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Biomedical subjects

D L Keefe

Publications and source records attributed to D L Keefe.

At least 37 records · Page 2Linked to original sources
PubMed2000

Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

Animals↗
PubMed2000

Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos.

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

Animals↗
PubMed2000

Estrogen modifies the temperature effects of progesterone.

To test the hypothesis that progestin-mediated increases in resting core temperature and the core temperature threshold for sweating onset are counteracted by estrogen, we studied eight women (24 +/- 2 yr) at 27 degrees C rest, during 20 min of passive heating (35 degrees C), and during 40 min of exercise at 35 degrees C. Subjects were tested four times, during the early follicular and midluteal menstrual phases, after 4 wk of combined estradiol-norethindrone (progestin) oral contraceptive administration (OC E+P), and after 4 wk of progestin-only oral contraceptive administration (OC P). The order of the OC P and OC E+P were randomized. Baseline esophageal temperature (T(es)) at 27 degrees C was higher (P < 0.05) in the luteal phase (37.08 +/- 0.21 degrees C) and in OC P (37.60 +/- 0.31 degrees C) but not during OC E+P (37.04 +/- 0.23 degrees C) compared with the follicular phase (36.66 +/- 0.21 degrees C). T(es) remained above follicular phase levels throughout passive heating and exercise during OC P, whereas T(es) in the luteal phase was greater than in the follicular phase throughout exercise (P < 0.05). The T(es) threshold for sweating was also greater in the luteal phase (38.02 +/- 0.28 degrees C) and OC P (38.07 +/- 0.17 degrees C) compared with the follicular phase (37.32 +/- 0.11 degrees C) and OC E+P (37.46 +/- 0.18 degrees C). Progestin administration raised the T(es) threshold for sweating during OC P, but this effect was not present when estrogen was administered with progestin, suggesting that estrogen modifies progestin-related changes in temperature regulation. These data are also consistent with previous findings that estrogen lowers the thermoregulatory operating point.

Adult↗
PubMed2000

Cardiovascular emergencies in the cancer patient.

Cardiovascular emergencies in oncology patients include all of the usual cardiac problems, as well as complications of cancer and its therapy. Pericardial effusions and tamponade, cardiac masses, and extrinsic compression of the heart and great vessels by tumor masses, or fluid collections may all occur. Certain tumors may secrete mediators that are directly toxic to the heart; for example, catecholamines are secreted by pheochromocytomas and serotonin is secreted by carcinoid tumors. Tumors can also cause arrhythmias due to the mediators they secret or to direct mechanical irritation of the heart or pericardium. Cancer therapy is also associated with cardiac emergencies. Perioperative myocardial ischemia or infarction, as well as arrhythmias, may complicate surgery. Pericardial effusions and tamponade can follow surgery, radiation, or chemotherapy. Chemotherapy with anthracyclines, mitoxantrone, and trastuzumab may prompt acute and chronic heart failure. 5-Fluorouracil causes coronary spasm in some patients, leading to angina, myocardial infarction, arrhythmias, and/or sudden death. Cyclophosphamide, particularly in high doses, may produce acute myopericarditis. Radiation may cause acute pericardial disease and late sequelae such as myocardial infarction, acute valvular insufficiency, or effusive constrictive pericarditis. Endocarditis also occurs in cancer patients in association with vascular access devices and immune compromise. This review will discuss each of these complications of cancer and its therapy.

Antineoplastic Agents↗
PubMed1999

The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes.

OBJECTIVE: To evaluate how well polar body location predicts spindle localization and to examine spindle morphology. DESIGN: Randomized, controlled animal study. SETTING: University-affiliated research laboratory. ANIMAL(S): Mature, female golden hamsters. INTERVENTION(S): After superovulation with pregnant mare serum gonadotropin and hCG, metaphase II oocytes were obtained and imaged under digital polarization microscopy. MAIN OUTCOME MEASURE(S): Identify the meiotic spindle in living, unfixed hamster oocytes and determine spindle location relative to the polar body. RESULT(S): Spindles were imaged in 30 oocytes and only in 5 of them could the polar body predict the spindle localization. In the remaining oocytes, the spindles presented a random distribution within the cytoplasm. CONCLUSION(S): These data show that the polar body location is not an accurate predictor for meiotic spindle location in mammalian oocytes.

Animals↗
PubMed1999

Microinjection of mitochondria into zygotes creates a model for studying the inheritance of mitochondrial DNA during preimplantation development.

OBJECTIVE: To determine the effect of mutant mitochondria on preimplantation embryo development and of preimplantation embryo development on the survival of mutant mitochondrial DNA. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): None. INTERVENTION(S): Mutant and wild-type mitochondria, fractionated from tissue obtained from a patient with MELAS syndrome, a mitochondrial disease, were microinjected into mouse zygotes. Control zygotes received either no injection or sham injection. MAIN OUTCOME MEASURE(S): Preimplantation embryo development and survival of mutant mitochondrial DNA as determined by polymerase chain reaction analysis. RESULT(S): After microinjection into zygotes, the MELAS mutation could be identified by polymerase chain reaction until the hatched blastocyst stage of embryo development. The survival of MELAS-injected zygotes, observed for 4 days after injection, did not differ from the survival of zygotes injected with wild-type mitochondria or from the survival of uninjected or sham-injected controls. CONCLUSION(S): It appears that preimplantation embryo development does not screen out mitochondrial DNA mutations introduced into fertilized oocytes, and low levels of mutant mitochondrial DNA do not disrupt early embryo development.

Animals↗
PubMed1999

Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova.

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.

Adenosine Triphosphatases↗
PubMed1999

Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol.

The oxidation of cellular sulfhydryl (SH) groups has been implicated in the induction of apoptosis in various types of cells and in the disturbance of the meiotic spindle of murine oocytes during aging. The objective of this study was to determine whether the SH-specific oxidant diamide could inhibit embryo development and induce cell death, and whether the antioxidant dithiothreitol (DTT) could counteract such effects. Exposure of mouse zygotes to diamide for 3 h at 25 or 50 microM (but not 12.5 microM) resulted in cell cycle arrest and cell death with evidence of apoptosis. At higher concentrations (100 or 200 microM), diamide induced necrosis as evidenced by propidium iodide-positive pronuclei within 24 h of treatment. Simultaneous addition of DTT at equimolar concentration prevented these effects. However, when DTT was added later, it was no longer protective. DTT also effectively protected against the thiol-oxidative damage caused by diamide in blastocysts. These results suggest that altering thiol-redox status in zygotes and blastocysts may result in cell cycle arrest, apoptosis, and/or cell death.

Animals↗
PubMed1999

Hormone replacement therapy may alleviate sleep apnea in menopausal women: a pilot study.

OBJECTIVE: The incidence of sleep apnea syndrome (SAS) in women increases after menopause. Progestins alone do not alleviate SAS in menopausal women. However, progestins may require concomitant estrogen administration and estrogen alone may stimulate breathing during sleep. To test these hypotheses, we studied the effects of estrogen alone and estrogen combined with progestin on SAS in menopausal women, using a prospective, cross-over, inception cohort study. DESIGN: In this pilot study, five women who developed SAS after menopause underwent 2 nights of polysomnography to obtain a baseline, then returned for polysomnography after 3-4 weeks of taking micronized 17 beta-estradiol (E2) and after 10-12 days of taking E2 combined with medroxyprogesterone acetate (E2 + P). Sleep stages were scored according to Rechtshaffen and Kales, frequency and length of apneas were recorded for each subject each night, and the data were analyzed by Student's t test. RESULTS: E2 and E2 + P both reduced the Respiratory Distress Index. E2 also raised the lowest oxygen desaturation associated with apneic episodes. Total minutes of rapid eye movement sleep increased, and the number of waking episodes decreased when the women were taking E2 and E2 + P, as previously reported. CONCLUSIONS: Within 1 month after initiating E2 or E2 + P, SAS was reduced in all patients. The Respiratory Distress Index decreased by 25%, and the addition of progestin brought the SAS reduction to 50% in this pilot study. A randomized study in a large group of patients is justified by the findings of this study. Because SAS increases the risk of cardiovascular disease and fatal accidents, the amelioration of SAS by sex steroid hormones could have significant implications for the health of menopausal women.

Aged↗
PubMed1999

Physiological variability of fluid-regulation hormones in young women.

We tested the physiological reliability of plasma renin activity (PRA) and plasma concentrations of arginine vasopressin (P[AVP]), aldosterone (P[ALD]), and atrial natriuretic peptide (P[ANP]) in the early follicular phase and midluteal phases over the course of two menstrual cycles (n = 9 women, ages 25 +/- 1 yr). The reliability (Cronbach's alpha >/=0.80) of these hormones within a given phase of the cycle was tested 1) at rest, 2) after 2.5 h of dehydrating exercise, and 3) during a rehydration period. The mean hormone concentrations were similar within both the early follicular and midluteal phase tests; and the mean concentrations of P[ALD] and PRA for the three test conditions were significantly greater during the midluteal compared with the early follicular phase. Although Cronbach's alpha for resting and recovery P[ANP] were high (0.80 and 0.87, respectively), the resting and rehydration values for P[AVP], P[ALD], and PRA were variable between trials for the follicular (alpha from 0.49 to 0.55) and the luteal phase (alpha from 0.25 to 0. 66). Physiological reliability was better after dehydration for P[AVP] and PRA but remained low for P[ALD]. Although resting and recovery P[AVP], P[ALD], and PRA were not consistent within a given menstrual phase, the differences in the concentrations of these hormones between the different menstrual phases far exceeded the variability within the phases, indicating that the low within-phase reliability does not prevent the detection of menstrual phase-related differences in these hormonal variables.

Adult↗
PubMed1999

Effects of oral contraceptives on body fluid regulation.

To test the hypothesis that estrogen reduces the operating point for osmoregulation of arginine vasopressin (AVP), thirst, and body water balance, we studied nine women (25 +/- 1 yr) during 150 min of dehydrating exercise followed by 180 min of ad libitum rehydration. Subjects were tested six different times, during the early-follicular (twice) and midluteal (twice) menstrual phases and after 4 wk of combined [estradiol-norethindrone (progestin), OC E + P] and 4 wk of norethindrone (progestin only, OC P) oral contraceptive administration, in a randomized crossover design. Basal plasma osmolality (P(osm)) was lower in the luteal phase (281 +/- 1 mosmol/kgH(2)O, combined means, P < 0.05), OC E + P (281 +/- 1 mosmol/kgH(2)O, P < 0.05), and OC P (282 +/- 1 mosmol/kgH(2)O, P < 0. 05) than in the follicular phase (286 +/- 1 mosmol/kgH(2)O, combined means). High plasma estradiol concentration lowered the P(osm) threshold for AVP release during the luteal phase and during OC E + P [x-intercepts, 282 +/- 2, 278 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O, for follicular, luteal (combined means), OC E + P, and OC P, respectively; P < 0.05, luteal phase and OC E + P vs. follicular phase] during exercise dehydration, and 17beta-estradiol administration lowered the P(osm) threshold for thirst stimulation [x-intercepts, 280 +/- 2, 279 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O for follicular, luteal, OC E + P, and OC P, respectively; P < 0.05, OC E + P vs. follicular phase], without affecting body fluid balance. When plasma 17beta-estradiol concentration was high, P(osm) was low throughout rest, exercise, and rehydration, but plasma arginine vasopressin concentration, thirst, and body fluid retention were unchanged, indicating a lowering of the osmotic operating point for body fluid regulation.

Adult↗
PubMed1998

Do attitudes toward disclosure in donor oocyte recipients predict the use of anonymous versus directed donation?

OBJECTIVE: To compare the demographic and psychological characteristics of oocyte recipients and to determine whether the issue of disclosure about the use of a donor is a correlate of the decision to use an anonymous or directed donor. DESIGN: Cross-sectional study. SETTING: University teaching hospital. PATIENT(S): Ninety consecutive recipients of donated oocytes (64 of whom used anonymous donors and 26 of whom used directed donors). INTERVENTION(S): Pretreatment psychosocial evaluation. MAIN OUTCOME MEASURE(S): Recipient opinions and attitudes regarding the choice of donor type and disclosure to others as determined through a semistructured interview. RESULT(S): There were no statistically significant differences with regard to demographic characteristics between recipients who used anonymous and directed donors. There were statistically significant differences between the groups with regard to the issue of disclosure. Recipients who used directed donors were more likely to have told others about using an oocyte donor and were more likely to indicate that they intended to inform the child about the nature of his or her conception. CONCLUSION(S): Oocyte recipients who use known donors differ significantly from those who use anonymous donors with regard to the issue of disclosure to others. Further studies are needed to determine the causal direction of this relation.

Adult↗
PubMed1997

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

Calcimycin↗