in progesterone levels (p < 0.05) compared with other groups. Moreover, progesterone receptor expression was lower than control and sham groups (p < 0.05). We postulate that pinealectomy leads to many morphological alterations of rat ovaries that are associated with functional changes in steroidogenesis and a decrease in progesterone receptor expression.

In vitro rapid organization of rabbit meniscus fibrochondrocytes into chondro-like tissue structures.

We described the behaviour of 120 days rabbit knee-meniscus cells in monolayer culture. The cells were grown forming cellular aggregates resembling true cellular nodules. Three stages of development of these nodules could be observed: formation of the cellular nodules between days 1 and 3; nodular growth, with their maximal at day 5; and nodular regression beginning at day 8. Ultrastructural analysis of the extracellular matrix of these cellular nodules was assessed on days 3, 5 and 8. At the formation stage, we could observe striated collagen fibrils and small bundles of tubular microfibrils either interspersed with very low quantities of amorphous elastin, being morphologically identical to elaunin fibers, or without only trace of elastin, being morphologically identical to oxytalan fibers. By day 5, fibrillar elements with 100 nm periodic ladder-like collagen VI fibrillar aggregates could also be detected. At day 8, the striated collagen fibrils and oxytalan fibers could not be observed. During this same period, there was an increase of a dense matrix comprised of collagen VI and mature elastic fibers. Chondroitin/dermatan sulfate proteoglycans were synthesized and became essential for the arrangement of collagen type VI, since chondroitinase ABC treatment of the culture disrupted collagen VI assembly, associated with the large spaces near the cell surface. In addition, the cells lost their fusiform morphology and changed into rounded cells. The results show that primary cultures of rabbit meniscus fibrochondrocytes maintain their capacity to form chondro-like structures in vitro. The organization process was rapid and uniform throughout the entire culture presuming that the normal signal transduction pathways are maintained intact and that essential factors in some phases of tissue organization are present.

Hepatic and renal effects of azidothymidine and acyclovir on pregnant rats.

The antiviral effect of azidothymidine (AZT) can be potentiated by acyclovir (ACV), and this drug association has been used in the management of HIV-infected patients. In the present study we examined the effects of this association on the livers and kidneys of both pregnant rats and their concepts. Previous data from this laboratory suggested that the deleterious effects of ACV on rat pregnancy are due to its extraplacental actions and these are, at least in part, counteracted by concomitant treatment with AZT. Kidneys and livers of pregnant rats were noticed to be much more sensitive to the toxic action of the drugs than those of their concepts, ACV eliciting much more evident morphological alterations than did AZT. Contrary to what was expected, in the group of rats treated with both drugs AZT was not able to diminish the severity of the alterations evoked by ACV. The proposed "protective" action of AZT against the abortive effect of ACV on rat pregnancy does not seem to be exerted through a renal or hepatic pathway.

[Ultrastructural features of cardiac muscle fibers of albino rats during the pregnancy and puerperal cycle]. / Aspectos ultra-estruturais da fibra muscular cardíaca de ratas albinas durante o ciclo gravídico-puerperal.

PURPOSE: In the present study we evaluated the morphology of left ventricular cardiomyocytes of albino rats during pregnancy and puerperium by means of transmission electron microscopy. METHODS: Once pregnancy was confirmed, 77 rats were randomly divided in two groups, respectively, gestation (G) and puerperium (P). The animals of the gestation group were divided into four subgroups, according to gestational age: 1st (G-A), 7th (G-B), 14th (G-C) and 21st (G-D) days of pregnancy. The group defined as puerperium was divided into three subgroups: 7th (P-A), 14th (P-B) and 21st (P-C) days of puerperium. In the end of each established period, the animals were sacrificed and fragments of the medium third of the left ventricle were resected and routinely prepared for electron microscopy analysis. RESULTS: The results obtained with transmission electron microscopy analysis revealed a gradative increase of the cardiomyocytes during pregnancy (increase of myofibrils, which are permeated by numerous mitochondria at the end of gestation). The blood capillary wall showed progressive thinning, with an increase of pynocytotic vesicles in endothelial cells, and intense sarcolemal folding at T-tubule level (capillary tunnels). In the puerperium group, there is a progressive regression in these alterations returning to pre-gestational level at the end of the puerperium cycle. These findings indicate the occurrence of hypertrophy of cardiomyocytes during pregnancy. CONCLUSION: The findings indicate the occurrence of hypertrophy of cardiomyocytes during pregnancy.

[Histomorphometric study of cardiomyocytes of the left ventricle of pregnant albino rats]. / Estudo histomorfométrico dos cardiomiócitos do ventrículo esquerdo das ratas albinas durante a prenhez.

PURPOSE: In the present study we evaluated, by light microscopy, and throughout morphometry, whether hypertrophy of cardiac striated muscular fibers of left ventricular occur in albino rat, during pregnancy. METHODS: After maiting, 12 nuliparous rats were divided into four groups with three animals for each group. The female rats corresponding to each group were killed at 1st, 7th, 14th and 21st days of pregnancy. RESULTS: Observation, on light microscopy (H.E) had at one view, did not display any alterations during pregnancy. However, the morphometry revealed that nuclei of cardiomyocytes are augmented in volume at 14th day of pregnancy and statistically significant data show the hypertrophy. CONCLUSION: The obtained data show the existence of a dinamic and reversible process of ventricular remodelling in consequence of adaptation physiological alterations of the cardiovascular normal pregnancy.

Aspectos ultra-estruturais da fibra muscular cardíaca de rats albinas durante o ciclo gravídico-puerperal / Ultrastructural aspects of cardiac muscle fibers of albino rats during pregnancy and puerperal cycle

Objetivo. No presente estudo avalianos, por intermédio da microscopia eletrônica de transmissão, a morfologia dos cardiomiócitos do ventrículo esquerdo de ratas albinas no decorrer do ciclo gravídico-puerperal. Métodos. Obtida a prenhez, 77 ratas foram divididas ao acaso em dois grupos denominados, respectivamente, de: gestação (G) e puerpério (P). Os animais pertencentes ao grupo gestação foram ainda subdivididos em quatro subgrupos, de acordo com a idade gestacional; 1§ (G-A), 7§ (G-B), 14§ (G-C) e 21§ (G-D) dias de prenhez. O grupo definido como puerpério foi também subdividido em três subgrupos; 7§ (P-A), 14§ (P-B) e 21§ (P-C) dias de puerpério. Findo o período estabelecido para cada subgrupo, os animais foram sacrificados, sendo coletados fragmentos do terço médio do ventrículo esquerdo, os quais após processamento apropriado, permitiram observação adequada à microscopia eletrônica de transmissão. Resultados. Os resultados hipertrofia gradativos dos cardiomiócitos no decorrer da prenhez (aumento das miofibrilas, que ao final da gestação apresentaram-se entremeadas de numerosas mitocôndrias). As paredes dos capilares sangüíneos, progressivamente, tornaram-se mais delgadas, com aumento das vesículas de pinocitose no interior das células endoteliais. Revelou ainda, acentuadas dobras do sarcolema em nível dos túbulos T (túneis capilares). No grupo puerpério, notamos gradativa regressão nessas alterações, com retorno às características pré-gestacionais. Os achados demonstram a ocorrência de hipertrofia no decorrer da gestação. Conclusão. Durante a prenhez há hipertrofia em cardiomiócito do ventrículo esquerdo com regressão durante o puerpério.

Estudo histomorfométrico dos cardiomiócitos do ventrículo esquerdo das ratas albinas durante a prenhez / Histomorphometrical study of cardiomyocytes of the left ventricle of albino rats during pregnancy

Objetivo. No presente estudo propusemo-nos a avaliar, por meio da mricroscopia de luz, os aspectos morfológicos e morfométricos dos cardiomiócitos do ventrículo esquerdo de ratas albinas durante a prenhez. Métodos. Acasalamos doze ratas virgens que foram divididas ao acaso em quatro grupos, de acordo com a idade gestacional. Os animais correspondentes a cada grupo foram sacrificados ao 1§(G-A), 7§(G-B), 14§(G-C) e 21§(G-D) dias de prenhez, sendo coletados fragmentos do terço médio do ventrículo esquerdo, os quais após processamento apropriado, permitiram observação adequada à microscopia de luz. A cariometria foi realizada mensurando-se os diâmetros maiores e menores dos cardiomiócitos com o auxílio de um tambor rotativo modelo K 8 X adaptando a um microscópio de luz. Resultados. O estudo em nível da microscopia de luz praticamente não mostrou alterações com o decorrer da prenhez. No entanto, a morfometria revelou que os volumes dos cardiomiócitos estão aumentados no 14§ dia da prenhez, mostrando-se estatisticamente significante quando comparado aos demais grupos estudados. Assim, nossos resultados demonstraram haver hipertrofia ventricular esquerda durante a gestação. Conclusão. Durante a gestação há um processo dinâmico reversível de remodelação ventricular em conseqüência das alterações adaptativas gravídicas.

Morphological aspects of the lamina propria of gingiva of persistent oestrous rats, or submitted to oophorectomy and treated with estrogen or dexamethasone

Los autores realizaron un estudio ultraestructural de la gingiva de ratas en estro permanente, ooforectomizadas y tratadas con estrógenos, con el objetivo de evaluar si esta estructura es un órgano blanco de las hormonas sexuales. Los resultados mostraron que la lámina propia de la gingiva de los animales en estro permnente y, en los ooforectomizados tratados con estrógenos, era rica en células y en fibras colágenas. En estos dos grupos se observaron grandes concentraciones de eosinófilos y fibroblastos activos, como también, de macrófagos y linfocitos, observándose además en el estroma, áreas electrotranslúcidas. En el grupo ooforectomizado se observó gran concentración de células y, el estroma denso contenía inmumerables fibras colágenas y substancias intercelular amorfa. En este grupo, los fibroblastos mostraron señales de atrofia y algunos macrófagos y leucocitos, señales degenerativas. El estroma gingival del grupo tratado con dexametasona, se mostró atrofiado, con gran número de células y fibras colágenas como de fibroblastos y fibrocitos. Fue observado un reducido número de macrófagos y de leucocitos. Se concluyó que la gingiva debe ser considerada como un órgano blanco de los estrógenos y que la ooforectomía no altera esa característica

The pheromonal restoration of cyclic activity in young estrogenized persistent estrus female rats is a vomeronasal effect.

Eight sham-operated (SO) and six vomeronasalectomized (VMNX) young adult female rats were used to demonstrate the participation of the vomeronasal organ (VNO) in the pheromonal restoration of cyclic activity (PRCA). All rats were normal four-day cycling before and after surgery and they received a single subcutaneous injection of 2 mg of estradiol benzoate (EB) to induce an anovulatory persistent vaginal estrus. Fifteen days after EB injection, they were treated by spraying in the nostrils, every 10 min for one hour on only one day, 50 muL of urine from young fertile males. SO rats showed PRCA in the following days, while VMNX did not. These results support the VNO as the receptor of the male urinary pheromone (or pheromones) responsible for PRCA, demonstrating that it is possible to improve the function of aging ovaries acting with an exteroceptive stimulus.

Hormonal pattern of the pheromonal restoration of cyclic activity in aging irregularly cycling and persistent-estrus female rats.

This study reports the hormonal pattern of pheromonal restoration of cyclic activity (PRCA) in irregularly cycling (IC) and in anovulatory persistent-estrus (PE) aging female middle-aged Wistar rats. Hypothalamic content of LHRH and plasma levels of LH, FSH, estradiol (E2), and progesterone (P) were measured by RIA methods, and prolactin (PRL) was examined by ELISA in 1) normal four-day cycling rats, 2) saline-treated IC and PE rats, and 3) IC and PE rats treated with sprays of male urine (50 microliters every 10 min for 1 h on only one day, at a distance of 1 cm from the nostrils). Group 1 showed low levels of LHRH and high levels of LH, FSH, E2, P, and PRL in the afternoon of proestrus (PR). In group 2, the pattern was not cyclical and the levels of the hormones were higher than basal for the cyclic rats, except for P, which was lower. Group 3 showed a depletion of the hypothalamic LHRH and an increase in plasma LH, FSH, E2, Pm, and PRL. FSH, E2, and P showed the strongest rise, leading to an elevated ratio of FSH to LH. Four-day estrous cycles reappeared in urine-treated rats on the succeeding days. The cyclic behavior of the PRCA hormones was, qualitatively, the same as in the normally cycling rats. According to the results, the origin of the PRCA effect is an olfactory input to the hypophysiotropic area of the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of diclofenac sodium on the reparation process of the surgically-perforated myometrium of pregnant albino rats: morphological and morphometrical evaluation.

1. The action of a non-hormonal anti-inflammatory agent on the reparation process of the surgically-perforated myometrium of pregnant albino rats was histologically evaluated. 2. On the 14th day after perforation, the muscular layers were still interrupted. 3. The morphological study was inconclusive. A morphometrical analysis, however, demonstrated that there was a meaningful diminution in the number of leucocytes and collagen fibers and an increase in the number of fibroblasts, probably with low synthetical power, in the group treated with diclofenac sodium. All these data indicate a delay in the reparation process of the uterine musculature.

Pheromonal male-induced diestrus and cyclicity in aging intact and young estrogenized female rats.

Reproductive aging in female rats is characterized by irregular estrous cycles, repeated pseudopregnancies, persistent estrus, and a decline in fertility. The persistently estrous rats do not ovulate or show cyclic increases of LH despite continued ovarian follicular development. These rats ovulate after mating with fertile males. On the other hand, a single injection of estradiol to young female cyclic rats causes persistent anovulatory estrus similar to that of aging rats, which has suggested that young cyclic estrogen-primed females may be a model for the study of reproductive senescence. We attempted to determine whether the male factor that elicits ovulation in aging females is pheromonal in nature. The effect of such a pheromonal factor on the persistent estrus of young estrogenized females was also tested. We observed the estrous cycle of aging (7-13-mo-old) and young estrogenized female rats daily. Nasal spraying of urine obtained from young adult male rats induced diestrus and regular cyclicity in both aging intact and young estrogenized female rats, probably because of due to pheromones contained in male urine. This fact suggests an important role of chemosensory communication in prolongation of the period with normal estrous cycles in the female's reproductive life.

[Ultrastructural aspects of the human uterine tube epithelium during menstrual cycle]. / Aspectos ultra-estruturais do epitélio da tuba uterina humana durante o ciclo menstrual.

PURPOSE: The authors perform an electron microscopy analysis of the human uterine tube epithelium during the menstrual cycle. TYPE OF STUDY: Prospective, experimental. Site: São Paulo School of Medicine. PATIENTS: Four eumenorrheic multiparous patients, two in the proliferating stage, and two in the secreting stage of the cycle. INTERVENTIONS: PATIENTS were submitted to bilateral salpingectomy because they presented a high reproductive risk. MEASUREMENTS AND RESULTS: The authors evaluated ultrastructural aspects of the epithelium in the isthmus, ampulla, and infundibulum. Two types of cells were identified: ciliated and non-ciliated or secretory. Secretory cells have more marked ultrastructural changes than ciliated cells during the cycle. In the luteal stage, secretory cells present quite developed and dilated endoplasmatic reticulum and Golgi apparatus, and numerous electro-lucent vesicles. They also verified that during the second stage of the cycle, the secretory cells in the ampullar region seems to release part of its cytoplasm in the tube lumen. CONCLUSIONS: The ampullar region of the uterine tube suffers greater hormonal influences than the other regions. Morphological changes occurring in the ampullar might have their role to play in the nutrition of the egg cell during the time it remains inside it.

Effect of nasal instillation of female urine or vaginal exudate on serum cortisol and prolactin levels in isolated and anesthetized male rhesus monkeys (Macaca mulatta).

Two male adult rhesus monkeys were used and caged individually. The room was temperature-controlled having a light-dark period of 12/12 hours. The animals were rapidly immobilized and immediately anesthetized with ketamine i. m. (10 mg/kg of body weight). They were bled four times at 15, 30, 45 and 60 mins after the ketamine injection, twice a week for 6 weeks. When necessary, maintenance doses of ketamine were administered. The serum levels of cortisol and prolactin after nasal instillation of a suspension of vaginal exudate showed lower values than in control conditions (nasal instillation of saline). The control levels of cortisol tended to increase up to 60 mins, whilst in experimental conditions (nasal instillation of female urine or vaginal exudate) did not show such an increase. Cortisol remained similar during the sampling and similar to the 15 mins control levels, but the difference is statistically significant only after instillation of vaginal exudate as compared with control. The levels of prolactin did not show significant variations during sampling either in control or after female urine instillation. However, the instillation of vaginal exudate decreased the prolactin levels at 15 mins which tended to recover the control levels up to 60 mins. These results support the hypothesis, discussed in a previous paper, that some chemical information from females suppresses or mitigates the effect of acute stress resulting from handling the animals before anesthesia.

Effect of nasal instillation of female urine or vaginal exudate on testosterone secretion in isolated and anesthetized male rhesus monkeys (Macaca mulatta).

Two male adult rhesus monkeys were individually placed in cages with a pulling device in order to immobilize the animals for anesthesia. The room was temperature-controlled having a light/dark period of 12/12 hours. The animals were rapidly immobilized and immediately anesthetized with ketamine i. m. (10 mg/kg of body weight). They were bled four times at 15, 30, 45, and 60 mins after the ketamine injection, twice a week during 6 weeks. When necessary, maintenance doses of ketamine were administered. The levels of serum testosterone in experimental conditions (nasal instillation of female urine or a suspension of vaginal exudate) showed significant lower values with respect to those in control conditions (saline instillation). The control levels of testosterone tend to increase up to 60 mins. The testosterone from samples obtained in experimental conditions did not show such an increase, remaining similar during the sampling and similar to the 15 min control levels that could be considered as basal. These results seem to point out some chemical information from females capable of modifying the pattern of secretion of testosterone of the males in the above mentioned experimental conditions.

[Ultrastructural aspects of the skeletal striated muscle, macrophages and fibroblasts during the regression of tadpole tails]. / Aspectos ultra-estruturais do músculo estriado esquelético, macrófagos e fibroblastos durante a regressão da cauda de girinos.

Striated muscle fibres and fibroblasts observed at electron microscope were entirely developed when the tail of tadpoles reached its maximum size. However, during resorption of the tail, striated muscle fibres showed signs of degeneration: rupture and disorganization of myofibrils, altered mitochondria and lipid droplets in the cytoplasm. A great amount of macrophages phagocyting myofibrils and fibroblasts containing collagen fibrils in several breakdown stages were also observed among degenerated muscle fibres.