Showing papers in "Journal of Experimental Zoology in 1982"

Differentiation of muscle fiber types in aneurogenic brachial muscles of the chick embryo.

Abstract: Cross-reinnervation studies performed ex ovo with newly hatched chicks demonstrate that peripheral motor neurons control the phenotypic characteristics of avian muscles. The present experiments were designed to determine whether or not nerves play a similar role during the initial expression of muscle fiber types. Previous experiments indicated that differentiation of specific fiber types occurs during the first week of embryogenesis, temporally coincident with the penetration of nerves within muscle masses. These observations suggested that peripheral nerves may be associated with the initial differentiation of fiber types. To test this hypothesis directly, anterior limb buds of the chick embryo were rendered aneurogenic by deletion of the brachial segment of the neural tube. To ensure a completely aneurogenic environment for developing brachial muscles, surgery was performed at day 2 in ovo before the exit of ventral root fibers. Experimental and control embryos from Stage (St) 25 (4.5 d) through St 45 (19d) were analyzed histochemically by a silver-cholinesterase reaction to detect nerves and by the myosin ATPase reaction, following alkali and acid preincubation, to determine the fiber type composition of the muscles. In addition, the total volume of aneurogenic and control muscles was compared. Results demonstrate that the characteristic myosin ATPase profiles of individual aneurogenic and innervated (control) muscles were identical throughout the entire period analyzed. Therefore, we conclude that these enzymic profiles are endogenously expressed and are not under neuronal control during early embryogenesis. Furthermore, the entire sequence of events from the migration of myogenic cells to the anterior limb bud through the division of the primary muscle masses to form individual brachial muscles proceeded on schedule in the absence of nerves. Since the growth of aneurogenic muscles was impaired, we conclude that during embryogenesis peripheral motor nerves are necessary initially for the proper growth of muscles and ultimately, for their survival. They are not involved, however, with either the initial formation or initial differentiation of individual brachial muscles.

insect vitellins: Identification, purification, and characterization from eight orders

Abstract: Vitellins were identified, purified, and analyzed from insects representing eight orders. The structures and polypeptide constituents of vitellins of Hyalophora cecropia, Tenebrio molitor, Rhodnius prolixus, Forficula auricularia, Periplaneta americana, and a mayfly were found to have common features. The native proteins had Mr of 385,000–470,000 (385–470 K) and were composed of high (100–180 K) and low (47–84 K) molecular weight polypeptides in equimolar proportions. The vitellins of Apis mellifera, a sphecid wasp, and Aedes aegypti, however, had lower Mr (200–350 K) and were composed of only large polypeptides (170–190 K). The higher Diptera form a distinct third group with vitellins made up entirely of small polypeptides of about 50 K.

Metabolic responses of striped bass (Morone saxatilis) to temperature acclimation. II. Alterations in metabolic carbon sources and distributions of fiber types in locomotory muscle

Abstract: The effects of temperature acclimation on (1) fiber-type distribution, and (2) alternate metabolic fuels for energy supply within different skeletal muscle fiber types in striped bass (Morone saxatilis) were examined Histochemical analysis demonstrated an increase in the proportional cross sectional area of oxidative red fibers from 903% to 1503% during cold acclimation (25°-5°C) The rate of oxygen consumption was 178 times greater in red muscle than white in 25°C acclimated fish This ratio increased to 232 in 5° fish Oxidation of both 14C-U-glucose and 14C-1-palmitate was significantly greater in red muscle than in white at both acclimation temperatures A lack of thermal compensation for metabolism of 14C-U-glucose was exhibited by both fiber types Red muscle showed a dramatic increase in rate of 14C-1-palmitate oxidation after cold acclimation Activities of glycolytic enzymes confirmed a high glycolytic capacity in white muscle; glycolytic capacities were dependent on acclimation temperature in both fiber types Only red muscle showed a positive compensation for activities of key enzymes from aerobic metabolism The results indicate an increasing reliance on aerobic metabolism of fat in the swimming musculature at cold acclimation temperatures The capacity for sustained swimming under these conditions is conserved by (1) an increase in the relative proportion of red fibers, and (2) an enhanced capacity for energy supply in the red muscle by oxidative metabolism of stored lipid

The leg flexor muscle of Carcinus. I. Innervation and excitatory neuromuscular physiology

Abstract: The innervation and neuromuscular physiology of the flexor muscles in the legs of Carcinus were investigated using a variety of histological and electrophysiological techniques. Slow muscle fibers were found in the proximal and distal regions of the muscle; intermediate- and fast-contracting fibers were found in most regions, but fast fibers predominated in the central regions. Muscle fibers were innervated by one to four excitatory axons. The extent of inhibitory innervation was not determined. The motor innervation showed significant departures from the previously described Brachyuran pattern. The results indicated the need for a thorough reexamination of the patterns of limb innervation of the crustacean tribes.

The developmental potential of mouse 16-cell blastomeres.

Abstract: Based on the criteria of relative size and cell surface polarization, subpopulations of outer (larger, polar) and inner (smaller, apolar) blastomeres have been isolated from mouse 16-cell morulae and reaggregated in groups of 16 cells, and the developmental potential of the aggregates has been assessed both in vitro and in vivo. Aggregates of outer, inner, or mixed groups of cells all formed blastocysts which outgrew and contained both characteristic trophectoderm and alkaline phosphatase positive inner cell masses in vitro. However, significant differences were observed in the timing of blastocyst formation, depending on cell type. Aggregates of outer cells recompacted more slowly, commenced fluid accumulation earlier, and contained more cells at the blastocyst stage than did inner cell aggregates. Aggregates containing both outer and inner cells were intermediate. Postimplantation development of blastocysts derived from aggregates of outer or inner cells, after transfer to pseudopregnant recipients, was normal and comparable to zona-intact control embryos. This relationship between the expression of different morphological and behavioural properties by the precursor cells of the trophectoderm and inner cell mass and the existence of totipotent cells in both outer and inner subpopulations is discussed.

Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture

Abstract: Osteoclasts were isolated from the endosteal surface of day 19 embryonic chick tibias by mild trypsinization. Osteoclast enrichment was achieved by passing cell suspensions through Nitex screening of selective sizes, including the eventual selective retention of osteoclasts on 12 micrometers polycarbonate filters or by sequential sieving through Nitex screens and fractionation on Percoll gradients. The enrichment procedures produced osteoclast populations of 50-75% based on morphological criteria with the latter isolation method providing populations with less matrix debris. The results of light microscopy, transmission and scanning electron microscopic observations indicate that osteoclasts can be maintained in culture for up to 10 days with retention of osteoclast morphology. This morphology includes a specialized ruffled plasma membrane, large numbers of mitochondria, lysosomes, as well as a multinucleated cytoplasm. Furthermore, acid phosphatase and butyrate esterase histochemical measurements support these morphological observations. In addition, chick hatchling circulating monocytes were isolated and purified by Ficoll-hypaque gradient centrifugation with subsequent adhesion to glass petri dishes. With time in culture, these cells form multinucleated cells, but lack the ultrastructural complexity of the isolated osteoclasts. This report describes a unique culture system to study osteoclast function and illustrates the similarities and differences of this system to the monocyte-to-giant cell culture system.

The leg flexor muscle of Carcinus. II. Distribution of muscle fiber types

Abstract: Three types of muscle fiber were recognized in the leg flexor mus- cle of Carcinus maenas on the basis of histochemical staining €or the oxida- tive enzyme NADHD and analysis of fiber cross-sectional area. The distribution of these fiber types within the muscle is described. The oxidative capacity and cross-sectional area of the fiber was correlated with the fiber type determined physiologically. Key words crab leg muscle, NADHD histochemistry, fiber types, oxidative capacity Investigation of the contraction time of crus- tacean muscle fibers has revealed a number of muscle fiber types. Two extremes can be eas- ily recognized: "slow" and "fast." Intermedi- ate types can also be identified (Atwood, '76), although they are better considered as pre- senting a continuum from one extreme to the other. A variety of histological methods have also been used to identify muscle fiber types. These include histochemical staining for adenosine triphosphatase (ATPase) and oxidative en- zymes, and measurements of sarcomere length. Traditionally, sarcomere length has been used as an indicator of fiber type (Atwood, '72, '76). Recently sarcomere length and muscle fiber type have come to be regarded as syn- onomous, with short sarcomere fibers being equated to fast fibers and long sarcomere fi- bers being equated to slow fibers (Lang et al., '80; Ogonowski et al., '80). Such extrapolation may not be valid in all cases. More recently, Lang et al. ('80) have used ATPase activity as well as sarcomere length to classify fibers. Some confusion seems to have arisen with attempts to correlate these morphological and histochemical measures with physiological results. This is well shown by the perplexing conclusion that "the oxida- tive capacity of the muscle fibers is not di- rectly correlated with muscle fiber type (based on adenosine triphosphatase activity and sar- comere length)" (Lang et al., '80). Biologically, it would appear to be more meaningful to re- late metabolic status to intrinsic physiological function. Close examination of the photomicro- graphs of Lang et al.'s ('80) histochemical sec- tions (the reproduction being admittedly poor) suggests that they could readily support such a correlation between physiological fiber type, oxidative capacity, and ATPase activity. That

Bovine serum albumin, sperm motility, and the “dilution effect”

Abstract: Epididymal spermatozoa from rabbit and ram were washed either once or twice using an efficient washing procedure and were then diluted in various media to a final concentration of approximately 1.4 x 10(7) cells/ml and incubated at 30 degrees C for up to 12 hours. Bovine serum albumin (BSA), either untreated or defatted, was found to be better than polyvinylpyrrolidone, ovalbumin, or alpha-lactalbumin, both at stimulating and maintaining motility levels and at reducing the tendency of the washed spermatozoa to stick to glass. BSA was effective in all media tested, being independent of Ca2+, PO4(3-), HCO3-, and ionic strength). BSA has a reversible stimulatory effect on motility. If BSA was added to sperm suspensions 3 1/2 hours after they had been washed and diluted in protein-free medium, motility was stimulated to levels not significantly lower than those observed in samples that had been washed and diluted in the presence of BSA. However, samples washed into BSA and then washed free of it behaved essentially as though they had never been in contact with protein. The motility, survival, and response to BSA of twice-washed spermatozoa were the same as those of once-washed spermatozoa, showing that epididymal plasma factors are not required for survival in vitro. It was concluded that dilution is not essentially detrimental to rabbit and ram spermatozoa. However, severe dilution of semen may result in levels of male reproductive tract fluids insufficient either to stimulate motility or to prevent sticking of motile cells to container surfaces. Few motile spermatozoa are recovered from samples of such diluted semen.

Gynogenesis caused by ultraviolet irradiation of salmonid sperm.

Abstract: Gynogenetic rainbow trout lines can be produced easily with the simple UV sperm irradiation technique detailed in this study. The dose effect on embryonic survival rate is called a pseudo "Hertwig effect" because of major differences with results of gamma irradiation: Some mixing of various karyotypes (from diploid to haploid number) were obtained with mean doses, probably because of the weak penetrating power of the ultraviolet rays. Irradiations lasting 4 minutes of more are required to produce homogeneous haploid progeny; heat shock applied to such fertilized eggs results in diploid fry of the maternal phenotype.

Sperm nuclear decondensation in mammals: role of sperm-associated proteinase in vivo.

Abstract: Previous studies from this (Zirkin et al., '80) and other (Marushige and Marushige, '78) laboratories have shown that proteinase associated with mammalian sperm nuclei is involved in thiol-induced sperm nuclear decondensation and protamine degradation in vitro. The results of these in vitro studies suggested the exciting possibility that the sperm nucleus itself might contribute proteinase involved in its subsequent in vivo decondensation during fertilization. In the present study, microinjection methods were used to test this possibility directly. Control hamster sperm nuclei, which exhibited proteinase activity, decondensed when incubated in vitro with disulfide reducing agent. As expected, these nuclei also decondensed when microinjected into ovulated hamster oocytes and formed morphologically normal pronuclei. When the proteinase associated with isolated sperm nuclei was removed with 0.5 M salt or inhibited with nitrophenyl-p-guanidinobenzoate, the nuclei were rendered incapable of decondensing in response to disulfide reducing agent in vitro. However, when these nuclei were microinjected into eggs, they decondensed and transformed into pronuclei. These results provide direct evidence that sperm-associated proteinase is not required for sperm nuclear decondensation and formation of the male pronucleus during fertilization.

Effects of rearing temperature on cuticle permeability and epicuticular lipid composition in Drosophila pseudoobscura

Abstract: The effects of larval and pupal thermal regime on adult transcuticular water loss rates and epicuticular hydrocarbon composition in Drosophila pseudoobscura were determined. Temperatures encountered by the larval stage had no effect, but flies emerging from pupae maintained at 24°C exhibited significantly lower cuticular permeabilities than those emerging from pupae maintained at 17°C. More than 50% of the variance in cuticular permeability was accounted for by variation in the proportion of n-pentacosadiene in the epicuticular hydrocarbons. High transcuticular water loss rates were correlated with higher proportions of relatively short-chain, methyl-branched alkanes and alkadienes. High proportions of longer chain lengths of both branched alkanes and alkadienes were associated with lower cuticular permeabilities. The molecular basis for these findings is discussed.

Studies of phospholipase A2 related to the hamster sperm acrosome reaction

Abstract: This report describes studies of innate and exogenous phospholipase A2 related to the acrosome reaction of golden hamster cauda epididymal sperm in vitro. Two acrosome reaction-inducing systems were used: a nonsyn-chronous system with epinephrine, and a synchronous system with the protonionophore FCCP. After a high percentage of sperm had undergone AR (in both systems) an increased phosphatidylcholine hydrolyzing activity was found in incubation media supernatants after partial purification by Affi-Gel Blue chromatography. A preliminary characterization demonstrates that the activity was stimulated by Ca2+, detectable at pH 7.4--7.8, but not at pH 4.5, and inhibited by p-bromophenacyl bromide and mepacrine. Thus, it appears that a phospholipase A2 was released as a result of the acrosome reaction. BSA purified by TCA precipitation and ethanol resolubilization (TCA-BSA), utilized in both types of incubations did not contain associated phospholipase A2 activity, but did contain an associated phosphatidylcholine hydrolyzing activity. The nonphospholipase A2 activity was removed by further purification of the TCA-BSA by Affi-Gel Blue chromatography, but the resulting highly purified TCA-BSA was still able to induce capacitation and acrosome reactions in hamster sperm at the same level as prior to column chromatography. Incubations of the sperm in the presence of a partially purified bee venom phospholipase A2 did not stimulate hamster sperm AR over control values in the presence of epinephrine, but did so in the absence of epinephrine (probably by mimicking the action of the innate sperm head phospholipase A2). These results provide further support to earlier studies from this laboratory suggesting the involvement of a sperm head phospholipase A2 as an important factor in the mammalian sperm acrosome reaction.

The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustaceans

Abstract: A sensitive pH-stat assay was used to determine the distribution in aquatic and terrestrial decapod crustaceans, correlation to osmoregulation, and selected kinetic properties of carbonic anhydrase. In all species the enzyme was concentrated in the gills, and specifically the posterior three or four gill pairs. Activity was highest in the gills of osmoregulating species, and the enzyme could be localized in the areas of the gill lamellae which contained a high density of salt absorbing cells. The enzyme from the blue crab, Callinectes sapidus, exhibits a number of characteristics remarkably similar to the Na+/K+ ATPase from that species. Both enzymes show the same inter- and intrabrachial distribution and the same reactions to altered environmental salinity. Branchial CA from both species was inhibited by high concentrations of NaCl and increasing pH. The enzyme showed a temperature optimum of 25°C. On the basis of these initial results, it appears that crustacean gill carbonic anhydrase plays an important role in the blood osmoregulatory process.

Simultaneous measurement of evaporative water loss, oxygen consumption, and thoracic temperature during flight in a carpenter bee

Abstract: The carpenter bee Xylocopa capitata has a high wing loading (425 mg cm−2 in females with empty crops) and exhibits a very high mass-specific oxygen consumption during flight (mean for free flight 52.3 ml O2 gm−1 hour−1). Evaporative water loss is also high during flight (26.6 mg gm−1hour−1) and is significantly correlated with rate of oxygen consumption (r = 0.73) as well as with temperature of the ambient air (r = 0.80) and the water vapor deficit (r = 0.66). No indication of active evaporative cooling was evident and the bees are true endotherms capable of rapid preflight thermogenesis. Analysis of the osmotic and ionic concentrations of the body fluids showed that no water stress was experienced by the bees. The use of suitable thermocouples and a sensitive capacitance-type humidity meter in an open flow-through system allowed instantaneous and simultaneous measurement of oxygen consumption, evaporative water loss, and thoracic temperature. All three variables fluctuated in close synchrony during preflight warmup, flight, and subsequent cooling.

Oocyte maturation in the amago salmon (Oncorhynchus rhodurus): In vitro effects of salmon gonadotropin, steroids, and cyanoketone (an inhibitor of 3β-hydroxy-Δ5-steroid dehydrogenase)

Abstract: The effect of partially purified chinook salmon gonadotropin (SG-G100) and a number of steroids on the induction of germinal vesicle breakdown (GVBD) in amago salmon (Oncorhynchus rhodurus) oocytes (with intact follicle layers) was investigated in vitro. SG-G100 was effective only at the highest concentration tested (1 microgram/ml). 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was the most potent maturation-inducing steroid tested, followed by 17 alpha-hydroxyprogesterone. Testosterone or deoxycorticosterone (DOC) enhanced the rate of GVBD in response to SG-G100. DOC also enhanced the response to 17 alpha,20 beta-diOHprog but testosterone was without effect, suggesting that DOC has a direct action on the oocyte while testosterone probably acts at the level of the follicle. Estradiol-17 beta had no effect on GVBD in response to SG-G100 or 17 alpha,20 beta-diOHprog. The action of SG-G100 was shown to be dependent on the synthesis of a second delta 4 steroidal mediator of maturation since cyanoketone, a specific inhibitor of 3 beta-hydroxy-delta 5-steroid dehydrogenase, completely abolished the maturational effects of the gonadotropin and pregnenolone but not delta 4 steroids. Radioimmunoassay of media in which oocytes were induced to mature in vitro with SG-G100 revealed significantly elevated levels of progesterone and 17 alpha,20 beta-diOHprog. Estradiol-17 beta levels, high in control media, were only elevated twofold by SG-G100. Levels of the two progestogens were extremely low or nondetectable in media in which oocytes were incubated with cyanoketone, while estradiol-17 beta levels remained high. These results are discussed in relation to other evidence indicating that 17 alpha,20 beta-diOHprog is the naturally occurring maturation-inducing steroid of amago salmon. The role of other steroid hormones, particularly the possible involvement of corticosteroids, in the control of final oocyte maturation in teleosts is explored.

Spontaneous activation of ovulated rat oocytes during in vitro culture

Abstract: Ovulated rat oocytes were observed to activate spontaneously during in vitro culture. The possible mechanisms involved in this activation were studied by culturing oocytes at various times (0-6 hr) after ovulation, in different media, and for different incubation periods (0-5 hr). Activated oocytes extruded the second polar body within 60 to 90 min of culture. Following 3 to 4 hr of culture chromosomes were scattered throughout the cytoplasm; however, no pronuclear formation was observed. Neither time after ovulation nor incubation in different media affected the rate of activation. The length of time the oviducts remained in the animal after cervical dislocation, however, significantly (P less than 0.01) affected the rate of activation. Oocytes obtained as rapidly as possible had an activation rate of 21% during in vitro culture, whereas oocytes obtained from oviducts which remained in the animal for 5 min after cervical dislocation had an activation rate of 94%. Therefore, exposure to changing oviductal conditions following cervical dislocation appears to be the critical factor influencing spontaneous activation of metaphase II rat oocytes during in vitro culture. Our studies demonstrate that rat oocytes can spontaneously activate during in vitro culture, a factor which may affect the fertilizability of the oocyte.

Fertile life of acrosome-reacted guinea pig spermatozoa.

Abstract: To study the fertile life of acrosome-reacted guinea pig spermatozoa, spermatozoa were induced to undergo a synchronous acrosome reaction after which vigorously motile acrosome-reacted spermatozoa were isolated from acrosome-intact ones. Acrosome-reacted spermatozoa thus prepared were incubated in vitro for various periods of time before they were mixed with zona-intact and zona-free eggs. Such spermatozoa retained their ability to fertilize zona-intact eggs for at least 2 hours after the acrosome reaction; thereafter they rapidly lost this ability. There was a close correlation between the decline and loss of the ability to penetrate the zona and the loss of hyperactivated motility by spermatozoa, indicating the importance of vigorous motility in passing through the zona. The ability of acrosome-reacted spermatozoa to fertilize zona-free eggs was maintained for many hours even after the spermatozoa had completely lost their capacity to fertilize zona-intact eggs. As long as acrosome-reacted spermatozoa are "alive," they seem to retain the capacity to fuse with the egg plasma membrane regardless of the quality of their motility.

Cell allocation in preimplantation mouse chimeras.

Abstract: The mechanism of cell allocation in the preimplantation mouse chimera was studied in aggregation chimeras of 4- and 8-cell stage embryos. To follow the fate of cells, some embryos were labeled by a 16-h incubation (at the 2-cell stage) with 3H-thymidine. At the 4-8-cell stage both labeled and unlabeled embryos were treated with pronase to remove the zona pellucida and then aggregated in various combinations: one labeled 8-cell and three unlabeled 4-cell embryos (labeled 8-cell chimeras); one labeled 4-cell and three unlabeled 8-cell embryos or two labeled 4-cell and two unlabeled 8-cell embryos (labeled 4-cell chimeras). Autoradiography of air-dried preparations of inner cell masses isolated from the chimeras at the blastocyst or late blastocyst stage by immunosurgery revealed that labeled 8-cell embryos contributed a disproportionately large number (over 60%) of their descendant cells to the inner cell mass of labeled 8-cell chimeras. By contrast, in labeled 4-cell chimeras only 8% (with one labeled 4-cell embryo) or 31% (with two labeled 4-cell embryos) of inner cell mass cells were derived from the labeled embryos. These results suggest that cell allocation in preimplantation mouse chimeras is not a random process and that cells of embryos developing ahead of others are preferentially allocated to the inner cell mass.

rRNA accumulation and protein synthetic patterns in growing mouse oocytes.

Abstract: The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.

Modalities of the action of temperature on sexual differentiation in field‐developing embryos of the European pond turtle Emys orbicularis (Emydidae)

Abstract: This paper is a study of sex ratios among turtle (Emys orbicularis) hatchlings and young from eggs incubated in the ground, as a function of soil temperature during the gonadal thermosensitive period. Five experiments were performed in Brenne (France) during the summers of 1974, 1975, and 1979. In all these experiments, the sex ratios were found to be skewed. In one experiment, the percentage of males was nearly 50%, whereas the number of females was reduced and the other individuals displayed an intersexual phenotype (the gonads were ovotestes). In the other experiments, the number of males was reduced, whereas the number of females approached 50% or was superior to 50% and the other animals displayed an intersexual (ovotestes) or indeterminate (thin atypical gonads) phenotype. The soil temperature was registered at the same depth as the eggs, and the temperature curves were analyzed for the 12 days following the beginning of the gonadal thermosensitive period (stage 16 of embryonic development). This analysis shows that the first half of this period is the most important for gonadal sexual differentiation. In the first experiment, temperature remained below or surpassed slightly the threshold temperature of 28.5°C (at which males and females, but also intersexes, are obtained). These conditions favored male differentiation. Then, the maxima were higher, allowing female differentiation which was either typical (2 cases of 21 only) or partial (intersexes which are probably genetic females). In the other experiments, the maxima were high, reaching 35 to 40°C, from the very beginning of the thermosensitive period, and although alternating with low minima (inferior to 25°C) they favored female differentiation and inhibited male differentiation. Therefore, intersexes, indeterminates, and probably some phenotypic females would correspond to genetic males.

Infertility in bitches induced by active immunization with porcine zonae pellucidae

Abstract: In a study designed to evaluate the contraceptive potential of anti-egg zona pellucida immunization, bitches were injected with isolated and solubilized zonae pellucidae of either the pig or the dog in saline and Freund's adjuvant or with saline and adjuvant alone (controls). They were boosted monthly, and serum samples were collected before the first injection and 10 days after each injection. The titers of anti-zona pellucida antibodies in each serum sample were measured by treating fresh canine oocytes with the serum, then evaluating antibody binding as indicated by indirect immunofluorescence, precipitation of the zona surface, and penetrability of the zonae by spermatozoa in vitro. The bitches were bred when they came into estrus. All three bitches immunized with porcine zonae developed high titers (1:10,000 or more by indirect immunofluorescence) of antibodies that cross-reacted with canine zonae to cause precipitation of the zona surface both in vivo and in vitro and that completely inhibited penetration of the zonae by spermatozoa in vitro. The two bitches immunized with canine zonae developed only low titers, and their sera had little or no effect on treated zonae. The two control bitches did not develop anti-zona antibody. None of the bitches immunized against porcine zonae became pregnant when bred, but one bitch immunized against canine zonae and one control did become pregnant. The bitches immunized with porcine zonae had somewhat abnormal cycles for unknown reasons. Thus, we could not establish with certainty whether the infertility resulted from specific interference with fertilization, as in vitro, or from alterations in ovarian function, or both.

Experimental studies of the origin and expression of metameric pattern in the chick embryo.

Abstract: On either side of Hensen's node of the fully extended primitive streak of the chick embryo (stage 4) the mesoderm is already organized into circular domains called somitomeres. As Hensen's node regresses, paraxial somitomeres are added in tandem and are early morphological representatives of metameric pattern in the mesoderm. These organized circular domains of mesenchyme cells are best visualized with stereo pair scanning electron microscopy. Experiments suggested that a prepattern of segmentation exists in and around the fully extended primitive streak. Streaks divested of Hensen's node can generate some paraxial somites, but only if surgically split down the midline. We assessed metameric pattern formation in nodeless streaks, both severed and unsevered down the midline. Operated blastoderms were cultured 15 hours, fixed, dissected, processed for scanning electron microscopy, and photographed in stereo. Split nodeless streaks produced a cranial to caudal sequence of somitomeric development. This sequence is similar to the sequential maturational events seen in the segmental plate of older embryos. The least mature somitomeres, toward the posterior end of the severed edge, appear as circular domains of radially oriented cells, looking much like the first somitomeres to emerge near Hensen's node of the stage 4 streak. More cranially along the severed edge, somitomeres are morphologically more mature, being more condensed, with cells oriented about a central myocoele. At the most cranial end of the severed piece, somitomeres are the most mature, having contracted about their centers to create intersomitomeric gaps that permit their identification with light microscopy as individual "somites." Embryos from which the node was removed, but the streak left intact, generated only the most primitive somitomeric pattern repetitively along either side of the primitive groove. We conclude that regression of Hensen's node provides for the timely initiation of morphogenesis of somitomeres from a prepattern of segmentation that already exists.

Acceleration of acrosome reaction in hamster spermatozoa by lysolecithin.

Abstract: When hamster spermatozoa were preincubated for 1–2 hr in Ca2+-containing medium and then exposed to lysolecithin (LC), large proportions of the spermatozoa underwent acrosome reactions within 15 min. These acrosome-reacted spermatozoa were capable of fertilizing eggs. The earliest visible sign of the reaction, wrinkling of the acrosomal cap, seen by 30 sec after exposure to LC. The reaction in many spermatozoa was completed within the next 30 sec.

Ammonia excretion by the gills of two marine teleost fish the importance of NH4+ permeance

Abstract: Varying the concentration of un-ionized ammonia (NH3) in the medium perfusing the gills of the isolated fish head while holding the total ammonia (NH4+ + NH3) concentration constant had no significant effect on gill ammonia excretion. In contrast, alteration in the medium NH4+ concentration at a fixed NH3 concentration produced a significant change in ammonia excretion. Thus, gill ammonia excretion was related to medium NH4+, not NH3, concentration.

Origin of melanosome structures and cytochemical localizations of tyrosinase activity in differentiating epidermal melanocytes of newborn mouse skin.

Abstract: The mode of differentiation of epidermal melanocytes was studied by ultrastructural cytochemistry in the skin of newborn mice of strain C57BL/10J. From observations of epidermal melanoblasts and melanocytes, stage I melanosomes, including both unit membranes and inner matrices, appear to be formed from Golgi vacuoles or rough endoplasmic reticulum (RER). Stage I melanosomes were positive to ammoniacal silver-nitrate reaction in the melanoblasts of 1-day-old mice. All stages of melanosomes were similarly positive in the differentiating melanocytes of 2-day-old mice. However, Golgi apparatus, RER, and vesicles were negative. Therefore, it is conceivable that structural proteins, originated from Golgi vacuoles or RER, are developed into specialized proteins and are detected by this reaction in stage I melanosomes. Stage I melanosomes were dopa-negative in the melanoblasts. Stage I and II melanosomes were similarly negative in the differentiating melanocytes. Thus, the melanoblasts are thought to begin production of stage I melanosomes prior to the onset of tyrosinase activity. In the differentiating melanocytes, dopa-melanin depositions were observed in stage III and IV melanosomes, trans Golgi saccules, and small vesicles derived from these saccules, but not in RER. These vesicles were in contact with, or fused to, melanosomes. These findings suggest that tyrosinase may be transferred by Golgi vesicles into stage I and II melanosomes originating from Golgi vacuoles or RER.

Characterization of spermatid/sperm basic chromosomal proteins in the genus Xenopus (Anura, Pipidae)

Abstract: Spermatid/sperm basic chromosomal proteins from 17 species and subspecies of the genus Xenopus (Anura, Pipidae) were compared. Electrophoresis on acetic acid/urea/Triton X-100 polyacrylamide gels revealed that each Xenopus species with a diploid chromosome number of 36, 72, or 108 showed multiple, diverse spermatid/sperm-specific basic chromatin proteins with mobilities greater than the somatic histones. The numbers and mobilities of these proteins were characteristic of each Xenopus species and each subspecies of Xenopus laevis. Cytochemical tests revealed that the sperm basic nuclear proteins of these Xenopus species and subspecies were rich in arginine and lysine and contained more arginine than the nuclear proteins of somatic cells. X. tropicalis (2n = 20) and X. sp. n. (Zaire) displayed spermatid/sperm-specific basic chromatin proteins which migrated within the histone H1 region of acetic acid/urea/Triton X-100 polyacrylamide gels. Cytochemically the sperm nuclei of these species resembled those of somatic cells. These observations suggest that spermatid/sperm basic nuclear proteins can be used as molecular markers for individual species and subspecies of the genus Xenopus.

The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans. I. Intraspecific responses to seasonal differences in temperature

Abstract: Total oxygen uptake in the blue crab Callinectes sapidus doubles with a rise in ambient temperature in the range 15–25°C, and increases precipitously (Q10=4.92) in the range 5–15°C. Both systemic and cellular phenomena are responsible for the pattern. At summer temperatures, where motor activities are maximal, the oxygen affinity of blue crab hemocyanin is low, and considerable volumes of oxygen are delivered to the tissues. At winter temperatures, where feeding and locomotion virtually cease, the hemocyanin-oxygen (HcO2) affinity is so high that little oxygen can be extracted by the tissue and O2 is transported entirely in the free form. At the transitional temperature of 15°C, a seasonal acclimation of HcO2 affinity permits appreciable oxygen delivery to the tissues in the spring, but curtails oxygen delivery in the fall. Both muscle and hepatopancreas utilize the hexose monophosphate shunt (HMS) in carbohydrate metabolism. Specific activities of the enzymes participating in the HMS are greater in hepatopancreas than muscle, and the sensitivity of O2 uptake to the glycolytic inhibitor iodoacetate (10−2 M) is smaller. The temperature dependence of O2 uptake in the range 15–25°C is much smaller in hepatopancreas than in muscle. The ratio of C-1/C-6 labeled glucose utilization indicates that, at low temperature, the activity of the HMS in hepatopancreas is five times greater than that of glycolysis. Thus, the low thermal sensitivity of O2 uptake in hepatopancreas appears to be due to a change in the relative contribution of the two pathways to total carbohydrate metabolism. There is no evidence of a compensatory response of intermediary metabolism in muscle, which comprises the major fraction of total biomass. The thermal dependence of O2 uptake by the animal exceeds that of either hepatopancreas or muscle, due to the large increase in HcO2 affinity, and hence a decrease in tissue O2 supply, at low temperature.

Specificity of sperm‐zona interaction

Abstract: The specificity of early sperm-zona interactions in vitro was studied using gametes from the mouse, rabbit, pig, and human. Mouse epididymal spermatozoa, capacitated in vitro, bound to the zonae of intact mouse oocytes and zona fragments removed mechanically from oocytes. When viewed with the scanning electron microscope (SEM), spermatozoa were found adherent more frequently to the external (cumulus) surface of the zonae than the internal (oocyte) surface. Noncapacitated mouse spermatozoa or spermatozoa incubated with zonae in the absence of external calcium did not bind to homologous zonae. by contrast, fresh, epididymal mouse spermatozoa and mouse spermatozoa capacitated in vitro appeared to attach equally well to both surfaces of zonae and zona fragments from rabbit and pig oocytes. Rabbit spermatozoa "capacitated" in vivo as well as noncapacitated rabbit spermatozoa, attached rapidly to both inner and outer surfaces of zonae from rabbit, mouse, and pig oocytes. This reaction was not dependent on external calcium. Human spermatozoa did not display any tendency to adhere to zonae from the rabbit, mouse, or pig. The results of these studies suggest that spermatozoa can associate with zona in a nonspecific as well as a specific manner. The use of heterologous systems in addition to homologous systems in sperm binding assays may be useful in distinguishing these two types of interaction.

Biochemical and immunohistochemical localization of alpha and beta keratin in avian scutate scales

Abstract: We have determined the distribution of alpha and beta keratins in avian scutate scales using SDS-PAGE and indirect immunofluorescence with non-cross-reacting rabbit antisera prepared to both alpha and beta keratins. Contrary to previous studies, we find that alpha keratin is definitely present in the epidermis of the outer scale surface as well as that of the inner surface and hinge region of the scale. In fact, all six alpha keratin polypeptides found in the whole scale epidermis are also present in epidermis taken exclusively from the outer scale surface. Three of these polypeptides demonstrate a specific enrichment in the epidermis of the outer scale surface when compared to the polypeptides of the entire scale epidermis. Immunofluorescence studies specifically localize the alpha keratins of the outer scale epidermis in the Stratum Basale and Stratum Intermedium, but the beta keratins are restricted to the Stratum Intermedium and Stratum Intermedium and Stratum Corneum.

In vitro synthesis and secretion of ecdysteroids by Drosophila melanogaster ovaries

Abstract: The ovaries of adult Drosophila melanogaster were shown to produce and secrete ecdysteroids in vitro by an ecdysteroid radioimmunoassay. The major secretory products were determined by HPLC/RIA analysis to be ecdysone, 20-hydroxyecdysone, and a highly polar fraction. Based on competitive binding studies, the secreted ecdysteroids contain 2–3 times more 20-hydroxyecdysone than ecdysone.