PubMed Health⌕ Search

Biomedical subjects

K A Zuelke

Publications and source records attributed to K A Zuelke.

10 recordsLinked to original sources
PubMed2006

Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression.

In cultured cumulus oocyte complexes (COC), FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and to identify candidate mRNAs involved. Experiment I: murine COC were cultured 4 h in the presence of FSH with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82 +/- 7%). When DRB was added at 0, 5, or 10 min after culture initiation, oocyte maturation was blocked (17 +/- 7, 14 +/- 6, and 21 +/- 6% GVBD, respectively). When DRB was added after 15, 20, or 30 min, progressively more COC underwent GVBD (37 +/- 6, 39 +/- 6, and 66 +/- 6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P < or = 0.01 and P </= 0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development, and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC.

Animals↗
PubMed1999

In vitro development and nutrient uptake by embryos derived from oocytes of pre-pubertal and adult cows.

The present study compared the developmental potential and uptake of nutrients by embryos from pre-pubertal and adult cows. Oocytes retrieved from ovaries of 5 to 7 month old calves and adult cows were matured and fertilized in vitro. Embryos were cultured in SOFaa to the blastocyst stage (7 days post-insemination). At successive stages of development, rates of glucose and pyruvate uptake were measured non-invasively by microfluorescence for individual embryos. Fertilization was equivalent in embryos from pre-pubertal and adult cows (P > 0.05), however development to blastocyst was significantly lower in embryos from pre-pubertal cows (9.8% versus 33.7%, respectively; P < 0.05). Total blastocyst cell number was not different between pre-pubertal and adult material (P > 0.05). Glucose uptake was exponential (pre-pubertal, r = 0.82; adult, r = 0. 82; P < 0.05), with an increase in uptake beyond the 8- to 16-cell stage. Glucose uptake was significantly lower in embryos from pre-pubertal cows at the 2- to 4-cell stages (1.5 versus 3.0 pmoles/embryo/hr; P < 0.05), but was equivalent to the adult cow at all other stages of development (P > 0.05). Pyruvate uptake was low until the blastocyst stage. Pyruvate uptake by embryos from pre-pubertal cows was significantly different to adult cows at the 1-cell stage (2.7 versus 4.6 pmoles/embryo/hr, respectively; P < 0. 05) and 2- to 4-cell stages (4.9 versus 3.6 pmoles/embryo/hr, respectively; P < 0.05). Pyruvate uptake was equivalent in the two groups in the later stages of development (P > 0.05). Perturbations in the uptake of nutrients by embryos from pre-pubertal cows were most likely due to the presence of a high proportion of developmentally incompetent embryos. Further, embryos from pre-pubertal cows that did develop to the blastocyst were as viable as blastocysts from adult cows with respect to nutrient uptakes and total cell number.

Animals↗
PubMed1998

Transgenic modification of cows milk for value-added processing.

The application of transgenic technologies in dairy cattle has been restricted largely to producing potential pharmaceutical or nutriceutical products in the mammary gland. Broader application of transgenesis in dairy cattle production will require identifying target traits that are both amenable to transgenic modification and economically important to the dairy industry. The casein proteins are the most valuable component of cows milk destined for value-added processing. The four bovine casein genes lie within a single, multi-gene locus of approximately 200 kb in length. The working hypothesis is that this multi-gene locus contains all of the DNA sequences required to regulate the coordinated expression of all four individual casein genes (i.e. a locus control region or LCR). The initial research aim is to clone the entire casein locus into a bacterial artificial chromosome (BAC) vector, thus preserving the extended 5'and 3' regions that flank the locus, as well as maintaining the spatial integrity of the four individual casein genes that comprise the locus. The author's laboratory has prepared a bacterial artificial chromosome (BAC) library of genomic DNA from elite dairy cattle. Partial, non-elite BAC clones of the casein gene locus are being tested in transgenic mice to establish proof of concept. Advances in nuclear transfer of transfected somatic cells should improve the efficiency of producing transgenic calves that possess a BAC casein construct introduced into an elite genetic background.

Animals↗
PubMed1997

Glutathione oxidation is associated with altered microtubule function and disrupted fertilization in mature hamster oocytes.

We hypothesized that depletion of glutathione (GSH) with diamide, a relatively specific GSH oxidant, may alter the meiotic spindle apparatus in mature hamster oocytes. Immunofluorescent analysis of oocytes exposed to diamide for 1.5 or 3 h revealed time- and concentration-dependent disruption of spindle morphology accompanied by chromosome clumping. In oocytes first cultured in diamide for 1.5 h and then in diamide-free medium for 1.5 or 3 h, microtubules appeared to repolymerize, but normal spindle structure was not regained. HPLC confirmed that diamide oxidized oocyte GSH under conditions identical to those associated with spindle-related abnormalities. Exposure of oocytes to 25 or 50 microM diamide before in vitro fertilization did not affect their ability to undergo fertilization. A significant proportion of the fertilized oocytes that had been exposed to 50 microM diamide before insemination exhibited abnormal multiple female pronuclei with an apparently normal male pronucleus. These observations indicate that mature hamster oocytes are susceptible to oxidative stress during the critical period that precedes fertilization and provide further evidence that GSH plays important roles in oocyte spindle function and pronucleus development.

Animals↗
PubMed1995

Carbendazim (MBC) disrupts oocyte spindle function and induces aneuploidy in hamsters exposed during fertilization (meiosis II).

Peri-fertilization exposure to Carbendazim (MBC; a microtubule poison) induces infertility and early pregnancy loss in hamsters. Presently, both in vivo and in vitro techniques were employed to characterize the effects of MBC on cellular aspects of fertilization in hamsters. Exposure to MBC during either in vivo or in vitro fertilization (IVF) induced identical morphological abnormalities in the maternal chromatin of zygotes and embryos. These abnormalities included either multiple second polar bodies (PB2), and/or multiple small female pronuclei (PN), or meiotic arrest. Multiple PB2, multiple female PN, multiple PB2 with multiple female PN, or meiotic arrest were exhibited by approximately 31%, 15%, 12%, and 2% of the in vivo zygotes; and 3%, 16%, 36%, and 20% of IVF zygotes, respectively. The effects of MBC persisted to day 2 of pregnancy as indicated by decreased (P < 0.05) embryo development to the two-cell stage and the presence of micronuclei in 6% of two-cell embryos from MBC-treated females. Immunofluorescence analysis of microtubules (MTs) confirmed that MBC disrupted spindle MTs during IVF. Numerical chromosome analysis revealed that a single dose of MBC administered during in vivo fertilization induced aneuploidy in the resulting pronuclear-stage zygotes. The present data point to two mechanisms by which peri-fertilization MBC exposure may induce early pregnancy loss: 1) arrested meiosis with no zygotic cleavage; or 2) induction of zygotic aneuploidy with subsequent developmental arrest.

Aneuploidy↗
PubMed1993

Increased glutamine metabolism in bovine cumulus cell-enclosed and denuded oocytes after in vitro maturation with luteinizing hormone.

The effects of LH on tricarboxylic acid (TCA) cycle activity within denuded and cumulus cell-enclosed bovine oocytes were investigated by measuring the metabolism of L-[14C(U)]glutamine and [2-14C]pyruvate to 14CO2 after in vitro maturation (IVM) with LH in serum-free, defined culture conditions. Initially, intact cumulus cell-enclosed oocytes or oocytectomized cumulus cell complexes were incubated for 24 h for IVM in control medium alone or supplemented with LH (10 micrograms/ml) before assessment of glutamine metabolism. Glutamine oxidation was increased (p < 0.01) only after IVM of intact cumulus cell-enclosed oocytes with LH when compared to either intact untreated controls, untreated oocytectomized controls, or oocytectomized cumulus cell complexes after LH treatment (0.23 +/- 0.02 vs. 0.13 +/- 0.01, 0.12 +/- 0.01, and 0.12 +/- 0.01 nmol 14CO2/micrograms protein/3 h, respectively). Glutamine metabolism was greater (p < 0.001) in denuded oocytes from cumulus cell complexes that were exposed to LH during IVM versus nontreated controls (6.4 +/- 0.3 vs. 5.1 +/- 0.2 pmol 14CO2/oocyte/3 h, respectively). Glutamine metabolism was increased (p < 0.05) in cumulus cell-enclosed oocytes after LH exposure during IVM but was unchanged after exposure to either FSH or thyroid-stimulating hormone when compared to control treatments (0.23 +/- 0.01, 0.20 +/- 0.02, 0.19 +/- 0.01 vs. 0.16 +/- 0.02 nmol 14CO2/micrograms protein/3 h, respectively). Pyruvate metabolism was not different between LH and control treatments. Evidence demonstrated that LH acts via the cumulus cells to increase glutamine metabolism within intact cumulus cell-enclosed oocytes and in mature oocytes denuded after LH exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effects of luteinizing hormone on glucose metabolism in cumulus-enclosed bovine oocytes matured in vitro.

The effects of LH on glucose metabolism within cumulus cell-enclosed bovine oocytes were determined. Cumulus cell-enclosed bovine oocytes were matured in vitro (IVM) in control medium alone or supplemented with LH, FSH, or TSH, then individually assayed for the metabolism of D-[5-3H]glucose, D-[1-14C]glucose, D-[6-14C]glucose, and D-[U-14C]glucose. Glycolytic activity was unchanged after IVM in 1 microgram LH/ml, but was greater (P < 0.05) after culture in 10 and 50 micrograms LH/ml than the control value (1.34 +/- 0.13, 1.87 +/- 0.20, and 1.63 +/- 0.14 vs. 1.19 +/- 0.13 nmol 3H2O/micrograms protein.3 h, respectively). Increased glycolytic activity was observed within cumulus cell-enclosed oocytes, but not in cumulus cell complexes from which the oocyte was removed. Also, no glycolysis was detected when denuded oocytes from any IVM treatment were assayed. Glycolytic activity was greater (P < 0.01) after IVM in LH (10 micrograms/ml) vs. TSH (0.5 microgram/ml), FSH (1.0 micrograms/ml), and control treatments (3.04 +/- 0.10, 2.44 +/- 0.10, 2.33 +/- 0.10, and 2.13 +/- 0.10 nmol 3H2O/micrograms protein.3 h, respectively). Treatment with TSH also increased (P < 0.05) glycolytic activity compared to control values. Relative to control values after IVM, pentose cycle activity was 73.6% less, 2.9% higher, and 33.9% higher with LH, FSH, and TSH treatments, respectively. Total 14CO2 generated from D-[U-14C]glucose did not differ between treatments. Glucose oxidation by the pentose cycle accounted for 30.5%, 29.7%, 40.7%, and 11.1% of the total 14CO2 production after IVM in control medium, FSH, TSH, or LH, respectively. Data indicate that IVM with LH results in increased glycolytic activity and mitochondrial glucose oxidation within cumulus cell-enclosed bovine oocytes, and that this may represent a mechanism by which LH enhances oocyte maturation.

Animals↗
PubMed1991

In vitro fertilization of goat oocytes.

Experiments were carried out to achieve fertilization (IVF) and initial embryonic development of goat oocytes in vitro. Oocyte/cumulus complexes were recovered from large follicles (greater than 7 mm) of hormonally treated doses and from 1-6-mm follicles of ovaries from hormonally superstimulated and nontreated goats. Three different sperm treatment/IVF media were used: defined medium (Brackett and Oliphant, Biol Reprod 1975; 12:260-274) with modifications (mDM); TALP (Bavister and Yanagimachi, Biol Reprod 1977; 16:228-237), as modified by Parrish et al. (Theriogenology 1986; 25:591-600), i.e. modified TALP (mTALP); and HEPES-buffered M199 with modifications (mH-M199). Immature oocytes (from 1-6 mm, small antral follicles) were cultured for in vitro maturation (IVM) in M199 buffered with bicarbonate and with modifications including supplementation with 20% (v/v) goat serum (mB-M199) with either (a) 100 micrograms LH/ml, (b) 5 micrograms FSH/ml, or (c) no added gonadotropin control. Insemination of (in vivo or in vitro) matured oocytes was performed with swim-up separated and heparin-treated freshly ejaculated sperm; additionally, caffeine was included in the mDM treatment. Use of mDM yielded better results than mTALP or mH-M199 (p less than .05). Results with oocytes after IVM were significantly better than those obtained with oocytes matured in vivo (68.4% vs. 45.5%, p less than 0.05). Presence of LH or FSH during oocyte maturation improved both the IVM and IVF results over those of the control (p less than 0.05). The highest proportion of fertilized oocytes (fertilization rate) was achieved by combining the use of mDM for sperm and IVF with IVM in the presence of LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Husbandry↗
PubMed1990

Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation.

Luteinizing hormone was shown to enhance maturation of immature oocytes obtained from slaughtered cattle as reflected by elevated proportions of oocytes that fertilized and reached blastocyst stages in vitro after in vitro fertilization (IVF). Higher proportions of ova were fertilized in vitro after in vitro maturation (IVM) in modified TCM-199 (TCM-199 + BSA + LH [USDA-bLH-B-5, 100 micrograms/ml]) than in TCM-199 alone (p less than 0.01). Further improvement in IVF (p less than 0.005) followed IVM when 20% proestrous (Day 20) bovine serum replaced the BSA, but similar proportions of inseminated ova (22.2% and 22.6%) developed into blastocysts. The positive LH effect was verified in defined conditions for IVM. Exposure of oocytes to the purified LH preparation (without any other added protein or biological substances) during IVM improved IVF (39.7% in TCM-199 vs. 73.5% in TCM-199 + LH; p less than 0.001) and blastocyst development (7.9% vs. 28.2%; p less than 0.005), respectively. Efforts to better define effective concentrations of LH revealed no difference in viability after IVM with 50 micrograms LH/ml vs. 100 micrograms LH/ml (27.0% vs. 28.3%, respectively); 10 micrograms LH/ml did not enhance viability when compared to TCM-199 alone (10.8% vs. 9.9%). Results demonstrate potential utility of this approach for investigation of factors influencing mammalian development by specific effects initiated during the interval of oocyte maturation.

Animals↗