Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis.
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Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species. (+info)
Purification and characterization of UDP-GlcNAc: GlcNAcbeta 1-6(GlcNAcbeta 1-2)Manalpha 1-R [GlcNAc to Man]-beta 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct.
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A new beta1,4-N-acetylglucosaminyltransferase (GnT) responsible for the formation of branched N-linked complex-type sugar chains has been purified 64,000-fold in 16% yield from a homogenate of hen oviduct by column chromatography procedures using Q-Sepharose FF, Ni(2+)-chelating Sepharose FF, and UDP-hexanolamine-agarose. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to tetraantennary oligosaccharide and produces pentaantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-6 arm. It requires a divalent cation such as Mn(2+) and has an apparent molecular weight of 72,000 under nonreducing conditions. The enzyme does not act on biantennary oligosaccharide (GnT I and II product), and beta1,6-N-acetylglucosaminylation of the Manalpha1-6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a beta1-4-linked GlcNAc residue only on the Manalpha1-3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R [GlcNAc to Man]-beta1,4-N-acetylglucosaminyltransferase VI. This is the only known enzyme that has not been previously purified among GnTs responsible for antenna formation on the cores of N-linked complex-type sugar chains. (+info)
Functional cross talk after activation of P2 and P1 receptors in oviductal ciliated cells.
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The presence of ATP and adenosine receptors and their role in controlling ciliary activity in oviductal ciliated cells was studied by measuring the ciliary beat frequency (CBF) in oviductal tissue cultures. ATP, adenosine, and related compounds increased the CBF in a dose-dependent manner. We established that P2 receptors of subtype 2Y(2) and P1 receptors of subtype A(2a) mediated the responses to ATP and adenosine, respectively. We found evidence to suggest that stimulation of ciliary activity by ATP requires D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] metabolism, intracellular Ca(2+) mobilization, and protein kinase C activation. On the other hand, the adenosine effect is mediated by activation of a G(s) protein-dependent pathway that enhances cAMP intracellular levels. To study the interaction between P2 and P1 receptors, cells were stimulated simultaneously with both agonists. We observed a synergistic increase of the CBF even at agonist concentrations (100 nM) that did not produce a significant response when added separately to the culture. Furthermore, a blocker of the cAMP pathway produced a reduction of the ATP response, whereas a blocker of the Ins(1,4,5)P(3) pathway also produced an inhibition of the adenosine response. Our evidence demonstrates that both ATP and adenosine receptors are present in a single ciliated cell and that a mechanism of cross talk could operate in the transduction pathways to control ciliary activity. (+info)
Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-D-glucosamine (GlcNAc): GlcNAcbeta 1-6(GlcNAcbeta 1-2)- manalpha 1-R[GlcNAc to man]beta 1,4N-acetylglucosaminyltransferase VI.
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A cDNA that encodes UDP-N-acetyl-d-glucosamine (GlcNAc):GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R[GlcNA c to Man]beta1, 4N-acetylglucosaminyltransferase VI (GnT VI), which is responsible for the formation of pentaantennary asparagine-linked oligosaccharides (N-glycans), has been cloned from a hen oviduct cDNA library based on the partial amino acid sequences of the purified enzyme. The isolated cDNA clone contained an open reading frame encoding 464 amino acids, including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. The primary structure was found to be significantly similar to human GnT IV-homologue, the gene for which was cloned from the deleted region in pancreatic cancer, and to human and bovine GnT IVs. Chicken GnT VI-transfected COS-1 cells showed a high GnT VI activity (26.8 pmol/h/mg protein), whereas nontransfected, mock-transfected, or human GnT IV-homologue-transfected COS-1 cells had no activity. Northern blot analysis using poly(A)(+) RNA from hen oviduct indicated that the size of GnT VI mRNA is 2.1 kilobases. Reverse transcription-polymerase chain reaction analysis showed that GnT VI mRNA was relatively highly expressed in oviduct, spleen, lung, and colon. (+info)
Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin.
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The interaction of Escherichia coli RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) with chick oviduct DNA and chromatin has been studied in order to quantify the RNA polymerase binding sites and rifampicin-resistant initiation sites. The number of high affinity binding sites at saturation was calculated from the number of molecules of enzyme bound to DNA or to chromatin. The number of rifampicin-resistant initiation sites was calculated from the total quantity and the number average chain length of RNA synthesized under the conditions in which only RNA polymerase already in a stable preinitiation complex could avoid inhibition by the drug. Using an assessment of the temperature requirement for the information of this preinitiation complex, we were able to differentiate initiations at single-stranded or nicked DNA regions from initiations at double-stranded DNA regions. This method enabled us to rule out the possibility that RNA chain initiation on chromatin was due to initiation at nicked or end regions of DNA. In addition, we also demonstrated that not all RNA polymerase molecules bound to DNA or chromatin are located at a site at which RNA chain initiation can immediately begin. This methodological approach should allow investigators to monitor the individual reactions and factors involved in in vitro transcription of eukaryotic DNA and chromatin. (+info)
Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin.
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Estrogen administration to chicks results in an increase in the chromatin template activity of oviduct target tissue as assayed under standard in vitro assay conditions. However, the results obtained by the simple measurement of template activity may be a complicated function of the number of available RNA polymerase initiation sites, the rate of RNA chain elongation, and the rate of reinitiation. In the present study, we have measured separately the change in both the number of chain initiations as well as the rate of RNA chain propagation under conditions in which reinitiation was eliminated. Chromatin prepared from either estrogen-treated or control oviducts both supported an RNA chain elongation rate of six nucleotides per s and a chain size of approximately 700 nucleotides. Thus, both the elongation rate and size of the average product remained relatively constant following estrogen stimulation. In contrast, within 8 hours after a single injection of estrogen to unstimulated chicks, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased to 150% in comparison to control values, and by 24 hours the level of polymerase bound to chromatin was twice that of the untreated control chick chromatin. With daily injections of estrogen, polymerase binding continued to rise. Coincident with the over-all increase in chromatin-bound polymerase was an increase in rifampicin-insensitive initiation sites and newly synthesized RNA chains. Unstimulated chick oviduct chromatin initiated 10,000 RNA chains/pg of DNA, while 24 hours of steroid treatment increased the number of initiated chains to 34,000 chains. These data demonstrate that the estrogen-induced increase in chromatin transcriptive activity was due to an increased number of polymerase binding and initiation sites on the chromatin template without a detectable change in the rate of RNA chain elongation. (+info)
Study of the functional anatomy of bovine oviductal mucosa.
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The oviducts of 31 cyclic cows were examined to study the structure and nature of the oviductal mucosa. The general distribution of spermatozoa within the oviductal mucosa was studied in five additional cows. The oviductal infundibulum is an asymmetric funnel-shaped structure surrounding the ostium. It is divided along the free boarder of the mesosalpinx and presents one wide and one narrow side. The mucosa of the wide side possesses a system of low interconnected cords that converge distally forming primary folds. The folds on the narrow side start sharply from the free margin and fuse toward the ostium abdominale. Areas between folds throughout the lumen of the oviduct show a high degree of complex organization. Interfold spaces are occupied by secondary and small interconnected folds which join to form a system of cul-de-sacs. In the infundibulum, these cul-de-sacs open toward the ovary, while cul-de-sacs present in the caudal isthmus and in the UTJ open toward the uterus. Marked variations were observed in the oviductal epithelium depending on the oviductal segment, basal or apical areas of the folds, and phase of the oestrous cycle. Near to the time of ovulation, numerous spermatozoa were found in the periphery of the caudal isthmus within pockets of basal interfold areas, as well as within pockets and cul-de-sacs of the tubo-uterine junction. Individual spermatozoa were also observed in peripheral areas of the ampullary-isthmic junction and ampulla. The topography of the oviduct provides a complex system of regulation which may influence not only the passage of gametes and/or embryos, but also movement of fluid within the oviductal canal. (+info)
Retinoic acid accelerates the development of reproductive organs and egg production in Japanese quail (Coturnix coturnix japonica).
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The effects of retinoic acid on the development of reproductive organs and egg production in female Japanese quail (Coturnix coturnix japonica) were investigated. Female quail were fed a diet containing retinoic acid at 4 mg/kg (RA) or two diets containing retinyl acetate at 5000 IU/kg (VA1) or 14 000 IU/kg (VA2) after being fed a vitamin A-free diet for 2 wk (experiment 1). The oviduct and ovary grew more rapidly (P < 0.05) in RA-treated quail than in VA-treated quail at 5 wk of age. In addition, the body weight of RA-fed quail was also greater (P: < 0.05) than that of VA-fed quail at 5 wk. The RA-treated quail laid their first eggs approximately 5 days earlier (P < 0.05) than the VA-treated quail. Furthermore, these RA-fed quail laid more eggs (P < 0.05) than those VA-fed quail during the experimental period. To confirm the results of experiment 1, a similar experiment was conducted to record the first egg and total eggs laid by quail fed VA2 or RA (experiment 2). The early onset of oviposition was again observed in the RA-treated group (P < 0.01). These results suggest that retinoic acid has a stimulating effect on the reproductive system of female Japanese quail, as has been previously shown in the reproductive system of male Japanese quail. (+info)