Fetotoxicity caused by the interaction between zinc and arsenic in mice

Arsenic is an environmental pollutant that induces congenital malformations in experimental models and can contribute to human birth defects. The environmental exposure to arsenic is relatively small when compared with the doses required to cause teratogenicity in mice and other laboratory animals. In order to study the action of zinc in the arsenic-induced teratogenicity, in the present work mice were either pretreated with zinc and later with arsenic or were treated simultaneously with zinc and arsenic in vivo and in vitro. Following administration of arsenate on gestation day 8, pregnant females were killed on the 17th day of gestation; maternal and fetal data were collected by laparotomy and used to calculate reproductive parameters. Fetuses were analyzed for the presence of external malformation and, after the appropriate processing, visceral and skeletal analyses were accomplished. Conceptuses were exposed in whole embryo culture to arsenicals on gestation day 8 (3-6 somite stage). After a 26 h culture period, morphological development was assessed. Neither pretreatment with zinc nor simultaneous administration of zinc prevented arsenic teratogenicity in these experimental models. (C) 2002 Wiley-Liss, Inc.

Reproductive effects in male rats exposed to diuron

Diuron is a ureic herbicide considered to have very low toxicity. The present study evaluated several aspects of reproductive toxicity of diuron in adult male rats. Diuron was diluted in corn oil and administered by oral gavage to groups of 18-20 rats at doses of 0, 125 or 250 mg/kg per day for 30 days; the control group received only the corn oil vehicle. At the end of the treatment period, approximately half the animals from each group were assigned to one of two terminal assessment lines: (1) reproductive organ, liver and kidney weights; measurement of diuron concentrations in liver and kidney; plasma testosterone determinations; evaluation of daily sperm production per testis; sperm number and sperm transit time in the epididymis; or (2) sexual behavior assessment during cohabitation with a receptive female; fertility and pregnancy outcome after natural mating; testicular, epididymal, kidney and liver histopathology; sperm morphology. After 30 days of oral diuron treatment, there were no treatment-related changes in body weights, but dose-related diuron residues were detected in the liver of all treated rats and absolute and relative liver weights were increased in both groups. There were no statistically significant differences between the treated and control groups obtained in plasma testosterone concentrations, or in parameters of daily sperm production, sperm reserves in the epididymis, sperm morphology or measured components of male sexual behavior. on the other hand, the number of fetuses in the litters from diuron-treated rats was slightly smaller than litters from control rats. Therefore, although the results did not indicate that diuron exposure resulted in direct male reproductive toxicity in the rat, they suggest that additional studies should be undertaken to investigate the possible effects on fertility and reproductive performance. (c) 2006 Elsevier B.V. All rights reserved.

Functional and Morphological Reproductive Aspects in Male Rats Exposed to Di-n-Butyl Phthalate (DBP) in Utero and During Lactation

The potential adverse reproductive effects, with emphasis on the epididymis, of in utero and lactational exposure to 100 mg/kg/d di-n-butyl phthalate (DBP) in adult male rat offspring were investigated. The fetal testis histopathology was also determined. The selected endpoints included reproductive organ weights, sperm motility and morphology, sperm epididymal transit time, sperm quantity in the testis and epididymis, hormonal status, fetal testis and epididymal histopathology and stereology, and androgen receptor (AR), aquaporin 9 (AQP9), and Ki-67 immunoreactivities. Pregnant females were divided into two groups: control (C) and treated (T). The treated females received DBP (100 mg/kg/d, by gavage) from gestation day (GD) 12 to postnatal day (PND) 21, while control dams received the vehicle. Some pregnant dams were killed by decapitation on GD20, and testes from male fetuses were collected for histopathogy. Male rats from other dams were killed at PND 90. Fetal testes from treated group showed Leydig-cell clusters, presence of multinucleated germinative cells, and increase of the interstitial component. Testosterone levels and reproductive organ weights were similar between the treated and control adult groups. DBP treatment did not markedly affect relative proportions of epithelial, stromal, or luminal compartments in the epididymis; sperm counts in the testis and epididymis; sperm transit time; or sperm morphology and motility in adult rats. The AR and AQP9 immunoreactivities and proliferation index were similar for the two groups. These results showed that fetal testes were affected by DBP as evidenced by testicular histopathologic alterations, but reproductive parameters and epididymal structure/function were not significantly altered in the adult animals exposed to 100 mg/kg DBP in utero and during lactation.

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Fertility of male adult rats submitted to forced swimming stress

We investigated whether stress interferes with fertility during adulthood. Male Wistar rats (weighing 220 g in the beginning of the experiment) were forced to swim for 3 min in water at 32ºC daily for 15 days. Stress was assessed by the hot-plate test after the last stressing session. To assess fertility, control and stressed males (N = 15 per group) were mated with sexually mature normal females. Males were sacrificed after copulation. Stress caused by forced swimming was demonstrated by a significant increase in the latency of the pain response in the hot-plate test (14.6 ± 1.25 s for control males vs 26.0 ± 1.53 s for stressed males, P = 0.0004). No changes were observed in body weight, testicular weight, seminal vesicle weight, ventral prostate weight or gross histological features of the testes of stressed males. Similarly, no changes were observed in fertility rate, measured by counting live fetuses in the uterus of normal females mated with control and stressed males; no dead or incompletely developed fetuses were observed in the uterus of either group. In contrast, there was a statistically significant decrease in spermatid production demonstrated by histometric evaluation (154.96 ± 5.41 vs 127.02 ± 3.95 spermatids per tubular section for control and stressed rats, respectively, P = 0.001). These data demonstrate that 15 days of forced swimming stress applied to adult male rats did not impair fertility, but significantly decreased spermatid production. This suggests that the effect of stress on fertility should not be assessed before at least the time required for one cycle of spermatogenesis.

Ramificação e distribuição do tronco pulmonar em relação aos brônquios, em fetos de bovinos azebuados

Mediante esta pesquisa, estudamos a ramificação e a distribuição das artérias pulmonares em 20 fetos de bovinos azebuados (13 machos e 7 fêmeas), com idades variando entre 6 e 9 meses. As peças foram fixadas em solução aquosa de formol a 10%, e o método de dissecação consistiu da abertura da cavidade torácica desses animais, com vistas à individualização dos vasos arteriais e brônquios. da artéria pulmonar direita observou-se que, mais comumente, 15 vezes (75%), parte como primeiro colateral o ramo ascendente, seguido do ramo descendente que acompanham os dois brônquios segmentares do brônquio traqueal, os quais irrigam, respectivamente, a porção cranial e caudal do lobo cranial do pulmão. Logo após este arranjo, surgem o ramo lobar acessório e em seguida o ramo lobar médio. O ramo lobar acessório pode emergir duas vezes (10%) depois do ramo lobar médio ou na mesma altura dele, cinco vezes (25%). Uma única vez (5%) aparecem dois ramos lobares médios. em cinco preparações (25%), os dois primeiros ramos da artéria pulmonar direita são vistos a nascer em tronco comum, o ramo lobar cranial. da artéria pulmonar esquerda nascem mais freqüentemente 18 vezes (90%) como 1º e 2º colaterais os ramos ascendente e descendente, e respectivamente duas vezes (10%) estes ramos provêm de um único colateral, o ramo lobar cranial. em cada lobo caudal (direito e esquerdo), penetra um ramo lobar caudal (direito e esquerdo) que emite número bastante variado de ramos arteriais (6 a 14) do lado direito e (7 a 16) do lado esquerdo.

Análise morfológica dos ovários de fetos bovinos da raça Nelore (Bos primigenius indicus) em diferentes fases de gestação

Vinte fetos fêmeas em diferentes fases de gestação da raça Nelore foram coletados para a realização desta pesquisa. Os ovários direitos e esquerdos foram dissecados e mensurados para verificação de seus comprimentos e larguras e em seguida, foram fixados inteiros em paraformaldeído tamponado a 4,00%, processados e incluídos em paraplástico. Os cortes com 5 mm foram submetidos à coloração com hematoxilina, tricrômio de Masson (para fibras colágenas), Verhoeff (para fibras elásticas) e com reticulina (para fibras reticulares). Os resultados mostraram que não há diferença significativa das mensurações entre os lados direito e esquerdo para os ovários dos fetos bovinos em diferentes fases de gestação, mas há correlação entre os valores obtidos das mensurações dos ovários em função da idade dos fetos, ou seja, o crescimento dos ovários acompanha o crescimento fetal. Os fetos em diferentes fases de gestação, apresentam epitélio germinativo, túnica albugínea e tecido conjuntivo no córtex e medula característicos da morfologia ovariana. Nos ovários de fetos com até 19 semanas de idade gestacional, é evidente a presença em grande quantidade de folículos primordiais e primários. A partir de 22 semanas de gestação, é evidente a presença em grande número, de folículos ovarianos em diferentes estágios de crescimento. A partir de 17 semanas de gestação, observa-se na medula, cordões com luz irregular revestidos por células cúbicas. As variações mais marcantes ocorreram a partir de 22 semanas de gestação.

Caracterização morfológica do útero e tubas uterinas de fetos bovinos da raça Nelore (Bos primigenius indicus) em diferentes fases de gestação

Para a realização desta pesquisa, foram coletados vinte fetos fêmeas em diferentes fases de gestação da raça Nelore. Os cornos uterinos e as tubas uterinas foram dissecados, mensurados e os fragmentos fixados em paraformaldeído tamponado a 4,00%, processados e incluídos em paraplástico. Os cortes com 5 mm foram corados com hematoxilina e eosina, tricrômio de Masson (para fibras colágenas), Verhoeff (para fibras elásticas) e com reticulina (para fibras reticulares). Os resultados mostraram que não há diferença significativa das mensurações entre os lados direito e esquerdo para os cornos uterinos e tubas uterinas, porém há correlação entre os valores obtidos das mensurações dos órgãos em função da idade dos fetos, ou seja, o crescimento dos órgãos acompanha o crescimento fetal. O epitélio de revestimento do útero não apresenta variações morfológicas evidentes no período analisado. A partir de 23 semanas de gestação, a mucosa uterina apresenta evolução marcante no desenvolvimento das projeções e não há aparecimento de glândulas endometriais na parede uterina no período analisado. A camada muscular apresenta a subcamada circular interna desenvolvida até 23 semanas de gestação e a partir de 24 semanas há presença das duas subcamadas. A camada serosa é típica e não mostra variabilidade no decorrer da gestação. As tubas uterinas apresentam diferenças de crescimento, principalmente dos pregueamentos que a partir de 23 semanas de gestação tornaram-se mais altos e ramificados, porém sem aparecimento de pregas terciárias. Com o desenvolvimento fetal, os cílios epiteliais do pregueamento tubárico são maiores em tamanho e em número. Até 32 semanas de gestação, a camada muscular das tubas uterinas apresenta apenas a subcamada circular interna. A camada serosa e o mesosalpinge são típicos e não apresentam variações importantes. As variações mais marcantes para os órgãos estudados ocorreram a partir de 23 semanas de gestação.

Quadro citológico vaginal, concentração plasmática de progesterona durante a gestação e medidas fetais em paca (Cuniculus paca Linnaeus, 1766)

Em 27 pacas (Cuniculus paca Linnaeus, 1766) objetivou-se descrever aos 30, 60 e 90 dias (D) de prenhez diagnosticada por ultrassonografia (US), os tipos celulares do epitélio vaginal em esfregaços vaginais, relatar as condições de abertura da vulva e as características do muco vaginal, determinar a concentração plasmática de progesterona (P4) por radioimunoensaio, e ainda, mensurar por ultrassonografia (US) o diâmetro biparietal (DBP) fetal aos 60 e 90 dias de prenhez. No D30, 40% das amostras exibiram células (com características estrogênicas) superficiais e presença de núcleos nus. Nos D60 e D90, células parabasais, intermediárias, superficiais e naviculares estavam presentes nas mesmas proporções, mas células endocervicais foram descritas em apenas 73,9% e 69% das amostras daqueles dias, respectivamente. No D30 a maior proporção de células naviculares e superficiais diferiu (p < 0,05) em relação aos outros tipos celulares presentes. O muco vaginal apresentou-se cristalino e fluido em 100% e em 70% das fêmeas nos D30 e D60, respectivamente. Observou-se o vestíbulo vaginal aberto em torno de 50% das fêmeas em todos os dias de exames. Valores mínimos detectáveis de P4 foram obtidos em 72% e em 83% das fêmeas, enquanto que as médias das medidas dos DBP foram 1,25 cm (± 0,16) e 2,34 cm (± 0,25) nos D60 e D90, respectivamente. O quadro citológico vaginal nos D30, D60 e D90 e o DBP fornecem elementos que contribuem para diagnóstico de gestação em pacas. A concentração de P4 demonstra a necessidade de maiores estudos da endocrinologia da gestação em pacas.

Influência do período de gestação e do tamanho da ninhada no crescimento ponderal intra-uterino de coelhos

Intrauterine growth of Norfolk rabbits was studied from the 20th day of gestation until the parturition (31st day). A theoretical equation was adjusted for fetuses body weight. The exponential model was tested by stepwise regression technique after linearization. Both stage of gestation (t) and number of fetuses (n) had significant effects on the growth rate. The following equation was proposed to describe the weight of the pups: W = exp. (-12,9772).n.exp. {(0,991263-0,014007.5-0,00039.n)t} (with R2 = 0,9932). The biological coherence and the interpretation of the coefficients permitted us to conclude that similar models could be used for other multiparous species. This model can also be used with or without the presence of different factors related to fetal growth.

Perinatal estrogen exposure: Later repercussion on the fertility of rats

The aim of the present study was to investigate the effects of perinatal estrogen exposure in the fertility of rats. Thus, rats were treated with estrogen on the 21st or 22nd day of intra-uterine life or treated with estrogen immediately after birth. It was observed that the testicular descent of males and beginning of puberty of females were advanced in all estrogen-treated groups. The females from estrogen-treated groups showed reduced frequency of estrous in 15 consecutive days of study, and there was an increase in estrous duration. Their fertility also were impaired and a reduction in the number of alive fetuses, as well as enhancement of pre- and postimplantation loss, mainly in the group treated with estrogen on the 21st day of intra-uterine life. However, the alterations observed in the fertility of estrogen-treated male rats were slighter and only females mated with male rats from the group treated with estrogen immediately after birth showed enhanced preimplantation loss. We suggest that the reproductive function is impaired by exposure to estrogen in the perinatal life of rats, and that the mechanisms involved in this effect are distinct for males and females. (C) 1997 Elsevier B.V.

Abortive and teratogenic effect of Acanthospermum hispidum DC. and Cajanus cajan (L.) Millps. in pregnant rats

Extracts of Acanthospermum hispidum and Cajanus cajan have been used by Brazilian people in an attempt to produce abortion. In order to evaluate the possible abortive and/or teratogenic effect of these plant extracts, female Wistar rats were treated with the aqueous extract (infusion, proportion C. cajan and A. hispidum 1:1.3). Doses of 0, 150, 300 and 600 mg/kg were daily administered by gavage during the organogenic period. The animals were sacrificed at term. There was no significant change in the mean weight of the fetuses, and no change in the percentage of post implantation loss in the treated groups. However, there was an increase in the number of external malformations, and this was related to dose. No internal malformations were observed in fetuses at term, but there was a significant incidence of fetuses with visceral anomalies. The tendency of the pregnancy to continue or terminate did not change with the treatment.

Experimental paracoccidioidomycosis in hamsters (Mesocricetus auratus): Gestational interactions

Paracoccidioides brasiliensis is a dimorphic fungus presenting specific steroid hormone receptors, both in the yeast and mycelial forms and estrogen inhibits the transition from mycelium to yeast. In the acute phase, the disease occurs with equal frequency in both sexes but in adults, females are spared. Placental fungal infection has been reported, but references to fetal infection have not been confirmed. We used 78 Syrian female hamsters divided into 3 groups: GI consisted of 30 infected mated females, GII of 20 infected unmated females and GIII of 28 uninfected mated females. Animals of group I were mated 4 weeks after infection and half of them were submitted to cesarean section on day 15 after successful mating; the other half was maintained and submitted to cesarean section and sacrificed 14 weeks after infection. Half of the animals of group II were sacrificed seven weeks and the other half 14 weeks after infection. Uninfected animals of group III were treated the same as the animals of group I. The animals were infected with strain 18 of P. brasiliensis by the intracardiac route. We evaluated the disease by the volume of granulomas in different organs, number of fungi in liver and spleen and the immunologic responses [ELISA, Double Immunodifusion (DID), Delayed Hypersensitivity Skin Test (DHT) and Macrophage Migration Inhibition (MMI)]. We studied the infection through the gestation by evaluation of the abortions, morphologic and clinic examinations of the fetuses. Our results showed that the infection did not transfer to the fetus through the placenta, but the number of abortions was larger among infected females. The newborns of GI females were smaller, weighed less and showed little vitality. The disease was more severe and disseminated in infected mated females, especially in the second sacrifice 14 weeks after inoculation, when the total volume of granulomas in them (56.3 mm) was much greater than in the infected unmated females (12 mm).

Study of the embryotoxic effects of an extract of rosemary (Rosmarinus officinalis L)

Extracts of rosemary, Rosmarinus officinalis L., have been used in folk medicine as a diuretic, an emenagogue, an antispasmodic and its aqueous extract does not present toxicity to man, presenting, however, abortive effects. In order to evaluate if this plant induces abortion and/or interferes with the normal development of the concepts, doses of 26 mg of a 30% (w/v) R. officinalis aqueous extract (13 mg solids/ml) made with leaves, flowers and stem were administered daily by gavage during two different periods of Wistar rat pregnancy. One group of animals (N = 12) received the extract from days 1 to 6 of pregnancy (preimplantation period) and another group (N = 14) received the same extract from days 6 to 15 of pregnancy (organogenic period). Control groups (N = 12) received saline in the same volume and during the same periods as their respective experimental groups. The animals were sacrificed at term. The treatment of the darns during either the preimplantation or the organogenic period did not cause significant changes in the postimplantation loss or in the number of anomalies or malformations of the term fetuses, which also showed a similar degree of development when compared with the respective controls. The percent of preimplantation loss in the group treated before embryo implantation increased, although the difference was not significant compared to the control. This result suggests that rosemary extract may present an anti-implantation effect without interfering with the normal development of the concept after implantation.

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)