Media optimization to promote rat embryonic development to the blastocyst stage in vitro.

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

Bcl-2 expression in a diabetic embryopathy model in presence of polyamines.

The frequency of congenital malformations is 3-5 times higher in mothers with pregestational diabetes mellitus than in general population. Apparently, this problem is due to change in the expression of apoptotic and antiapoptotic genes induced by the oxidative stress derived from the diabetes/hyperglycemia. One of these genes is Bcl-2, which is associated with the control and inhibition of apoptosis. The purpose of the present work was to study the effect of polyamine addition over expression of Bcl-2 gene in a model of diabetic embryopathy. For this, gestational day 10.5 (GD10.5) rat embryos were incubated at 37°C for 24 h in control medium, medium with high glucose, or medium with high glucose and supplemented with spermidine or spermine. Post-cultured embryos were harvested and observed to obtain morphological scores; some of them were subjected to molecular biology studies: DNA isolation plus conventional PCR or RNA isolation plus RT-PCR; other embryos were fixed with paraformaldehyde and used for immunohistochemical detection of Bcl-2 protein. Although Bcl-2 mRNA was similarly expressed in all rat embryo treatments, Bcl-2 protein was found only in control-incubated embryos. In conclusion, it seems that the inhibition of Bcl-2 gene expression induced by glucose was not reversed by polyamines.

BMP-2 and BMP-4 signalling in the developing spinal cord of human and rat embryos.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors implicated in multiple biological events. Studies on mice, chickens and other experimental animals have shown that BMP signalling plays critical role in embryonic development, in particular in the neural patterning. In our study we comparatively evaluated BMP-2 and BMP-4 protein expression in the developing spinal cord of human and rat embryos. The human and rat embryos of Carnegie stages 14, 18 and 20 were embedded in paraffin and cut serially in transversal direction. BMP-2 and BMP-4 were detected by immunohistochemical staining. Spatial and temporal expression pattern of BMP-s during early stages of spinal cord development was similar in human and rat embryos. Higher expression of BMP-s was seen in the dorsal and lower expression in the ventral part of the developing spinal cord both in human and rat embryos. However, temporal difference in the expression of BMPs in the non-neural ectoderm between human and rat embryos was noted. Staining of BMP-s in the non-neural ectoderm adjacent to the developing spinal cord in the human embryos seemed to have a tendency to decrease from earlier to later developmental stages, while in rat embryos there was an opposite tendency.

Comparison and improvement of three methods for primary culture of rat embryonic cortical neurons / 中国实验动物学报

Objective To compare three methods for culture of fetal cortical neurons of SD rats and find out the suitable culture conditions of fetal cortical neurons in vitro.Methods The cortex of 16-18-day embryonic rat was used for culture in this study.Mechanical dissociation, trypsin digestion and papain digestion were applied respectively to the neuron culture.The morphological characteristics of neuronal cells at different time points were observed and neuron purity was identified by immunofluorescence staining assay.Results High purity of the fetal rat cortical neurons was successfully achieved by all the three culture methods, and each had distinct morphological characteristics at different time points.The purity of neurons was 96.28%, 95.63%and 97.34%, respectively, with no significant differences among the three groups (P>0.05).Conclusions The three culture methods are improved in our study.Stable neurons with high purity can be obtained by all the three methods respectively, and each of these methods has distinct characteristics.

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos.

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures. A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600 s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos.

AN ATRIOPEPTIN IMMUNOHISTOCHEMICAL STUDY OF ATRIAL MUSCLE CELLS OF RAT EMBRYOS AND NEONATES / 解剖学报

The occurrence and distribution of the atrial muscle cells containing atriopeptinimmunoreactive granules were studied in rat embryos and newborn rats with immunohistochemistry. The results showed that immunoreactive granules occurred in a few atrial muscle cells of embryos at 13 days old but they were not found in those cells at 11 and 12 days. With development of the embryos, the number of cells containing immunoreactive granules in atrium increased and their granules became more abundant and located mainly around the nucleus. Most of the granulated atrial muscle cells distributed in trabecular structure of the luminal surface of atria. They gradually decreased in number towards the pericardial side. The nongranulated atrial muscle cells mainly located near the pericardium and in atrial septum. The results suggested that the specific differentiation of atrial muscle cells occurred at early period of rat embryos, some cells became atriopeptin immunoreactive positive cells, while the others remained as negative cells. The feature of the differentiation of atrial muscle cells may reflect the functional development of the atria.

A STUDY OF LOCALIZATION OF ATRIOPEPTINLIKE SUBSTANCE IN HEART VENTRICLES OF RAT EMBRYOS / 解剖学报

In this paper the distribution of the atriopeptin-like immunoreactive substance in the ventricles of rat embryos of 13-19 days old was investigated by immunohistochemistry and electron microscopy. The results showed that atriopeptin-like immunoreactive granules were located around the nucleus in some cardiocytes of the ventricles of rat embryos. Most of these cells were distributed in the pectinated or trabecular structures in the luminal surface of left ventricle and a few of them in the myocardium of left and right ventricles. In the same embryo ventricle muscle cells contained less immunoreactive granules than those in the atria. Under electron microscope the atrial specific granule-like granules were found mainly near the Golgi complex. Some cells were devoid of such granules in cytoplasm. In the ventricles the distribution of the muscle cells containing atrial specific granule-like granules corresponds to the sites of muscle cells containing atriopeptin-like substance from the immunohistochemical study. The results suggest that the so-called "atriopeptin" is also present in some ventricular myocytes in rat embryos. The presence of atriopeptin-like substance may be related to the unique type of embryonic circulation.

A HISTOCHEMICAL OBSERVATION ON ISOLATED BONE CELLS FROM CALVARIUM OF RAT EMBRYOS CULTURED IN VITRO / 解剖学报

An experimental study was carried out on the isolated bone cells, from the embryonic calvarium of Sprague-Dawley rats which were taken out in vitro culture in the later period of gestation. Observation of the early stage of culture was carried out with phase contrast microscope, H. E. staining and AcP and AlP technique. It was found that both the osteogenic cells of the cambium layer of the periosteum and the undifferentiated cells from the bone marrow could develop into large squamous-shaped cells. These cells possessed many processes connected with the adjacent cells and stained light red color with Sirius Red but revealed no birefringency under polarized microscope. This indicates that these cells are precursors of collagen formation. The negative reaction with the alkaline phosphatase staining pointed out that these cells still belong to the osteogenic cells.

AN ULTRASTRUCTURAL STUDY OF THE DIFFERENTIATION AND DEVELOPMENT OF ATRIAL MUSCLE CELLS OF RAT EMBRYOS / 解剖学报

The development and differentiation of myocardial cells in the atria of rat embryos from 11 to 19 days and neonates were studied by electron microscopy, particularly with reference to the occurrence and distribution of atrial specific granules in muscle cells. The results were as follows:Atrial specific granules were invisible in muscle cells of 11 days embryos and occurred only in a few muscle cells in 12 days embryos. Since then their size and number in muscle cells increased with development. The granules persisted during mitosis.In atria of embryos from the 12th day on two kinds of myocardial cells could be distinguished, the one containing specific granules and the other without. The former showed well developed Golgi complex and abundant rough endoplasmic reticulum. As development proceeds both types of cells showed increasing amount of myofilaments and mitochondria in their cytoplasm and following the same trend approaching maturity.