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PubMed2003

Deviation of the blastocyst axis from the first cleavage plane does not affect the quality of mouse postimplantation development.

Several researchers have suggested recently that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst is orthogonal to the first cleavage plane of the two-cell embryo. To determine the universality of this relationship, we used embryos of two different genotypes, F1 (C57BL/6 x DBA/2) and CD-1. The position of the first cleavage plane in the early blastocyst was determined by labeling a blastomere with the fluorescent lineage tracer DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) at the two-cell stage. Approximately one quarter of the blastocysts from both genotypes possessed an Em-Ab axis that respected the orthogonal relationship with the first cleavage plane. However, the remainder of the blastocysts deviated from the orthogonal relationship. This result indicates that the orthogonal orientation of the Em-Ab axis to the first cleavage plane is not a universal phenomenon. We also tested whether the angular relationship between the Em-Ab axis and first cleavage plane influences postimplantation embryo development. We sorted the blastocysts that had the Em-Ab axis orthogonal to the first cleavage plane from the ones that did not. These two types of blastocysts were transferred separately into surrogates, and fetal development was examined in late gestation. The results revealed that both types of blastocysts produced normal fetuses at a similar frequency. Thus, the relationship of the blastocyst axis to the first cleavage plane does not significantly influence later development.

Animals↗

PubMed1984

Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer.

The birth of a rhesus monkey resulting from in vitro fertilization is reported. Oocytes recovered at laparoscopy from five gonadotropin-stimulated donors were inseminated in vitro with sperm preincubated with caffeine and dibutyryl cyclic AMP. After insemination, oocytes were cultured for 33-46 hr. Twenty-two embryos were transferred nonsurgically into 11 recipient females. One recipient showed signs of implantation but did not carry to term. A second female became pregnant after receiving one 4-cell and one 6-cell embryo fertilized in vitro. The subsequent course of early pregnancy and embryonic and fetal development were characteristic of normal singleton pregnancies. A healthy term male infant was delivered by Cesarean section 176 days after fertilization. This birth has validated our procedures for in vitro fertilization of rhesus monkey gametes and provides an experimental model for studies of early embryonic development in primates.

Animals↗

PubMed2003

Maternal-fetal distribution and prenatal toxicity of 2,2,4-trimethyl-1,2-dihydroquinoline in the rat.

BACKGROUND: Polnoks R (poly-2,2,4-trimethyl-1,2-dihydroquinoline) is used as an antioxidant in elastomer processing. It is an embryotoxic and fetotoxic agent. This chemical given per os to female rats induces also teratogenic effect but only at doses toxic to the mother. The aim of the study was to evaluate prenatal development and tissue distribution in rats exposed to 2,2,4-trimethyl-1,2-dihydroquinoline (TMDHQ), a monomer of Polnoks R. METHODS: Females were exposed orally to unlabeled TMDHQ during organogenesis at doses 50-400 mg/kg to asses prenatal toxicity and to radiolabeled 14C monomer at a dose 210 mg/kg to evaluate tissues distribution. RESULTS: TMDHQ administered to pregnant females per os at doses 100 mg/kg and higher produced teratogenic effect (cleft palate, wavy ribs, kyphoscoliosis, exencephaly, external hydrocephalus, hydronephrosis, and renal hypoplasia). Peak 14C-radioactivity was found in mothers' plasma about 10 hr after administration of this compound at dose 210 mg/kg. The accretion of 14C proceeded with a kinetic constant of 0.35 hr(-1) and a half-life of 53.3 hr. Kidneys are the main organs of monomer excretion. The highest concentration of 14C in maternal tissues 24 hr after oral dosing was found in adipose tissue, sciatic nerve, muscles, kidneys, and liver. Radiocarbon retention in fetuses was the highest in kidneys at all time points after dosing. CONCLUSIONS: This study has demonstrated that transplacental exposure to Polnoks R monomer is teratogenic in rats. 14C retention in placenta, amniotic fluid, and fetal tissues indicates that this compound or its metabolites penetrate into placenta to the fetus.

Abnormalities, Drug-Induced↗

PubMed1996

Developmental Modulation of a beta myosin heavy chain promoter-driven transgene.

The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

Adenosine Triphosphatases↗

PubMed1992

Prenatal growth pattern of the human maxilla.

Regarding maxillofacial morphogenesis there has been a long debate on the growth of the maxillary structure. Using 120 normal fetal maxillae of gestational ages from 16 to 41 weeks, palatal radiograms and frontal histologic sections were made. We have observed two pairs of accentuated growth areas in the fetal maxillae and named them primary growth centers to formulate the maxillary trapezoid (MT) by radiologic image. The MT is formed by four primary growth centers that are best demonstrated by palatal radiograms of the fetal maxilla as well as by frontal histologic sections. The dimensional increase in the MT during the fetal period is documented and statistically analyzed. From this series of results, we have suggested that the growth centers which demarcate the MT are the basic structures of the developing human maxilla. It was also found that the four primary growth centers are the most active sites for maxilla formation until 20 weeks of gestation and thereafter the growth of the maxilla is enhanced by the participation of the intramembranous bone formation along the periphery. This was in contrast to the central primary growth centers that have already finished maturation in the early fetal period and remain only as a peripherally radiating arrangement of thick trabecular bones.

Bone Development↗

PubMed1985

The internal milieu of the pregnant rat is not growth promoting for maternal tissues and it reduces growth of fetal paw transplants.

We have investigated whether the internal milieu of the pregnant female rat is growth promoting to fetal or adult tissues. Adult female littermates were divided into two groups, one of which remained virginal while the other was bred repeatedly. Four successive pregnancies did not affect skeletal growth (i.e. tail length increase) or BW gain. Three-month-old females were hypophysectomized (HX) or sham HX on day 13 of pregnancy. Age-matched virgins were treated similarly. Eight days after surgery, the width of the tibial epiphysial plate of the HX virgins had decreased from 146.2 +/- 5.6 mu to 98.4 +/- 2.5 mu. In the pregnant rats, the width of the plate in the shams was reduced to 120.8 +/- 7.6 mu, and hypophysectomy tended to further reduce its width to 103.2 +/- 8.4 mu. Paws from 14- to 15-day fetal rats were transplanted under the kidney capsule of adult nonpregnant or pregnant hosts and grown there for 6 or 11 days. In the nonpregnant hosts the paws grew substantially during both periods, but in gravid females their growth was inhibited by about 35%. Paws were also transplanted to the kidney of HX pregnant and HX nonpregnant females to determine whether the internal milieu of the gravid female retains more growth-promoting/growth-supporting activity after pituitary removal than does that of HX virgin hosts. In both types of HX hosts, transplant growth was inhibited to the same degree (65-70%) relative to growth in intact hosts. The growth-inhibiting properties of the internal environment of the gravid hosts were not due to ovarian steroids because injections of estradiol plus progesterone had no effect on transplant growth. We also tested the effects of placental transplants and placental extracts on the growth of transplanted paws in juvenile female hosts. Both placental treatments caused inhibition of transplant growth by 25-30%. Overall, our results indicate that the internal milieu of the pregnant rat is not somatotrophic for her tissues, and it actually suppresses growth of fetal tissue transplants. The placenta may be responsible for this growth-inhibitory effect.

Animals↗

PubMed1994

Reproductive capacity of the nucleus of the male gamete after completion of meiosis.

PURPOSE: Our purpose was to investigate the possibility of achieving fertilization and subsequent normal embryonic development by injecting round spermatid nuclei into rabbit oocytes. RESULTS: Two-to four-cell-stage embryos developed after round spermatid nuclear injections into rabbit ooplasma could further develop in vitro up to the expanding blastocyst stage or in vivo up to complete gestation. CONCLUSION: The current findings show that the haploid set of chromosomes of round spermatid can pair with the chromosomes of the ootid to participate in complete fertilization and subsequent embryonic and fetal development. In addition, we suggest that postmeiotic modifications of the round spermatid are not required for the pairing of male gamete chromosomes with those of the ootid.

Animals↗

PubMed2003

Abnormal regulation of DNA methyltransferase expression in cloned mouse embryos.

Cloning by somatic cell nuclear transfer is inefficient. This is evident in the significant attrition in the number of surviving cloned offspring at virtually all stages of embryonic and fetal development. We find that cloned preimplantation mouse embryos aberrantly express the somatic form of the Dnmt1 DNA (cytosine-5) methyltransferase, the expression of which is normally prevented by a posttranscriptional mechanism. Additionally, the maternal oocyte-derived Dnmt1o isoform undergoes little or none of its expected translocation to embryonic nuclei at the eight-cell stage. Such defects in the regulation of Dnmt1s and Dnmt1o expression and cytoplasmic-nuclear trafficking may prevent clones from completing essential early developmental events. Furthermore, aberrant Dnmt1 localization and expression may contribute to the defects in DNA methylation and the developmental abnormalities seen in cloned mammals.

Animals↗

PubMed1987

Studies on the growth of the fetal guinea pig. Effects of reduction in uterine blood flow on the plasma sulphation-promoting activity and on the concentration of insulin-like growth factors-I and -II.

The effects of prenatal growth restriction caused by uterine artery ligation at midgestation has been studied in pregnant guinea pigs. Ligation of a uterine artery at day 30 of pregnancy commonly caused a reduction in fetal growth of greater than 45% by days 40-65 of gestation. This was associated with substantial delays in the development of a number of fetal tissues and in particular that of the skeleton which remained cartilagenous for longer than normal. Hence normally by day 50 of pregnancy clear evidence of epiphyseal ossification in the long bones of the fore- and hindlimbs was present, but in growth retarded fetuses of less than 50% of normal size such evidence was sparce. Delayed skeletal development and the slowing of fetal growth rate correlated well with marked depression of plasma sulphation-promoting activity. Indeed plasma from fetuses that were less than 40% of normal size inhibited sulphate incorporation into pig costal cartilage. This indicated the presence of inhibitory factors in the plasma of such fetuses, an interpretation that was re-inforced by the observation that plasma IGF-II concentrations were 2-4 times above normal. In contrast plasma IGF-I concentration was depressed upto 50% by growth retardation in line with the fall in fetal plasma insulin concentration. The changes in plasma sulphation-promoting activity and of IGF-I are consistent with slowing of DNA, RNA and protein synthesis and of gene expression in tissues of the growth-retarded fetus. The elevated fetal plasma IGF-II concentration provided further evidence that in the fetal guinea pig this hormone has a potentially glyconeogenic action and maintains essential glycogen stores.

Animals↗

PubMed1998

Effects of the in vitro chemical environment during early embryogenesis on subsequent development.

The development of the preimplantation embryo seems morphologically very simple, and embryologists previously assumed that an embryo that developed to the blastocyst stage was fully capable of normal development after transfer to the uterus of a recipient female. This complacency was disturbed by reports that exposure of early embryos to mutagens such as methylnitrosourea led to fetal abnormalities, decreased birth rates, and decreased life-span. Even more disturbing are recent reports that culture of early embryos in supposedly benign conditions can adversely affect their subsequent development. Techniques have been developed for the production of cattle and sheep embryos by in-vitro fertilization and by cloning. Such embryos must be cultured for several days before they can be transferred, and, in some cases, this has been related to abortion, very high birthweight, physical abnormalities and peri-natal mortality of the calves and lambs. This syndrome may result from an unbalanced development of the trophoblast relative to the inner-cell mass, possibly related to the presence of serum, glucose, or ammonium in the culture medium. An analogous phenomenon has been observed in human in-vitro fertilization where babies from single pregnancies have below-normal birth-weight. There is also evidence to suggest that the in-vitro environment of the gametes before fertilization can affect subsequent embryonal and fetal development. Exposure of mouse oocytes to vitrification solutions has been shown to lead to fetal malformations, and treatment of bull sperm with glutathione improves early embryo development. The common thread in these diverse observations is that development can be affected by events that occur long before any defect is apparent. Consequently, the production of a morphologically normal embryo is no guarantee that fetal development and post-natal life will be normal. This is of immediate concern in human reproductive medicine due to the increasing use of sperm injection for fertilization, and the emergence of in-vitro oocyte maturation. Further development and application of reproductive techniques would benefit from a toxicological evaluation of risk factors and exposure limits.

Animals↗

PubMed1991

A teratologic evaluation of continuous-wave, daily ultrasound exposure in unanesthetized pregnant rats.

Pregnant Sprague-Dawley rats were trained to remain immobile when placed in water in an ultrasound exposure tank and exposed to 0, 0.1, 2.0, or 30.0 W/cm2 ISPTA (spatial peak, temporal average), 3.0-MHz continuous wave (cw) ultrasound on embryonic (E) days 4-19 for approximately 15 min/day. On E20 fetuses were removed; weighed; examined for external, skeletal, and visceral malformations; and uteri were examined for resorptions. Analyses revealed no increase in pre-implantation loss and no effects on maternal body weight, food, or water consumption. No increase in skeletal or visceral malformations was found, in fact exposed groups had a lower incidence of defects than controls. A significant increase in resorptions in the lowest exposure group (0.1 W/cm2) was obtained, but the effect was isolated, non-dose dependent and not credible as a treatment-related effect. No reduction in fetal weight was obtained, in fact the lowest (0.1-W/cm2) and middle (2.0-W/cm2) exposure level groups weighed slightly more than controls. The immobility procedure succeeded in avoiding anesthetization or forced restraint of the dams, thereby eliminating these factors as potential confounders. The results demonstrated that in unanesthetized, unrestrained rats in utero exposure to incident intensities of ultrasound of up to 30.0 W/cm2 cw ultrasound (or estimated internal exposures of 4-21 W/cm2, depending on body orientation to the incident beam) produced no evidence of embryotoxicity based on fetal necropsy data.

Analysis of Variance↗

PubMed1984

The effect of protein on preimplantation mouse embryo development in vitro.

The effects of supplementing culture medium with protein for the culture of mouse embryos from two cells to blastocysts were examined in vitro. The proportion of embryos developing was affected by the type of protein and concentration. The highest rates were obtained in protein-free medium, fetal calf serum, and A Grade bovine serum albumin at all concentrations tested (2-16 mg/ml). Reduced rates of embryo development were observed in proteins of human origin, particularly at the highest concentrations tested. Purification of human and bovine serum albumin resulted in a marked reduction of embryo development. Significantly more normal fetuses were found in pregnant mice receiving transferred embryos grown in protein free-medium than in medium containing fetal calf serum. It is concluded that protein supplementation of culture medium may adversely affect embryo development and viability. These observations could have important implications for human in vitro fertilization.

Animals↗

PubMed2006

Development of the protympanum.

The protympanum, the only opening of the tympanic cavity, has enormous significance in the pathogenesis of middle ear inflammation. In our study, we analyzed 70 macerated temporal bones stereomicroscopically to understand the development of the protympanum. Details of this embryonic process were attained using histological sections made in frontal and horizontal planes. In contrast to recent descriptions, the present work reveals that almost the entire protympanum develops from different processes of the petrous bone. The tympanic bone takes part in forming only the posterior border of the lateral wall of the protympanum. Consequently the tympanic plate, which forms the inferior half of the mandibular fossa, is formed by two anlages. The anterosuperior quarter is formed by the petrous bone and the remainder by the tympanic bone. The main structures of the medial wall of the protympanum are the carotid canal with the internal carotid artery (ICA). The carotid canal starts to develop as two laminae of the petrous bone in the 18th fetal week. In cases of incomplete closure of these laminae the carotid canal occupies dehiscences increasing the danger of injury to the ICA. The presence of the ICA is necessary for the development of the carotid canal. If the artery takes an aberrant pathway in the tympanic cavity, or is absent, there is no sign of the carotid canal.

Adult↗

PubMed1997

Developmental changes in the myocardial architecture of the chick.

BACKGROUND: Numerous studies describing myocardial architecture have been performed on the adult heart but considerably fewer have been made during embryonic or fetal development. To serve as a basis for interspecies comparison of ventricular morphology, and as a reference for studying the effects of experimental perturbations, we examined the development of chick throughout the entire incubation period. METHODS: Chick hearts from stage 14 (day 2) to stage 46 (day 21) were perfusion-fixed, and sectioned in transverse, frontal and sagittal planes. The ventricular myocardial architecture was examined and photographed in the scanning electron microscope. RESULTS: At embryonic stage 16 and earlier, the smooth-walled heart loop had an outer myocardial mantle, cardiac jelly, and endocardium. From stage 18, there was an outer compact and inner trabeculated myocardium. Trabeculated myocardium could be subdivided into the outer (basal) portion adjacent to the compact layer and the central (luminal) part. The outer basal layer could be distinguished from the inner luminal by shorter and finer trabeculae with small, round intertrabecular spaces. From stage 24, the patterns of trabeculae and intertrabecular spaces were ventricle-specific. Between stages 24 to 31, abundant trabeculations were present throughout both ventricular cavities. The trabeculae were initially radially arranged, but later adopted a spiral course, which persisted in a simplified form into adulthood. CONCLUSIONS: The ventricular myocardium undergoes distinctive morphogenesis, characterized by changes in trabecular patterning and orientation. We speculate that the embryonic trabecular architecture reflects the directions of the main stresses. Unlike fetal and adult hearts, which rely mostly on the compact myocardial layer, the trabeculae play a crucial role in the contractile function of the embryonic heart.

Animals↗

PubMed1994

Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves.

Bovine inner cell mass (ICM) cells were used as donor nuclei in nuclear transfer procedures to determine their totipotency. ICMs were isolated by immunosurgery from expanded Day 7-9 blastocysts that had been produced in vitro. Each individual ICM cell was transferred into an enucleated oocyte. Oocytes were checked for enucleation with Hoechst dye to ensure that all DNA was removed, thus eliminating the possibility of parthenogenetic development. ICM cell-oocyte units were fused by a brief electrical pulse (110 V DC, 15 microseconds in a 500-microns chamber), and the resulting zygotes were placed into CR1 bovine embryo culture medium supplemented with amino acids and fetal calf serum. The nuclear transfer embryos were scored for development to the blastocyst stage on Day 7 (day of fusion = 0). A total of 948 nuclear transfers were completed in 25 trials. In 12 of the trials, development to the blastocyst stage (5%, 30 of 629) was observed. This resulted in an overall developmental rate of 3% for all trials. Twenty-six of the ICM-derived blastocysts were transferred. The initial pregnancy rate at 30 days was 23% with six pregnant recipients. Two pregnancies were lost after 60 days, and four calves were born, two of which were stillborn. These results demonstrated that nucleic of ICM cells from expanded bovine blastocysts were pluripotent, if not in fact totipotent, since these nuclei after nuclear transfer to enucleated oocytes could direct embryonic and fetal development resulting in live offspring.

Animals↗

PubMed1995

Receptor tyrosine kinases and the regulation of hematopoiesis.

The ongoing production of mature blood cells during the lifetime of an animal is vital for survival. Hematopoiesis is the complex process whereby a small population of pluripotential stem cells give rise to mature cell types with specialized functions. The development of mature blood cells proceeds in a hierarchical fashion originating from a self-renewable stem cell population that gradually becomes committed to lineage-restricted differentiation. Two major themes concerning the regulation of hematopoiesis have emerged. First, the proliferation, survival and differentiation of immature progenitor cells depends on extracellular signals produced by cells within the hematopoietic microenvironment. Second, protein tyrosine phosphorylation is the major biochemical mechanism by which the extracellular signals are transmitted and interpreted. In this review we will concentrate on the role of receptors with intrinsic tyrosine kinase activity (RTKs) in hematopoietic regulation. RTKs form part of a highly conserved signaling mechanism that plays an important role in the development of evolutionary diverse organisms. During hematopoiesis, RTKs are a central component of the mechanism by which hematopoietic stem cells receive extracellular signals and interpret these signals to direct the lineage restricted differentiation of multipotential progenitors. In addition, RTKs may also play an important regulatory role in the ontogeny of the hematopoietic system during embryonic and fetal development. Taken together, RTKs are an important component of the mechanisms that regulate the development and behavior of hematopoietic stem cells.

Animals↗

PubMed1991

Dynamic histomorphometric evaluation of human fetal bone formation.

We have evaluated dynamic and static parameters of bone formation in femoral metaphyses collected from two human fetuses at 19 weeks of gestation. Tetracycline was administered to the mother at set intervals (2-5-2 day schedule) before interruption of pregnancy. Labels were distinct and sharply linear, suggesting a well organized calcification front at this early stage of mineralization. Mineral apposition rate (MAR) was fastest (4.1 +/- 0.3 microns/d) in the periosteal (Ps) envelope, and about half that value in the endosteal envelopes (endocortical: 2.5 +/- 0.1, cancellous 2.1 +/- 0.1 microns/d). Because cellular activities may vary throughout the metaphyseal area, sections were arbitrarily separated in 0.75 mm layers starting from the growth plate. Three measured parameters decreased rapidly with increasing distance from the physis: Ps MAR: 4.9 to 2.3 microns/d, trabecular osteoid thickness: 5.9 to 1.2 microns, and cartilage volume (CgV/TV): 5.4% to 1.2%. Others did not vary significantly along the metaphysis. Comparison of several static parameters with those measured in five autopsy specimens from full-term infants showed that bone and cartilage volume, and trabecular thickness increased while osteoid thickness and parameters of resorption decreased in the second half of the gestation period. The study indicates that fetal bone matrix mineralization is already highly organized at mid-gestation, and validates the use of histomorphometry to assess bone maturation during early skeletal development.

Bone Development↗

PubMed1978

Cell ploidy in molar placental disease.

Molar diseases of the placenta is associated with cystic change in the villi. The cysts may be from 5-20+ mm in diameter. This disease has been described in association with triploid and diploid cell lines and with and without an accompanying embryo or fetus. It may be followed by malignant change and invasive chorio-carcinoma. In order to investigate the association between cell ploidy, embryonic development and subsequent malignancy, a detailed study of 30 conceptuses with molar disease was made, with the accompanying maternal history and follow-up. The cell ploidy was determined by measurement of nuclei by a cytoscan light microscope connected to a computer program as has been previously described. Diploid cell lines were not found with embryonic or fetal development. Triploid cell lines were always associated with an embryo or fetus. Triploidy is not associated with hyperplastic changes in the trophoblast. These results are presented and discussed.

Aneuploidy↗