Showing papers in "Journal of Experimental Zoology in 1969"

Cell lineage of the imaginal discs in Drosophila gynandromorphs

Abstract: A collection of 379 drawings of D. simulans claret gynandromorphs, kindly supplied to the authors by Dr. A. H. Sturtevant, was used in order to analyze the cell lineage of the adult cuticular structures. The marker phenotypes forked bristles or yellow body color were used to trace the male tissue. The degree of mosaicism within the different imaginal discs was used to estimate the number of blastoderm nuclei whose descendants form the primitive imaginal discs on each side. The estimates are 23 for the compound-eye-antennal discs, 12 for the wing disc, several for the first and second leg discs and perhaps 1 for each of the prothoracic, third leg and abdominal discs. The frequency with which two parts are separated by sex, one male and one female, is assumed to vary inversely with the closeness of their embryonic descent. In this way were constructed the morphogenetic maps of the relative locations on one side of the embryo of the presumptive head, thoracic and abdominal adult structures. Locations of the presumptive parts within the morphogenetic maps are the same as the relative positions of the parts on the adult surface. Arguments are presented to place the morphogenetic map on the three-dimensional surface of the egg. The data are consistent with the model that the morphogenetic map of the prospective adult structures occupies a medial band along both sides of the embryo surface. Poulson's ('50) ground plan of the egg also places the presumptive larval hypodermis in this location. This model is considered relative to the experimental evidence marking the early embryo stages.

An ultrastructural and cytological study of preimplantation development of the mouse.

Abstract: Each stage of preimplantation development in the mouse from the fertilized egg to the blastocyst stage (including the unfertilized egg) was studied cytologically and ultrastructurally. Observations were made on the appearance and elaboration of several cellular organelles, inclusions and cell surface specilizations. The fertilized egg exhibits many intranuclear annulate lamellae, an increase in cytoplasmic vesicle number when compared to the unfertilized egg, and small amounts of crystalloids; mitochondria are vacuolated and small. The 2-cell stage is very similar to the fertilized egg but shows an increase in the number of cytoplasmic vesicles. The 4-cell stage is characterized by many changes: functional nucleoli appear, vacuolated mitochondria enlarge, cytoplasmic vesicles continue to increase in number, rough endoplasmic reticulum appears (as mitochondria-associated sacs), and some ribosomes are localized near the plasma membrane. At the 8-cell stage, large numbers of free ribosomes are observed in the cytoplasm, but clusters (polysomes) predominate at the 16-cell stage (morula). Morulae develop junctional complexes and exhibit differences in cytoplasmic basophilia between cells, which may be a prelude to differentiation. At the blastocyst stage, nucleoli change to an elongate form and differences in cytoplasmic background density can be observed ultrastructurally. Observations suggest that the contents of the blastocoel may be derived from the cytoplasmic vesicles, which increase in number and size subsequent to fertilization and discharge their contents into the intercellular spaces; the blastocoel arises as these fluid-filled spaces become confluent and enlarge.

In vitro acrosome reaction and capacitation of golden hamster spermatozoa by bovine follicular fluid and its fractions

Abstract: Unheated bovine follicular fluid is very toxic to hamster spermatozoa. This toxicity is due to the action of globulin or globulin-like substances in the fluid. When the fluid is pre-treated at 56°C for about 30 minutes these spermicidal substances are detoxified, and the fluid becomes capable of inducing the acrosome reaction and capacitation of the spermatozoa. Follicular fluid contains two factors responsible for the induction of the acrosome reaction and capacitation of spermatozoa, namely the sperm-stimulating factor and the acrosome reaction-inducing factor. An efficient sperm acrosome reaction and capacitation can be accomplished only in the presence of these two factors. The sperm-stimulating factor is dialyzable and heat-stable (at 90°C). The acrosome reaction-inducing factor, on the contrary, is non-dialyzable and heat-labile (at 90°C). The relationship between sperm acrosome reaction and capcitation is discussed.

Correlation of structure, speed of contraction, and total tension in fast and slow abdominal muscle fibers of the lobster (Homarus americanus)

Abstract: The fibers in the deep and superficial extensor muscles of the lobster abdomen differ in structural and physiological characteristics. The superficial extensor muscle fibers have relatively long (> 6 μ) sarcomeres, in which thin and thick myofilaments overlap for about 3 μ per half-sarcomere at rest length. The deep extensor muscle fibers have relatively short (< 4 μ) sarcomeres, in which the region of overlap is about 1.5 μ per half-sarcomere at rest length. The ratio of thin to thick filaments is 6:1 in the superficial muscle fibers and 3:1 in the deep muscle fibers. Contraction and relaxation are much more rapid in the deep muscle fibers. The superficial muscle fibers develop substantially more tension than the deep muscle fibers in high K+, in caffeine, in high K+ plus caffeine, and during prolonged spikes in solutions containing tetraethylammonium chloride. The results are consistent with the predictions of the sliding filament hypothesis of muscle contraction.

The stabilization of cartilage properties in the cartilage-forming mesenchyme of the embryonic chick limb.

Abstract: Experiments have been done to determine the time in the development of the embryonic chick limb when the cells in the cartilage-forming area become “stabilized.” The particular criterion for stability used in this work was the ability of the cells to resist the influences in the limb which cause some limb mesenchyme cell to form cartilage and other limb mesenchyme cells to form soft tissue. Blocks of cartilage-forming mesenchyme were transplanted from the cartilage-forming area of a second limb to the prospective soft tissue area of a second limb. The host limb was permitted to develop for 48 hours, and then examined to determine if the implanted cells had formed cartilage outside of the normal cartilage pattern of the host limb. The embryos used as a source of the implanted blocks varied in stage from stage 22 to stage 27, the hosts also varied in stage from stage 22 to stage 27. It was found that a block of cartilage-forming mesenchyme generally would conform with the host limb pattern if the donor was stage 24 or younger and the host was stage 24 or younger. A block of cartilage-forming mesenchyme generally did not conform with the host limb pattern if the donor was stage 25 or older whatever the stage of the host. We conclude that the cartilage-forming cells become stabilized by this criterion between stage 24 and stage 25.

Some aspects of tail regeneration in the lizard, Anolis carolinensis. I. A description based on histology and autoradiography.†,‡

Abstract: The histology of the tail and its autotomy planes in Anolis carolinensis follows the same basic plan as that described for other lizards. Variations in detail which are of importance to the present paper are described. The normal regeneration process was studied using histologic and autoradiographic techniques. These studies suggest: (1) that the muscle makes little if any contribution to the regenerate, (2) that the source of cells for the regenerate is primarily from the various connective tissue elements and (3) that the tail has no apical accumulation blastema. It is proposed that the term blastema as applied in amphibian limb regeneration cannot be applied in lizard tail regeneration. The growth of a normal regenerate results from extensive interstitial growth in the differentiating tissues and from subapical growth in the less differentiated areas of these tissues.

Amylase variants in Drosophila melanogaster: linkage studies and characterization of enzyme extracts.

Abstract: The amylases of D. melanogaster were characterized by several parameters. Starch-iodine and 3,5-dinitrosalicylic acid reduction methods were employed to determine activity. Conditions were defined under which activity, in crude aqueous extracts could be restricted to α-amylase(s) of fly origin. Biochemical properties of the α-amylases from eleven homozygous Amy strains were found to be very similar in temperature stability, pH optimum, substrate specificity and the effects of various activators and inhibitors. All were activated by chloride ions and showed a pH optimum of about 7.4. Relative efficiencies on several substrates were tested: soluble starch, amylopectin, β-limit dextrins, glycogen and amylose. EDTA completely inhibited all extracts, presumably by the removal of calcium required for activation. The specific α-amylase inhibitor from wheat grain also completely inhibited activity, as did reduced glutathione. PCMB had no discernible effects on activity. Eight of the 11 strains tested are known to produce different electrophoretic banding patterns for amylases. The total maximum activity of each strain may also be used to characterize it and, accordingly, three strains with amylases of the same electrophoretic mobility were distinguished by differences in their specific activities. Sexual dimorphism in amylase activity was defined, as well as activity in heterozygotes between strains. Aside from the distinctions between strains already noted, four strains differed in their relative susceptibility to heat and to α-amylase inhibitor. Linkage experiments indicated that the Amy region lies at 77.3 on the genetic map of the second chromosome and to the right, but near, section 52F on the salivary chromosome map.

Photoperiodic control of antler cycles in deer. I. Phase shift and frequency changes

Abstract: When subjected to reversed light cycles, deer antlers are shed and regenerated six months out of phase with respect to the outdoor environment. They respond solely to the lighting conditions, not to temperature changes. Deer exposed to accelerated years grow antlers more frequently than normal, but in no cases do they produce them more often than every three months. When the annual light cycle is prolonged to 24 months, deer tend to grow antlers every other year in accordance with the artificial cycle. In general, deer forced to grow antlers more frequently than normal produce stunted outgrowths owing to the abbreviated years. Those maintained on extra long light cycles, however, do not grow extra large antlers. Analysis of these data suggests that the onset of antler growth is entrained by increasing day lengths in deer previously sensitized by decreasing days. However, older animals can sometimes express an endogenous yearly antler growth cycle irrespective of certain prevailing artificial lighting conditions.

An ultrastructural study of lens invagination in the mouse.

Abstract: Early lenses of mouse embryos were studied with the electron microscope to determine whether their cells contain organelles that can be causally related to the invagination of the lens placode. The cells of the placode and early lens cup are joined at their apical ends by junctional complexes. At the level of the zonula adhaerens, favorable sections show cytoplasmic filaments, 35 A to 50 A wide, arranged about the apex of a cell. Groups of filaments appear to originate on or near the plasma membrane and then to extend across the cytoplasm, parallel to the apical cell surface, to terminate on or near the plasma membrane some distance away. They are nearly always straight; in contrast, nearby lateral and apical cell membranes are twisted into irregular folds and projections. We present a model to explain lens invagination in which the filaments are considered to be contractile. On the basis of their location in the cells and their presumed function, these organelles are proposed to be a major component in the mechanism that leads to early morphogenesis of the lens.

Lactate dehydrogenase isozymes of the flatfish, Pleuronectiformes: kinetic, molecular and immunochemical analysis.

Abstract: At least two genes are responsible for lactate dehydrogenase (LDH) synthesis in the tissues of eighteen species of flatfish (Pleuronectiformes). The relative activity of these genes is markedly skewed with LDH‐A being present in much larger amounts and in a wider variety of tissues than LDH‐B. Certain flatfish exhibit a single LDH‐A while others have five forms of this tetramer. Molecular hybridization experiments demonstrate that these five tetramers have two distinguishable subunits. These subunits were purified and subjected to amino acid analysis, peptide mapping, kinetic and immunochemical analysis and shown to be very similar, perhaps identical, in primary structure. Treatment with reducing reagents failed to interconvert them. LDH‐B was partially purified and shown to be kinetically and immunochemically distinct from LDH‐A. As is true for many other fish, additional LDH isozymes are obseved in eye and brian tissues. These results show that the flatfish genetic information for LDH synthesis is similar to that of other fish, and flatfish LDH A and B subunits are homologous to those of other vertebrates. Copyright

Adenosine deaminase phenotypes among sexual and parthenogenetic lizards in the genus Cnemidophorus (Teiidae).

Abstract: Electrophoresis of liver homogenates has revealed distinct adenosine deaminase (ADA) phenotypes in each of four sexual species belonging to the Cnemidophorus sexlineatus group. A fifth sexual species, C. tigris, shares one of these phenotypes with a species of the C. sexlineatus group, but is distinguished from this species by other genetic and biochemical characters. Hence, knowledge of ADA phenotypes permits discrimination among each of the five sexual species suspected of involvement in interspecific hybridizations leading to the origin of parthenogenetic Cnemidophorus species. Combinations of two or three ADA phenotypes characteristic of sexual species are found in each parthenogenetic species studied. This finding supports the theory of hybrid origin of parthenogenetic species, while the combination of particular alleles in each parthenogenone permits the implication of specific sexual species in its origin.

Photoperiodic control of antler cycles in deer. II. Alterations in amplitude

Abstract: Deer native to temperate zones grow new antlers each year in synchrony with the annual photoperiodic cycle. When the amplitude of this cycle is reduced from normal winter and summer solstices (at 42° latitude) of nine and fifteen hours to as little as eleven and one-half and twelve and one-half hours (equivalent to about 9° latitude), sika deer still tend to grow antlers on schedule, even when that schedule is accelerated. Deer exposed to days of constant length for prolonged periods of time fail to replace their antlers when the light:dark ratio is 12L/12D. When held on unchanging short (8L/16D) or long (16/8D; 24L/OD) days, however, they continue with their antler cycles at irregular intervals averaging about 85% of the siderial year. These results are interpreted in terms of an inherent circannian rhythm, the duration of which can be adapted to the frequency of the prevailing photoperiodicity, but which reverts to approximately a year on constant long or short days and disappears altogether on simulated equatorial photoperiods.

Cytological aspects of fertilization in the lamellibranch, Mytilus edulis I. Polar body formation and development of the female pronucleus

Abstract: Aspects of fertilization encompassing the meiotic events of the maternal chromatin, the formation of the polar bodies, and the development of the female pronucleus have been investigated in the lamellibranch, Mytilus edulis. The cromosomes of the mature egg of Mytilus edulis at the time of fertilization are organized on the first metaphase plate of meiosis. The first meiotic metaphase figure, oriented normal to the egg's cortex, consists of two asters, each of which contains paired centrioles, microtubules, and some smooth endoplasmic reticulum. Subsequent to sperm incorporation, the first polar body is produced by a process akin to cytokinesis following the movement of the chromosomes at anaphase. Later, the metaphase plate of the second meiotic apparatus is formed. Morphologically, the second metaphase figure is similar to the first; however, each aster appears to contain only one centriole. The second polar body, produced in the same manner as the first, remains associated with the zygote via a cytoplasmic bridge which persists up to the time of the first cleavage. Both polar bodies contain the same cellular inclusions as the zygote; however, only the second polar body has its chromatin delimited by a perforated nuclear envelope. After the formation of the second polar body, the maternal chromatin is organized into chromosomal vesicles. The chromosomal vesicles later fuse to form the female pronucleus.

Activation of ribosomal RNA synthesis in initiation of Wolffian lens regeneration.

Abstract: Tissue engaged in the transformation of iris into lens, triggered by lens removal in the adult newt (Triturus viridescens), was labeled in vitro with 14C-uridine, and the sedimentation profiles of RNA synthesized by the tissue were studied in sucrose density gradients. The radioactivity in the 18S and 28S areas, which is insignificant in the normal iris, increases significantly during 2 to 10 days after lens removal, and subsequently retains a high level until the completion of lens regeneration. These data are interpreted as indicating activation of ribosomal RNA (rRNA) synthesis by lens removal, and are discussed in connection with the ultrastructural modification of the nucleoli. Further data from the radioactive profiles are consistent with the notion that the level of control of rRNA production in this system is the transcription of the rRNA precursor rather than the post-transcriptional processing of the precursor. The activation of rRNA production described in this paper is the earliest event so far detected in this system, preceding activation of DNA replication, cell replication, and dedifferentiation.

Activation of DNA replication in the iris epithelium by lens removal.

Abstract: The pattern of DNA replication during the initial stages of Wolffian lens regeneration has been examined by an autoradiographic study of tritiated thymidine uptake in zero to five-day regenerates (Triturus viridescens). Scanning of complete sets of serial sections for each iris revealed the absence of labeled cells in normal, one-, and two-day iris epithelia, and a very infrequent occurrence of labeled iris epithelial cells in some three-day regenerates. After this stage, cell labeling is seen in all iris epithelia. Cell labeling frequency increases markedly by four days, and is at a very high level in five-day regenerates. Further data indicate that labeling begins in the dorsal sector of the iris epithelium. By five days after lens removal, however, it reaches similar levels in dorsal, lateral, and ventral sectors. Thus, DNA replication is induced throughout the iris epithelium after lens removal, even though the lateral and ventral sectors of the iris epithelium do not participate in actual lens formation. Comparison between the outer and inner laminae of the iris epithelium shows that during all stages studied the great majority of labeled cells are in the inner lamina.

Physiological studies on supercooled killifish (Fundulus heteroclitus). I. Serum inorganic constituents in relation to osmotic and ionic regulation at subzero temperatures.

Abstract: Physiochemical properties (serum osmolality and blood pH), serum inorganic ions (sodium, potassium, calcium, magnesium, chloride, phosphate and bicarbonate) and tissue water were studied in parallel groups of adult male Fundulus heteroclitus acclimated to various temperatures (30°C, 20°C, 10°C, 4°C, 2°C, −1°C and −1.5°C) in salt water under otherwise constant laboratory conditions. When the acclimation temperature was lowered from 20°C to −1.5°C, serum osmolality increased by 20%. This increase was not indicative of osmoregulatory failure, however, since the proportion of total serum osmolality accounted for by inorganic electrolytes dropped from 98% at 20°C to 93% at −1.5°C. Serum electrolytes such as sodium, chloride, calcium, magnesium and bicarbonate increased in the subzero cold by 12%, 17%, 30%, 33% and 11% respectively whereas serum potassium and inorganic phosphate levels were unchanged. Blood pH was significantly higher at 10°C than at any other temperature. The water content of liver, testes and muscle decreased by 8% in the subzero cold. These changes in serum electrolytes and tissue water did not indicate osmoregulatory failure, however, since the new levels, once established, were maintained as long as the fish were alive. Although osmotic and ionic regulation were not as effective in the cold, they were by no means so poor as to cause death by osmotic imbalance.

Nucleic acid synthesis in preimplantation rabbit embryos. I. Quantitative aspects, relationship to early morphogenesis and protein synthesis.

Abstract: The ability of rabbit embryos to incorporate tritiated uridine and thymidine into acid-insoluble material in vitro has been quantitated by liquid scintillation counting at daily intervals throughout the preimplantation period. The ribonucleic acid content of the embryos has been determined by the orcinol reaction. It is shown that, in spite of a low level of Actinomycin D-sensitive uridine incorporation during cleavage, there is no net synthesis of ribonucleic acid until immediately prior to blastocyst formation. The acceleration of ribonucleic acid synthesis which occurs at that time precedes, by some 36 hours, the acceleration of protein synthesis. This finding is discussed from the standpoint of genetic expression in the preimplantation rabbit embryo, and with reference to the possibilities for post-transcriptional control of protein synthesis.

Hormonal and nutritional requirements for limb regeneration and survival of adult newts.

Abstract: Newts which were fed daily for two weeks prior to hypophysectomy survived significantly longer and regenerated limbs better than newts fasted for two weeks prior to hypophysectomy. Since limbs were amputated five days after hypophysectomy, regeneration was initiated in the complete absence of pituitary hormones. Newts which were hypophysectomized 14 days after limb amputation were found to possess blastemas significantly smaller at eight and also at 16 days after the operation than the blastemas of sham-operated newts. Thus, hypophysectomy, performed after limb regeneration had progressed through the wound healing and dedifferentiation phases, resulted in a significant retardation in the growth of the blastema. To determine which hormones are essential to normal limb regeneration and survival, newts were hypophysectomized and treated with various combinations of hormones, or grafted with pituitaries from newts or from axolotls (Ambystoma mexicanum). A prolactin-thyroxine combination, growth hormone, and ectopic pituitary grafts from newts or axolotls, significantly prolonged the life of hypophysectomized newts. These newts also regenerated limbs in a normal fashion. Thyroxine alone, prolactin alone, thyroxine + ACTH, ACTH, or saline were not effective in restoring the health of hypophysectomized newts and were not effective in restoring normal limb regeneration ability.

Ontogeny of the immune response to skin allografts in relation to lymphoid organ development in the amphibian Xenopus laevis Daudin.

Abstract: The response of larval and adult Xenopus to first set skin allografts was studied by examination at regular intervals of the external appearance of the grafts and of serial sections through the graft region. Operations were performed on all stages of larvae ranging from those with rudimentary to those with mature lymphoid organs. Larvae received single allografts from either larval or adult donors, skin from the latter being a good morphological marker: adults received adult skin only. Control autografts were also applied to both larval and adult toads. All autografts were accepted; no incompatible phenomena were observed. Both adults and larvae, even those operated at the most immature stage of development, were able to repond immunologically to the skin allografts. The response was similar in larval and adult toads and included a lymphocytic invasion of the graft which disappeared when destruction of the graft was near completion. This invasion was correlated with the state of development of the lymphoid organs of the host: if these organs were immature at the time of application of the graft, the invasion was delayed but eventually occurred when the host reached a more advanced larval stage. The capacity to invade a graft with lymphocytes could be correlated with the lymphoid maturation of the thymus. These results support the view that the allotransplantation of tissues in immature amphibian larvae, before acquisition of immunological competence, does not necessarily preculude the possibility of an immune response to the same tissues in more mature larvae.

RNA in Cecropia moth ovaries: Sites of synthesis, transport, and storage†

Abstract: Azure B staining of Cecropia moth ovarian follicles indicated that the pattern of RNA concentrations characteristic of vitellogenic oocytes is disrupted by a gross redistribution and dispersion, beginning with the final discharge of the cytoplasm of the nurse cells into the oocyte. The cytoplasmic redistribution is largely complete two days later when chorion formation begins, the germinal vesicle breaks down, and the first meiotic spindle forms. At this stage the follicle contains 30 μg of RNA, of which 27 μg are in the follicle cells and only 3 μg in the oocyte itself. The proportionately large amount of follicle cell RNA apparently reflects the synthetic functions of these cells in the formation of yolk and the secretion of chorion. Of the 3 μg in the oocyte, only 1 μg is incorporated into the early embryo while the remainder is found in the yolk cells and serosa. Autoradiography of follicles labeled with H3-uridine either in vivo or by a “pulsechase” procedure in vitro indicated that, as in other polytrophic insect ovaries, the nurse cell nuclei are the most conspicuous source of oocyte RNA. The germinal vesicle incorporated labeled uridine in previtellogenic follicles. In vitellogenic and later stages neither a germinal vesicle nor a follicle cell contribution to oocyte RNA was detected by the procedures used.

Cytological aspects of fertilization in the lamellibranch, Mytilus edulis. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes.

Abstract: The events of fertilization involving the modifications of the incorporated spermatozoon and the association of the paternally and maternally derived pronuclei have been studied by techniques of light and electron microscopy in the lamellibranch, Mytilus edulis. At the site of gamete plasma membrane fusion a small fertilization cone is produced. As the spermatozoon moves through the fertilization cone, its nuclear envelope degenerates. The degeneration of the nuclear envelope is followed by the dispersion of the sperm chromatin and the formation of the pronuclear envelope. The sperm aster, produced during the formation of the male pronucleus, consists of fasicles of microtubules and endoplasmic reticulum which radiate from a centrosphere region containing two centrioles. When the pronuclei complete their migration toward each other they are structurally similar and contain a fibro-granular nucleoplasm. Eventually, the pronuclei become closely apposed, their proximal surfaces flatten, and produce an array of interdigitating nucleoplasmic projections. Concomitantly, the chromatin condenses and forms large reticular aggregations. Association of the parentally derived chromosomes is effected when the two pronuclear envelopes are dismantled. Microtubules that comprise a portion of the mitotic spindle become associated with the chromosomes as they move together in preparation for the first cleavage division. Hence, like many vertebrates, but unlike sea urchins, no zygotic nucleus is produced.

Degeneration and regeneration in abdominal flexor motor neurons in the crayfish.

Abstract: Electrophysiological studies of sectioned crayfish motor axons of the abdominal flexor muscle have demonstrated that the distal stumps, isolated from their central connections for up to several months, are remarkably resistant to degeneration, as previously shown in the crayfish claw opener preparation. These distal stumps remained electrically excitable and intracellular recording showed that their neuromuscular junctional properties were indistinguishable from normal ones in such properties as waveform and amplitudes of the evoked junctional potentials. Furthermore, repetitive stimulation of the distal stumps evoked jp's which showed facilitation as in normal preparations. When degeneration finally occurred, electrical stimulation of the nerve no longer evoked jp's, and no progressive deterioration of junctional properties was observed. Regeneration ultimately occurred in these nerves but required at least three weeks or more. The electrical properties of these regenerated junctions were not different from normal ones. The evidence from time-course and correlated histological studies of degeneration and regeneration suggest that regeneration of these crayfish nerves occurs by axonal fusion rather than by outgrowth of an entirely new distal apparatus from centrally located cell bodies, as is the mechanism in vertebrates and some invertebrates.

Epithelial metaplasia induced on extraembryonic membranes. I. Induction of epidermis from chick chorionic epithelium.

Abstract: The induction of typical epidermis from the chick chorionic epithelium (CE) was studied with the following methods: (a) Grafting of 15-day scale dermis (tarsometatarsal dermis) directly onto 9-day host-chorioallantoic membrane (CAM). (b) Grafting of a recombinant composed of 9-day CE and 15-day scale dermis onto 9-day host-CAM. (c) Grafting of a recombinant composed of 9-day CE and 15-day scale dermis inside a Millipore filter chamber placed on top of host-CAM and (d) Organ culture of 9-day CE in contact with 15-day scale dermis on a liquid culture medium. Examination of the explants resulting from the four different explantation methods mentioned above has shown that the CE is highly plastic and is readily transformed into typical scale epidermis. There is no epidermal induction when a non-dermal tissue (muscle) is placed in contact with 9-day CE. These observations together with other results obtained in the control series, support the idea that a specific dermal influence is involved in the metaplasia of the CE to epidermis. Metaplastic changes of the CE leading to epidermal differentiation are described. Evidence is presented indicating that the sole origin of the induced epidermis is from the homogeneous cell population belonging to the inner, basal cells of 9-day CE. Environmental conditions in which CE metaplasia proceeds influence the degree of normality of the induced epidermis. The limitations and the advantages of each transplantation method and its possible application to a more general problem concerning epithelial metaplasia and tissue interactions in skin development are discussed.

Physiological studies on supercooled killifish (Fundulus heteroclitus) II. Serum organic constituents and the problem of supercooling

Abstract: To gain insight into the mechanism permitting Fundulus heteroclitus to survive subzero temperatures in a supercooled state, previously published data on inorganic components of the serum were complemented by studies on serum organic constituents in parallel groups of adult male killifish acclimated to various temperatures (30°C, 20°C, 10°C, 4°C, 2°C, −1°C and −1.5°C) in salt water under otherwise constant laboratory conditions. When the acclimation temperature was lowered from 20°C to −1.5°C, there was little or no change in levels of serum total non-protein nitrogen, total amino acids, urea and non-glucose free carbohydrates. Total serum protein levels were usually unaffected by the cold, but occasionally showed a decrease. Serum total cholesterol levels increased by 69% in the subzero cold, presumably due to an increased lipid metabolism. Whereas most serum constituents (organic and inorganic) showed only minor changes in supercooled F. heteroclitus, serum glucose levels increased from three- to sixfold. Serum glucose levels of 68 mg% in fish at 20°C increased to 460 mg% in sexually regressed fish at −1.5°C but to only 280 mg% in supercooled mature fish. It was suggested that the elevation of serum glucose in the subzero cold permitted survival in a supercooled state, presumably by retarding ice crystal growth on any ice nuclei that might form spontaneously in the blood.

Meiosis of mouse eggs before and after sperm penetration.

Abstract: Mature mice were induced to ovulate by injections of PMS and HCG and their eggs were prepared, using an air-dry technique, for the examination of chromosomes during the first and second meiotic divisions Sixty percent of 72 eggs recovered from follicles between 10 and 85 hours before ovulation were at prophase, whereas 91%–64% of 139 eggs were at metaphase I from 7 to 3 hours before ovulation Anaphase I and telophase I were seen in 17 eggs 45–0 hours before ovulation The bivalents were clumped together in a single mass at the earliest stage of diakinesis Separate, extended bivalents which underwent contraction up to metaphase I were observed later Counts of bivalents in 82 eggs at diakinesis and metaphase I revealed no deviation from the haploid number (n = 20) The dyads in the first polar body were usually extended and somewhat diffuse in appearance, whereas those in the egg were not A total of 295 eggs were examined from 24 females inseminated 15 hours after ovulation At 3, 35, 45 and 55 hours after ovulation 3%, 20%, 26% and 43% of recovered eggs respectively were penetrated by sperm, indicating that a certain length of time may be required for capacitation of mouse sperm Approximately 90% of eggs with an intact sperm head (Type I) were at metaphase II and nearly 100% of eggs with a swollen sperm head (Types III and IV) were at telophase II Four distinct configurations of metaphase II, “spread,” “clumped,” “compact,” and “granular” were seen in 384 unfertilized eggs recovered from 36 females at various times after induced ovulation The haploid number of dyads was observed in all of 70 eggs counted at metaphase II in the “spread” configuration The proportion of unfertilized eggs with a “compact” metaphase II increased from 2% at 5–7 hours to 12% at 24–315 hours after ovulation, while the proportion of eggs with a “granular” metaphase II increased from 11% to 47% over the same period The second meiotic metaphase exhibited these degenerative changes mainly at the end of the fertilizable period of the egg

Chronic allograft rejection in Lumbricus terrestris.

Abstract: To determine the capacity of earthworms to reject tissue transplants, first-set orthotopic allografts of body wall were exchanged between 280 adult Lumbricus terrestris. In addition, 30 worms were autografted as controls. Both auto- and allogeneic tissue transplants healed in promptly; autografts survived permanently while allografts showed varying degrees of incompatibility. Intrapopulation allografts in worms from Canada showed 22% permanently surviving transplants while 95% were not destroyed in the Oregon group; the remaining allografts in both groups were completely destroyed after prolonged chronic periods or showed incomplete rejection after observations during eight and one-half months. No intrapopulation Oregon transplants were rejected completely while only 1% were in the Canadian group. About 5.5% of interpopulation allografts were destroyed on Oregon hosts and 15.3% on Canadian recipients between 38 and 153 days after grafting. Therefore, recognition and rejection of tissue allo-antigens are characteristic of the primitive immune response of annelid worms.

Aster-equatorial surface relations and furrow establishment.†

Abstract: Flattened eggs of the sea urchin Hemicentrotus pulcherrimus were used in experiments designed to determine whether furrow establishment depends upon any particular geometrical relationship between the asters and the equatorial surface. In normal eggs at first cleavage the asters are 29.5 μ apart and the distance from the spindle center to the equatorial surface is 47.3 μ. In flattened eggs, the mitotic apparatus was pushed to an excentric position. The maximum spindle-to-surface distance that permitted furrowing was 62.5 μ. At second cleavage the spindle-to-surface distance is normally 36.2 μ but furrows develop when the distance is as great as 47.5 μ. The interastral distance and spindle-to-surface distance were varied simultaneously in flattened, dispermic eggs containing four asters. The activity of supernumary sperm asters was not detectably different from that of the mitotic apparatus. If the interastral distance was 32.5 μ furrows formed when the spindle-to-surface distance was between 32.5 μ and 47.5 μ. When the interastral distance is increased to 35 μ or more at the same spindle-to-surface distances, furrowing was rare. Very short spindle-to-surface distances were produced by perforating the flattened cells. They allowed study of combinations involving large interastral distances and short spindle-to-surface distances. Under this circumstance asters 35 μ or farther apart invariably elicited furrows when the spindle-to-surface distance was 20 μ or less. Decreasing the spindle-to-surface distance remedies a deficiency occasioned by abnormally great interastral distances. These results are considered to support the view that furrow establishment requires conjoint action of a pair of asters upon equatorial cell surface.

Clonal distribution of melanocytes in piebald-spotted and variegated mice.

Abstract: Experimental observations of mice support the hypothesis that the complete pigmentation of most normal mammals and birds develops by the expansion and merger of clones of pigment cells from the same centers that appear as pigmented spots against a white background in piebald-spotted mutants. Specific piebald-spotted mutants, bt and s, having nearly normal pigment coverge in certain parts of their bodies were selected for progressive restriction of those areas until the center of each area was located. Repeated reversion of the same pigment areas in variegated Miwh, Wa, and Va heterozygotes indicated that each of the areas was a clone of cells derived from a single primordial melanoblast that migrated from the neural crest to the location of the center during embryogenesis. White regions completely separated cells of different clones into differently pigmented spots when piebald-spotting mutants, bt or s, were combined with variegated Miwh/+. Because the separation of clones was so complete, it appears that no pigment cells exist in the intervening white regions at any stage of development. Additional evidence also indicates that restricted proliferation of pigment cells is a common end effect in all piebald-spotting mutants, even though more direct effects may be on the pigment cell, the neural crest or the tissue environment of the skin.

Some aspects of tail regeneration in the lizard, Anolis carolinensis. II. The role of the peripheral nerves,

Abstract: The work reported here affirms that the ependyma supports tail regeneration and demonstrates that the peripheral nerve supply makes no essential contribution to the regenerative process. The normal peripheral nerve supply in the tail of Anolis carolinensis was found to be 4.5 fibers per (100 μ)2. In tails in which the peripheral nerve supply was reduced to 0.34 fibers per (100 μ)2 normal regeneration occurred if the ependyma was present. In the absence of the ependyma (i.e., the central nervous system) but with 50–75% of the peripheral nerves present no normal regenerative outgrowth occurred; however, three or four myomeres were regenerated.

Electrophoretic variations of tyrosinase in follicular melanocytes during the hair growth cycle in mice

Abstract: Multiple forms of tyrosinase separable by acrylamide-gel electrophoresis are demonstrable in extracts of hair bulbs prepared on the fifth (anagen IV) through the thirteenth days (anagen VI) of a hair growth cycle induced in C57BL/6J mice by plucking of quiescent hairs. The enzymic bands visualized by incubation of the gels in L-DOPA have been designated as T1, T2 and T3. Although T1 has been observed through day 19, the T2 and T3 components disappear by day 15 of the hair growth cycle. Tyrosinase activity is not demonstrable in skin extracts prepared during telogen or on days 1–3 post-plucking (anagen I–III). The slow development of a single DOPA-melanin band in such samples has been demonstrated to represent non-specific oxidation by hemoglobin that migrates at a slightly slower rate than the T3 component. The precise state of tyrosinase within follicular melanocytes prior to day 5 of the hair growth cycle remains to be determined. The functional significance of the multiple forms of tyrosinase also is yet to be established.