Analysis of heart development in cultured rat embryos

The long-range goal of this research is to establish an in vitro system that will permit pertubation of mammalian heart development and in situ examination of the cellular and molecular events underlying cardiac morphogenesis. Rat embryos at 9.5-11.5 days of gestation were placed in culture bottles containing rat serum and Tyrode's solution. Embryos cultured for 24 and 48 h were compared to age-matched in vivo controls for morphological score, morphometric analysis of heart development, and confocal and electron microscopic analysis of myofiber pattern formation. Morphological scores indicated that embryos cultured for 24 h from day 9.5 to 10.5 had essentially normal development when compared to age-matched embryos allowed to develop in vivo. Development of embryos maintained for 48 h in culture was slightly delayed at 66-68% of age matched in vivo embryos. Analysis of hearts from embryos allowed to develop 9.5-11.5 days in vivo plus 24 and 48 h in culture showed that the ventricular thickness and height, as well as the truncal, atrial and ventricular diameters were equivalent to those of hearts from age-matched in vivo controls. Hearts from embryos allowed to develop from 11.5-12.5 days in vitro and cultured for 24 and 48 h had smaller left ventricular and atrial dimensions than controls. Cardiac myofibrillogenesis and myofibrillar pattern formation in embryos cultured from 9.5 days of in vivo development for 48 h were also normal. These studies indicate that the rat whole embryo culture system is a useful model to study several critical periods in mammalian heart development.     

Functional analysis of a rat sodium channel carrying a mutation for insect knock-down resistance (kdr) to pyrethroids

As a model for establishing an optimized medium for human in vitro fertilization (IVF), modified human tubal fluid (HTF) media containing amino acids at concentrations found in human serum and follicular fluid were prepared, and the effect of the media on development of random-bred (ICR) and F1 hybrid (CBF1) mice embryos was studied. The total concentrations of amino acids found in serum and follicular fluid were about one-third to one-half the concentrations present in two conventional media used in human IVF: Ham's F-10 and Eagle's minimal essential medium (MEM). When ICR mouse embryos were cultured in the HTF medium containing 21 amino acids at concentrations found in follicular fluid, the number of embryos developing to morulae at 72 h and to blastocysts at 96 h increased in comparison with those cultured in HTF medium. When HTF containing amino acids at concentrations found in serum was used, only induced morula formation at 72 h was enhanced. The number of hatching blastocysts at 96 h also increased when CBF1 mouse embryos were cultured with HTF supplemented with amino acids at concentrations found in follicular fluid. When ICR mouse embryos were cultured in modified HTF media containing concentrations of amino acids found in Ham's F-10 and MEM that contained higher concentrations of glutamine, embryo development was inhibited. The amount of ammonium produced during incubation for 3 days was significantly less when embryos were cultured in media containing concentrations of amino acids found in follicular fluid compared with when Ham's F-10 or MEM was the culture medium. Ammonium is produced by the breakdown of glutamine in the culture medium during incubation with or without embryos. These results suggest that the concentrations of amino acids found in follicular fluid are more effective and safer for embryo culture than those in other media currently in use.     

Survival and neurite formation of mesencephalic trigeminal neurones of the rat in vitro

In order to study the development and functional properties of single, isolated, rat mesencephalic trigeminal neurones, a cell-culture procedure was developed for these specific primary sensory neurones. Mesencephalic trigeminal neurones were isolated from the brainstem of 16-day-old rat embryos. Various factors thought to promote the survival and growth of these neurones in vitro were examined. Outgrowth and maintenance of mesencephalic trigeminal neurones in vitro appeared to be stimulated by a muscle-derived factor, present in muscle-conditioned medium or in muscle extract. Of the neurotrophic factors examined, brain-derived neurotrophic factor and neurotrophin-3, but not nerve-growth factor, promoted the survival of rat mesencephalic trigeminal neurones. Optimal survival of these neurones was found to occur on a monolayer of astrocytes, an effect mediated through direct cell-to-cell interactions.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.     

A substance secreted by rat Sertoli cells induces feminization of embryonic chick testes in vitro

Male and female gonads from 7- to 9-day-old chick embryos were cultured for 6 days in Sertoli cell-conditioned medium or in serum-free medium to investigate the possible effect of substances secreted by rat Sertoli cells on chick gonad development. Histological analysis showed that whereas all female gonads proceed through normal ovarian development in both culture media, most of male gonads showed clear feminization only when cultured in Sertoli cell-conditioned medium; male gonads cultured in serum-free medium developed as normal testes. Because the only substance detected in our conditioned medium with the potential to cause these effects was sex-specific antigen (Sxs), our results provide further evidence that Sxs antigen may play a role in sexual differentiation in birds, and probably in mammals.     

In vitro analysis of ovarian differentiation and the initiation of meiosis in the rat

Intact, cranial, and caudal portions of fetal rat gonads on days 13, 14, 15, 16 and 17 of gestation were cultured singly on Nucleopore filters in Eagle's MEM and McCoy's 5A media (both supplemented with fetal calf serum and glutamine) to evaluate the role of the mesonephros in mammalian gonadogenesis. After 2--7 days in culture, in vitro and in vivo control tissues were examined by light and electron microscopy to determine the extent of morphological differentiation and gonium maturation. The onset of morphological differentiation and meiotic activity, as determined by synaptonemal complex formation, was found to be independent of the mesonephric epithelium. Ovaries and testes were also cultured as heterologous pairs on opposite sides of collagen-coated Nucleopore filters for 4--6 days in McCoy's 5A media. In the absence of transfilter cellular migration and establishment of cell-to-cell contact, the in vivo pattern of sexual dimorphic onset of meiosis was observed. This finding lends support to the concept that local somatic-germ cell associations as opposed to extragonadal factors, are capable of providing the necessary environment for regulation of meiosis in the fetal rat.     

Differential regulation of mouse embryo development and viability by amino acids

The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.     

Cell protein degradation in cultured rat embryo fibroblasts. Suppression by vinblastine of the enhanced proteolysis by serum-deficient media

Rat embryo fibroblasts grown in Eagle's minimal essential medium with 10% serum were labeled with L-[14C]leucine. After a 24 h cold chase, rates of proteolysis were evaluated by measuring the appearance of trichloroacetic acid-soluble 14C in the media. Cells remaining in minimal essential medium with 10% serum (basal) showed a proteolysis rate of 1% per h, whereas cells placed in minimal essential medium alone (serum-deficient) showed a stimulation of proteolysis to 3--4% per h. This enhanced proteolysis was transitory, occurring only for the first 4--8 h after cells were placed in the serum-deficient media. Vinblastine 10-5 M inhibited the enhanced proteolysis 40% but had no effect on basal proteolysis. Control experiments showed no detectable hydrolysis of extracellular proteins, nor did vinblastine affect the rate of protein synthesis. These data suggest that basal and enhanced proteolysis have at least partially distinct mechanisms in the cell and that only enhanced proteolysis involves microtubules.     

Studies on the cultivation of isolated murine renal glomeruli (author's transl

The rate of growth of isolated murine renal glomeruli was studied in vitro. Separation by screening was followed by cultivation in Carrel flacks using Eagle's medium, and the number of utilizable tests was 104. It was possible to cultivate glomeruli for up to 27 days and to observe the growth of cells. These were epithelial capsule cells, endothelial capillary cells and fibroblasts. The existence of mesangial cells not be convincingly demonstrated experimentally. The problems associated with the cultivation of isolated renal glomeruli are discussed with reference to data reported in the literature. Possible uses of this particular model are pointed out in some detail.     

Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin-1 system during embryonic development

In the study reported here, we localized at the protein level the major components of the interleukin (IL)-1 system in the human embryo, and we investigated the endometrial factors influencing their secretion during embryonic development. To localize these components, we performed immunohistochemical experiments in 44 oocytes and 78 embryos. The following primary antibodies were used: monoclonal mouse anti-human IL-1 receptor type I (IL-1R tl), monoclonal mouse anti-human IL-1 beta, and polyclonal rabbit anti-human IL-1 receptor antagonist (IL-1ra). For embryo culture, human embryos at different developmental stages were cultured in 100-microliters drops of Ham's F-10 medium + 4 mg/ml BSA (n = 33), in 100-microliters drops of Menezo B2 culture medium (n = 18), or in wells with 1 ml of Menezo B2 culture medium (n = 8). For embryo coculture, endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) were isolated from human secretory endometrium and cultured until confluence in 75% Dulbecco's Modified Eagle's Medium and 25% MCDB-105 containing antibiotics and supplemented with 10% charcoal-Dextran-treated fetal bovine serum. Individual human embryos were cocultured with experimental EEC and ESC (n = 23 and n = 4, respectively) for 5 days in 600-microliters drops of Menezo B2 medium, and conditioned medium was removed every 24 h. Human embryos were also cultured with EEC-conditioned medium (n = 9). IL-1 alpha, IL-1 beta, and IL-1ra levels were determined by ELISA in the 24-h culture- or coculture-conditioned media. Immunostaining confirmed the presence of IL-1 beta, IL-1ra, and IL-1R tl in oocytes and embryos in all stages analyzed, with no statistical differences. IL-1 alpha, IL-1 beta, and IL-1ra were absent in conditioned media of cultured embryos and embryos cocultured with ESC. However, when human embryos were cocultured with EEC or with EEC-conditioned medium alone, two different populations of embryos were observed: IL-1 producers (57% and 56%) and IL-1 nonproducers (43% and 44%, respectively). Finally, the IL-1 profile of a single human embryo cocultured with maternal EEC which successfully implanted and developed is presented, this pattern being similar to that described in the IL-1 producer population. These results demonstrate the presence of the IL-1 system in the human embryo. However, the selective release of IL-1 only when embryos were cocultured with EEC or EEC-conditioned medium indicates an obligatory role of the endometrium in the regulation of the embryonic IL-1 system. Furthermore, the differential embryonic production of IL-1 may be related to the implantation capability of the embryos.     

Culture of pre-implantation Chinese hamster (Cricetulus griseus) embryos in vitro

Pre-implantation embryos from Chinese hamsters (Cricetulus griseus) were cultivated under completely defined conditions. The embryos were placed in drops of chemically defined medium under liquid paraffin and cultured in an atmosphere of 10% CO2 in air. By this method, development will proceed in vitro from the two-cell stage up to the blastula within 72 h. It is possible to stop the cultivation at different stages of development, to fix the embryos and analyse the chromosomes. The method described in detail seems to be appropriate for examination of the induction of genetic defects during the first days of embryogenesis.     

Assessment of teratogenic effects of lidocaine in rat embryos cultured in vitro

BACKGROUND: Lidocaine has been reported to cause neural tube closure defects in vitro in mice at clinically relevant concentrations. However, no studies have been conducted to further investigate this potentially hazardous effect of lidocaine. This study was aimed to reassess teratogenic effects of lidocaine in vitro in rats. METHODS: Sprague-Dawley rat embryos were explanted at 8:00 AM on gestational day 9 and were cultured in medium containing various concentrations of lidocaine. (Embryos in the control group were cultured without lidocaine). After 50 h of culture, they were evaluated for growth size and morphology, including the neural tube closure. RESULTS: In the presence of 250 microM of lidocaine, embryos showed a increased incidence of situs inversus compared with control group but were otherwise normal. At 375 microM, embryos showed slight growth retardation but no significant morphologic abnormalities. At 500 microM, all viable embryos showed severe morphologic abnormalities. However, morphologic abnormalities were so-called nonspecific types and neural tube closure defects were not observed. CONCLUSIONS: Results from the current study indicate that lidocaine causes teratogenic effects in vitro in rats only at concentrations much higher than clinically relevant concentrations. Furthermore, lidocaine did not cause neural tube closure defects at any concentrations evaluated.     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

A note on assessing the superiority of a combination drug with a specific alternative

The gas atmosphere and medium composition are critical factors in the in vitro development of one- and two-cell embryos of several species. The present study evaluated the effect of different O2/CO2 concentrations (2/5, 2/10, 5/2.5, 5/5, 5/10, 10/10 and 21/5) on pig one- and two-cell embryo development. The embryos were individually cultured, for 6 days at 39 degrees C in a medium rich in bicarbonate and glutamine and containing pyruvate and lactate but lacking glucose. When the CO2 levels increased from 2.5% to 10%, the pH of the medium decreased from 8.2 to 7.5 and the development of the embryos was affected, but this depended mainly on the O2 levels. Pig embryo development was inhibited by 2 and 21% O2 levels. The optimum level for pig embryo development was 5% O2 and 5% CO2, whatever the criteria used to evaluate embryo development. At these optimal levels, the mean number of cells per embryo was 26 +/- 1.7 (ls mean +/- SE), and 50% of the one- and two-cell embryos developed to blastocysts. The substitution of 0.5% bovine serum albumin (BSA) in the medium by 0.3% polyvinyl-pyrrolidone (PVP) significantly decreased the one- and two-cell embryo development. When the calcium and chloride contents of the medium with PVP were reduced, however, the embryo development was similar to that observed in the medium containing BSA. Pig embryo development in vitro was found to be optimal under an atmosphere of 5% O2 and 5% CO2 and PVP could replace BSA as the high molecular weight supplement.     

Effects of fluoroquinolones and magnesium deficiency in murine limb bud cultures

Quinolone-induced arthropathy is probably caused by a lack of functionally available magnesium in immature joint cartilage. We used an in vitro assay to study the effects of fluoroquinolones on cartilage formation in mouse limb buds from 12-day-old mouse embryos in regular and in magnesium-deficient medium. Omission of magnesium from the medium had no adverse effect on the outcome of the culture: limb buds grew and differentiated well in regular and in magnesium-deficient Bigger's medium. Lack of calcium, however, severely impaired the development of the explants; this result was even more enhanced when both minerals (magnesium and calcium) were omitted. Electron microscopy revealed cell necrosis and deposition of electron-dense material in the vicinity of chondrocytes from limb buds after 6 days in a magnesium-free medium. A series of seven fluoroquinolones was tested at 30, 60, and 100 mg/l medium. At a concentration of 30 mg/l sparfloxacin only had a slight effect on limb development. At concentrations of 60 and 100 mg/l sparfloxacin, temafloxacin and ciprofloxacin impaired limb development in vitro concentration-dependently. The effects were enhanced in a magnesium-deficient medium (concentration of magnesium <10 micromol/l). Fleroxacin, lomefloxacin and ofloxacin impaired limb development only slightly; no significant differences were recognizable between the outcome in regular and in magnesium-deficient medium. Pefloxacin did not show any effect on limb development in both media. Using electron microscopy, very similar alterations as described above for the limbs cultured in magnesium-deficient medium were observed with ofloxacin at a concentration of 30 mg/l, which had no effect on the growth of the explants when evaluated macroscopically. The affinity of six fluoroquinolones to magnesium was determined by the use of a fluorescence assay. The affinity to magnesium correlated with the activity of the drugs in the limb bud assay. We conclude that fluoroquinolones have no effect on murine limb development in vitro at concentrations that are achieved under therapeutic conditions (peak concentrations approx. 1-5 mg/l in plasma). Effects at higher concentrations (60 and 100 mg/l) are slightly enhanced (factor 2) if the magnesium concentration in the medium is low. Macroscopically, limbs develop regularly in a magnesium-free medium, but ultrastructurally typical alterations are exhibited (e.g. cell necrosis and pericellular deposition of electron-dense material).     

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.     

Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.     

Ethoxyresorufin-O-deethylase (EROD) inducing potencies of planar chlorinated aromatic hydrocarbons in primary cultures of hepatocytes from different developmental stages of the chicken

In vitro induction of ethoxyresorufin O-deethylase (EROD) activity in cell cultures is an extensively validated tool for measuring overall potencies of mixtures of halogenated aromatic hydrocarbons (HAHs) in samples from the abiotic or biotic environment. For risk assessment with special attention to effects in wild birds, an assay was developed that makes use of chicken embryo hepatocytes. However, it was questioned whether compound-specific responses are consistent at the various developmental stages. The results of our present study show that there are considerable differences between early and late embryonal and post-hatching stages. The induction of EROD was measured in primary chicken hepatocyte cultures. The cells were isolated at day 14 and day 19 of embryonal development and at day 1 post hatching. Hepatocytes were exposed in vitro to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126, IUPAC nomenclature) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118). The respective compounds were chosen as representatives for dioxins, furans, non-ortho PCBs, and mono-ortho PCBs. These groups of chemicals have been identified as environmental contaminants with major dioxin-like effects that are mediated by a common receptor, the arylhydrocarbon (Ah) receptor. At all developmental stages, TCDF was more potent than TCDD. Relative potencies (RP = EC50TCDD/EC50HAH) decreased in the order TCDF < TCDD < PCB 126 < PCB 118. Depending on the developmental stage, TCDF was 1.2 to 3.4 times more potent than TCDD. PCB 126 was equipotent or less potent by a factor of 3 than TCDD. PCB 118 was 100 to 300 times less potent than TCDD. Both the mean effective concentration (EC50) and the maximum EROD activity (Ymax) of all compounds were lower in hepatocyte cultures from 14-day-old embryos than those from 19-day-old embryos or 1-day-old hatchlings. RPs were comparable in 19-day-old embryos and in hatchlings, but significantly different in 14-day-old embryos.