bluuebunny/crossref_metadata_2025 huggingface dataset:
Major vitelline envelope proteins in pipefish oocytes originate within the follicle and are associated with the Z3 layer
Authors: Begovac Paul C. , Wallace Robin A.
Published: June 2005

A method is described for purifying salt‐washed vitelline envelopes of pipefish eggs. Approximately 72% of the envelope dry weight is represented by 2 extractable proteins with molecular masses of 109 and 98 kDa. The 2 proteins are present in a 1:2 ratio, respectively, on 1‐dimensional electrophoretic gels; the 98 kDa protein can be resolved further into a family of peptides by 2‐dimensional gel electrophoresis. A monoclonal antibody raised against vitelline envelope protein recognizes both groups of protein and no other component of the oocyte, plasma, or liver. Immunocytochemistry indicated that the recognized antigen first appears in late cortical alveolistage oocytes, coincident with formation of the Z3 layer of the vitelline envelope; immunogold staining of thin sections confirmed antigen localization to the Z3 layer. Incubation of intact follicles with [35S]methionine in vitro followed by 2‐dimensional gel electrophoresis and immunoblotting demonstrated the synthesis of the vitelline envelope proteins by such preparations. However, isolated follicle cells or denuded oocytes failed to yield convincing evidence of a similar nature. We conclude from these observations that the principal vitelline envelope proteins originate from within the follicle rather than elsewhere, such as the liver, and that the structural integrity of the follicle may be necessary for their elaboration.
Intestinal transfer of growth hormone into the circulatory system of the rainbow trout, Salmo gairdneri: Interference by granule cells
Authors: Le Bail Pierre‐Yves , Sire Marie‐France , Vernier Jean‐Marie
Published: June 2005

To test if growth hormone, well known for its strong effect on fish growth, could be incorporated into the diet, bovine growth hormone (bGH) was introduced into the lumen of the digestive tract of trout yearling (Salmo gairdneri). Radioimmunoassay (RIA) and radioreceptoras‐say (RRA) were used to assess plasma concentration of bGH. In our experimental conditions, the concentration of bGH peaks after 1 hour, in treated fish only, attesting to the intestinal hormone transfer (coefficient of absorption is about 0.2%). Immunocytochemistry is used to follow the hormone transfer process. Most bGH molecules absorbed by enterocytes of the distal segment are found in the vacuoles (phagolysosomes) of the apical hyaloplasm. A small part escapes the intracellular degradation and is found in the intercellular space and in the interstitial space of the lamina propria. At this level, the granule cells trapped the antigenic hormone, as did immune cells.

The results of this study indicate that oral administration of GH can be potentially used in fish culture.
Leucine pool sizes in the fat body of ovariectomized and vitellogenically active adult Acheta domesticus (L.)
Authors: Bradley James T. , Harber Gregory J.
Published: June 2005

Hemolymph and intracellular fat body leucine were extracted from 10‐day‐old ovariectomized adults and sham‐operated females and were isolated by thin layer chromatography. Leucine pool sizes in both tissues were estimated by a double label‐dansylation procedure. Ovariectomy had no effect on the hemolymph leucine pool size, but the total intracellular fat body leucine pool size was greater in intact than in ovariectomized females. The postinduction rates of vitellogenin synthesis in the fat bodies of ovariectomized and sham‐operated females are discussed in the context of these data. A mechanism responsible for the lowered leucine pool size in the fat body of ovariectomized crickets remains to be identified.
Effects of 17α,20β‐dihydroxy‐4‐pregnen‐3‐one on the in vitro production of 11‐ketotestosterone by testicular fragments of the common carp, Cyprinus carpio
Authors: Barry Terence P. , Aida Katsumi , Hanyu Isao
Published: June 2005

Testicular fragments of the common carp were incubated with a range of doses of 17 α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20‐P, 3, 30, 300, and 3,000 ng/ml) in combination with either silver carp gonadotropin (GtH, 0.5 μg/ml), 17α‐hydroxy‐4‐pregnen‐3‐one (17‐P, 100 ng/ml), testosterone (T, 100 ng/ml), or Ringer alone (control). The production of T, 11‐ketotestosterone (11‐KT), 17‐P, and 17,20‐P were measured after 18 hr incubation by specific radioimmunoassay. Gonadotropin, 17‐P, and T all significantly stimulated 11‐KT production above control levels in the absence of 17,20‐P. Addition of 17,20‐P inhibited 11‐KT production in response to these agents in a dose‐dependent manner; doses 30 ng/ml and higher completely eliminated the stimulatory effects of GtH and 17‐P, and only 3 ng/ml 17,20‐P significantly inhibited T‐stimulated 11‐KT production. In the control incubations, there was a trend of decreasing 11‐KT production with increasing dose of 17,20‐P. In the incubations treated with GtH and 17‐P, decreasing 11‐KT levels were not accounted for by corresponding increases in the levels of T. In all incubations, the levels of 17‐P increased in response to 17,20‐P, although this may have been due to the conversion of 17,20‐P to 17‐P. The results indicate that at least one of the two enzymes that converts T to 11‐KT, 11β‐hydroxylase and 11β‐hydroxysteroid dehydrogenase, is inhibited by physiological levels of 17,20‐P. In addition, the activities of other enzymes involved in androgen biosynthesis, such as C‐17,20‐lyase, may also be inhibited by 17,20‐P.
Cell intercalation during notochord development in Xenopus laevis
Authors: Keller Ray , Cooper Mark S. , Danilchik Mike , Tibbetts Paul , Wilson Paul A.
Published: June 2005

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high‐resolution time‐lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamel‐liform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid‐ to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well‐defined geometric and mechanical environment.
Pre‐axonogenesis migration of afferent pioneer cells in the grasshopper embryo
Authors: Bentley David , Toroian‐Raymond Alma
Published: June 2005

In insects, afferent neurons arise primarily from the ectodermal epithelium in the periphery and differentiate at the site of their precursor mitosis. Here we describe ectodermally derived cells that migrate away from their site of origin and initiate axonogenesis at a distant location. In embryonic grasshopper limb buds, the first two pairs of afferents to differentiate are the pair of Ti1 pioneers at the limb tip and the pair of Cx1 cells found at the base of the limb. While the Ti1 pioneers arise from the mitosis of a pioneer mother cell at the limb tip, the Cx1 cells are shown to emerge from the epithelium at circumferential positions that are approximately 150° apart and that belong to different embryonic compartments. The cells migrate into contact with each other before initiating axonogenesis, and their axons then extend in a new direction that is orthogonal to the route of cell migration.
Different subsets of axonal guidance cues are essential for sensory neurite outgrowth to cutaneous and muscle targets in the dorsal ramus of the embryonic chick
Authors: Tosney Kathryn W. , Hageman Martha S.
Published: June 2005

The dorsal ramus nerve diverges dorsally from each spinal nerve to innervate the epaxial muscle and dermis that are derived in situ from each dermamyotome. The outgrowth of both the sensory and motor components of this nerve are sensitive to the proximity of the dermamyotome. Motoneurons display a direct target response that is not dependent upon the concurrent outgrowth of sensory neurites (Tosney: Dev. Biol. 122:540–588, 1987). Likewise, the outgrowth of sensory neurites could be directly dependent on the dermamyotome. Alternatively, sensory neurites could be dependent on motor axons that in turn require the dermamyotome for outgrowth. To distinguish between these possibilities, motor outgrowth was abolished by unilateral ventral neural tube deletion and the patterns of subsequent sensory neurite outgrowth were assessed. The cutaneous nerve branch formed in all cases. In contrast, neither of the epaxial muscle nerves formed in the absence of epaxial motoneuron outgrowth. Furthermore, sensory neurites could not be detected diverging into muscle from the cutaneous nerve or entering muscle via other novel routes. We conclude that motoneurons are essential for sensory outgrowth to epaxial muscle but not to cutaneous targets. It is clear that different subsets of navigational cues guide sensory afferents to muscle and to cutaneous destinations.
Regional identity is established before gastrulation in the Xenopus embryo
Authors: Turner A. , Snape A. M. , Wylie C. C. , Heasman J.
Published: June 2005

Regional differences in cell recognition properties along the animal‐vegetal axis of Xenopus laevis embryos were investigated by using an in vitro cell sorting assay. Dissociated cells were obtained from defined regions of blastula‐ and early gastrula‐stage embryos. Binary combinations of cells from different regions, or from the same region at different ages, were aggregated in stationary culture. Labelling of one population in each pair with a cell‐autonomous dye allowed the degree of sorting out in the resulting aggregates to be scored. In combinations of cells from animal caps (prospective ectoderm) and vegetal masses (prospective endoderm), sorting was detectable at the equivalent of late blastula stage and increased with developmental age to the gastrula stage.

In addition, cells from the same region but different stages sorted from each other, indicating temporal changes in regional identity. Marginal cells (prospective mesoderm) sorted strongly from vegetal cells but only weakly from animal cells.

These results indicate the presence of regional identity in the form of specific recognition properties in cells of the Xenopus blastula. We suggest that these properties act to establish and maintain coherent cell populations, corresponding to the primary germ layers, prior to gastrulation. These results account for the gradual restriction in developmental capacity of blastomeres seen previously in single‐cell transplantation experiments from this laboratory.
Invagination of the otic placode: Normal development and experimental manipulation
Authors: Hilfer S. Robert , Esteves Ruth A. , Sanzo James F.
Published: June 2005

The inner ear forms from paired ectodermal primordia that lie to either side of the developing hindbrain. Initially each primordium forms a shallow depression in the ectodermal surface. Invagination to form an otic pit coincides with the formation of several deep folds in the epithelial surface. An initial fold appears parallel to the embryonic axis and at the junction of the rhombencephalon with somitomeric mesoderm. This is followed by formation of cranial and caudal folds perpendicular to the axis and minor folds that are within the pit formed by earlier folding. The central region of the otic primordium remains in close apposition to the lateral surface of the neural tube during the process of fold formation, until the otic pit becomes quite deep. At that time, mesenchymal cells penetrate between the two layers.

Experimental analysis of invagination supports the conclusion that otic invagination is controlled differently from that of similar organ primordia, such as the eye and thyroid. Whereas these other primordia can be stimulated to undergo normal morphogenetic shape changes precociously by treatments that presumably activate motile processes in the cytoskeleton, the same conditions have little effect on the otic placode. Similarly, neither inhibitors of calcium transport nor inactivators of calmodulin activity prevent otic pit formation, while these drugs block invagination of other primordia. These results suggest that otic invagination may be caused by changes in the surrounding tissues rather than by an activation of motility within the primordium.
Aromatic alcohol oxidase: A new membrane‐bound H2O2‐generating enzyme in alimentary tissues of the slug Arion ater
Authors: Mann Vincent , Large Andrew , Khan Sultan , Malik Zulfiqar , Connock Martin J.
Published: June 2005

We employed a sensitive spectrophotometric technique to detect H2O2‐generating enzymic activities capable of utilizing aldehydes or alcohols as substrates. We analysed tissue homogenates and subcellular fractions of the terrestrial slug Arion ater. Of the tissues tested, those of the alimentary system (especially crop and digestive gland) had the greatest activity and generated H2O2 when supplied with a variety of alcohol and aldehyde substrates. Aldehyde oxidase activity was soluble and had high apparent affinity for benzaldehyde. This activity is probably analogous to mammalian aldox (aldehyde oxidase; E.C.1.2.3.1), since it could use an artificial electron acceptor system and greatly preferred benzaldehyde to hypoxanthine. However, it differed from guinea pig liver aldehyde oxidase in that salicylaldehyde was a potent inhibitor rather than a substrate. Alcohol oxidase activity was particulate but not localised in mitochondria or peroxisomes. In digestive gland, it was localised differently from mannitol oxidase. In crop, the subcellular sites are either the same or inseparable by isopycnic density gradient centrifugation in sucrose gradients. Activity was at least ten times greater with aromatic alcohols than with ethanol, propanol, or butanol. No activity was detected with methanol, and the enzyme could not utilise an artificial electron acceptor system or pyridine nucleotides. Aromatic alcohol oxidase differed from mannitol oxidase with respect to temperature and pH sensitivity and in exhibiting greater apparent affinity for its substrates. The alcohol oxidase accepted a wide variety of aromatic alcohols as substrates. In several respects it resembled the corresponding enzyme of wood‐rot fungi, implying that the slug utilises dietary lignin or lignin precursors. Possible dietary, microbicidal, and detoxifying roles are considered for slug aldehyde and alcohol oxidases.
Neuropeptides influencing in vitro pituitary hormone secretion in hatchling turtles
Authors: Denver Robert J. , Licht Paul
Published: June 2005

Static culture techniques were used to examine the effects of neuropeptides on thyrotropin (TSH), growth hormone (GH), and luteinizing hormone (LH) secretion by turtle pituitaries (Pseudemys scripta). Pituitaries were exposed to 2.5–2,500 nM thyrotropin‐releasing hormone (TRH), ovine corticotropin‐releasing hormone (oCRH), sauvagine (SV), carp urotensin I (UI), rat growth hormone‐releasing hormone (rGHRH), human GHRH (hGHRH), carp GHRH‐like immunoreactive peptide (cGHRH‐LIP), vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), salmon gonadotropin‐releasing hormone (sGnRH) or chicken II GnRH (cGnRH). Medium concentrations of TSH, GH, and LH were estimated by RIA. Significant dose responses in TSH secretion were observed with TRH, oCRH, rGHRH, and hGHRH; VIP was also a potent stimulator of TSH, but a dose response was not observed. The two CRH‐like molecules, SV and UI, also stimulated TSH at the single dose tested (25 nM). All these neuropeptides had similar potencies. In one test in which PRL was measured, it was stimulated by 25 nM TRH but not by an equivalent dose of the CRH‐like neuropeptides. GH secretion was elevated by TRH, rGHRH, hGHRH, and VIP. oCRH at the highest dose (250 nM) stimulated GH, but SV and UI had no effect on GH at the single dose tested (25 nM). Neither AVT, cGHRH‐LIP, nor the two GnRH molecules had any effect on TSH or GH secretion. Data obtained by bioassay of TSH in representative samples were consistent with results of RIA.

In contrast to the diversity of neuropeptides affecting pituitary TSH and GH, only GnRH stimulated significant and marked increases in LH secretion. AVT produced a small increase in LH output at the higher doses tested (250 and 2,500 nM), and all other neuropeptides were inactive at all doses. Taken together with other comparative data, these results suggest that in ectothermal vertebrates, TSH and GH responses are generally not as specific for neuropeptides as they are in normal mammals but may resemble mammals in certain abnormal states. However, there is not a clear phylogenetic pattern in which neuropeptides may be stimulatory to TSH or GH. The gonadotropes in both ectotherms and mammals appear to be relatively specific for molecules of the GnRH family.
Adolescence in Euplotes octocarinatus
Authors: Kuhlmann Hans‐Werner , Heckmann Klaus
Published: June 2005

Ten mating types are known for the hypotrich ciliate Euplotes octocarinatus. The mating types are determined by four codominant alleles that control the production of four gamones. The gamones induce mature cells of certain other mating types to get ready for conjugation. That the cells respond only to gamones that they do not synthesize themselves is rationalized by assuming that mating competence is specified by gamone‐specific receptors and that cells express receptors only for those gamones they do not synthesize themselves.

Here we show that exconjugant clones are initially composed of cells that neither release gamones nor respond to gamones. If cells are heterozygous and therefore capable of producing two gamones, they start at a clonal age of about 15 fissions to synthesize first one gamone and five to ten fissions later a second gamone. Later still, their capacity to respond to gamones develops, first for one gamone and then five to ten fissions later for a second gamone. Cells responding in maturity to three gamones often acquire this capacity in three steps. These results support our assumption that in E. octocarinatus the gamones are perceived by gamone‐specific receptors. We propose that the genes coding for these gamone receptors are derepressed individually in clonal development. The same is assumed for the derepression of the genes that code for the four gamones. We further propose that the cells have the capacity to synthesize receptors for all four gamones, but they down‐regulate those receptors to which they synthesize a corresponding gamone.
The temporal pattern of vitellogenin synthesis in Drosophila grimshawi
Authors: Kambysellis Michael P. , Hatzopoulos Polydefkis , Craddock Elysse M.
Published: June 2005

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S‐methionine and 3H‐amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13–15 days. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two‐phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day‐old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7–9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13–15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 μg of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 μg vitellogenins/h. About 2 μg of this are synthesized in the fat body, and about 4 μg in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.
Functional organization of cerebral microvasculature in a marsupial, the Northern Quoll (Dasyurus hallucatus)
Authors: Snyder Gregory K. , Gannon Bren , Baudinette R. V. , Nelson John
Published: June 2005

Capillaries within the central nervous system (CNS) of eutherian mammals form meshworks with numerous anastamoses, whereas capillaries in the CNS of marsupials consist entirely of hairpin‐like loops, without anastamotic interconnections. Counter‐current blood flow in capillary loops may have been important in the evolutionary development of a cerebral vascular supply. However, loops are not found in eutherian mammals, perhaps because of a limited benefit to the diffusive conductance of gases.
Comparative study of ureogenesis in freshwater, air‐breathing teleosts
Authors: Saha N. , Ratha B. K.
Published: June 2005

The ureogenic potential of five species of freshwater, air‐breathing teleosts was studied. The activity of all five enzymes of the ornithine‐urea (c‐u) cycle could be assayed in four out of the five species. The enzymatic activities were generally high. Physiological levels of ammonia and urea and of enzymes of the o‐u cycle have been considered to be associated with the potential for terrestrial life in different species. The species having an efficient o‐u cycle had low levels of ammonia and high levels of urea in various tissues. Ammonia was the major excretory product in the aquatic medium in all five air‐breathing species. However, the rate of excretion of urea was greater than that of exclusively freshwater teleosts reported earlier. There was apparently no diurnal variation in the rates of excretion of ammonia. The rate of excretion of urea was significantly higher during the night than during the day in only two of the species examined, namely, Heteropneustes fossilis and Anabas testudineus. These observations strengthen our earlier suggestion that active ureogenesis through a functional o‐u cycle does exist in some freshwater, air‐breathing teleosts.
Effects of the organophosphate insecticides diazinon and parathion on bobwhite quail embryos: Skeletal defects and acetylcholinesterase activity
Authors: Meneely Gail A. , Wyttenbach Charles R.
Published: June 2005

Bobwhite quail eggs were injected at 48 or 72 hr of incubation with various doses of the organophosphate (OP) insecticides diazinon or parathion and the embryos were examined after an additional 48 hr of incubation by both histological and cartilage‐staining methods. Bobwhite embryos did not display the notochordal folding or vascular enlargement reported for OP‐injected chicken embryos. Cartilage staining of embryos injected with insecticide at 72 hr of incubation and recovered at day 12 of incubation revealed severe shortening and contortion of the vertebral axis, as well as tibiotarsal, rib, and sternum defects. Parath on was more potent in causing skeletal defects than diazinon. No type I defects (micromelia, parrot beak) were detected. Radiometric acetylcholinesterase (AChE) assays of whole embryo homogenates were performed for day 6, 9, and 12 diazinon‐injected and control embryos. Diazinon effected drastic reductions in AChE activity. Although the AChE and axial skeletal responses of bobwhite embryos to OP injection are similar to those reported in the literature for other species, some major differences in the bobwhite response were noted: namely, the absence of notochordal folding in the young bobwhite embryo and the absence of type I defects at day 12. These differences suggest that further studies with the bobwhite quail would be useful in clarifying the mechanisms involved in OP‐induced teratogenesis.
De novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture
Authors: Buhi W. C. , Vallet J. L. , Bazer F. W.
Published: June 2005

The objective of the present study was to identify and characterize in a limited manner the major de novo oviductal secretory proteins (OSP) synthesized and released by the porcine oviduct. Oviductal tissue was collected on various days of the estrous cycle (EC) and early pregnancy (EP) and cultured in a modified minimal essential medium supplemented with 100 μCi L‐[3H]‐leucine. Oviductal secretory activity, as measured by the rate of incorporation of 3H‐leucine (dpm/mg wet tissue weight) into nondialyzable macromolecules, was greatest(P < .01) between days 0 and 2 and reached its lowest levels on days 10 to 15. There was no difference between left and right side or pregnancy status. This increased rate of incorporation at proestrus and estrus is temporally associated with elevated levels of estrogen. Incorporation rate for ampulla was greater than for the isthmus. Analysis of oviductal culture medium by one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and fluorography revealed three protein bands of relative molecular weight (Mr) 335,000, 115,000, and 85,000, which were associated with proestrus, estrus, and metestrus and were not detectable on other days. All three proteins also incorporated 3H‐glucosamine. The 115,000 Mr band was the major 3H‐glucosamine‐labeled protein. Two protein bands (Mr 60,000 and 20,000) were expressed with increasing progesterone during diestrus. Other de novo synthesized protein bands appear to be present throughout the EC and EP with little modulation by estrogen or progesterone. Thus, this study demonstrates that for the porcine oviduct, the increase in the incorporation rate of 3H‐leucine into OSP by both whole oviduct and ampulla and de novo synthesis and secretion of three glycoproteins, Mr 335,000,115,000, and 85,000, were associated with proestrus and estrus when events such as fertilization and early cleavage stages of embryo development occurred.
In vitro fertilization of gonadotropin‐stimulated leopard cat (Felis bengalensis) follicular Oocytes
Authors: Goodrowe Karen L. , Miller Anne M. , Wildt David E.
Published: June 2005

Based on techniques developed for the domestic cat, in vitro fertilization (IVF) studies were conducted in the taxonomically related leopard cat (Felis bengalensis). Adult females received pregnant mares' serum gonadotropin (PMSG) followed 80 or 84 h later by human chorionic gonadotropin (hCG) on two to four occasions over a 40‐day to 27‐month interval. Oocytes were collected laparoscopically from ovarian follicles 25–27 h after hCG and co‐cultured with processed, homologous spermatozoa (37°C, 5% CO2 in air, humidified atmosphere) for 30–36 h. There was no apparent ovarian refractoriness to repeated treatments with exogenous gonadotropins. Overall, the mean number of mature follicles present and the total number of oocytes and proportion of immature oocytes collected did not differ (P > 0.05) between the 80 h (4.9 ± 0.9; 4.7 ± 1.2; 14.9%, respectively) and 84 h (5.6 ± 1.4; 5.4 ± 1.7; 22.2%, respectively) gonadotropin interval groups. However, the proportion of mature leopard cat oocytes fertilized in vitro, as determined by embryonic cleavage, was increased (P < 0.005) by extending the interval between PMSG and hCG from 80 (17.5%) to 84 (52.4%) h. These data 1) demonstrate that, compared to the domestic cat, the ovaries of the leopard cat are less responsive to a given PMSG/hCG treatment; 2) indicate that leopard cat follicular oocytes can be recovered readily by laparoscopy and are capable of becoming fertilized in vitro; and 3) suggest that IVF may be a viable approach for producing embryos and perhaps enhancing captive propagation of rare Felidae.
Human chorionic gonadotropin (hCG) induces gonad reversal in a protogynous fish, the bluehead wrasse, Thalassoma bifasciatum (teleostei, labridae)
Authors: Koulish S. , Kramer C. R.
Published: June 2005

Female to male successive hermaphroditism (protogyny) is common in several groups of marine fish. Thalassoma bifasciatum, the bluehead wrasse (Labridae), found in the reefs of the Caribbean normally undergoes sex reversal after receiving behavioral cues. This report deals with the successful use of human chorionic gonadotropin (hCG) in inducing gonad reversal in this species. Eighty percent (n = 40) of the treated fish showed signs of reversal in 1–6 weeks; only 11% (n = 54) of the control (nontreated) group showed signs of reversal during the same period. The number of fish undergoing reversal increased with the length of the treatment period, 55% after 1 week, 100% after 6 weeks. A bluehead color pattern, typical of the terminal male phase, also appeared more frequently in the longer treated groups. To determine the efficacy of hCG in inducing gonad reversal, morphological criteria for reversal had to be established. For the majority of treated fish, the presence of both spermatogenic cysts and degenerating oocytes was sufficient to label these gonads unambiguously as undergoing reversal. However, at the extremes of this process, i.e., onset (early) and endpoint (late) stages, ambiguity could arise in identifying a gonad as undergoing reversal, and, therefore, key criteria were established for these stages. In our hands, the most consistent and reliable criterion for the early stage reversal was appearance of male germ line cells, clusters of “spermatogonial‐like” cells. For the late stage, recognizable remnants of late‐state oocytes had to be present. Some details of the histological changes that occur during early, middle, and late stages of reversal are also described. It is suggested that these results with hCG shed new light on the endocrine control of gonad reversal in T. bifasciatum.
Cold shock induces actin reorganization and polyspermy in sea urchin eggs
Authors: Santella Luigia , Monroy Alberto
Published: June 2005

The effect of low temperature on the cortical actin system and polyspermy in sea urchin eggs was studied. Eggs cold‐shocked at 3°C for 1 hour formed a cortical system of polymerized actin and were polyspermic when fertilized. These effects were completely reversible following a 7–20 minute recovery period at room temperature. The present data raises the possibility that actin, or components of the egg cytoskeleton, regulate sperm entry in sea urchin eggs.
The Role of Refractoriness to Long Daylength in the Annual Reproductive Cycle of the Female Bennett's Wallaby (Macropus rufogriseus rufogriseus)
Authors: Curlewis J. D. , Loudon A. S. I.
Published: June 2005

The beginning of the breeding season of the female Bennett's wallaby occurs when seasonal quiescence terminates 1–2 months after the summer solstice. In this study, the role of photoperiod in terminating seasonal quiescence was examined. One week before the summer solstice, five non‐lactating wallabies were transferred from natural to artificial summer solstice daylength for 5 months. The beginning of the breeding season in these animals as indicated by births, matings, and peripheral progesterone profiles was not different from that of five control animals maintained on natural photoperiod. The following year, three animals were transferred from natural to summer solstice daylength on February 25 and were held on the artificial photo‐period until September 30. Changes in plasma progesterone concentrations indicative of the beginning of the breeding season occurred on June 12–30 (range), which was significantly (P < 0.01) advanced by 29 days when compared with six control animals. These results indicate that the decrease in daylength that occurs after the summer solstice is not required to induce the termination of seasonal quiescence at the beginning of the breeding season. Further, the beginning of the breeding season can be advanced by transferring animals to long daylength early in seasonal quiescence. Photorefractoriness to long daylengths may therefore be important in the initiation of the breeding season in this species. In further experiments, groups of six animals were transferred from natural to artificial summer solstice daylength on September 26 and December 9 and pouch young were removed 7 days after the transfer. In September, reactivation of the quiescent corpus luteum followed soon after removal of pouch young (RPY) indicating that exposure to long day‐length had not induced a transition into seasonal quiescence. In December, RPY was not followed by reactivation of the quiescent corpus luteum indicating that animals were in seasonal quiescence. These results suggest that the female Bennett's wallaby may need to experience a period of shortening days after the summer solstice before exposure to long days can again initiate seasonal quiescence.
Comparison of semilunar cycles of spawning activity in Fundulus grandis and F. heteroclitus held under constant laboratory conditions
Authors: Hsiao Shyh‐Min , Meier Albert H.
Published: June 2005

Two closely related killifish, Fundulus grandis of the Gulf coast and F. heteroclitus of the Atlantic coast, were held under standard laboratory conditions and monitored for spawning activity. Daily egg collections for 4–5 months during summer revealed semilunar cycles of spawning activity in both F. grandis (three aquaria) and F. heteroclitus (five of six aquaria). The mean periods were near 13.7 days for F. grandis and near 14.8 days for F. heteroclitus. The shorter duration in F. grandis closely approximates the tidal cycle in the Gulf of Mexico, which is an expression of the declinational cycle of the moon (tropical month: 27.3 days). The longer duration in F. heteroclitus closely approximates the tidal cycle along the Atlantic coast, which, along with the moonlight cycle, is an expression of the synodic month (29.5 days). Unless the fish respond to subtle exogenous cues that can influence spawning under isolated laboratory conditions, these results indicate that both killifish have endogenous cycles with very precise periods. These cycles free run for several months in specific phase relationships with tidal (F. grandis) and tidal/moonlight cycles (F. heteroclitus) present in their respective natural habitats.
Effects of extracellular pH and D‐lactate efflux on regulation of intracellular pH during isotonic contractions in a molluscan muscle: A 31p‐nuclear magnetic resonance study
Authors: Wiseman Robert W. , Ellington W. Ross , Rosanske R. C.
Published: June 2005

Isotonic contractions were electrically induced in isolated radula protractor muscles of the whelk Busycon canaliculatum, mounted and superfused under anoxic conditions in a special 5 mm phosphorus nuclear magnetic resonance (31P‐NMR) probe. 31P‐NMR spectra showed that there was a decrease in intracellular pH (pHi) from 7.29 to 7.01 during the time course of 30 min of contractile activity. Subsequent biochemical analysis of the tissue and superfusion medium showed that D‐lactate and, to a much lesser extent, alanopine and octopine were the major glycolytic end products. Approximately 75% of the total D‐lactate produced was released into the medium. When the superfusate pH was changed from 7.8 to 7.2, a much more pronounced intracellular acidification took place, as the pHi fell to 6.80. However, there was no significant effect on the extent of release of D‐lactate from the muscle. Elevation of the superfusate pH to 8.2 had no significant effect on final pHi or D‐lactate efflux. D‐lactate efflux was reduced when α‐cyanohydroxycinnamic acid or L‐lactate was added to the superfusate at pH 7.8. These treatments had no significant effect on the final pHi. Movement of proton equivalents and D‐lactate efflux are not tightly linked in this muscle system.
Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta
Authors: Watson R. Douglas , Thomas Mark K. , Bollenbacher Walter E.
Published: June 2005

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules.

A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head‐ligated larvae. However, big PTTH was ∽10‐fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH.

The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last‐instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)‐ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA‐detectable ecdysteroids in prothoracic‐gland‐conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA‐detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity.

The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.
Ecdysteroid synthesis by crab Y‐organs
Authors: Lachaise Fabienne , Carpentier Gilles , Sommé Ghislaine , Colardeau Jacqueline , Beydon Philippe
Published: June 2005

Y‐organs of the crab Carcinus maenas synthesize two ecdysteroids—ecdysone and 25‐deoxyecdysone—which are the precursors of 20‐hydroxyecdysone and ponasterone A, respectively; 25‐deoxyecdysone was detected by using two methods: (1) radiolabeled cholesterol injection into premolt crabs, followed by in vitro incubation of their Y‐organs and high‐performance liquid chromatography (HPLC) analysis of the various ecdysteroids formed, and 2) identification of ecdysteroids secreted by incubated crab Y‐organs by means of two successive HPLC purifications, followed by radioimmunoassay (RIA) and mass spectrum analysis. The only sterols found in the Y‐organs are 7‐dehydrocholesterol and cholesterol. These are probably the ecdysone and 25‐deoxyecdysone precursors. The biological activity of 25‐deoxyecdysone, tested by its induction of the transcription of gene P1 in Drosophila fat body, was the same as that of 20‐hydroxyecdysone.
Spermiogenesis in Chaetoderma sp. (Aplacophora)
Authors: Buckland‐Nicks John , Chia Fu‐Shiang
Published: June 2005

The fine structure of spermiogenesis in Chaetoderma sp. has been studied with light and electron microscopy. In early spermiogenesis the Golgi body secretes a pro‐acrosomal granule posteriorly that migrates along the plasma membrane to the anterior of the spermatid. The Golgi body moves to an anterior depression in the nucleus and secretes granular vesicles that form the apical horn. A large vesicle accumulates next to the Golgi body, and an apical dense tube is produced around the apical horn that projects anterio‐laterally, over the large vesicle, and eventually contacts the pro‐acrosome. Both centrioles produce flagella, which extend anteriorly through the cytoplasm. The distal flagellum circumnavigates the nucleus and exits posteriorly. The proximal flagellum exits anteriorly alongside the apical dense tube, which further elongates. The chromatin condenses first in granules, leaving large lacunae centrally, and later into an almost homogeneous dense mass. No manchette of microtubules was observed during condensation, but individual microtubules were visible in the cytoplasm. The residual cytoplasm flows out anteriorly along the proximal flagellum and is eliminated together with the Golgi body and large vesicle. The distal flagellum breaks free from the cytoplasm and unwinds, finally exiting centrally from the mid‐piece. In the mature sperm the acrosome is at the tip of a long apical dense tube that projects anterio‐laterally from the apical horn. The nucleus terminates in the posterior end of the apical horn. The mid‐piece is composed of five mitochondria which surround the centrioles. The flagellum is harnessed to the mid‐piece by a centriolar satellite complex that is attached to the plasma membrane by the annulus. The end‐piece is filamentous and contains two central microtubules. This sperm type may be unique among Mollusca, because its structure and morphogenesis exhibit numerous differences even from its relatives, the chitons.
Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration
Authors: Krotoski Danuta , Bronner‐Fraser Marianne
Published: June 2005

We have examined the distribution in Xenopus embryos of β1 subunits of integrin, as recognized by cross‐reactive antibodies against the avian integrin beta1 subunit. These antibodies recognize a doublet of bands of approximately 120 kD in Xenopus embryos. The distribution pattern of these integrin cell surface receptors was compared with that of two possible ligands, fibronectin and laminin, in the extracellular matrix during the time of neural crest cell migration. Integrin immunoreactivity in the early neurula was observed lightly outlining somite and epidermal cells and the notochord. The integrin immunostaining increased with developmental age and was observed on most cell types in the embryo but was particularly notable in the intersomitic clefts through which motoraxons grow. The immunoreactivity in this region was not, however, wholly on the axon surfaces, since intersomitic integrin remained detectable in embryos in which the neural tube had been ablated. Fibronectin and laminin were more extensively distributed than integrin at all stages examined. Immunoreactivity for both was observed around the neural tube, notochord, somites, epidermis, dorsal mesentery, and lateral plate mesoderm. The distribution of laminin and fibronectin around the somites was particularly interesting since it was non‐uniform and similar to that of integrin. Strongest staining was observed in the intersomitic clefts, and weakest staining was observed on the medial surface of the somites, which faces the neural tube and notochord. The major differences in distribution pattern between the fibronectin and laminin immunoreactivities were that only fibronectin was detected in the mesenchyme of the dorsal fin. Our results demonstrate that a molecule homologous to avian integrin is present in Xenopus embryos during neural crest cell migration and motoraxon outgrowth. Its presence in the intersomitic clefts and on the surface of many embryonic cell types together with the abundant distribution of its ligands are consistent with a potentially important developmental function in neurite outgrowth and/or muscle development.
Prolactin counteracts effects of short day lengths on pelage growth in the meadow vole, Microtus pennsylvanicus
Authors: Smale Laura , Lee Theresa M. , Nelson Randy J. , Zucker Irving
Published: June 2005

To test whether growth of the winter coat in short day lengths is contingent on suppression of plama prolactin (Prl) levels, female meadow voles (Microtus pennsylvanicus) were kept in short day lengths for 12 weeks and were injected daily with saline or Prl; long‐day animals were treated with either the dopamine agonist, bromocryptine (bromo), bromo plus Prl, or saline. Prl treatment prevented the growth of the winter coat normally observed after 12 weeks in short day lengths, but bromocryptine did not stimulate pelage growth in long‐day voles. Pelage growth in short day lengths appears contingent upon decreased plasma prolactin levels.
Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid
Authors: Van Winkle Lon J. , Haghighat Nasrin , Campione Allan L.
Published: June 2005

Two‐cell mouse conceptuses were cultured in media that contained various concentrations of inorganic ions and amino acids. Substrates of the amino acid transport system Gly were detrimental to development at slightly hyposmotic concentrations of other ions. In contrast, these amino acids increased the frequency at which two‐cell conceptuses developed into blastocysts at total ion concentrations of 355 to 405 mM. Data reported elsewhere is consistent with the possibility that the total ion concentration in oviductal fluid exceeds 360 mM, whereas the concentration of glycine in this fluid may be on the order of 10 mM. Therefore, a high ion concentration and glycine may counteract the potentially harmful effects of each other in situ. Like some marine organisms, preimplantation mouse conceptuses may use glycine as an intracellular osmolite because accumulation of inorganic ions could perturb the activities of some enzymes.
Preparation of nucleate and anucleate fragments of hamster and mouse eggs by centrifugation
Authors: Yanagimachi R. , Yang C. H.
Published: June 2005

Nucleate and anucleate fragments of hamster and mouse eggs can be prepared by centrifuging zona‐free eggs in a Percoll gradient. These fragments are fertilizable and can be used for various studies of nucleus–ooplasm interactions.
Immunocytochemical localization of a calcium‐binding phosphoprotein in hemocytes of heterodont bivalves
Authors: Marsh M. E.
Published: June 2005

Calcium‐binding phosphoprotein particles are the most abundant extracellular proteins in the hemolymph of heterodont bivalves, and granular hemocytes are the most abundant cells in the same fluid. In this study, the hemocytes of Rangia cuneata were examined ultrastruc‐turally and probed with anti‐phosphoprotein IgG to demonstrate that the granulocytes are a probable source of the hemolymph phosphoprotein. The granulocyte cytoplasm is laden with large vesicles containing an amorphous homogeneous matrix and variable numbers of electron‐dense particles; the latter are ultrastructurally similar to the extracellular phosphoprotein. The vesicle particles and matrix are related forms of the hemolymph phosphoprotein as evidenced by heavy gold labeling when Lowicryl sections were sequentially treated with rabbit‐anti‐phosphoprotein IgG and colloidal gold‐anti‐rabbit IgG. The vesicles may be the loci for posttranslational phosphory‐lation and eventual secretion of the calcium‐binding phosphoprotein, or alternatively the vesicles may be digestive structures which degrade internalized phosphoprotein.