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PubMed2005

Type II keratins precede type I keratins during early embryonic development.

We and others have recently demonstrated that the keratin (K) gene family in mammals is even more complex than previously thought [Eur. J. Cell Biol. 83, 19-26]. To address the function of keratins during early development, precise information on their spatio-temporal expression is required. Here, we examined the expression of selected mouse keratins from pre-implantation to mid-gestational embryonic stages using RT-PCR and immunofluorescence. At E0.5, transcripts encoding K5, K6, K7, K8, K14, K15, K18, and K19 are apparently absent. We report on a post-transcriptional regulation of type I keratins, preventing filament formation in 8- to 16-cell stage embryos. In these embryos, mRNAs coding for K7, K8, K18, and K19 are present, but only K7 and K8 are translated into protein which is deposited in aggregates. Following the accumulation of K18 protein at E3.5, keratin filaments are formed. Delayed onset of type I keratin protein expression was additionally observed in later embryonic stages for K5 and K14. K5 protein expression starts in the forelimb surface ectoderm as early as E9.25, while the expression of its partner, K14, begins at E9.75. From E9.25 to E9.75, K5 forms atypical filaments with K18. Remarkably, in embryonic K5-/- mice, K14 formed normal filaments until E12.5 despite the absence of its partner K5, due to the presence of K8. Following periderm formation, K14-containing filaments disappeared and K14 became localized in aggregates in basal keratinocytes. Despite the absence of a keratin cytoskeleton, there was no cytolysis. We suggest that the formation of the first embryonic cytoskeleton from soluble keratins is regulated by unknown mechanisms. Whether the premature expression of type II keratins relates to their proposed role in TNF- and Fas-mediated signalling is presently unknown.

Animals↗
PubMed2005

Importance of sperm genotype (indicus versus taurus) for fertility and embryonic development at elevated temperatures.

Heat stress has negative effects on bovine reproduction, particularly for European breeds (Bos taurus taurus) that are less thermotolerant than zebu cattle (Bos taurus indicus). Here, the evidence that spermatozoa and oocyte both contribute to early embryonic resistance to heat shock is demonstrated. In addition, the use of reproductive biotechnologies to improve bovine thermotolerance, are outlined by comparing data from taurus, indicus and crossbred genotypes.

Animals↗
PubMed2005

Neurotoxic effects of alcohol and acetaldehyde during embryonic development.

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Expression of the glycinergic system during the course of embryonic development in the mouse spinal cord and its co-localization with GABA immunoreactivity.

To understand better the role of glycine and gamma-aminobutyric acid (GABA) in the mouse spinal cord during development, we previously described the ontogeny of GABA. Now, we present the ontogeny of glycine-immunoreactive (Gly-ir) somata and fibers, at brachial and lumbar levels, from embryonic day 11.5 (E11.5) to postnatal day 0 (P0). Spinal Gly-ir somata appeared at E12.5 in the ventral horn, with a higher density at the brachial level. They were intermingled with numerous Gly-ir fibers reaching the border of the marginal zone. By E13.5, at the brachial level, the number of Gly-ir perikarya sharply increased throughout the whole ventral horn, whereas the density of fibers declined in the marginal zone. In the dorsal horn, the first Gly-ir somata were then detected. From E13.5 to E16.5, at the brachial level, the density of Gly-ir cells remained stable in the ventral horn, and after E16.5 it decreased to reach a plateau. In the dorsal horn, the density of Gly-ir cells increased, and after E16.5 it remained stable. At the lumbar level, maximum expression was reached at E16.5 in both the ventral and dorsal horn. Finally, the co-localization of glycine and GABA was analyzed, in the ventral motor area, at E13.5, E15.5, and E17.5. The results showed that, regardless of developmental stage studied, one-third of the stained somata co-expressed GABA and glycine. Our data show that the glycinergic system matures 1 day later than the GABAergic system and follows a parallel spatiotemporal evolution, leading to a larger population of glycine cells in the ventral horn.

Animals↗
PubMed2006

The impact of oocyte maturation media on early bovine embryonic development.

Successful production of high quality blastocysts in vitro depends on the use of a culture system that ensures the acquisition of developmental competence by the maturing oocyte. It is now clear that the in vitro maturation environment has a major influence on the oocyte's ability to acquire the potential to develop into blastocysts. In this work we examine the impact of oocyte culture media on the quality of blastocysts by comparing developmental rates, cell number and their allocation to embryonic cell lineages, apoptosis, and expression of developmentally important genes. Higher total cell count and ICM:TCN ratio, which are indicative of embryo viability, were observed in embryos derived from oocyte maturation in TCM-199 supplemented with serum when compared to blastocysts derived from oocyte maturation in SOF BSA. Moreover, oocyte maturation in TCM-199 supplemented with serum-generated embryos of higher morphological quality and producing higher levels of Interferon Tau transcripts when compared to embryos derived from oocyte maturation in SOF BSA. In conclusion, the oocyte maturation regimen affected the morphological feature of blastocysts, including total cell count and allocation of cells to trophectoderm (TE) and inner cell mass (ICM) lineages and the expression profiles of genes involved in various embryo functions such as early embryonic growth, regulation of gene transcription, trophoblast differentiation and function, embryo-maternal communication, and stress response. Our results show that the oocyte culture media have strong impact on the quality of embryos produced in vitro and emphasize the need for more in depth evaluation of oocyte maturation protocols.

Animals↗
PubMedDate not supplied

Eyelid fusion and epithelial differentiation at the ocular surface during mouse embryonic development.

PURPOSE: To investigate epithelial differentiation at the ocular surface of the developing mouse eye by examining temporal and spatial changes in the expression of specific keratins. METHODS: Ocular tissues, including the entire eyeball, conjunctiva, and eyelid, of mouse embryos at embryonic day (E) 12.5 to E18.5 as well as of adult mice were examined by hematoxylin-eosin staining and by immunohistochemistry with antibodies to keratins K4, K10, K12, and K14. RESULTS: Hematoxylin-eosin staining revealed that eyelid fusion occurred at E17.5. Keratin immunohistochemistry demonstrated that: (1) K4 was expressed before K12, which in turn was expressed before K10; (2) expression of K4, K12, and K14 was spatially heterogeneous in the epithelia of the eyelid invaginations before eyelid fusion, but thereafter was continuous and homogeneous in the entire conjunctival epithelium, corneal epithelium, and basal cell layer of the surface epithelia, respectively; and (3) K10 immunoreactivity was not detected before eyelid fusion but was apparent in the epidermis of the eyelid thereafter. CONCLUSIONS: Eyelid fusion is a critical period for differentiation of the ocular surface ectoderm into the epithelia of the conjunctiva, cornea, and eyelid skin. The conjunctival epithelium differentiates before the corneal epithelium, which in turn differentiates before the eyelid epidermis.

Animals↗
PubMed1984

Preimplantation human embryonic development from polypronuclear eggs after in vitro fertilization.

The occurrence of grossly normal-appearing morula stage human embryos (8- to 12-cell) that developed from eggs fertilized in vitro containing three pronuclei is described. Serial section analysis by transmission electron microscopy demonstrated that 25% of the blastomeres were multinucleate, with as many as five deoxyribonucleic acid-containing nuclei (determined by fluorescence microscopy) in a single cell. Light and electron microscopy indicated that normal cortical and zona reactions had taken place. Fine-structural development of the cytoplasm was characteristic of morula stage human embryos. The results suggest the need to determine pronuclear number prior to syngamy and are discussed with respect to the notion that human embryos may have the capacity to develop normally in spite of the presence of abnormal cells.

Blastomeres↗