bluuebunny/crossref_metadata_2025 huggingface dataset:
Antisense Targeting Protein Kinase A Type I as a Drug for Integrated Strategies of Cancer Therapy
Authors: TORTORA GIAMPAOLO , CIARDIELLO FORTUNATO
Published: January 2004

Abstract: We have studied the role of protein kinase A (PKA) and its type I isoform (PKAI) in the transduction of mitogenic signaling, apoptosis, and angiogenesis. We have contributed to the development of selective inhibitors of PKAI, including a hybrid DNA/RNA mixed backbone oligonucleotide (AS‐PKAI). We, and others, have demonstrated that AS‐PKAI has a cooperative antitumor effect with a selected class of cytotoxic drugs and with radiotherapy in vitro and in vivo and that these effects can also be obtained following oral adinistration. Previously, we developed a series of therapeutic models based on the pleiotropic role played by PKAI in cell proliferation, apoptosis, and angiogenesis. On the basis of our former demonstration of functional and structural interactions of PKAI and the activated epidermal growth factor receptor (EGFR), we have shown that the combined blockade of both signaling molecules by AS‐PKAI and either the monoclonal antibody C225 (erbitux) or the small molecule ZD1839 (gefitinib), results in a marked cooperative antitumor effect in a variety of human tumor models. A further cooperative antitumor effect can be obtained when AS‐PKAI is used in combination with both EGFR inhibitors and either cytotoxic drugs or radiotherapy. The antitumor activity is associated with inhibition of growth factors and angiogenic factors production and to induction of apoptosis. In light of the recently demonstrated role of PKAI on the bcl‐2‐dependent apoptotic pathway, we have recently shown a synergistic antitumor, antiangiogenic, and proapoptotic effect of AS‐PKAI in combination with antisense bcl‐2 (oblimersen) or with a bispecific bcl‐2/bcl‐xL second generation antisense. A connection between COX‐2, EGFR and PKAI was established, and we demonstrated that the combination of AS‐PKAI with gefitinib and a COX‐2 inhibitor, all adminstered orally, can result in a potent antitumor and antiangiogenic activity. These studies support the development of AS‐PKAI as a novel anticancer agent and suggest its potentially relevant role when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.
Targeting Protein Kinase C‐alpha (PKC‐α) in Cancer with the Phosphorothioate Antisense Oligonucleotide Aprinocarsen
Authors: LAHN MICHAEL , SUNDELL KAREN , MOORE STEPHANIE
Published: January 2004

Abstract: Antisense oligonucleotides (ASOs) offer a novel pharmacological platform to develop highly specific drugs. As shown by the clinical development of aprinocarsen, an ASO directed against protein kinase C‐alpha (PKC‐α), this platform has made a remarkable advance from the bench to the bedside. This review summarizes the rationale of the early development of aprinocarsen and current clinical experience.
Effect of a Cognitive Behavioral Intervention on Reducing Symptom Severity During Chemotherapy
Authors: Given Charles , Given Barbara , Rahbar Mohammad , Jeon Sangchoon , McCorkle Ruth , Cimprich Bernadine , Galecki Andrzej , Kozachik Sharon , Brady Albert , Fisher-Malloy Mary Jo , Courtney Kathy , Bowie Elizabeth
Published: January 2004

Purpose

To describe a randomized trial of a cognitive behavioral intervention on reducing symptom severity among patients diagnosed with solid tumors and undergoing a first course of chemotherapy and to determine whether the intervention had an additive or interactive effect on symptom severity in the presence of supportive care medications.

Patients and Methods

Patients (N = 237) were accrued from comprehensive and community cancer centers, interviewed, and randomly assigned to either the experimental intervention (n = 118) or conventional care (n = 119). A symptom severity index, based on summed severity scores across 15 symptoms, was the primary outcome. Each patient’s site of cancer, stage at diagnosis, chemotherapy protocols, and use of supportive medications were learned from medical records.

Results

Groups were equivalent at baseline, and attrition by characteristics by group was not different. The proportion of patients not receiving chemotherapy at 10 and 20 weeks did not differ by group. At the 10- and 20-week observations, there was a significant interaction between the experimental group and baseline symptom severity. Patients in the experimental group who entered the trial with higher symptom severity reported significantly lower severity at 10 and 20 weeks. Controlling for chemotherapy treatment status at follow-up and supportive care medications did not alter the effect of the experimental intervention.

Conclusion

Compared with conventional care alone, the experimental intervention was effective among patients who entered the trial with higher levels of symptom severity. Age, sex, site or stage of cancer, and supportive medications did not modify the effect of this cognitive behavioral intervention on symptom severity.
Antioxidants and Cancer Therapy: A Systematic Review
Authors: Ladas Elena J. , Jacobson Judith S. , Kennedy Deborah D. , Teel Katherine , Fleischauer Aaron , Kelly Kara M.
Published: January 2004

Purpose

Many patients with cancer take antioxidant nutritional supplements during cancer treatment to alleviate treatment toxicities and to improve long-term outcomes, but little is known about the efficacy and safety of antioxidant use during cancer treatment. We reviewed English-language manuscripts published in the biomedical literature, reporting the results of observational studies of antioxidant status and cancer outcomes and of intervention trials of antioxidants among patients receiving chemotherapy with or without radiation for various malignancies.

Methods

We searched the Medline database and the bibliographies of the retrieved manuscripts, reviews, and books on antioxidants and cancer. The retrieved studies are grouped by study design, malignancy, and end points.

Results

More than 100 citations were retrieved; 52 met our criteria, 31 were observational studies, and 21 were intervention trials. The studies varied in study design, timing of observation/intervention, intervention protocol, malignancy, and anticancer regimen.

Conclusion

These inconsistencies preclude a definitive conclusion as to the effect of chemotherapy on antioxidant status in patients undergoing anticancer therapy. However, our review suggests that total antioxidant status (measured by total radical antioxidant parameter) declines during cancer treatment. Adequately powered trials or observational studies among patients with a specific cancer diagnosis receiving a specific treatment regimen are needed to address patients' and physicians' concerns regarding these associations.
Efficacy of Oral Adjuvant Therapy After Resection of Colorectal Cancer: 5-Year Results From Three Randomized Trials
Authors:   Meta-Analysis Group of the Japanese Society for Cancer of the Colon and Rectum and the Meta-Analysis Group in Cancer
Published: January 2004

Purpose

Adjuvant therapy of colorectal cancer with oral fluorinated pyrimidines is attractive because of its ease of administration and good tolerability. The purpose of this meta-analysis is to assess the survival and disease-free survival benefits of treating patients after surgical resection of a primary colorectal tumor with oral fluoropyrimidines for 1 year.

Patients and Methods

This meta-analysis was performed on individual data from three randomized trials conducted by the Japanese Foundation for Multidisciplinary Treatment for Cancer involving a total of 5,233 patients with stages I to III colorectal cancer.

Results

The overall hazard ratio in favor of oral therapy was 0.89 for survival (95% CI, 0.80 to 0.99; P = .04), and 0.85 for disease-free survival (95% CI, 0.77 to 0.93; P < .001). Thus oral therapy reduced the risk of death by 11% and the risk of recurrence or death by 15%. There was no significant heterogeneity between trials, nor did the benefit of oral therapy depend on tumor stage (I, II, or III), tumor site (rectum or colon), patient age, or patient sex.

Conclusion

Oral fluoropyrimidines improve disease-free survival and survival of patients after resection of early-stage colorectal cancer. These observations support the use of these agents alone after resection of early-stage disease, as well as further testing of oral agents in combination with new drugs that have recently shown antitumor activity in advanced colorectal cancer.
Thymidylate Synthase Expression and Prognosis in Colorectal Cancer: A Systematic Review and Meta-Analysis
Authors: Popat Sanjay , Matakidou Athena , Houlston Richard S.
Published: January 2004

Purpose

A number of studies have investigated the relationship between thymidylate synthase (TS) expression and survival in colorectal cancer (CRC) patients. Although most have reported poorer overall and progression-free survival with high TS expression, estimates of the hazard ratio (HR) between studies differ wildly. To derive a more precise estimate of the prognostic significance of TS expression, we have reviewed published studies and carried out a meta-analysis.

Materials and Methods

Twenty studies stratifying overall survival and/or progression-free survival in CRC patients by TS expression status were eligible for analysis. The principal outcome measure was the HR. Data from these studies were pooled using standard meta-analysis techniques.

Results

Thirteen studies investigated outcome in a total of 887 cases with advanced CRC, and seven studies investigated outcome in a total of 2,610 patients with localized CRC. A number of methods were used both to assess TS expression and to assign TS status. Sample sizes varied greatly, small sample sizes being a feature of the advanced disease studies. The combined HR estimate for overall survival (OS) was 1.74 (95% CI, 1.34 to 2.26) and 1.35 (95% CI, 1.07 to 1.80) in the advanced and adjuvant settings, respectively, but there was evidence of heterogeneity and possible publication bias.

Conclusion

Tumors expressing high levels of TS appeared to have a poorer OS compared with tumors expressing low levels. Additional studies with consistent methodology are needed to define the precise prognostic value of TS.
Prognostic Factors for Survival in Previously Treated Patients With Metastatic Renal Cell Carcinoma
Authors: Motzer Robert J. , Bacik Jennifer , Schwartz Lawrence H. , Reuter Victor , Russo Paul , Marion Stephanie , Mazumdar Madhu
Published: January 2004

Purpose

To describe survival in previously treated patients with metastatic renal cell carcinoma (RCC) who are candidates for clinical trials of new agents as second-line therapy.

Patients and Methods

The relationship between pretreatment clinical features and survival was studied in 251 patients with advanced RCC treated during 29 consecutive clinical trials between 1975 and 2002. Clinical features were first examined in univariate analyses, and then a stepwise modeling approach based on Cox regression was used to form a multivariate model.

Results

Median survival for the 251 patients was 10.2 months and differed according to year of treatment, with patients treated after 1990 showing longer survival. In this group, the median overall survival time was 12.7 months. Because the purpose of this analysis was to establish prognostic factors for present-day clinical trial design, prognostic factor analysis was performed on these patients. Pretreatment features associated with a shorter survival in the multivariate analysis were low Karnofsky performance status, low hemoglobin level, and high corrected serum calcium. These were used as risk factors to categorize patients into three different groups. The median time to death in patients with zero risk factors was 22 months. The median survival in patients with one of these prognostic factors was 11.9 months. Patients with two or three risk factors had a median survival of 5.4 months.

Conclusion

Treatment with novel agents during a clinical trial is indicated for patients with metastatic RCC after progression to cytokine treatment. Three prognostic factors for predicting survival were used to categorize patients into risk groups. These risk categories can be used in clinical trial design and interpretation.
Urinary VEGF and MMP Levels As Predictive Markers of 1-Year Progression-Free Survival in Cancer Patients Treated With Radiation Therapy: A Longitudinal Study of Protein Kinetics Throughout Tumor Progression and Therapy
Authors: Chan Linda W. , Moses Marsha A. , Goley Elizabeth , Sproull Mary , Muanza Thierry , Coleman C. Norman , Figg William D. , Albert Paul S. , Ménard Cynthia , Camphausen Kevin
Published: January 2004

Purpose

To determine the predictive value of urinary levels of two angiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMPs), in a longitudinal study to determine their correlation with 1-year progression-free survival in patients with cancer.

Patients and Methods

VEGF and MMP levels were measured in the urine of 65 cancer patients at first evaluation, during therapy, and at follow-up (n = 242); normalized by creatinine levels; and compared with 16 healthy controls. The correlation of initial levels and trends of VEGF and MMPs with 1-year progression-free survival was assessed using two-sample tests and stepwise logistic regression.

Results

Urinary VEGF levels at presentation were different between patients with local-regional cancer and normal controls, and between patients with metastatic prostate cancer and local-regional disease (P = .04 and .01, respectively). Similar results were found with MMP measurement (P = .03 and .0001, respectively). Of those patients subsequently treated with radiation, VEGF levels at presentation between patients with no evidence of disease (NED) after radiation and those who had persistent or recurrent disease after radiotherapy were also different (P = .039). The comparison between angiogenic factor levels taken at least 1 month postradiotherapy and the last level taken during treatment was the strongest predictor of patient 1-year progression-free survival (P = .004). Similarly, the overall MMP trend was also significantly associated with 1-year progression-free survival, as was the individual MMP-2 trend (P = .004 and .001, respectively). Stepwise logistic regression revealed that the VEGF trend comparing postradiation levels with last level taken during treatment was an independent predictor of progression-free survival (P = .02).

Conclusion

This small exploratory study suggests that the angiogenic urinary trends of VEGF and MMPs may be useful predictive markers for progression-free survival in cancer patients after the completion of radiotherapy.
Eligibility and Outcomes Reporting Guidelines for Clinical Trials for Patients in the State of a Rising Prostate-Specific Antigen: Recommendations From the Prostate-Specific Antigen Working Group
Authors: Scher Howard I. , Eisenberger Mario , D'Amico Anthony V. , Halabi Susan , Small Eric J. , Morris Michael , Kattan Michael W. , Roach Mack , Kantoff Philip , Pienta Kenneth J. , Carducci Michael A. , Agus David , Slovin Susan F. , Heller Glenn , Kelly William Kevin , Lange Paul H. , Petrylak Daniel , Berg William , Higano Celestra , Wilding George , Moul Judd W. , Partin Alan N. , Logothetis Christopher , Soule Howard R.
Published: January 2004

Purpose

To define methodology to show clinical benefit for patients in the state of a rising prostate-specific antigen (PSA).

Results

Hypothesis. A clinical states framework was used to address the hypothesis that definitive phase III trials could not be conducted in this patient population. Patient Population. The Group focused on men with systemic (nonlocalized) recurrence and a defined risk of developing clinically detectable metastases. Models to define systemic versus local recurrence, and risk of metastatic progression were discussed. Intervention. Therapies that have shown favorable effects in more advanced clinical states; meaningful biologic surrogates of activity linked with efficacy in other tumor types; and/or effects on a target or pathway known to contribute to prostate cancer progression in this state can be considered for evaluation. Outcomes. An intervention-specific posttherapy PSA-based outcome definition that would justify further testing should be described at the outset. Reporting. Trial reports should include a table showing the number of patients who achieve a specific PSA-based outcome, the number who remain enrolled onto the trial, and the number who came off study at different time points. The term PSA response should be abandoned. Trial Design. The phases of drug development for this state are optimizing dose and schedule, demonstration of a treatment effect, and clinical benefit. To move a drug forward should require a high bar that includes no rise in PSA in a defined proportion of patients for a specified period of time at a minimum. Agents that do not produce this effect can only be tested in combination. The preferred end point of clinical benefit is prostate cancer–specific survival; the time to development of metastatic disease is an alternative.

Conclusion

Methodology to show that an intervention alters the natural history of prostate cancer is described. At each stage of development, only agents with sufficient activity should be moved forward.
Low-Dose Thalidomide Ameliorates Cytopenias and Splenomegaly in Myelofibrosis With Myeloid Metaplasia: A Phase II Trial
Authors: Marchetti Monia , Barosi Giovanni , Balestri Francesca , Viarengo Gianluca , Gentili Sara , Barulli Sara , Demory Jean-Loup , Ilariucci Fiorella , Volpe Antonio , Bordessoule Dominique , Grossi Alberto , Le Bousse-Kerdiles Marie Caroline , Caenazzo Andrea , Pecci Alessandro , Falcone Antonietta , Broccia Giorgio , Bendotti Cesarina , Bauduer Fredric , Buccisano Francesco , Dupriez Brigitte
Published: January 2004

Purpose

A phase II dose-escalation trial was conducted to ascertain low-dose thalidomide safety and response in patients with advanced myelofibrosis with myeloid metaplasia (MMM).

Patients and Methods

Thalidomide was administered together with current therapy to 63 patients, starting at 50 mg daily and increasing to 400 mg as tolerated.

Results

Half of the patients sustained daily doses more than 100 mg and the drop-out rate was 51% at 6 months: the drop-out rate was lower in patients with high baseline fatigue score. At efficacy analysis, anemia was ameliorated in 22% of the patients and transfusions were eliminated in 39% of transfusion-dependent patients. Platelet count increased by 50 × 109/L or more in 22% of patients with an initial count lower than 100 × 109/L. Splenomegaly decreased by more than 50% of the initial size in 19% of patients. Reduction of an overall disease severity score occurred in 31% of patients and was associated with a significant reduction of fatigue. Disease severity amelioration was independently predicted by a high baseline myeloproliferative index (ie, large splenomegaly, thrombocytosis, or leukocytosis).

Conclusion

Low-dose thalidomide displays an acceptable toxicity profile and provides an objective and subjective advantage to a relevant portion of MMM patients.
Nuclear translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle kinases
Authors: Longshaw Victoria M. , Chapple J. Paul , Balda Maria S. , Cheetham Michael E. , Blatch Gregory L.
Published: January 2004

The co-chaperone murine stress-inducible protein 1 (mSTI1), an Hsp70/Hsp90 organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. The mSTI1 protein can be phosphorylated in vitro by cell cycle kinases proximal to a putative nuclear localization signal (NLS), which substantiated a predicted casein kinase II (CKII)-cdc2 kinase-NLS (CcN) motif at position 180-239 and suggested that mSTI1 might move between the cytoplasm and the nucleus under certain cell cycle conditions. The mechanism responsible for the cellular localization of mSTI1 was probed using NIH3T3 fibroblasts to investigate the localization of endogenous mSTI1 and enhanced green fluorescent protein (EGFP)-tagged mSTI1 mutants. Localization studies on cell lines stably expressing NLSmSTI1-EGFP and EGFP demonstrated that the NLSmSTI1 was able to promote a nuclear localization of EGFP. The mSTI1 protein was exclusively cytoplasmic in most cells under normal conditions but was present in the nucleus of a subpopulation of cells and accumulated in the nucleus following inhibition of nuclear export (leptomycin B treatment). G1/S-phase arrest (using hydroxyurea) and inhibition of cdc2 kinase (using olomoucine) but not inhibition of casein kinase II (using 5,6-dichlorobenzimidazole riboside), increased the proportion of cells with endogenous mSTI1 nuclear staining. mSTI1-EGFP behaved identically to endogenous mSTI1. The functional importance of key residues was tested using modified mSTI1-EGFP proteins. Inactivation and phosphorylation mimicking of potential phosphorylation sites in mSTI1 altered the nuclear translocation. Mimicking of phosphorylation at the mSTI1 CKII phosphorylation site (S189E) promoted nuclear localization of mSTI1-EGFP. Mimicking phosphorylation at the cdc2 kinase phosphorylation site (T198E) promoted cytoplasmic localization of mSTI1-EGFP at the G1/S-phase transition,whereas removal of this site (T198A) promoted the nuclear localization of mSTI1-EGFP under the same conditions. These data provide the first evidence of nuclear import and export of a major Hsp70/Hsp90 co-chaperone and the regulation of this nuclear-cytoplasmic shuttling by cell cycle status and cell cycle kinases.
Integrin activation
Authors: Calderwood David A.
Published: January 2004

The ability of cells to regulate dynamically their adhesion to one another and to the extracellular matrix (ECM) that surrounds them is essential in multicellular organisms. The integrin family of transmembrane adhesion receptors mediates both cell-cell and cell-ECM adhesion. One important, rapid and reversible mechanism for regulating adhesion is by increasing the affinity of integrin receptors for their extracellular ligands (integrin activation). This is controlled by intracellular signals that, through their action on integrin cytoplasmic domains, induce conformational changes in integrin extracellular domains that result in increased affinity for ligand. Recent studies have shed light on the final intracellular steps in this process and have revealed a vital role for the cytoskeletal protein talin.
Strategies for Developing Biomarkers of Heart Failure
Authors: Jortani Saeed A , Prabhu Sumanth D , Valdes Roland
Published: February 2004

Background: Heart failure (HF) is a devastating disease with increasing prevalence in elderly populations. One-half of all patients die within 5 years of diagnosis. The annual cost of treating patients with HF in the US is more than $20 billion, which is estimated to be greater than that of myocardial infarction and all cancers combined. Given the complex pathophysiology and varied manifestations of HF, interest has intensified in developing biological markers to predict susceptibility and aid in the early diagnosis and management of this disease.

Methods: We searched Medline via Ovid for studies published during the period 1966–2003 regarding various biomarkers suggested for HF. Our review focused on developing strategies for discovering and using new biomarkers, particularly those potentially linked to pathophysiologic mechanisms. We also point out strategic advantages, limitations, and methods available for measuring each of the currently proposed markers.

Results: Biomarkers reviewed include those released from the heart during normal homeostasis (natriuretic peptides), those produced elsewhere that act on the heart (endogenous cardiotonic steroids and other hormones), and those released in response to tissue damage (inflammatory cytokines). The concept of using a combination of multiple markers based on diagnosis, prognosis, and acute vs chronic disease is also discussed. In view of recent advances in our understanding of molecular biochemical derangements observed during cardiac failure, we consider the concept of myocardial remodeling and the heart as part of an endocrine system as strategies.

Conclusion: Strategically, biomarkers linked to mechanisms involved in the etiology of HF, such as dysregulation of ion transport, seem best suited for serving as early biological markers to predict and diagnose disease, select therapy, or assess progression.
Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro
Authors: Bone W , Jones NG , Kamp G , Yeung CH , Cooper TG
Published: January 2004

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.
Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers
Authors: Oussaid B , Lonergan P , Khatir H , Guler A , Monniaux D , Touze JL , Beckers JF , Cognie Y , Mermillod P
Published: January 2004

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.
Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability
Authors: Pintado B , de la Fuente J , Roldan ER
Published: January 2004

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.
Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida
Authors: McEvoy TG , Coull GD , Broadbent PJ , Hutchinson JS , Speake BK
Published: January 2004

Cattle, pig and sheep oocytes isolated from healthy cumulus-oocyte complexes were pooled, within species, to provide samples of immature denuded oocytes with intact zona pellucida (n = 1000 per sample) for determination of fatty acid mass and composition in total lipid, constituent phospholipid and triglyceride. Acyl-containing lipid extracts, transmethylated in the presence of a reference penta-decaenoic acid (15:0), yielded fatty acid methyl esters which were analysed by gas chromatograph. Mean (+/- SEM) fatty acid content in samples of pig oocytes (161 +/- 18 micrograms per 1000 oocytes) was greater than that in cattle (63 +/- 6 micrograms; P < 0.01) and sheep oocytes (89 +/- 7 micrograms; P < 0.05). Of 24 fatty acids detected, palmitic (16:0; 25-35%, w/w), stearic (18:0; 14-16%) and oleic (18:1n-9; 22-26%) acids were most prominent in all three species. Saturated fatty acids (mean = 45-55%, w/w) were more abundant than mono- (27-34%) or polyunsaturates (11-21%). Fatty acids of the n-6 series, notably linoleic (18:2n-6; 5-8%, w/w) and arachidonic acid (20:4n-6; 1-3%), were the most abundant polyunsaturates. Phospholipid consistently accounted for a quarter of all fatty acids in the three species, but ruminant oocytes had a lower complement of polyunsaturates (14-19%, w/w) in this fraction than pig oocytes (34%, w/w) which, for example, had a three- to fourfold greater linoleic acid content. An estimated 74 ng of fatty acid was sequestered in the triglyceride fraction of individual pig oocytes compared with 23-25 ng in ruminant oocytes (P < 0.01). It is concluded that the greater fatty acid content of pig oocytes is primarily due to more abundant triglyceride reserves. Furthermore, this species-specific difference, and that in respect of polyunsaturated fatty acid reserves, may underlie the contrasting chilling, culture and cryopreservation sensitivities of embryos derived from pig and ruminant (cattle, sheep) oocytes.
Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro
Authors: Leonardsen L , Stromstedt M , Jacobsen D , Kristensen KS , Baltsen M , Andersen CY , Byskov AG
Published: January 2004

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.
Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus
Authors: Norman SJ , Poyser NL
Published: January 2004

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.
Heterogeneity in the bioactivity of LH secreted by peripubertal rats
Authors: Leigh AJ , Poyser NL , Bonney RC , Whitehead SA , Wilson CA
Published: January 2004

Treatment of immature rats with 5 iu equine chorionic gonadotrophin (eCG) on day 25 typically stimulates a preovulatory surge of LH on day 27 and ovulation on day 28. In rats weighing > 60 g at the time of treatment, an LH surge and ovulation occurred in 75% of the animals but, in rats weighing < 60 g, only 13% ovulated even though 69% showed an LH surge. Previous findings have shown that exogenous LH can stimulate ovulation in the rats < 60 g, indicating that the anovulation was not due to ovarian immaturity, but rather to an abnormal form of LH. Thus, it was important to determine whether the bioactivity of LH released at the time of the surge differs in rats < 60 g compared with rats > 60 g. Experiments showed that LH from both groups of eCG-treated animals were equipotent in stimulating testosterone production from incubated Leydig cells and progesterone production from cultured granulosa cells. Similarly the surge of progesterone in vivo, which occurs co-incident with the LH surge, was of similar magnitude in both groups of animals. Since prostaglandin synthesis increases at the time of ovulation and is also stimulated by LH, it was investigated whether the activity of ovarian phospholipase A2, the rate limiting enzyme in prostaglandin synthesis, and ovarian prostaglandin E2 concentrations differed in the animals > 60 g and < 60 g. Phospholipase A2 activities were similar in both groups of animals at the time of the LH surge, as were the prostaglandin E2 concentrations. However, in all animals that ovulated (15/20 in rats > 60 g and 2/15 in rats < 60 g), there was a threefold increase in ovarian prostaglandin E2 concentrations. The results show that, in underweight animals, the bioactivity of LH, in terms of its ability to stimulate steroidogenesis and phospholipase A2 activity, is similar to that released by animals > 60 g; however, the LH produced by the underweight animals fails to induce ovulation by failing to increase, either directly or indirectly, prostaglandin E2 production. Comparison of the profiles of plasma LH collected at the time of the LH surge on an anionic ion exchange column indicates that the LH from rats < 60 g possesses significantly less of the neutral or basic glycoform of LH than that from rats > 60 g. This finding provides a further index that the biopotency of LH produced by underweight animals is different from that of rats > 60 g.
Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice
Authors: Nielsen M , Bjornsdottir S , Hoyer PE , Byskov AG
Published: January 2004

The distribution of nuclear oestrogen receptor alpha (ER-alpha) in the sex organs of fetal and newborn mice has been investigated immunohistochemically. There was no visible ER-alpha immunoreaction in the sexually undifferentiated gonads, whereas a faint immunoreaction was detected in a few cells surrounding the sex ducts, the Mullerian and Wolffian ducts. After sex differentiation, the immunoreaction of ER-alpha was observed in various cell types, with the exception of both the male and female germ cells. In the fetal ovary, immunoreaction was restricted to the surface epithelium and a few stroma cells without any preferential localization. In the testis, the number of ER-alpha-immunopositive cells, identified as Leydig and peritubular cells, increased with age. Immediately after sex differentiation, cells surrounding the sex ducts were ER-alpha-immunopositive, but no immunoreaction was detected in the epithelium in either sex. During development, the epithelium of the sex ducts attained a topographic difference in ER-alpha immunoreaction. In females, immunoreaction was detected in the epithelium of the oviduct, but not in the uterus. In males, the immunoreaction of ER-alpha was intense in the epithelium of the efferent ducts, weak in the epididymis and absent in the vas deferens. ER-alpha immunoreaction in the cells surrounding the sex duct differed between the sexes, being high in all these cells in females, but of varying intensity in males. ER-alpha may not play an important role in the development and function of ovarian cells, but may be involved in the development of Leydig and peritubular cells. Furthermore, substances that react with ER-alpha may influence the male germ cells indirectly through the ER-alpha-immunopositive peritubular cells. In addition, in both sexes, ER-alpha-immunopositive cells surrounding the sex ducts may be involved in the mediation of growth and functional differentiation of the ductal epithelium.
Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos
Authors: Ernst CA , Rhees BK , Miao CH , Atchley WR
Published: January 2004

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).
Effects of an organophosphate pesticide, quinalphos, on the hypothalamo-pituitary-gonadal axis in adult male rats
Authors: Sarkar R , Mohanakumar KP , Chowdhury M
Published: January 2004

The effects of chronic sub-lethal doses (7-14 mg kg-1 a day for 15 days) of quinalphos were evaluated in adult male rats for changes in testicular morphology, circulatory concentrations of hormones (LH, FSH, prolactin and testosterone), activities of acetylcholinesterase (AChE) and angiotensin converting enzyme (ACE) as well as metabolism of biogenic amines (dopamine, noradrenaline and 5-hydroxytryptamine (5-HT)) in the hypothalamus and pituitary. Hormones were assayed by radioimmunoassay or chemiluminescent immunoassay (testosterone). The enzymes were estimated after spectrophotometry and the biogenic amines by HPLC-electrochemistry. Sub-lethal chronic administration of quinalphos resulted in: decreased testicular mass and AChE activity in central as well as peripheral organs; increased serum LH, FSH, prolactin and testosterone concentrations; decreased pituitary or increased testicular ACE activity; severe disruption of spermatogenesis with increasing doses of pesticide; and no significant effects on dopamine, noradrenaline or 5-HT concentrations in the hypothalamus or pituitary. Administration of oestradiol (50 micrograms per rat a day) during pesticide treatment resulted in: a significant decrease in the mass of the testis and accessory sex organs; decreases in serum LH, FSH, testosterone concentrations; an increase in prolactin concentration; and a decrease in dopamine or an increase in noradrenaline and 5-HT in the hypothalamus or pituitary. Oestradiol had a marked effect: in pesticide-treated animals, the pesticide effects were significantly reversed. This indicates that in pesticide toxicity, the hypothalamo-pituitary-gonadal axis is operational. Since many of the observed pesticide effects could be inhibited by oestradiol, it is suggested that the pesticide acts directly on the gonadotrophins. In conclusion, quinalphos decreases fertility in adult male rats by affecting the pituitary gonadotrophins.