Two different pathways for the transport of primitive and definitive blood cells from the yolk sac to the embryo in humans

During the early human embryonic period nutrients and blood cells are temporarily provided by the extraembryonic yolk sac (YS). The YS before week six is involved not only in primitive but also in definitive erythropoiesis. While the destiny of primitive erythroid cells that fill the blood vessels of the YS is well known, the final destination of erythrocytes present in the endodermal vesicular system is unknown. In the present study we have investigated, step by step, the destiny of the erythrocytes present in the endodermal vesicles during the embryonic period. Twelve human YSs and their corresponding yolk stalks were analyzed between weeks 4 and 7 of embryonic age by light and scanning electron microscopy. It is shown that erythrocytes (according to their size and morphological features) located within the endodermal vesicles of the YS wall are pulled out through endodermal pits into the YS cavity, from where they reach the lumen of the primitive gut of the embryo through the vitelline duct, a temporary pathway communicating both compartments. During the study period no erythrocytes were seen within the embryo's vascular network where only primitive erythroblasts were identified. Our results indicate that the vitelline duct plays an important transient role as a pathway for the transport of nutrients and blood cells between the YS and the embryo before week five of embryonic development that ends just at the time when YS‐embryo circulation becomes established. Microsc. Res. Tech. 73:803–809, 2010. © 2010 Wiley‐Liss, Inc.     

The role of the gastrointestinal epithelium as a possible pathway for the transfer of nutrients to the embryo's circulation

The endodermal cells of the human yolk sac (YS) produce non‐nucleated erythrocytes (NNEs) and numerous serum proteins that are transiently storage within the YS cavity. After their transfer via the vitelline duct to the embryo gastrointestinal lumen, the nutrients’ final fate is unknown. With the aim of investigate how erythroid cells and nutrients are conveyed to embryo circulation, we studied, using a morphological and immunohistochemical approach, the embryo anatomy and the serum protein α‐fetoprotein (AFP) presence, in 15 human embryos and their YS, collected from tubal pregnancies from 4 to 8 wpf. We observed at 5 wpf, a strong AFP staining in the endodermal cells of the YS, thereafter AFP was only present in the YS cavity and the gastrointestinal lumen. During 7 wpf, AFP expression declined and disappeared, concomitant with YS regression. Between 5 and 7 wpf, NNEs were observed in the gastrointestinal cavity, where they accumulate in the stomach. Here, the cells were attached to the endodermal epithelial cells or were free in the lumen. By scanning electron microscopy, we identified signs of NNEs phagocytized by endodermal cells. Those NNEs free in the lumen, after hemolysis, were probably removed by endocytosis (cell debris). Taking all together, we postulate that after reaching the endodermal epithelial cells of the stomach, nutrients are transferred to the embryo by a phagocytic/endocytic mechanism that is operative until the end of 6 wpf. After absorption, NNEs are probably degraded within phagosomes, nutrients delivered to the cell cytoplasm and then transported towards the embryonic circulation. Microsc. Res. Tech. 78:500–507, 2015. © 2015 Wiley Periodicals, Inc.     

A morphological analysis of the transition between the embryonic primitive intestine and yolk sac in bovine embryos and fetuses

The yolk sac (YS) is the main source of embryonic nutrition during the period when the placenta has not yet formed. It is also responsible for hematopoiesis because the blood cells develop from it as part of the primitive embryonic circulation. The objective of this study was to characterize the transitional area between the YS and primitive gut using the techniques of light microscopy, transmission electron microscopy, and immunohistochemistry to detect populations of pluripotent cells by labeling with Oct4 antibody. In all investigated embryos, serial sections were made to permit the identification of this small, restricted area. We identified the YS connection with the primitive intestine and found that it is composed of many blood islands, which correspond to the vessels covered by vitelline and mesenchymal cells. We identified large numbers of hemangioblasts inside the vessels. The mesenchymal layer was thin and composed of elongated cells, and the vitelline endodermal membrane was composed of large, mono‐ or binucleated cells. The epithelium of the primitive intestine comprised stratified columnar cells and undifferentiated mesenchymal cells. The transitional area between the YS and the primitive intestine was very thin and composed of cells with irregular shapes, which formed a delicate lumen containing hemangioblasts. In the mesenchyme of the transitional area, there were a considerable number of small vessels containing hemangioblasts. Using Oct4 as a primary antibody, we identified positive cells in the metanephros, primordial gonad, and hepatic parenchyma as well as in YS cells, suggesting that these regions contain populations of pluripotent cells. Microsc. Res. Tech. 76:756–766, 2013. © 2013 Wiley Periodicals, Inc.     

Development of the avian lymphatic system

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.     

Immunolocalization indicates that both original and regenerated lizard tail tissues contain populations of long retaining cells,putative stem/progenitor cells

The regeneration of the tail in lizards is likely sustained by stem/progenitor cells located in the stump after amputation of the tail. This microscopic and ultrastructural study shows the localization of 5‐bromo‐deoxy‐uridine (5BrdU)‐long retaining labeled cells in different tissues of the tail stump. These putative stem/progenitor cells are sparsely detected in the epidermis of scales, adipose tissue, intermuscle connective septa, myosatellite cells, and perichondrion of the vertebrae. Most of 5BrdU‐labeled cells are present in the bone marrow of vertebrae as hemocytoblasts and reticulate cells, whereas more numerous myelocytes and polychromatophilic erythroblasts show a variable level of nuclear labeling. 5BrdU and tritiated‐thymidine labeled and unlabeled hemopoietic cells are seen in circulating vessels and in the blastema where their maturation is completed. This observation indicates that the entire differentiation span of both white and red blood cells, at least during tail regeneration, lasts longer than 4 weeks. Labeled polychromatophilic erythroblasts and heterophilic and basophilic myelocytes are present in the synusoidal vessels of the regenerating tail. This study indicates that extravasating blood cells involved in immunity make large part of the forming blastema cell population, but are replaced by mesenchymal cells of different origin. The presence of long retaining labeled cells in tissues of the tail stump is likely connected to the production of blastema mesenchymal cells. Although no direct cell‐lineage study has been done, histological, immunocytochemical, and autoradiographic studies have indicated that it is from these tissues that proliferating cells appear mainly localized after tail amputation and blastema formation. Microsc. Res. Tech. 78:1032–1045, 2015. © 2015 Wiley Periodicals, Inc.     

Morphological changes in mouse embryos cryopreserved by different techniques

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.     

Surface and internal structure correlation: high-voltage and scanning electron microscopies of wholemount alveolar walls of human lung

We found that the high-voltage electron microscope (HVEM) operating at 1–5 MeV was able to transilluminate and form a focused transmission image of whole-mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph. Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features. Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 μm thick were delineated with the high-voltage electron microscope. Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other. HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.     

Visualization of proteoglycans and link protein in embryonic chick limb cartilage via cryofixation, freeze-substitution and immunochemical techniques

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30–34 chick embryos was high-pressure frozen, freeze-substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin-6-sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron-dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron-dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin-6-sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested from our results.     

Fluorescence microscopy of rat embryo sections stained with haematoxylin–eosin and Masson's trichrome method

The fluorescence pattern induced by haematoxylin–eosin (HE) and Masson's trichrome (MT) staining methods on paraffin sections of rat embryos (from 13 to 18 days old) has been studied. Using optimal excitation (green light, 545 nm), HE- or MT-stained sections showed a selective red emission of the acidophilic tissue components, which was due to eosin Y in the case of HE and to acid fuchsin and/or xylidine ponceau in the case of MT. The fluorescence intensity induced by these anionic dyes was variable and related to the substrate nature and the embryo age. Whereas in young embryos only the immature red blood cells showed a noticeable fluorescence, in the oldest embryos there were also other tissue components that selectively fluoresced with these dyes, in particular fibre lens cells, elastic fibres, zymogen granules and muscle cells. Spectrofluorometric studies on free dyes and densitometric analysis of protein blots confirmed microscopical observations. Our results indicate that the standard HE and MT staining methods can be used in recognizing the appearance of specific protein structures in embryonic tissues by means of fluorescence microscopy.     

Microscopic evaluation of vesicles shed by erythrocytes at elevated temperatures

The images of human erythrocytes and vesicles were analyzed by a light microscopy system with spatial resolution of better than 90 nm. The samples were observed in an aqueous environment and required no freezing, dehydration, staining, shadowing, marking, or any other manipulation. Temperature elevation resulted in significant concentration increase of structurally transformed erythrocytes (echinocytes) and vesicles in the blood. The process of vesicle separation from spiculated erythrocytes was video recorded in real time. At a temperature of 37°C, mean vesicle concentrations and diameters were found to be 1.50 ± 0.35 × 10⁶ vesicles per microliter and 0.365 ± 0.065 μm, respectively. The vesicle concentration increased approximately threefold as the temperature increased from 37 to 40°C. It was estimated that 80% of all vesicles found in the blood are smaller than 0.4 μm. Accurate account of vesicle numbers and dimensions suggest that 86% of the lost erythrocyte material is lost not by vesiculation but by another, as yet, unknown mechanism. Microsc. Res. Tech. 76:1163–1170, 2013. © 2013 Wiley Periodicals, Inc.     

Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane

It has generally been assumed that tumors do not induce lymphangiogenesis and only very recently animal models have been presented showing tumor-induced lymphangiogenesis. We have grown two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the chorioallantoic membrane (CAM) of chick and quail embryos. The suspended tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. When grown on the CAM of quail embryos intratumoral endothelial cells could be specifically stained with the QH1 antibody. In C6 gliomas the vascular pattern was more regular than in 10AS carcinomas. The vessels often grew radially into the glioma and many of them were invested by smooth muscle alpha-actin-positive periendothelial cells. Lymphatics, which were identified by vascular endothelial growth factor receptor-3 (VEGFR-3) in situ hybridization were absent from C6 gliomas, although a weak expression of the lymphangiogenic growth factor, VEGF-C, could be detected in the C6 cells by Northern blot analysis. In contrast, 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with BrdU-labeling rates of about 9% of lymphatic endothelial cells. Our studies demonstrate the heterogeneity of interactions of tumor cells with blood vessels and lymphatics and show that sufficient quantities and/or quality of lymphangiogenic growth factors are crucial for the induction of lymphatics in tumors.     

Preparation of fetal rat brains for light and electron microscopy

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: (1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. (2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11–20 days (E11–E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11–E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11–E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s-collidine buffer, followed by osmium tetroxide in s-collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.     

Live imaging of fluorescent proteins in chordate embryos: from ascidians to mice

Although we have advanced in our understanding of the molecular mechanisms intrinsic to the morphogenesis of chordate embryos, the question of how individual developmental events are integrated to generate the final morphological form is still unresolved. Microscopic observation is a pivotal tool in developmental biology, both for determining the normal course of events and for contrasting this with the results of experimental and pathological perturbations. Since embryonic development takes place in three dimensions over time, to fully understand the events required to build an embryo we must investigate embryo morphogenesis in multiple dimensions in situ. Recent advances in the isolation of naturally fluorescent proteins, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos using optical imaging. These technologies allow direct visual access to complex events as they happen in their native environment, and thus provide greater insights into cell behaviors operating during embryonic development. Since most fluorescent protein probes and modes of data acquisition are common across species, we have chosen the mouse and the ascidian, two model organisms at opposite ends of the chordate clade, to review the use of some of the current genetically-encoded fluorescent proteins and their visualization in vivo in living embryos for the generation of high-resolution imaging data.     

New methods for chicken embryo manipulations

The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here, we present a novel way of adapting the well‐known EC culture of whole chick embryos to time‐lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable, because it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Erythrophagosomal haemolytic degradative pathway in rat brown adipocytes induced by hyperinsulinaemia: an ultrastructural study

The phagocytosis and degradation of erythrocytes were studied in brown adipose tissue of experimentally hyperinsulinaemic rats. We found that insulin induces intensive erythrophagocytosis by brown adipocytes and their degradation by haemolytic pathway. Ultrastuctural study revealed that haemolytic degradation of erythrophagosomes was characterized by progressive and uniform decrease of erythrocyte matrix density. At the beginning of the degradative process small, clear vesicles resembling primary lysosomes were visible inside the erythrophagosome. With time, the erythrocyte structure totally disappeared and transformed into a fine, granular material within the erythrophagosomal vacuole. Finally, the erythrocyte membrane detached from the phagosomal and clumped into the vacuolar space forming one or several small myelin‐like figures. In conclusion, brown adipocytes are capable of performing intensive erythrophagocytic activity when brown adipose tissue is stimulated and blood flow is enhanced. The molecular basis for favouring a haemolytic instead of more common granular erythrophagosomal degradative pathway remains unknown.     

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.     

Spatial distribution of Reissner's fiber glycoproteins in the filum terminale of the rat and rabbit

The subcommissural organ secretes into the third ventricle glycoproteins that condense to form the Reissner's fiber (RF). At the distal end of the central canal of the spinal cord, the RF-glycoproteins accumulate in the form of an irregular mass known as massa caudalis. Antibodies against RF-glycoproteins and a set of lectins were used at the light and electron microscopic level to investigate the spatial distribution of the massa caudalis material in the rat and rabbit filum terminale. In the sacral region of the rat, the central canal presents gaps between the ependymal cells through which RF-glycoproteins spread out. The bulk of massa caudalis material, however, escapes through openings in the dorsal wall of the terminal ventricle. In the rabbit, the massa caudalis is formed within the ependymal canal, at the level of the second coccygeal vertebra, it accumulates within preterminal and terminal dilatations of the central canal, and it escapes out through gaps in the dorsal ependymal wall of the terminal ventricle. The existence of wide intercellular spaces and a large orifice (neuroporous) in the dorsal ependymal wall of the terminal ventricle, and the passage of RF-material through them, appear to be conserved evolutionary features. After leaving the terminal ventricle of the rat and rabbit, RF-glycoproteins establish a close spatial association with the numerous blood vessels irrigating the filum terminale, suggesting that in these species the blood vessels are the site of destination of the RF-glycoproteins escaping from the central canal, thus resembling the situation found in lower vertebrates. When passing from the RF stage to the massa caudalis stage, the rabbit RF-glycoproteins lose their sialic acid residues, exposing galactose as the terminal residue. Since this sialic acid-galactose modification of RF-glycoproteins had also been described in lamprey larvae, it may be regarded as a conserved evolutionary feature associated with the formation of the massa caudalis.     

Development beyond the gastrula stage and digestive organogenesis in the apple-snail <i>Pomacea canaliculata</i> (Architaenioglossa,Ampullariidae)

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenteron’s lake, from where it is accumulated into the large cells forming the midgut wall (“giant cells”). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.     

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-F10 culture medium during 24 h 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-F10, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 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 96 h 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, CM10 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.