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Biomedical subjects

T G Cooper

Publications and source records attributed to T G Cooper.

At least 19 recordsLinked to original sources
PubMed1999

Overlapping positive and negative GATA factor binding sites mediate inducible DAL7 gene expression in Saccharomyces cerevisiae.

Allantoin pathway gene expression in Saccharomyces cerevisiae responds to two different environmental stimuli. The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e. g. asparagine or glutamine) is provided. Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i) UAS(NTR) element associated with the transcriptional activators Gln3p and Gat1p, (ii) URS(GATA) element associated with the repressor Dal80p, and (iii) UIS(ALL) element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription. Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g. DAL5 and DAL3). The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using the DAL7 (encoding malate synthase) as a model. We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS(NTR), two UIS(ALL), and three URS(GATA) elements. The two GATA-containing UAS(NTR) elements are coincident with two of the three GATA sequences that make up the URS(GATA) elements. The remaining URS(GATA) GATA sequence, however, is not a UAS(NTR) element but appears to function only in repression. The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observed in vivo.

Allantoin↗
PubMed1999

Genome-wide transcriptional analysis in S. cerevisiae by mini-array membrane hybridization.

Access to the powerful micro-array analytical methods used for genome-wide transcriptional analysis has so far been restricted by the high cost and/or lack of availability of the sophisticated instrumentation and materials needed to perform it. Mini-array membrane hybridization provides a less expensive alternative. The reliability of this technique, however, is not well documented and its reported use has, up to this point, been very limited. Our objective was to test whether or not mini-array membrane hybridization would reliably identify genes whose expression was controlled by a specific set of genetic and/or physiological signals. Our results demonstrate that mini-array hybridization can correctly identify genes whose expression is known to be controlled by the GATA-factor regulatory network in S. cerevisiae and in addition can reliably identify genes not previously reported to be associated with this nitrogen control system.

Amino Acid Sequence↗
PubMed1999

Morphological characterization of ejaculated cynomolgus monkey (Macaca fascicularis) sperm.

The aim of this study was to give reference values for the frequency of morphological sperm abnormalities present in the semen from non-experimental cynomolgus monkeys as well as for the dimensions of sperm heads. Spermatozoa from the liquid portion of electroejaculates from 14 cynomolgus monkeys were air-dried as smears, fixed, and stained with Harris's Haematoxylin and subjected to visual analysis of morphology and computer-aided analysis of ten morphometric variables. The majority (83%) of sperm were morphologically normal. Tail defects were the most common (11%), and showed the highest variation between individuals, the values ranging between 4 and 23%. Head abnormalities consisted of large, tapering, and amorphous forms but were not frequent (0.4%), the values ranging between 0 and 1.3%. Midpiece imperfections were found in all the individuals; the mean percentage was 5%, and the range varied between 3 and 9%. Tail plus midpiece was the only multiple abnormality observed, with a mean value of 1.5% and a range between 0 and 8%. The majority of these double defects consisted of a coiled tail together with a coiled midpiece. Mean values for the morphometric parameters characterizing sperm heads were as follows: area 17.2 microm2, perimeter 15.2 microm, length 5.8 microm, width 4.0 microm, L/W ratio 1.5, gray-level 98, ellipticity 0.2, first shape factor 0.9, second shape factor 1.4, and third shape factor 1.1. Overall coefficients of variation for the majority of parameters were below 7%, showing the great homogeneity in the dimensions of cynomolgus sperm heads. Most useful parameters for sperm characterization, according to their low variability, were perimeter, length, width, L/W ratio, and shape factors. Differences in these parameters were, however, observed between monkeys.

Animals↗
PubMed1999

Responses of monkey epididymal sperm of different maturational status to second messengers mediating protein tyrosine phosphorylation, acrosome reaction, and motility.

The maturation of various aspects of sperm function have been demonstrated in monkey and human epididymal sperm, including the ability to undergo the acrosome reaction. The present study aimed to investigate the maturational changes in non-human primate sperm in the signal transduction mechanisms leading to the acrosome reaction involving cyclic AMP, Ca(2+) influx, protein kinase C, and protein tyrosine phosphorylation. Sperm from the caput, corpus, and cauda epididymidis of cynomolgus monkeys were incubated in a complete medium for 2.5 hr, followed by 30 min stimulation with 1 mM dibutyryl cAMP and 1 mM caffeine, 50 microM 1, 2-dioctanoyl-sn-glycerol (DOG), and 50 microM Ca(2+)-ionophore A23187. Quantitative Western blotting revealed little difference in tyrosine phosphorylated proteins among the caput, corpus, and cauda sperm without stimulation. Incubation with cAMP increased the amount of tyrosine phosphorylated proteins up to 10-fold in the corpus and cauda sperm, but to a lower extent in the caput sperm. Ca(2+)-ionophore attenuated the cAMP stimulation but had no effect on its own. Such responses in tyrosine phosphorylated proteins were in great contrast to the responses in the acrosome reaction, where A23187 was the strongest stimulant, resulting in induction of the reaction in 50 +/- 5%, 11 +/- 5%, and 8 +/- 4% cauda, corpus and caput sperm, respectively (mean +/- sem, n = 6). DOG and cAMP in combination induced acrosome reactions in about 10% of viable cells in the cauda and corpus but not caput sperm. Caput sperm responded to cAMP with increases in percentage motility without forward progression whereas cauda sperm displayed marked kinematic changes expected of hyperactivation. Comparisons of responses suggest that the major tyrosine phosphorylated proteins detected are unlikely to be involved immediately in the precipitation of the acrosome reaction, but more related to flagellar motion. Development of signal transduction pathways is part of the epididymal maturational process.

Acrosome Reaction↗
PubMed1999

A re-appraisal of the post-testicular action and toxicity of chlorinated antifertility compounds.

Some 30 years ago, alpha-chlorohydrin and some analogues were considered as close to the ideal contraceptive which acted rapidly and reversibly on the post-testicular maturation of spermatozoa. Despite their early promise, research funding was withdrawn only 5 years later because of what were considered to be unacceptable side-effects in primates. The literature on the toxic effects of these contraceptive agents was reviewed and was found to be wanting in respect to the rigour of scientific methods applied (impure compounds were used, inappropriate target populations were studied, excessive doses were employed, abstracts were cited from which no full publications subsequently arose). These compounds remain the closest approach yet to non-hormonal contraceptives for males and have led to the synthesis of related compounds which have a similar antifertility action but with much diminished toxicity. If toxicity remains a problem, a range of other compounds now known to have a similar antifertility action, should be investigated.

Animals↗
PubMed1999

Experience with external quality control in spermatology.

Results are presented from participation in an external quality control (EQC) programme for semen analysis (UK NEQAS). Formalin-fixed semen samples and videotapes of motile spermatozoa were distributed four times a year over a 3-4 year period. Over the entire period there was close agreement for sperm concentration with, initially, the average of values from the other groups participating in the scheme, and later, values designated as reference values obtained from six laboratories of several chosen that consistently agreed with each other. The initial underestimation of the percentage of normal forms was abolished at the time of change in derivation of designated values and this largely eliminated the difference to establish closer agreement with the designated values. A consistent bias in the assessment of different categories of progressive sperm motility appeared to be resolved by a conscious decision to consider most spermatozoa as grade b and the exceptions as grade a, rather than the converse. Feedback of results to the technicians of the laboratory participating in an external quality control programme leads to reappraisal of subjective evaluation and to harmonization of results between laboratories.

Fixatives↗
PubMed1999

Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study.

In a randomized, placebo-controlled, double-blind study we investigated whether high-dose oral treatment with vitamins C and E for 56 days was able to improve semen parameters of infertile men. Ejaculate parameters included semen volume, sperm concentration and motility, and sperm count and viability. Thirty-one patients without genital infection but with asthenozoospermia (< 50% motile spermatozoa) and normal or only moderately reduced sperm concentration (> 7 x 10(6) spermatozoa/ml) (according to WHO criteria) were examined. To investigate the influence of the epididymal storage period on semen parameters, the patients were asked to deliver two semen samples with abstinence times of 2 and 7 days both before and at the end of vitamin treatment. After randomization, the patients received either 1000 mg vitamin C and 800 mg vitamin E (n = 15) or identical placebo capsules (n = 16). No changes in semen parameters were observed during treatment, and no pregnancies were initiated during the treatment period. Combined high-dose antioxidative treatment with vitamins C and E did not improve conventional semen parameters or the 24-h sperm survival rate. Prolonged abstinence time increased ejaculate volume (P < 0.05), sperm count (P < 0.05), sperm concentration (P < 0.05) and the total number of motile spermatozoa (P < 0.05).

Administration, Oral↗
PubMed1999

Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms.

Homozygous c-ros knockout male mice that lack prepubertal differentiation of the epididymal initial segment are healthy but sterile, despite normal sperm production and mating. Detailed computerized analysis of the motility of spermatozoa maturing in the epididymis revealed only minor defects. However, the majority of motile mature sperm released from the cauda epididymidis showed various extents of flagellar angulation that could not be corrected by raising extracellular osmolality. Measurement of the osmolality of cauda epididymal fluid showed no difference from the wild type. Studies in wild-type mice indicated a maturational change in the ability of motile sperm to maintain straight flagella during incubation, but angulation was induced in cauda sperm by the volume-sensitive ion channel blockers quinine, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and BaCl(2), or by exposure to hypotonic media. Flagellar angulation, induced in the wild type or intrinsic to the knockout, was relieved upon demembranation by Triton X-100, confirming that it was a cell swelling phenomenon. A lack of response of immature wild-type sperm and mature knockout sperm to the channel blockers suggests that there is normally a development of the volume regulatory mechanisms upon maturation that is defective in sperm from the knockout animal. The resultant flagellar angulation may account for the reduction in sperm numbers in the oviduct of mated females and the failure to fertilize in vivo.

Animals↗
PubMed1999

The dual-specificity protein phosphatase Yvh1p acts upstream of the protein kinase mck1p in promoting spore development in Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen. We show that yvh1 disruption causes a defect in spore maturation; overexpression of MCK1 or IME1 suppresses this yvh1 phenotype. While mck1 mutations are epistatic to those in yvh1 relative to spore maturation, overexpression of MCK1 does not suppress the yvh1 slow-vegetative-growth phenotype. We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth.

Dual-Specificity Phosphatases↗
PubMed1999

Synergistic operation of the CAR2 (Ornithine transaminase) promoter elements in Saccharomyces cerevisiae.

Dal82p binds to the UIS(ALL) sites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression. CAR2, which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators of CAR2 transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of the CAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2 expression and, if so, of ascertaining their relative contributions to CAR2's expression and control. We show that Rap1p binds upstream of CAR2 and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest that CAR2 gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of the CAR2 promoter and its operation closely follow the model proposed for CAR1.

5' Untranslated Regions↗
PubMed1999

Control of nitrogen catabolite repression is not affected by the tRNAGln-CUU mutation, which results in constitutive pseudohyphal growth of Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to nitrogen availability in several ways. (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR). Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell's doubling time. (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth. (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G1. A specific allele of the SUP70/CDC65 tRNAGln gene (sup70-65) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR. Our data confirm that pseudohyphal growth occurs gratuitously in sup70-65 mutants cultured in nitrogen-rich medium at 30 degrees C. However, we find neither any defect in NCR in the sup70-65 mutant nor any alteration in the control of YVH1 expression, which has been previously shown to be specifically induced by nitrogen starvation.

Gene Expression Regulation, Fungal↗
PubMed1998

Molecular cloning and expression of rat contraception associated protein 1 (CAP1), a protein putatively involved in fertilization.

Epididymal fluid from rats rendered infertile by oral administration of ornidazole contains a protein CAP1 (contraception-associated protein 1) that is absent from epididymal fluid, but present on epididymal sperm, from fertile vehicle-treated rats. The gene for CAP1 has been isolated from a rat testis cDNA library and its expression investigated in different tissues. The deduced protein sequence of CAP1 contains 189 amino-acid residues and database searches revealed a high degree of homology (83-95%) with human and mouse DJ-1 at the nucleotide and amino acid levels. Northern blot hybridisation from different rat tissues indicated that CAP1 is encoded by a 1.6-kilobase RNA transcript and seems to be ubiquitiously expressed in the rat with a high level of expression in the testis.

Adult↗
PubMed1998

Efficient PCR-based random mutagenesis of sub-genic (100 bp) DNA fragments.

Here we describe a method for performing a PCR-driven random mutagenesis of 100 bp DNA fragments that yields mutations at a useful frequency. The method is a modification of the manganese ion substitution PCR technique, and neither creates 'hot-spots' nor favors transition mutations over transversions.

DNA, Fungal↗
PubMed1998

Creatine kinase activity in human spermatozoa and seminal plasma lacks predictive value for male fertility in in vitro fertilization.

OBJECTIVE: To examine the predictive value of creatine kinase in human spermatozoa and seminal plasma from patients treated in an IVF program. DESIGN: Prospective, blind clinical study. SETTING: Male infertility clinic. PATIENT(S): Seventy-three patients and 32 fathers (age, <45 years). INTERVENTION(S): Determination of creatine kinase activity in seminal plasma, washed spermatozoa, and swim-up purified spermatozoa from patients treated in an IVF program. MAIN OUTCOME MEASURE(S): Creatine kinase activity in seminal plasma, washed spermatozoa, and swim-up purified spermatozoa. RESULT(S): Creatine kinase activity in washed spermatozoa correlated significantly with normal sperm morphology. No significant correlations were found between creatine kinase activity or creatine kinase isoenzyme ratio in seminal plasma, washed spermatozoa, or swim-up purified spermatozoa and success in the IVF program. The percentage of spermatozoa with normal morphology was significantly lower in patients whose sperm did not fertilize than in patients whose sperm did fertilize oocytes and in the control group of proven fathers. CONCLUSION(S): Total creatine kinase activity and creatine kinase isoenzyme distribution are not sperm function markers for prediction of male fertility in IVF treatment.

Adult↗
PubMed1998

Influence of sperm surface antibodies on spontaneous pregnancy rates.

OBJECTIVE: To determine the rates of spontaneous pregnancies among women whose infertile male partners had sperm surface antibodies. DESIGN: Retrospective analysis. SETTING: Infertility clinic of a university referral center. PATIENT(S): One hundred fifty-seven infertile couples; the male partner had IgA and/or IgG sperm surface antibody concentrations of >10%. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Spontaneous pregnancy rates (PRs) over 6 years. RESULT(S): Spontaneous PRs correlated negatively with antibody concentrations. CONCLUSION(S): Although the chance of spontaneous pregnancy among women whose partners had sperm antibody concentrations of <50% was good, intracytoplasmic sperm injection should be recommended to patients with concentrations of >90%.

Female↗
PubMed1998

Standardization of sampling and staining methods for the morphometric evaluation of sperm heads in the Cynomolgus monkey (Macaca fascicularis) using computer-assisted image analysis.

Automated sperm morphology analysis (ASMA) technology has improved the assessment of sperm morphology, but the results depend on the use of adequate and standardized procedures. In this study the Sperm-Class Analyzer (SCA) ASMA system was used to assess sperm head morphometry in the Cynomolgus monkey and to evaluate the influence of sample size, intraslide variation, and the use of three staining techniques on the accuracy of image processing and sperm head morphometry. Haematoxylin is the staining technique of choice for Cynomolgus spermatozoa, as optimum contrast of sperm heads with the surrounding background allows efficient segmentation, i.e. sperm head boundary detection, making the image analysis process more accurate. The analysis of 100 spermatozoa is recommended since a larger sample size did not result in more accurate sperm head morphometry. There were no differences in either the percentage of correctly binarized sperm heads or sperm head dimensions among samples obtained from different zones of the slides, although differences in stain intensity (grey level) were detected. The measurements made on Haematoxylin, Diff-Quik and Hemacolor-stained slides yielded different values for all of the sperm head parameters under consideration. This result demonstrates that the procedures of fixation and staining significantly affect the dimensions of sperm heads.

Animals↗
PubMed1998

The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens.

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.

Animals↗