Relative growth rates of maxillary mesenchyme in the chick embryo

Chick embryos were injected with [3H]-thymidine at days 3-7 of incubation and were fixed and embedded in plastic. The embryos were divided into three stage groupings of development [Hamburger and Hamilton: J Morphol 88:49-92, 1951], and labeling indices were determined for each of the following delineated regions within the maxillary process at each stage: region 1, subepithelial mesenchyme located at the medial side of the maxillary process adjacent to the roof of the stomodeum; region 2, subepithelial mesenchyme at the ventral tip of the maxillary process (as seen on cross section); region 3, subepithelial mesenchyme at the lateral portion of the maxillary process below the eye; and region 4, interior mesenchyme defined as the central portion of the maxillary process and separated from the epithelium by the three other regions. Results indicated that differences exist among the regions examined and that these differences were stage specific. At stages 19-21 and stages 24-25 1/2, growth rates were higher in subepithelial regions than interiorly. At stages 28-29, however, a statistically significant difference among the regions was not found. These results suggested that there is an association between growth rates in the maxillary process mesenchyme and its proximity to the overlying epithelium and that these effects are related to the stage of development.     

Developmental deficiencies of the upper facial skeleton due to partial elimination of mesencephalic neural crest cells in the chick embryo

With the aim to test the hypothesis that cells derived from the mesencephalic portion of the neural crest, are involved in the process of differentiation of various upper facial bones, in 41 chick embryos of the 6-somite stage (approx. 26 hours of incubation) the anterior and middle thirds of this part of the neural crest were partially eliminated by micro-laser irradiation, either unilaterally or bilaterally. Of the 14 embryos sacrificed at the age of 12 days, a number of 6 proved to have developed harelip and/or cleft palate conditions. In these embryos, in addition a reduction or absence of the maxillary, palatal, jugale and quadrato-jugale was observed. On the contrary, other facial bones as well as the first and second branchial arch cartilages proved to have developed normally. From these results the conclusion may be drawn that (a sufficient number of) cells from the anterior and middle thirds of the mesencephalic neural crest are indispensable for a normal differentiation of the maxillary, palatal, jugale and quadrato-jugale.     

Lithium Chloride and Trypan Blue Induce Abnormal Morphogenesis by Suppressing Cell Population Growth

Full primitive streak stage chick embryos were cultured in vitro for 20 hrs and monitored every 4 hr for morphology, cell number and blastoderm area. In normal embryos, the cell population growth is exponential and correlates directly with Increasing morphological rank. The chick blastoderm area expands in two waves, one immediately after gastrulation and another after 16 hr in culture, while cell population growth is predominant between 4–16 hr. Trypan blue and LiCI inhibit cell population growth, epiboly and shaping of organ primordia. Both teratogens induce a similar spectrum of abnormalities although the severity of abnormal development is greater with LiCl for the given dose. In most abnormal embryos the cell population size and blastoderm area are inhibited most, which is detectable already after 12 hr of culture. We have established that the cell population growth, morphogenesis and area expansion constitute a parametric hierarchy with the cell population growth as the most independent parameter in regulating normal morphogenesis.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

Silver Staining, Featuring Rapid Reduction, For Whole Mounts of Retina and Optic Pathways in Chick Embryos

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5-7 hr with hourly changes of distilled water or with running tap water for 4-6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16-48 hr. (4) Impregnate with 1.5% AgNO₃ for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2-5 sec. Wash the eye with distilled water 30-60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20-30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.     

Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy

Isotretinoin ingestion during the first trimester of human pregnancy can induce malformations of the skull, ears, face, central nervous system, eyes, palate, lungs, circulatory system, limbs, and digits. A single oral dose of isotretinoin on day 8 of gestation in hamsters induces a similar syndrome of congenital malformation. The present study concerned scanning electron microscopic (SEM) observation of embryonic and fetal hamster craniofacial structures at 4, 8, 12, 24, 48, and 72 hr after administration of an oral dose of 50 mg/kg isotretinoin or an equivalent volume of the vehicle. The variability in development among control embryos recovered 4 hr after treatment precluded objective assessment of pathologic change by SEM at very early time points. Craniofacial damage was obvious within 8-12 hr of isotretinoin treatment, and it included hypoplasia of the maxillary and mandibular processes of the first branchial arch, a rudimentary second arch, and apparent collapse of the forebrain. Equivalent fusion between the lateral nasal process and the maxillary process and between the medial nasal process and the maxillary process in treated and control embryos accounts for the very low incidence of cleft lip observed in fetuses. The terminal microstomia was not associated with excessive merging or overgrowth of the first arch components. Hypoplasia of the first arch can account for retinoid-induced macrostomia and microstomia.     

Growth and differentiation schedule of mouse embryos obtained from delayed matings.

We previously showed that digit formation in mouse embryos from early morning mating seemed to progress faster than those from overnight mating. In this study, to confirm this phenomenon, we examine whether the embryos from normal (0 hr from ovulation to fertilization) and delayed matings (3, 6, or 9 hr from ovulation to fertilization) respond differently to some acute teratogens when they are treated at the same time point from mating. Five mg/kg of cytosine arabinoside (Ara-C) was given to pregnant mice intraperitoneally at 246, 249, or 252 hr (day of gestation (dg) 10) after mating. The patterns of Ara-C induced digit malformations in embryos from the delayed mating groups were those of more advanced stages, when compared with normal mating groups with the same time intervals from mating to Ara-C treatment. In other words, oocytes fertilized up to 9 hr after the presumed time of ovulation could grow similarly to those of normally fertilized oocytes. This catch-up phenomenon suggests that the ovulation clock should be used for the startpoint of the time scale of the growth and differentiation of embryos rather than fertilization clock.     

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.     

Vegetalization of Sea Urchin Larvae Induced with Cycloheximide

The embryos, kept at 20°C for 3 hr–6 hr from the time of fertilization (at the morula stage), were cultured in sea water containing cycloheximide (10–16 mM) for successive 3 hr and then transferred to normal sea water. The embryos, thus treated, became vegetalized larvae. With the same treatment performed at a developmental stage prior to 3 hr of fertilization, most of embryos developed to small blastulae filled with mesenchyme-like cells. The treatment at a stage after 6 hr of fertilization yielded normal plutei. From the embryos exposed to both ¹⁴C-leucine and ³H-thymidine during the treatment, labelled chromatin was isolated. Only in the presumptive vegetalized embryos obtained by the cycloheximide treatment of morulae, ratio of ¹⁴C-radioactivity found in proteins of chromatin to ³H-radioactivity in DNA was markedly lower than that observed in chromatin from control embryos. The rate of ³H-radioactivity-decrease by DNase I treatment was higher in chromatin isolated from the presumptive regetalized embryos than that observed in chromatin isolated from control ones. Probable failure of chromatin structure formation, due to cycloheximide-inhibition of chromatin protein synthesis, seems to disturb the determination in the embryos at the morula stage, resulting in an induction of vegetalized embryos.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Survival of 4-cell mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing

Four-cell mouse embryos obtained by fertilization in vitro were frozen ultrarapidly, immediately after one-step exposure to a highly concentrated solution (modified VS1) at room temperature, and later thawed in a 37 degrees C waterbath. The proportion of morphologically normal embryos on thawing was 95.7% (198/207), and 83.8% (166/198) of these embryos developed to morulas and early blastocysts after 24 hr in culture. All frozen-thawed embryos that developed to morulas and early blastocysts were transferred to the uterine horns of recipients on day 3 of pseudopregnancy and 47.0% (78/166) of transferred embryos developed to normal young.     

Preimplantation embryo development in the mouse: Role of histidine decarboxylase

The present study determines the effect of a specific and an irreversible inhibitor of histidine decarboxylase (HDC), α-fluoromethylhistidine (α-FMH) on the mouse preimplantation embryo development in vitro. The embryo culture technique was used to assess the effect of α-FMH. Embryos recovered at 0800–0900 hr (AM) on day 3 of pregnancy were 4–8 cells, whereas those recovered at 1600–1630 hr were mostly 8-cell compacted embryos. Of the day 3-AM embryos, 81.3 ± 4.3% developed to blastocysts within 48 hr when cultured in the medium alone, but addition of α-FMH (0.19 or 0.38 mM) drastically reduced the blastocyst formation to 26.6 ± 7 or 16.8 ± 4.3%. Most of them were arrested before the compaction stage. Addition of L-histidine, the substrate for HDC, did not alter the inhibition of blastocyst formation in the presence of α-FMH (37.2 ± 10.9%). Of the day 3-PM embryos, 99.3 ± 0.7% developed to blastocyst stage when cultured in the medium alone and addition of α-FMH (0.19 or 0.38 mM) did not affect the embryo development (92.1 ± 4.3 or 81.9 ± 9.9% developed to blastocysts). The birth of healthy young following transfer of these blastocysts into pseudopregnant mice indicates normal development of the embryos under this condition. The results suggest that histamine synthesis may be required for the process of compaction and thus the formation of blastocyst.     

APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC AND CHEMICAL TREATMENT

Embryos of the brown alga Fucus vesiculosas L. were grown as populations in glass petri dishes in seawater at 15 C in continuous low-intensity unilateral fluorescent illumination for periods up to 2 weeks. A quantitative estimate of increase in nuclear number was made from acetocarmine squash preparations of samples taken at 12-or-24 hr intervals. Over the period of 2-6 days embryos showed a doubling time of about 12-18 hr. Under normal seawater culture conditions each embryo formed a single rhizoid. When grown in seawater supplemented with sugar concentrations above 0.4 m , Fucus embryos developed as multicellular spherical embryos lacking rhizoids. In 0.6 m sucrose-seawater, 97% of the embryos were apolar at 2 days; only 37% were apolar at 4 days, many having recovered from the sucrose inhibition. Some embryos remained apolar after growth in 0.6 m sucrose for 2 weeks. Nuclear counts showed that sucrose-seawater markedly inhibited the rate of cell division. Other sugars including D-glucose, D-fructose, D-galactose and the sugar alcohol D-mannitol were also effective. When apolar embryos grown in sucrose-seawater were returned to seawater, embryo growth resumed at the normal seawater rate, judged from nuclear counts. Such embryos formed multiple rhizoids, varying from two to eight rhizoids per embryo, which developed on the embryo quadrant or half away from the unilateral light. Each of the multiple rhizoids originated from a single small cell in the periphery of the multicellular spherica embryo. Thus the rhizoid-forming stimulus apparently had been subdivided among a number of the cells of the apolar embryos. The implications of this finding are discussed. Attempts to produce multiple rhizoids by treatment of embryos with indoleacetic acid or 2,4-dichlorophen-oxyacetic acid failed. However, embryos treated with 10⁻⁴ M or 5 × 10⁻⁵ m 2,3,5-triiodobenzoic acid formed 40 and 30% multiple rhizoids, respectively, suggesting that some chemical, perhaps hormonal, mechanism is involved in polarization and rhizoid initiation in Fucus embryogenesis.     

Improved development of rat embryos in culture during the period of craniofacial morphogenesis

To study the mammalian craniofacial development, the culture conditions of rat whole embryo during the period of major craniofacial morphogenesis were examined. The improved rotating apparatus which is gassed continuously was used. Rat embryos explanted at 11.5 days (plug day 0) developed in vitro for up to 72 hr, that is, throughout the period of major craniofacial morphogenesis, and cultured embryos showed normal facial formation. The medium was equilibrated with a gas mixture of 95% 02, 5% CO2. The 100% rat serum improved the protein content of embryos cultured for 48 hr compared with the medium consisting of 50% rat serum and 50% Tyrode solution, although somite number was not altered. Furthermore, 100% rat serum containing 2 mg/ml glucose was the best medium for supporting growth of embryos when it was measured by protein content. Thus, the best culture medium was pure rat serum containing 50 units/ml penicillin, 50 micrograms/ml streptomycin, and 2 mg/ml glucose. Protein content, body weight, craniofacial formation, and somite number of embryos cultured for 48 hr with continuous gassing were much better than those cultured with noncontinuous gassing.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Cryopreservation of mouse half-morulae and chimeric embryos by vitrification.

Mouse half-morulae were cryopreserved less than or equal to 1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2-propanediol as cryoprotectant. The developmental rates of the frozen-thawed half-embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty-one of the half-embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half-morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen-thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half-morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half-embryos of the components.     

The effect of stratification on in vitro protein synthesis in seeds of Pinus lambertiana

Incorporation of ¹⁴C-phenylalanine in $\text{in vitro}$ systems from sugar pine ($\text{Pinus lambertiana}$) seeds was studied. Embryo ribosomes from both dry and stratified seeds supported incorporation (431 and 326 pmoles, respectively, of phenylalanine per mg ribosome) when combined with an embryo pH 5 fraction from stratified seeds. Female gametophyte ribosomes from dry seeds were active (302 pmoles phenylalanine incorporated per mg ribosome) but lost 61 percent of their capacity to support protein synthesis after 35 hours' stratification. The pH 5 fraction from embryos increased in capacity to support incorporation as stratification progressed up to 60 days (398 pmoles phenylalanine per mg ribosome when ribosomes were from 90-day stratified embryos) while the pH 5 fraction from female gametophytes was never active.     

Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the trophoblast cells. These are believed to be involved in facilitating the accumulation of fluid into the blastocyst cavity. We have investigated the ability of mouse embryos to regulate AQP gene expression in response to different treatments expected to affect the passage of water across the trophoblast cells using real-time PCR. In the first experiment 8-cell embryos were allowed to develop to blastocysts in media from 300 to 400 mOsm. Blastocyst formation was unaffected by media made hyperosmolar by glycerol, whereas blastocyst formation was significantly reduced in sucrose-based 350 and 400 mOsm media. AQP 8 mRNA levels were reduced when embryos were cultured in glycerol-based hyperosmolar media. The mRNA levels of AQP 3, 7, 9, and 11 were not significantly affected by hyperosmolar media. In the second experiment blastocysts were punctured (0 hr) and allowed to re-expand. AQP mRNA levels were examined after 2, 6, and 10 hr. Compared to control embryos, the expression of AQP 3, 7, and 9 were upregulated after 2 hr. Upregulation was sustained only for AQP 9 and this was sustained up to 6 and 10 hr after puncture. In the third experiment we compared expression of AQPs between in vitro cultured and in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.     

Freezing of bovine embryos: Effects of embryo quality,time from thawing to transfer and number frozen per vial

Over a ten-month period 736 embryos were collected from 103 cows induced to superovulate. Embryos were frozen in 10% glycerol and, after thawing, 655 appeared viable and were transferred nonsurgically to synchronous recipients. When embryos did not appear degenerative before freezing and transfers were performed within 3 hr of thawing, 40% of the embryos resulted in pregnancies. If embryos showed signs of degeneration before freezing, only 27% resulted in pregnancies after thawing and transfer. Similarly, when non-degenerative embryos were kept in Casou straws for more than 3 hr after thawing, fewer produced pregnancies (25%) following transfer. The number of embryos frozen in each vial ranged from one to 18 and this had no significant effect on success. By selecting embryos to be frozen and transferring them soon after thawing, the proportion of embryos surviving the freeze-thaw process can be very high.