bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of actinomycin D and cycloheximide on transcript levels of IGF‐I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin
Authors: Johnson Thomas R. , Trojan Jerzy , Rudin Susan D. , Blossey Betty K. , Ilan Judith , Ilan Joseph 
Published: February 2005

AbstractThe stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin‐like growth factor I transcripts in primary cultures incubated in serumfree medium. The levels of IGF‐I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF‐I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7‐fold over 7 hours. The half‐lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF‐I transcripts, but not actin transcripts, is controlled in part by an actinomycin D‐sensitive process.
Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight‐cell embryos: Evidence for the “Crabtree effect”
Authors: Seshagiri Polani B. , Bavister Barry D. 
Published: February 2005

AbstractThe development of hamster eight‐cell embryos is inhibited by glucose in culture medium containing inorganic phosphate (Pi). This is hypothetically attributed to the “Crabtree effect,” in which enhanced glycolysis inhibits respiratory activity and oxidative metabolism. To examine this hypothesis, oxygen consumption of hamster eight‐cell embryos was measured using a microelectrode. A two‐ to three‐fold decrease in oxygen consumption was observed in embryos cultured with glucose and Pi. Oxidizable substrates and intermediates of the Krebs cycle supported embryo development only in the absence of glucose and Pi; Krebs cycle inhibitors (fluoroacetate and arsenite) arrested embryo development. Under anaerobic conditions, pyruvate and lactate did not support embryo development. Inhibition of both respiration and oxidative activity in cultured hamster embryos by glucose and Pi is consistent with the existence of a Crabtree effect and indicates that the metabolic properties of preimplantation embryonic cells differ markedly from those of most somatic cells and resemble some cancer cells.
Changes in maturation‐promoting activity in the cytoplasm of pig oocytes throughout maturation
Authors: Mattioli M. , Galeati G. , Bacci M. L. , Barboni B. 
Published: February 2005

AbstractMaturation‐promoting factor (MPF) was examined in maturing pig oocytes by electrofusing them with germinal vesicle (GV) oocytes. Oocytes containing high levels of MPF (MI or MII stages) induced the breakdown of the GV introduced by fusion and the formation of the metaphase plate in 1 hr. A similar effect was seen when two or three GV oocytes were fused with a MII oocyte and then incubated for 1 hr in the presence of cycloheximide (a specific protein synthesis inhibitor), indicating that high levels of preformed MPF are present at the metaphase stage. During the maturation in vitro of cumulus‐enclosed oocytes, a first sharp rise in MPF was seen between 26 and 29 hr of culture (MI stage); MPF declined after 2 hr (AI‐TI stages) and again reached high levels at 35 hr, where it remained for the rest of maturation. Denuded oocytes showed a similar behavior, but MPF appeared 9 hr earlier and the rise, due to the asynchronous maturation of these oocytes, was not as sharp as in cumulus enclosed oocytes. Cycloheximide was used to study protein synthesis requirements for oocyte maturation. Intact GV were observed after 44 hr of culture when cycloheximide was added at 26 hr or earlier, and chromosome decondensation and pronuclear formation were observed when the drug was added at 32 hr. Transcriptional requirements were investigated by treating the oocytes with α‐amanitin, an RNA polymerase inhibitor. This drug could completely inhibit the maturation of cumulus‐enclosed oocytes, but this was a somatic cell‐mediated effect since denuded oocytes were insensitive to this treatment. Enucleated oocytes exhibited an increase in MPF after 24 hr of culture, and low levels of this factor were seen after 40 h of maturation. These data indicate that both rises in MPF require active protein synthesis, whereas transcription is not necessary for the resumption of meiotic cycle. Nuclear activity may be required for the second rise in MPF.
Ion channels in boar sperm plasma membranes: Characterization of a cation selective channel
Authors: Cox Thomas , Campbell P. , Peterson R. N. 
Published: February 2005

AbstractPlasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high‐pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.
Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm
Authors: Martin Renee H. , Chernos Judy E. , Rademaker Alfred W. 
Published: February 2005

AbstractThe effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.
Analysis of a murine male germ cell‐specific transcript that encodes a putative zinc finger protein
Authors: Höög Christer , Schalling Martin , Grunder‐Brundell Eva , Daneholt Bertil 
Published: February 2005

AbstractA cDNA species, corresponding to a gene with testis‐specific expression (TSGA), was isolated from a testis cDNA library. The temporal and spatial expression of TSGA was studied by in situ hybridization as well as RNA filter hybridization. In tissue sections, the TSGA sequence was confined to cells within the seminiferous tubules. For filter hybridization, RNA was isolated from testis of prepubertal rats of different ages as well as from enriched populations of various germ cell types. It was found that TSGA is expressed only in male germ cells and that the steady‐state level of TSGA transcripts reaches a maximum during the meiotic and the postmeiotic stages of germ cell development, suggesting a meiotic or postmeiotic function for the encoded protein. TSGA encodes a putative protein having 1,214 amino acids and contains a zinc finger, a structure that previously has been shown to mediate binding to nucleic acids.
Activation of a two‐cell stage‐specific gene following transfer of heterologous nuclei into enucleated mouse embryos
Authors: Latham Keith E. , Solter Davor , Schultz Richard M. 
Published: February 2005

AbstractZygotic gene activation occurs at the two‐cell stage in the mouse embryo, resulting in the appearance of many new proteins, including a stage‐specific family of related proteins of Mr 70,000. The mechanisms that regulate the stage‐specific expression of these proteins were examined by transplanting nuclei from oocytes, four‐cell‐stage blastomeres, inner cell mass cells, cultured embryonic stem cells, or differentiated endoderm‐like PYS2 cells to enucleated one‐cell embryos. Although none of these cell types synthesizes the 70 kDa complex, all were able to direct the synthesis of the 70 kDa complex following transplantation and overnight culture to the two‐cell stage. These results suggest that the embryonic cytoplasm can exert a dominant, positive regulatory influence on a variety of heterologous nuclei that results in the transcription of a stage‐specific gene. In addition, these results indicate that activation of the gene(s) coding for the 70 kDa complex is not dependent on prior programming during oogenesis and oocyte maturation, and that repression of the gene(s) coding for this complex after the two‐cell stage does not involve irreversible gene inactivation.
Liposome‐Mediated DNA uptake by sperm cells
Authors: Bachiller Daniel , Schellander Karl , Peli Janos , Rüther Ulrich 
Published: February 2005

AbstractTo investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.
Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss)
Authors: Penman D. J. , Iyengar A. , Beeching A. J. , Rahman A. , Sulaiman Z. , Maclean N. 
Published: February 2005

AbstractThere have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish.Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.
Cryopreservation of mouse half‐morulae and chimeric embryos by vitrification
Authors: Nagashima Hiroshi , Kobayashi Kazuhiko , Yamakawa Hirohito , Ogawa Shyoso 
Published: February 2005

AbstractMouse half‐morulae were cryopreserved ≤1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2‐propanediol as cryoprotectant. The developmental rates of the frozen‐thawed half‐embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty‐one of the half‐embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half‐morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen‐thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half‐morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half‐embryos of the components.
Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models
Authors: Molinia F. C. , Swan M. A. 
Published: February 2005

AbstractThe organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X‐100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X‐100 demembranated sperm while 0.01% and 0.05% Triton X‐100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1% Triton X‐100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X‐100 permeabilized sperm models. Reactivated motility of 0.05% Triton X‐100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.
Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing
Authors: Métézeau Philippe , Cotinot Corinne , Colas Guy , Azoulay Martine , Kiefer Héléne , Goldberg Michel E. , Kirszenbaum Marek 
Published: February 2005

AbstractFlow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of (1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or (2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa “optically” oriented.
Growth and DNA replication in rabbit blastocysts
Authors: Lawitts Joel A. , Butler James E. , Kiessling Ann A. , Biggers John D. 
Published: February 2005

AbstractDNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24‐hr intervals over the period of days 4–7 postcoitum (pc). Median DNA content increased 53 times over the 72‐hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37°C) increased 393‐fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by34‐fold and 6,078‐fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within‐litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6‐ and 7‐day‐old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.
Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents
Authors: Breed W. G. , Leigh C. M. 
Published: February 2005

AbstractThe distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD‐phallacidin‐positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD‐phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.
Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells
Authors: Liu Qiangyuan , Wang Linfang , Miao Shiying , Zhao Min , Zong Shudong , Yan Yuan‐Chang , Koide S. S. 
Published: February 2005

AbstractA general mammalian expression vector designated pSV2‐EP was reconstructed by inserting an oligonucleotide fragment into pSV2‐dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2‐EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly‐A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from Àgt11 expression libraries. A recombinant plasmid (pSVRS‐1) was constructed by inserting RSD‐1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2‐EP vector. Chinese hamster ovarian (CHO) dhfr‐negative cells were contransformed with pSV2‐dhfr and pSVRS‐1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD‐1. One of these transformed cell lines stained intensely with anti‐rSMP‐B antibodies, demonstrating that the RSD‐1 was expressed in the transformed CHO cells.
Bovine embryos produce a urokinase‐type plasminogen activator
Authors: Berg Debra A. , Menino Alfred R. 
Published: February 2005

AbstractThe type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12–14 embryos were collected from estrus‐synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre‐cultured for 2 days in Minimum Essential Medium Alpha (MEMα) with 10% heat‐inactivated fetal calf serum, washed in serum‐free MEMα, and cultured individually for 5 days in 50 μl microdrops of MEMα with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at −20°C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at −20°C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase‐type PA (anti‐uPA) and tissue‐type PA (anti‐tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P < 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P < 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P < 0.05) activity than from ED. Treatment with anti‐uPA decreased PA activity (P < 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti‐tPA. Amiloride completely eliminated PA activity (P < 0.05) in tissues, medium, and BF. These results suggest that day 12–14 bovine embryos secrete a urokinase‐type PA.