Quantitative change in fibronectin in cultured müllerian mesenchymal cells in response to diethylstilbestrol and müllerian-inhibiting substance

I report on the synthesis of fibronectin in the developing chick Müllerian duct mesenchymal cells. Before the differentiation of female chick Müllerian duct, the amount of fibronectin in the cells of the right duct is 44% lower than in the left duct. While after differentiation, the amount of fibronectin in the right duct is 29% lower, as compared to the left duct. Estrogenic hormone diethylstilbestrol (DES) treatment was carried out at the 5th day of incubation when both female Müllerian ducts were undifferentiated. Three days after DES treatment, the regression of the right duct was prevented, and the amount of fibronectin was induced by 89%, while induction in the left duct was 11%. Eight days after DES administration, the amount of fibronectin in the right and left Müllerian duct was induced by 150 and 76%, respectively. After DES treatment in the male embryo, both Müllerian ducts were retained, and the capacity for fibronectin synthesis was preserved. Application of the indirect immunocytochemical labeling technique revealed Müllerian-inhibiting substance (MIS) binding sites on the membrane of the Müllerian mesenchymal cells. The addition of chick MIS in the culture medium reduced the amount of detectable fibronectin in the cultured mesenchymal cells. The synthesis of fibronectin in intestinal mesenchymal cells was not affected by DES or MIS.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.     

Intraneural vascular response to defective development of the chick mesencephalon

The development of intraneural vessels was studied in response to an induced hypermorphosis of neural tissue inthe midbrains of 38 chick embryos ranging in age from three days through 14 days of incubation. The pattern of vascularization was compared with that of normal chick embryos at comparable stages of development. In the experimental embryos, the increase in mitotic figures along the ventricular borders of the mesencephalon is accompanied by the establishment of an endoneural plexus approximately one day earlier than is the case during normal vascularization of the midbrain. This plexus also penetrates more deeply and extensively into the ependymal layer. Surface vessels and intraneural vascular elements are dilated, and the cerebrospinal fluid contains varying amounts of blood released from large intraneural vessels which protrude into the ventricle. The most prominent cerebrovascular effects seem to occur between the fourth and eighth days of incubation. Thereafter, the cerebrovascular pattern becomes more normal except for relatively few isolated hemorrhagic areas.     

In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage.

The sequential processes of in vitro development of whole mouse embryos were classified by stages according to the in vivo criteria of E. Witschi (1972, “Biology Data Book,” Part II: “Rat,” L. Altman and D. S. Dittmer, eds., 2nd ed., Vol. 1, pp. 178–180, Federation of American Societies for Experimental Biology, Bethesda, Md.) and K. Theiler (1972, “The House Mouse,” Springer-Verlag, Berlin/New York). The mouse embryos which developed in vitro in each day of culture were then classified into stages according to the characteristics of mouse embryos developed in vivo. A series of 10 blastocysts were inoculated into 35-mm plastic culture dishes (30–50 blastocysts per experiment). Developing embryos were scored on the fourth, sixth, and eighth days and classified into stages. Among the total of 118 blastocysts cultured in three repeated experiments, 100 mouse embryos had attached and developed in culture dishes. Ninety-four percent of the attached mouse embryos developed to the early egg cylinder stage after 4 days of incubation, and 87% grew to the stage of late egg cylinder after 6 days of culture. An average of 62% of the embryos reached the early somite stage with heart beating after 8 days in culture with frequent medium change. In two separate experiments single mouse blastocysts were placed individually in culture dishes in 2 ml of culture medium. The development of each embryo was followed every day. Each of 10 blastocysts had attached in its respective culture dish and had developed to the early egg cylinder stage after 4 days of culture. About 50 to 70% of each of these 20 individually isolated mouse embryos developed in vitro to the early somite stage after 8 days of culture.     

Production of vigorous,desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor

Summary This report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20–50 M abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.Plant Research Centre contribution No. 1523     

Insulin synthesis in chick embryo retinas during development

Retinas of chick embryos contain insulin (1) and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th–18th day) with [³H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14–15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [³H]insulin was released from explants into the medium. This was evident also after 6–8 days in culture.     

In vitro culture and interferon-tau secretion by ovine blastocysts

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-τ) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO₂ in air or DMEM without BSA in 5% CO₂, 7% O₂, and 88% N₂ at 39 °C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-τ secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0±50.25 μm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357±43.75 μm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038±155.8 μm. An anti-viral assay and a ELISA for IFN-τ were developed. There was a considerable variation in the time of onset and amount of IFN-τ secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-τ after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-τ after 1 day in culture. The mean concentration of IFN-τ secreted by day-8 embryos after 1 day in culture (10.99±2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8±1.75 ng/ml).     

Influence of egg size on embryos and larvae of Fundulus heteroclitus (L.)

Egg size for Fundulus heteroclitus (L.) populations is concordant with the distribution of the two F . heteroclitus subspecies, i.e. F. h. heteroclitus eggs are considerably larger than F. h. macrolepidotus eggs. The influence of egg size on survival of embryos during incubation and survival and growth of newly-hatched larvae was estimated for four populations representing both subspecies along the Atlantic coast of the United States and in Delaware Bay. Survival of embryos was determined for incubation periods of 14, 21 and 28 days. Greatest differences in survival were detected following the longest incubation period where less than 50 per cent of the smaller F. h. macrolepidotus eggs survived while little or no mortality was detected among the larger F. h. heteroclitus eggs . Influence of egg size on larval survival was also greatest among those larvae hatched after 28 days where F. h. macrolepidotus larvae survived without food, for an average of 6 days, while F. h. heteroclitus larvae survived 11–12 days. F. h. heteroclitus larvae were significantly larger at hatching than F. h. macrolepidotus larvae. Larval growth rates were the same (0.4 mm day⁻¹) in both subspecies. As a result, size differences at hatching were still maintained after 42 days of growth. The differences in egg size along with other morphological and reproductive characteristics of F. heteroclitus populations probably represent genetically based adaptations to environmental conditions, of which the length of the spawning season is one of the major components stimulating the coevolution of these traits.     

Hypoxic adaptations of hemoglobin in Tibetan chick embryo: high oxygen-affinity mutation and selective expression

Tibetan chicks (Gallus gallus) survived with high hatchability (35.0%) and Recessive White Feather broilers (RWF) from low elevations survived rarely and with a low hatchability (3.0%) after simulated incubation under hypoxia of 13% O2. The functional mutation of Met-32D(B13)-Leu of alpha(D) globin chain was related with hypoxia based on allele distribution, homology model building and oxygen affinity assay. Whole embryos on days 3-8 and whole blood on days 9-18 were collected to investigate the stage expression profiles of all seven globins and HIF-1alpha by real-time PCR. Under hypoxia (12.0% O2) on days 3-8, HbE was overexpressed, HbA was expressed earlier and HbP expression was restricted, which completely overturned the expression profile under normoxia. The amount of hemoglobin expression in Tibetan chicks was remarkably higher than that of RWF. HIF-1alpha expression peaked early in both breeds, with. In conclusion, the special hypoxic expression profile on days 3-8 certainly is a common molecular mechanism of hypoxia tolerance in surviving Tibetan chick and RWF embryos; the mutation Met-32D(B13)-Leu and increasing hemoglobins are important mechanisms of hypoxia adaptation in Tibetan chick embryos, and we suggest that HIF-1alpha could be responsible for the hypoxic expression profile.     

Teratogenic effects of dilantin on thoraco-abdominal organs of developing chick embryos

Congenital anomalies on some viscera like heart, liver and kidney have been investigated in chick embryos after a single injection of dilantin (3 mg/egg), a known antiepileptic drug, on 4th day of incubation. On 19th day of incubation, chick embryos were collected to observe the gross malformations and histological changes in heart, liver and kidney. On gross examination, visceroptosis (29%), thin anterior abdominal wall (28%), ectopia cordis (10%) and dextrocardia (1%) were observed. Histological examination of the kidney revealed glomerular degeneration in kidney while in liver, dilated central veins with degenerated hepatocytes were present. Longitudinal section of the heart showed thicker musculature specially of ventricles with a narrower lumen in comparison to that of the control. The results indicate teratogenicity of dilantin in developing chick embryos.     

In vitro reformation of the perichondrium from perichondrial-free Meckel's cartilages of the embryonic chick

Perichondria were removed from Meckel's cartilages of chick embryos of Hamburger and Hamilton stages 34, 38, or 39 (8, 12, or 13 days of incubation) and cultured, either at the air-medium interface or submerged, under standard organ culture conditions, for 7 to 21 days. Meckel's cartilages formed a new fibrous perichondrium by the 10th day of culture. Perichondria both formed earlier and were thicker in those cartilages cultured at the air-medium interface than in those cultured submerged. Histological and ultrastructural analysis indicated that the outermost layer of Meckelian chondrocytes dedifferentiated into fibrous cells to form the new fibrous perichondrium; i.e., the fibrous perichondrium can arise from superficial chondrocytes.     

Development of spontaneous motility in chick embryos. The spinal and supraspinal component of the inhibitory effect of glycine and GABA.

Development of the effect of glycine and gamma-aminobutyric acid [GABA] on spontaneous motility was studied in 11- to 19-day-old chick embryos under normal conditions and after acute and chronic decapitation. Chronic decapitation was performed on the 2nd day of incubation. Glycine (100 mg/kg egg weight) and GABA (103 mg/kg egg weight) (applied onto the shell membrane) demonstrably inhibited spontaneous motility only from the 15th day of incubation, the inhibitory effect increasing with the embryo's age. When administered together in half doses, glycine and GABA completely inhibited spontaneous motility for the first time in 19-day-old embryos. Neither amino acid influenced depression of motility immediately after decapitation, but 24 and 48 hours after, in 17- and 19-day-old embryos, they had a paradoxical effect, i.e. they transiently activated motor activity and even caused motor paroxysms. After chronic decapitation, both glycine and GABA again had a mild, protracted inhibitory effect. A comparison of spontaneous motility in normal and chronically decapitated embryos showed that the role of supraspinal factors in spinal motor output increases significantly with development of the chick embryo from the 15th day of incubation and that inhibition of these supraspinal factors plays the decisive role in the effect of glycine and GABA.     

Pulmonary stenosis with ventricular septal defect, common aorticopulmonary trunk, and dextroposition of the aorta: morphologic and qualitative physiologic effects in caffeine-treated chick embryos

Effects of caffeine administration to Hamburger-Hamilton stage 19 chick embryos (3 days of incubation) were investigated. A morphologic study of the effect of caffeine on cardiogenesis showed that caffeine produced total cardiac malformations in the chick in a dose-related fashion. A maximum frequency of 70.6% was observed with 4.7 mg caffeine. Major malformations included common aorticopulmonary trunk and dextroposition of the aorta accompanied by ventricular septal defect with/without pulmonary stenosis. Qualitative analysis of cinegraphs following exposure of embryos to a single teratogenic dose of caffeine (3.5 mg/egg) produced marked alterations in cardiac function when compared with chick Ringer's controls. Within 3 minutes after exposure to caffeine, dilation of the common ventricle and weak ventricular contractility were observed and persisted for 1 hour. Dose-response data and microcinematographic observations suggest that caffeine induced cardiac anomalies by a direct toxic effect on the embryo rather than by altering cardiac cell function. Our data also suggest that pathophysiologic changes in cardiac function may play an important role in the pathogenesis of caffeine-induced cardiac anomalies in the chick embryo.     

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.     

Opioid elements in development of the spontaneous motility of chick embryos

The effects of the acute and chronic administration of a pure opioid antagonist--naltrexone--was studied in chick embryos from the 4th to the 19th day of incubation. In acute administration, naltrexone (40 mg/kg egg weight) induced paroxysmal activation of spontaneous motility in both normal and spinal embryos from the 13th-15th day of incubation. Activation attained 3- to 4-fold the resting activity of chick embryos of the same ages. The chronic administration of naltrexone (7.46 +/- 1.18 mg/kg e.w. per 24 h) from the 4th to the 16th day of incubation was not manifested either in the embryos' somatic development or in the weight of the brain hemispheres, but it depressed the development of spontaneous motility to 26.1-75.8% of the activity of the control embryos. This developmental effect was not demonstrably correlated either to the length of time for which naltrexone was administered, or to when, in the course of incubation, it was administered to the chick embryos. The results are evaluated as evidence of the participation of opioid elements in the development and effectuation of central motor input functions in the early stages of ontogenetic development.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Volume contents volume 38 (1985)

Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l nahpthaleneacetic acid (NAA). Embryogenic responses were observed at cultured temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7–54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium.     

Antibodies to alpha-fetoprotein disrupt histogenesis in cultured chick retinae

alpha-Fetoprotein (AFP) is an oncofetal antigen believed to play an important role in normal development and carcinogenesis but very little is known about such a role. We have investigated here the role of AFP in neural retina development by selectively neutralising AFP in vitro. AFP has been immunohistochemically located in different cells and layers of the retina during its development, 4-day-old embryos being the earliest developmental stage when AFP is detected in the growing ganglion cell layer. Seven-day-retinae treated with antibodies to AFP in organ culture for 3 days did not continue to develop in the same way that they do in the egg. Neither the plexiform layers nor the buds of photoreceptor cells were observed after this culture period. In contrast, 7-day retinae cultured in the presence of non-immune serum developed in a manner similar to retinae in ovo. We present here the first evidence, derived by selectively blocking AFP in vitro with specific antibodies for an essential role of AFP in the normal development of the chick retina.     

The control of the differentiation of female embryonic germ cells in the bird

The left gonad from female chick embryos at 4–12 days of incubation was cultured in vitro as pieces of intact gonad, pieces of isolated cortex, and groups of pure germ cells. All cultures were maintained for a time equal to 17 days in ovo. At the end of the culture period, a cytological and quantitative study was made on the germ cells.The results show that some germ cells in pieces of intact 6-day gonad and pieces of 6-day cortex complete their normal developmental sequence and enter zygotene. This shows that the factors that control the differentiation of the germ cells reside in the cortex of the gonad and their expression does not depend upon the pituitary and the medullary estrogens after 6 days of incubation.Germ cells that are cultured as isolated cells do not attach to the tissue culture substrate, do not divide mitotically, and do not enter zygotene. Evidence is presented that suggests 12-day germ cells do enter zygotene when cultured with pieces of 12-day cortex. These data suggest the differentiation of the female germ cells is regulated by the somatic cells of the cortex.     

Changes of creatine concentration during development of chick embryo

Changes of creatine concentration during development of chick embryo. Acta physiol. pol., 1985, 36 (3): 208-215. Investigations were carried out on embryos of Star Cross chicken after 6, 8, 10, 12, 14, 16, 18 and 20 days of incubation. It was found that the concentration of total creatine increased from 0.18 to 0.67 mg/g of embryo weight. The increase of creatine concentration at the time of development of the embryo was proportional to the increase in nitrogen concentration, and the share of creatine nitrogen in the pool of total nitrogen was about 1% throughout the whole period of embryonal development and during the first two days after hatching. The amount of creatine in fresh egg and in the yolk sac of the newly hatched chicken was about 1.5 mg. It was estimated that chick embryos during their development synthesized, on the average, 18 mg (6 mMol) of creatine. The course of changes in creatine concentration in the developing chick embryo is very similar to the course of changes in the rate of heat production.