Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34+ HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34+ cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34+ cells, but not CD34- cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80+ macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

Culture of ovine IVM/IVF zygotes in isolated mouse oviduct: effect of basal medium

The basal medium that supports Isolated Mouse Oviduct [IMO] is important for supporting embryo development and quality. The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos [p>0.05]. The hatching rate, total and trophectoderm cells number in IMO groups' blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system [SOFaaBSA alone] but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone

[Survival and development of mouse 8-cells embryos after vitrification in glycerol-sucrose and ethylen glyco based solutions]

Nowadays gamete and embryo freezing is an appropriate approach for preserving of genetic traits in laboratory animals, rare and endangered species. Frozen cells are suitable replace for actively breeding animals colony. The aim of this study was to preserve laboratory mouse embryo, using vitrification method and comparing effect of two cryoprotectants, glycerol sucrose [GS] and ethylene glycol-ficoll-sucrose [EFS40] on 8-cells and morula stage embryos of the mouse. Following mice superovulation 258[73.5%] out 351 embryos were in 8-cell and moroula stages. 188 morphologically intact embryos were exposed in the GS and EFS40 drops and then each 4 of them transferred to one special micro tube and after ends sealing, finally were cooled up to- 196[degree sign] C with liquid nitrogen vapors and immediately plunged into liquid nitrogen. One to three months later, embryos were thawed, recovered and cultured. The recovery rate of post-thawing embryos from EFS group [90%] was more than percentage of embryos recovered from GS [85%] group. In also survival rate of embryos undergoing further cleavage post-culturing to blastocyte stage, from EFS and GS groups were 53/7% and 19/6% respectively. This difference was significant at p<0.001. However difference between EFS group with fresh embryos, un-frozen embryos, for achieve to blastocytes stage which was 68/6% for second group, wasn't significant [p<0.05], but this item was significant between fresh embryo ang GS groups [p<0.001]. Generally results of our study show that, use of vitrification of 8-cell and morula NMRI mouse strain embryos using EFS as cryoprotectant is a suitable, easy and economical method for preservation of mouse embryos

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Time course of degradation and deadenylation of maternal c-mos and cyclin A2 mRNA during early development of one-cell embryo in mouse

Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributed to a change in the translational activity or stability of mRNA in the egg and early embryo. As a result, translational control is critical for a variety of developmental decisions, including oocyte maturation and initiation of preimplantation development. In this study, using real-time RT-PCR method, we defined the time course of degradation and deadenylation of an oocyte specific gene [c-mos] more precisely and a gene that is re-synthesized after ZGA [cyclin A2]. Our data indicate that oocyte-specific transcript, c-mos, degrades rapidly while cyclin A2 mRNA does not and the deadenylation of c-mos mRNA precedes the process of degradation. Our findings suggest that time-dependent elimination of different maternal mRNA is a way for regulation of translation in early development of mouse embryos

Sobrevivência de blastocistos Mus domesticus domesticus vitrificados em meio contendo 9.0M de etileno glicol na presença de sacarose / Survival rate of Mus domesticus domesticus blastocysts vitrified in 9.0M of ethylene glycol with sucrose

Os experimentos realizados tiveram como objetivo determinar a taxa de sobrevivência in vitro e in vivo de blastocistos Mus domesticus domesticus vitrificados em meio contendo 9,0M de etileno glicol e 0,3M de sacarose. As soluçöes testadas foram denominadas de I e IS, quando a adiçäo do crioprotetor foi realizada em duas etapas, e II e IIS, quando a adiçäo deste foi realizada em apenas uma etapa. Na soluçäo de vitrificaçäo I, foi realizada inicialmente uma desidrataçäo prévia dos embriöes por um período de 2 minutos em soluçäo de 1,8M de EG em PBS + 6 por cento de BSA e, logo após, eles foram transferidos, por um período de 30 segundos, para a soluçäo composta por uma associaçäo de 9,0M de EG em PBS + 6 por cento de BSA, antes da imersäo em nitrogênio líquido. Na soluçäo de vitrificaçäo IS, o procedimento foi idêntico ao da soluçäo I, apenas com o acréscimo de 0,3M de sacarose às soluçöes crioprotetoras. Na soluçäo de vitrificaçäo II, os embriöes foram expostos diretamente a uma soluçäo composta por uma associaçäo de 9,0M de EG em PBS + 6 por cento de BSA, onde permaneciam por 30 segundos antes da imersäo em nitrogênio líquido. Na soluçäo de vitrificaçäo IIS, o procedimento foi idêntico ao da soluçäo II, apenas com a adiçäo de 0,3M de sacarose à soluçäo crioprotetora. No experimento I, foi determinada a taxa de sobrevivência de 299 blastocistos após a exposiçäo às soluçöes crioprotetoras, näo sendo observada diferença estatística entre os tratamentos e o grupo controle. O experimento II permitiu avaliar a sobrevivência embrionária in vitro (taxa de eclosäo) após a vitrificaçäo de 330 blastocistos, onde as soluçöes I e IS foram estatisticamente superiores às demais, apresentando taxas de eclosäo de 49 e 40 por cento, respectivamente. No experimento III, realizou-se a transferência de 141 blastocistos vitrificados, após a exposiçäo às soluçöes I e IS, para fêmeas receptoras. Näo houve diferenças estatísticas entre as taxas de sobrevivência embrionária determinadas aos 14 dias de prenhez, tanto para implantaçöes (37 e 32 por cento) quanto para fetos (27 e 27 por cento), respectivamente. A presença de sacarose na soluçäo de vitrificaçäo näo proporcionou uma maior sobrevivência de blastocistos Mus domesticus domesticus vitrificados em uma soluçäo contendo 9,0M EG.

Transferencia embrionaria en modelo ratón / Embryo transfer in mice, experimental model

La transferencia embrionaria (TE) se utiliza para optimizar la fertilización in vitro (FIV). Se trata de establecer un modelo experimental de TE que permitía la implantación y desarrollo de embriones. Se emplearon 53 ratones hembras adultas, 23 donadoras y 22 receptoras. Diez donadoras recibieron estimulación ovárica con gonadotrofina coriónica intraperitoneal (i.p., 1UI/ratón). Catorce receptoras recibieron estimulación endometrial con gonadotrofina menopáusica humana (i.p, 2 UI/ratón). Se comprobó la gestación con Papanicolaou en el frotis vaginal. La obtención de embriones fue mayor (p<0.05) en animales con estimulación ovárica comparados con los que no recibieron este tratamiento, tanto en obtención de embriones de 48 h (8.8 ñ 1.6 y 6.4 ñ 0.6, respectivamente) como en embriones de 72 h (6.1 ñ 0.8 y 4.0 ñ 0.7, respectivamente). El porcentaje de éxito de fertilización fue semejante en animales receptores con o sin estimulación endometrial independiente de la edad del embrión (50 por ciento con y 37 por ciento sin estimulación). La duración de la gestación fue mayor (p<0.05) en animales con estimulación endometrial (9.4 ñ 0.7 días) comparada con los que no recibieron tratamiento (6.3 ñ 0.3 días). Aun cuando el porcentaje de éxito de la FIV fue bajo, se logró establecer un modelo experimental. Se concluye que el número de embriones recuperados fue mayor con estimulación ovárica, siendo su sobrevida intrauterina algo más prolongada en animales receptores con estimulación endometrial

Quantitative and topographic analysis of sulphur in the zona pellucida from mouse, hamster and rabbit oocytes

Mediante la técnica del microanálisis de rayos X, se investigó la cantidad y distribución del azufre en la zona pelúcida de los ovocitos de ratón, hamster y conejo. La cantidad de azufre varía entre 2853 y 10178 mmol/Kg de peso seco. La cantidad más alta de azufre se encontró en los ovocitos de hamster y la más baja en los ovocitos de conejo; siendo la cantidad encontrada en los ovocitos de ratón, intermedia entre ambas. Se detectó una disminución gradual del azufre desde la región interna hacia la región externa de la zona pelúcida, en los ovocitos de ratón y conejo. Esto es coincidente con las asimetrías morfológica y/o bioquímica descritas para la zona pelúcida de estas especies. Aunque se han descrito asimetrías bioquímica y funcional en la zona pelúcida de los ovocitos de hamster, en el presente análisis no se detectó una distribución asimétrica del azufre. Sin embargo, resultó evidente una estratificación de este elemento en la zona pelúcida de los ovocitos de hamster