Use of zebrafish ( Danio rerio ) embryos as a model to assess effects of mercury on developing skeletal muscle: a morphometric and immunohistochemical study / Uso de embriones de pez cebra ( Danio rerio ) como modelo para evaluar los efectos del mercurio en el desarrollo del músculo esquelético: un estudio morfométrico e inmunohistoquímico

Exposure to mercury in the environment continues to be a significant worldwide concern, especially for developing embryos and fetuses. While extensive research effort has focused on the effects of mercury on the developing nervous system, much less is known concerning adverse effects of mercury on other organ systems, including the development of skeletal muscle. We exposed developing zebrafish embryos to a range of concentrations of mercuric chloride (100 to 400 µg/liter or ppb) and compared them to control embryos (0 µg/L mercuric chloride). Embryos were examined at 48 hours post fertilization (hpf) for morphometry and morphological deformities of skeletal muscle fibers in the trunk and tail. Embryos exposed to 400 ppb mercuric chloride showed decreased trunk and tail areas compared to control embryos. A dose-dependent reduction in muscle fiber length was observed, and exposure to all concentrations of mercuric chloride used in this study resulted in decreased muscle fiber immunohistochemical staining with anti-myosin antibodies. Irregular muscle fiber diameters, twisted muscle fibers, and degenerated muscle fibers were observed in sections of embryos stained with eosin at the higher exposure concentrations. Evidence presented in this study suggests that exposure to even low concentrations of mercuric chloride adversely affects skeletal muscle fiber development or muscle fiber integrity, or both.

La exposición al mercurio en el medio ambiente sigue siendo una preocupación mundial importante, especialmente para el desarrollo de embriones y fetos. Si bien un amplio esfuerzo de investigación se ha centrado en los efectos del mercurio en el sistema nervioso en desarrollo, se sabe mucho menos sobre los efectos adversos en otros sistemas orgánicos, incluido el desarrollo del músculo esquelético. Expusimos embriones de pez cebra en desarrollo a un rango de concentraciones de cloruro de mercurio (100 a 400 mg / l o ppb) y los comparamos con embriones de control (0 mg / L de cloruro de mercurio). Los embriones se examinaron a las 48 horas después de la fertilización (HPF) pararealizar la morfometría y verificar las deformidades morfológicas de las fibras del músculo esquelético en el tronco y la cola. Los embriones expuestos a 400 ppb de cloruro de mercurio mostraron una disminución de las áreas del tronco y la cola en comparación con los embriones de control. Se observó una reducción dependiente de la dosis en la longitud de la fibra muscular, y la exposición a todas las concentraciones de cloruro de mercurio utilizadas en este estudio, dio como resultado una tinción inmunohistoquímica de fibra muscular disminuida con anticuerpos anti-miosina. Se observaron diámetros irregulares de fibras musculares, fibras musculares retorcidas y fibras musculares degeneradas en secciones de embriones teñidos con eosina en las concentraciones de exposición más altas. La evidencia presentada en este estudio sugiere que la exposición incluso a bajas concentraciones de cloruro mercúrico afecta negativamente el desarrollo de la fibra del músculo esquelético o la integridad de la fibra muscular, o ambas.

[Teratogenic effects of Origanum Vulgare in mice fetals]

A number of studies on reproduction have mentioned Origanum Vulgare extract's ability to reduce mortality rates and improve fertility rates. However, other studies have suggested that it is possible to use Origanum Vulgare extract to induce abortion. The aim of this study was to investigate the effect of different doses of Origanum Vulgare on embryo survival and macroscopic abnormalities in mice. In this study, 24 mice Balb/c female weighting approximately 25-30 g were divided into 4 groups. Origanum Vulgare extract was prepared; different concentrations [2.5, 12.5, and 25 mg in 0.25 ml distilled water] were administered, by oral gavage, to three experimental groups of mice between day 6 [starting gastrulation] until day 15 of pregnancy [end of organogenesis]. The control group consisted of six mice that received 0.25 ml of distilled water daily. On day 16 of study, pregnant mice were anesthetized by chloroform and fetuses were removed and stained with Alcian Blue, Alizarin Red s and microwave irradiation. Morphological and skeletal abnormalities were investigated by light and stereomicroscopes. The results of this study showed that high doses of the Origanum Vulgare extract significantly decreased the mean number of embryos [10 +/- 0.5, P>0.05], mean number of live embryos [7 +/- 0.5, P>0.05] in each mouse and resulted in significant reduction in mean weight[1184 +/- 8 mg, P>0.05] and crown-rump length[11.9 +/- 0.23 mm, P>0.05] and the overall size of fetuses compared to control group, whereas there was no significant difference between the groups receiving low dose of Origanum Vulgare extract with control group. In addition, under the effect of the Origanum Vulgare extract the subcutaneous bleeding seemed [2 +/- 0.1, P>0.05] significantly more frequent compared to the control group. Origanum Vulgare extract did not have any positive effect on fetal development; and high dosages led to an increased incidence rate of abortion and fetal malformations in the fetuses of women who received it.

Estudio retrospectivo sobre las posibles complicaciones maternofetales de la exposición a cabergolina / Retrospective Study of any potencial Adverse Effects of Cabergoline on Pregnancy and Embryofetal Development

Objetivos: Estimar la frecuencia de complicaciones maternofetales en mujeres que se embarazaron durante el tratamiento con cabergolina (CAB). Estimar la frecuencia de patología detectada posnacimiento en los niños producto de dichos embarazos. Material y métodos: Estudio retrospectivo y multicéntrico de 86 embarazos en 78 mujeres con hiperprolactinemia idiopática (7) o tumoral (44 micro y 27 macro), en tratamiento con CAB en el momento de la concepción. Edad: 20 a 45 años; PRL inicial: 30 a 1429 ng/ml; duración del tratamiento previo al embarazo 1 a 120 meses; dosis: 0.125 a 4 mg/semana. El rango de exposición embriofetal a la CAB fue de 3 a 27 semanas, el 96.39% de las pacientes la recibió durante el primer trimestre y el 3.61% hasta el segundo. Resultados: No hubo complicaciones mayores durante el embarazo. Se registraron 7 abortos espontáneos (8.1%) y 75 partos, de los cuales 49 fueron vaginales y 26 cesáreas. Se registraron 69 recién nacidos, 63 fueron a término y 6 pretérmino (8.8%), ninguno bajo peso para la edad gestacional. En 3 (5.2%) recién nacidos se observó: 1 malformación mayor (Síndrome de Down) y 2 menores (hernia umbilical e inguinal). Se obtuvo seguimiento de 42 recién nacidos; se diagnosticó epilepsia refractaria en uno y un trastorno generalizado del desarrollo en otro. No se halló una mayor frecuencia de complicaciones en los embarazos ni en los recién nacidos expuestos a CAB que en la población normal. Sería necesario mayor número de pacientes para concluir sobre la seguridad de CAB durante el embarazo.

Objectives: To assess the rate of any potential adverse effects on pregnancy and embryo-fetal development in women who became pregnant under treatment with cabergoline (CAB). To follow up medical data of children who were born from mothers exposed to Cab in early weeks of gestation. Material and methods: Observational, retrospective and multicenter study on 86 pregnancies in 78 women with idiopathic or tumoral hyperprolactinemia. All patients were under Cab at conception. The average age was 29 (range: 20-45). Pituitary images at diagnosis showed 44 microadenomas, 27 macroadenomas and 7 were normal. Serum PRL at baseline was between 30 and 1429 ng/ml. Duration of therapy before pregnancy ranged from 1 to 120 months. Maternal and fetal exposure to cabergoline and doses ranged from 0.125 to 4 mg/week. The mean serum PRL level under which patients achieved pregnancy was 17 ng/ml. Fetal exposure ranged from 3 to 27 weeks; 96.39% of patients received CAB during the first trimester of pregnancy and 3.61% until the second one. Results: No significant complications during pregnancy were found. Seven women (8.1%) had spontaneous abortions. Term deliveries were recorded in 63/69, preterm in six (8.8%), none of them with low weight for gestational age. Neonatal abnormalities were observed in 3 (5.2%): 1 major (Down syndrome) and 2 minor malformations (umbilical and inguinal hernia). Two out of 42, developed abnormalities during the follow- up, one of them was a refractory epilepsy during the second month of life, the other presented a Pervasive Developmental Disorder diagnosed in the third year of life. Conclusion: No significantly higher frequency of complications was found in pregnancies and/or offspring exposed to CAB than in normal population. Larger series of patients are needed to asses the safety.

Effect of antiretroviral drugs on maternal CD4 lymphocyte counts, HIV-1 RNA levels, and anthropometric parameters of their neonates

OBJETIVOS: Estudar o efeito das drogas anti-retrovirais sobre a quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV-1 e parâmetros antropométricos de seus neonatos. MÉTODOS: Estudo prospectivo avaliando 57 gestantes e seus neonatos em três grupos: Grupo AZT, gestantes portadoras do HIV utilizando zidovudina (n=20); Grupo TT, mães utilizando zidovudina+lamivudina+nelfinavir (n=25), e Grupo Controle, mulheres saudáveis (n=12). A quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV foi analisada em dois períodos durante a gestação. O prognóstico perinatal levou em consideração as taxas de pré-termos, restrição de crescimento intra-útero, mortalidade perinatal e transmissão vertical do HIV-1. Os dados foram analisados utilizando-se testes não paramétricos de qui-quadrado, Mann-Whitney, Friedman, Kruskal-Wallys e Wilcoxon para amostras pareadas, considerando-se significativos valores associados a p<0,05. RESULTADOS: Observou-se homogeneidade entre os dados demográficos e antropométricos de realce. A carga viral, inicialmente elevada (14.370 cópias/ml), reduziu-se significativamente no grupo com tratamento tríplice , chegando a 40 cópias/ml. Quanto à contagem de linfócitos CD4, observou-se recuperação significativa nas pacientes do grupo TT, no final da gestação, sendo esse valor significativamente diferente em comparação ao grupo AZT (p = 0,0052). Não se observou diferença entre os grupos quanto à duração da gestação, aos índices de Apgar, e à classificação antropométrica neonatal. Não houve nenhum caso de transmissão vertical do HIV-1. CONCLUSÕES: Os resultados obtidos na presente casuística demonstram eficiência e sugerem segurança no uso de anti-retrovirais na gestação sobre parâmetros antropométricos dos neonatos.

Effects of Protein Source and Energy Substrates on the In Vitro Development of Bovine Embryos in a Two-step Culture System

In this study, we examined the effects of a two-step culture system, which involves the use of different culture media for early cleavage and later stage embryos, on the in vitro development of bovine embryos. We also investigated the effect of glucose, phosphate and citrate on the in vitro early developmental period of bovine embryos in a two-step culture system. Moreover, the supplementation of different protein sources (BSA-V, BSA-FAF and FBS) during IVC did not affect the frequency of blastocyst development. Using two-step culture, embryos were cultured in protein-free media for an initial 5 days. This was then followed by the same culture media or an FBS supplemented media. The developmental rates of blastocysts in the FBS containing group were significantly higher than in the replaced with no serum containing group. Embryos cultured in mSOF supplemented with 1.5 mM glucose plus 1.2 mM phosphate were significantly inhibited. The inhibition of developmental competence by glucose plus phosphate was consistent with the existence of 0.5 mM sodium citrate. This study indicates that a two-step culture system, which applies different conditions for early cleavage embryos, i.e., serum-free media, vs. later stage embryos, with serum containing media, may be effective for in vitro production systems. In addition, the developmental competence of bovine embryos was depressed in the presence of glucose plus phosphate as compared to either alone or the absence of both. Therefore, the avoidance of this negative effect should allow more optimal conditions to be developed for in vitro production.

Comparison of embryonic development in cleavage stage mouse embryo biopsy between acid Tyrode's solution and laser assisted techniques.

The study was carried out to determine the effectiveness and safety of the infrared 1.48 microm laser in cleavage stage mouse embryo biopsy, compared to the conventional acid Tyrode's solution. One hundred and thirty cryopreserved cleavage stage mouse embryos were included in the study. Fifty embryos were biopsied by acid Tyrode's solution. Forty-seven embryos were biopsied by the infrared 1.48 microm laser. Thirty-three embryos were incubated without biopsy as the control group. Thirteen of 50 embryos in the acid Tyrode's group and 16 of 47 in the laser assisted group became cavitating morulae on day 4, meanwhile 23 of 33 in the control group reached this stage. The blastocyst formation of acid Tyrode's, laser assisted and control group were 94.0, 97.8 and 100.0 per cent, respectively. The hatching rate of acid Tyrode's solution, laser assisted and control group were 78.7, 84.7 and 63.6 per cent, respectively. No significant difference in blastocyst formation and hatching rate was found. The percentage of grade I blastocysts in control group (96.9%) was significantly higher than those in acid Tyrode's solution (68.0%) and the laser assisted group (76.0%). There was no significant difference in the percentage of grade 1 blastocysts between the acid Tyrode's solution and the laser assisted group. In conclusion, the infrared 1.48 microm wavelength laser may be an alternative to acid Tyrode's solution in embryo biopsy.

Effect of IP3 and ryanodine treatments on the development of bovine parthenogenetic and reconstructed embryos

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.

Effect of titanium exposure on embryonic development during pre-implantation period in rats.

Doses of titanium trichloride (1/10th and 1/5th of LD50) were administered once and daily to pregnant rats to assess their effect on embryonic development. 1/5th dose of TiCl3 administered once orally on 1st, 2nd and 3rd day post-coitum. Similarly 1/10th of LD50 was administered daily. Results revealed that 1/10th LD50 dose of TiCl3 was more effective during pre-implantation period as number of 4 and 8-celled embryos decreased as compared to 1/5th. Delayed hatching of the blastocysts on day 5 was registered in TiCl3 treated dam.

Pre-eclamptic toxemia: potential new therapy based on animal studies.

OBJECTIVE: Pre-eclamptic toxemia (PET) affects 4 to 8% of human pregnancies. Presently, reliable specific therapies to treat this disorder are not available. This study was designed to develop a new therapeutic approach in the management of PET using an animal model. DESIGN: Pregnant rats (5/group) infused with 50 mg L-NAME daily via osmotic mini pumps from day 17 of gestation developed a PET-like syndrome. Systolic blood pressure (BP) was monitored daily during pregnancy and up to 7 days postpartum by the tail cuff method. Pup weight and mortality were recorded immediately after delivery. We examined the effect of CGRP to ameliorate L-NAME-induced hypertension during pregnancy, and the efficacy of CGRP and progesterone in combination to inhibit L-NAME-induced hypertension during the post-partum period. RESULTS: Blood pressure in L-NAME-treated rats was significantly elevated (P < 0.01) throughout pregnancy (141 +/- 3 to 166 +/- 10 mm Hg). CGRP 10 micrograms/day did not cause hypotension, the values being similar to controls which received only saline. On the other hand, CGRP infusion inhibited L-NAME-induced hypertension to normotensive levels (116 +/- 3 to 122 +/- 2) during pregnancy (up to day 22 of gestation), but not during postpartum period (137 +/- 8 to 148 +/- 2). During the post-partum period, neither progesterone nor CGRP by itself was effective in lowering L-NAME-induced hypertension. The combination of CGRP with progesterone decreased BP to control levels in the post-partum period, and also significantly improved foetal mortality and growth (P < 0.05). CONCLUSIONS: CGRP inhibited L-NAME-induced hypertension during pregnancy and not during postpartum period. The same phenomenon was evident in the presence of adequate levels of progesterone in the post-partum period. We believe that CGRP regulates vascular adaptations during pregnancy and these effects may be progesterone-dependent. This combination treatment of CGRP plus progesterone may be a promising therapy in the management of PET in humans.

Development effect of technical dimethoate in rats: maternal and fetal toxicity evaluation.

Technical dimethoate was administered orally to pregnant rats through day 6-20 of gestation at doses 3.75, 7.5, 15 and 30 mg/kg/day. Dose of 30 mg/kg/day produced high mortality rate in dams and was not considered for developmental toxicity evaluation. Dimethoate produced enzymatic changes in liver of dams associated with mild pathomorphological changes in liver and brain. Significant fetotoxic effects were not observed at the tested dose levels as evidenced by total number of implantations, percentage resorption, and live fetuses except reduction in fetal weight. Reduced acetylcholinesterase activity in fetal brain and placenta at higher dose levels indicated possible transmigration of dimethoate from dams to fetuses. The absence of anomalies in fetal gross, visceral morphology and skeleton suggests technical dimethoate as non teratogenic in rat at tested dose levels.

Embryonic/fetal mortality after exposure to tritiated water in pregnant Swiss albino mice during different gestation periods.

Pregnant Swiss albino mice were given a priming injection(im) of tritiated water (HTO) at the dose rate of 2.3 and 5 microCi/ml body water (74, 111 or 185 KBq/ml body water) at 0, 6 and 14 day post conception (d.p.c.) and were subsequently maintained on tritiated drinking water ad libitum during preimplantation (0-5 d.p.c.), organogenetic (6-12 d.p.c.) or fetal (14-18 d.p.c.) period, respectively. On day 18 of gestation the females were sacrificed by cervical dislocation to record the implant sites per dam and embryonic/fetal mortality. Significant reduction was observed in average implant sites per dam when the females were exposed to any of the three doses during the preimplantation period due to embryonic resorption before implantation. However, the same was found to be within the normal range when mothers were exposed during the organogenetic or fetal period. Prenatal mortality (embryonic resorption/fetal death) was higher after in utero exposure to different doses during preimplantation period as compared to organogenetic period, but mortality did not occur after exposure to any of the doses during the fetal period. Occurrence of mortality was found to be dose dependent.