Changes in the content of cyclic AMP and cyclic GMP in eggs of Mactra solidissima during the second cleavage cycle.

The levels of cyclic AMP and cyclic GMP during the second cleavage cycle of eggs of Mactra solidissima have been measured. Cyclic GMP levels vary reciprocally with cyclic AMP levels, the cyclic GMP level increasing up to 4 to 5-fold and the cyclic AMP level falling to 50 percent at the onset of mitosis.     

Exogenous peroxidase in the eggs of rats during cleavage]

The presence of exogenous peroxidase of the rat's eggs during cleavage at the 1-, 2-, 8-cellular stages and the blastocysts were studied after 30 minute incubation with horseradish peroxidase (molecular weight 40.000, approximate size of the molecule 40 A). The activity of exogenous peroxidase in the area of zona pellucida was established in the perivitelline space, in the cytoplasm - in the pinocytosis vesicles, in certain greater vacuoles and in certain secondary lysosomes. At the stage of blastocyst the penetration of exogenous protein was greater than at earlier stages. The reaction product was found here in the intercellular space and in the blastocyst cavity. The obtained data suggest possible penetration of the protein from the surrounding area into the egg during its passing the uterine tube. The substances influencing the differentiation of the egg or teratogens may penetrate the egg in the same way.     

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.     

A eutherian X-linked gene, PDHA1, is autosomal in marsupials:A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA)for the pyruvate dehydrogenase E1α subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.     

Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs

The oviducts of both oestrous and pseudopregnant rabbits can be used for the successful culture of mammalian embryos for short periods. This has alowed some selection to be made on the embryos as they are examined on at least two occasions before final transfer. Not only have pregnancy rates been normal, but in some instances they have been higher following a limited period (2–3 days) in the rabbit oviduct. It would appear that these higher pregnancy rates result from a more intensive selection of embryos at the time of transfer rather than from some substance acquired during storage in the oviduct. However, the system is not without disadvantages. There is some loss of embryos (15–30%) in the oviduct and all embryos recovered may not have developed at the normal rate.The rabbit oviduct has been used as a site of xenogenous fertilization. Initial reports indicate that success in that area is lower than when using large animals as the site of fertilization. With more widespread interest in the use of microsurgery in embryos, the rabbit oviduct has been used for the short term storage of agar cylinders and has been found to be unsuitable because of the high rate of degeneration of agar chips. However, the rabbit oviduct is still useful as an experimental tool in the manipulation of embryos from the domestic species.     

The participation of the embryonic genome during early cleavage in the rabbit

Actinomycin D and the mushroom toxin α-amanitin similarly inhibit ribonucleic acid synthesis in the rabbit zygote. Actinomycin D also causes an immediate arrest of cleavage, whereas α-amanitin allows limited further development. The decreasing rate of amino acid incorporation caused by continuous exposure of cleaving rabbit embryos to α-amanitin suggests that a relatively homogeneous embryonic RNA is involved in the support of early protein synthesis and is turning over with a half-life of approximately 24 hr, or three cell generation times.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

Inter-strain differences in the development of the pattern of periplasm distribution during cleavage of honeybee eggs

During cleavage of honeybee eggs two peaks arise in the longitudinal distribution pattern of the periplasm which coincide with the site of the differentiation center and the site of a mesodermal center. A very similar pattern is exhibited by the dorsal plasmstrip, a narrow band of thicker periplasm which is formed during cleavage along the dorsal midline of the egg. The present paper describes the development of the dorsal plasmstrips of two inbred strains of honeybees during early cleavage stages. Differences between the two strains reside in the total size of their dorsal plasmstrips and in the timing of the formation of the anterior peak which coincides with the site of the differentiation center. The bearing of these findings upon embryological studies is discussed.     

D quadrant specification in the leech Helobdella: Actomyosin contractility controls the unequal cleavage of the CD blastomere

The unequal division of the CD blastomere at second cleavage is critical in establishing the second embryonic axis in the leech Helobdella, as in other unequally cleaving spiralians. When CD divides, the larger D and smaller C blastomeres arise invariantly on the left and right sides of the embryo, respectively. Here we show that stereotyped cellular dynamics, including the formation of an intercellular blastocoel, culminate in a morphological left-right asymmetry in the 2-cell embryo, which precedes cytokinesis and predicts the chirality of the second cleavage. In contrast to the unequal first cleavage, the unequal second cleavage does not result from down-regulation of one centrosome, nor from an asymmetry within the spindle itself. Instead, the unequal cleavage of the CD cell entails a symmetric mitotic apparatus moving and anisotropically growing rightward in an actomyosin-dependent process. Our data reveal that mechanisms controlling the establishment of the D quadrant differ fundamentally even among the monophyletic clitellate annelids. Thus, while the homologous spiral cleavage pattern is highly conserved in this clade, it has diverged significantly at the level of cell biological mechanisms. This combination of operational conservation and mechanistic divergence begins to explain how the spiral cleavage program has remained so refractory to change while, paradoxically, accommodating numerous modifications throughout evolution.     

Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.     

Localization of gamma-tubulin and cyclin B during early cleavage in physiologically polyspermic newt eggs

To understand the mechanism of the very slow block to polyspermy in physiologically polyspermic eggs of the newt Cynops pyrrhogaster, we used confocal laser microscopy to determine the distribution of gamma-tubulin and cyclin B1 in fertilized eggs. More gamma-tubulin was localized in the animal hemisphere than in the vegetal. The centrosomes of the principal sperm nucleus and the zygote nucleus had much accumulated gamma-tubulin, but little gamma-tubulin was associated with the centrosomes of the accessory sperm nuclei. These results are consistent with observations that the largest sperm aster is associated with the principal sperm nucleus. More cyclin B1 appeared in the animal hemisphere than in the vegetal at the end of interphase. The zygote nucleus had much accumulated cyclin B1, but little cyclin B1 was associated with the accessory sperm nuclei. Cyclin B1 disappeared earlier around the zygote nucleus at metaphase than around the accessory sperm nuclei. These findings correspond well with the earlier entry and exit into metaphase in the zygote nucleus than in the accessory sperm nuclei in newt eggs, supporting our maturation-promoting factor (MPF) model that accounts for the mechanism of nuclear degeneration in physiologically polyspermic eggs. Cyclin B1 began to accumulate in the nucleus during interphase in synchronous cleavage, and its greatest expression was in the centrosomes and the nucleus at prometaphase.     

Fundamental motor patterns of the mammalian fetus.

Techniques that permit direct observation of fetuses in vivo recently have expanded our understanding of prenatal behavioral development in mammals. Although fetal motor activity seems to lack the dynamic, goal-directed character of postnatal behavior, the dimensions that define behavioral organization after birth are applicable to the movements expressed by fetuses. Fetal activity exhibits temporal, sequential, and spatial organization that emerges between the inception of movement and term. Fetal rodents, for example, exhibit coordinated motor patterns antecedent to postnatal righting, locomotion, suckling, maternal-infant communication and grooming behavior, while other action patterns appear to be functional adaptations to the intrauterine niche. Fetuses also are behaviorally responsive to sensory stimulation and changes in environmental conditions in utero. Expression of these behavioral properties emphasizes continuity between prenatal and postnatal life while implying an adaptive role for behavior before birth.     

Indel evolution of mammalian introns and the utility of non-coding nuclear markers in eutherian phylogenetics

Nuclear DNA intron sequences are increasingly used to investigate evolutionary relationships among closely related organisms. The phylogenetic usefulness of intron sequences at higher taxonomic levels has, however, not been firmly established and very few studies have used these markers to address evolutionary questions above the family level. In addition, the mechanisms driving intron evolution are not well understood. We compared DNA sequence data derived from three presumably independently segregating introns (THY, PRKC I and MGF) across 158 mammalian species. All currently recognized extant eutherian mammalian orders were included with the exception of Cingulata, Dermoptera and Scandentia. The total aligned length of the data was 6366 base pairs (bp); after the exclusion of autapomorphic insertions, 1511 bp were analyzed. In many instances the Bayesian and parsimony analyses were complementary and gave significant posterior probability and bootstrap support (>80) for the monophyly of Afrotheria, Euarchontoglires, Laurasiatheria and Boreoeutheria. Apart from finding congruent support when using these methods, the intron data also provided several indels longer than 3 bp that support, among others, the monophyly of Afrotheria, Paenungulata, Ferae and Boreoeutheria. A quantitative analysis of insertions and deletions suggested that there was a 75% bias towards deletions. The average insertion size in the mammalian data set was 16.49 bp +/- 57.70 while the average deletion was much smaller (4.47 bp +/- 14.17). The tendency towards large insertions and small deletions is highlighted by the observation that out of a total of 17 indels larger than 100 bp, 15 were insertions. The majority of indels (>60% of all events) were 1 or 2 bp changes. Although the average overall indel substitution rate of 0.00559 per site is comparable to that previously reported for rodents and primates, individual analyses among different evolutionary lineages provide evidence for differences in the formation rate of indels among the different mammalian groups.