Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.     

Desialylation of core type 1 O-glycan in the equine embryonic capsule coincides with immobilization of the conceptus in the uterus

During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry a sialylated core type 1 O-linked glycan, Neu5Ac-(2-->3)-Gal-(1-->3)-GalNAc-(1-->Ser/Thr. By comparison, analysis of post-fixation capsules (days 17-19) revealed a desialylated core type 1, Gal-(1-->3)-GalNAc-(1-->Ser/Thr. The equine embryonic capsule also furnished 4-substituted GlcNAc, 4-substituted Glc and 2,3,4,6-tetrasubstituted Glc residues, the concentrations of which did not change between pre- and post-fixation stages. The loss of sialic acid from the sialylated core type 1 in the capsule appears to be directly related to successful fixation of the conceptus, and thus critical to the continuance of pregnancy in horses.     

Phylogeny and Phytogeography of Eupatorium (Eupatorieae, Asteraceae): Insights from Sequence Data of the nrDNA ITS Regions and cpDNA RFLP

Eupatorium were examined by sequencing the internal transcribed spacers (ITS) of nuclear ribosomal DNA and restriction site analysis of chloroplast DNA. Molecular data provided strong evidence that (1) this genus originated in North America, (2) the genus diverged into three morphological species groups, Eutrochium, Traganthes and Uncasia in North America, and (3) one of the North American Uncasia lineages migrated into temperate Europe and eastern Asia over the Bering land bridge. The estimated divergence times support a late Miocene to early Pliocene migration from North America to Eurasia via the Bering land bridge. A European species was sister to all of the eastern Asian species examined. The disjunct distribution pattern of the genus Eupatorium is incongruent with the classical Arcto-Tertiary geoflora concept. Received 13 September 1999/ Accepted in revised form 4 January 2000     

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ±1 capsules separated media in the “donor” chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the “recipient” chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P_app) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.     

Embryo transport through the mare's oviduct depends upon cleavage and is independent of the ipsilateral corpus luteum.

Two experiments were conducted using 14 mares. In Exp. 1, mares were inseminated with semen treated with TEPA, which, in other species, has been shown to lead to an arrest in ovum cleavage at 2--4 cells. The oviducts and/or uterus were then flushed 7--10 days after ovulation in 6 mares (Group A) or 2--6 days after ovulation in 5 mares (Group B). Fresh eggs were found in the oviduct flushes of 5 Group A and 5 Group B mares: 9 of the 10 eggs appeared to have cleaved, but none had developed beyond 16-cells. Seven eggs contained spermatozoa and 3 of 4 eggs from each group showed evidence of fertilization when examined ultrastructurally. Group A mares had thus retained fertilized eggs in the oviduct beyond the time at which they would normally have entered the uterus (6 days), indicating that development beyond at least the 2- to 4-cell stage is necessary for normal transport. In Exp. 2, 5 attempts were made to recover the embryo within 4 days of ovulation and transfer it to the contralateral oviduct. A single pregnancy resulted, indicating that a unilateral interaction with the corpus luteum was not necessary for the transport of the embryo to the uterus.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride- sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)- channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.      

Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development

Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with D-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and D-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with D-[1-14C]glucose, and L-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.     

CONSTRAINTS ON MAMMALIAN FORELIMB DEVELOPMENT: INSIGHTS FROM DEVELOPMENTAL DISPARITY

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark‐based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo‐wide constraint.